Acute Kidney Injury and Chronic Kidney Disease
Transcript of Acute Kidney Injury and Chronic Kidney Disease
University of South FloridaScholar Commons
Graduate Theses and Dissertations Graduate School
4-4-2017
Acute Kidney Injury and Chronic Kidney DiseaseJin WeiUniversity of South Florida, [email protected]
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Scholar Commons CitationWei, Jin, "Acute Kidney Injury and Chronic Kidney Disease" (2017). Graduate Theses and Dissertations.http://scholarcommons.usf.edu/etd/6780
Acute Kidney Injury and Chronic Kidney Disease
by
Jin Wei
A dissertation submitted in partial fulfillment of the requirements for the degree of
Doctor of Philosophy Department of Molecular Pharmacology and Physiology
College of Medicine University of South Florida
Major Professor: Ruisheng Liu, M.D., Ph.D. Byeong Jake Cha, Ph.D.
Javier Cuevas, Ph.D. Alfredo M Peguero-Rivera, M.D.
Daniel Kay-Pong Yip, Ph.D.
Date of Approval: March 30, 2017
Keywords: Acute Kidney Injury, Ischemia Reperfusion Injury, Chronic Kidney Disease, Renal Hemodynamic
Copyright © 2017, Jin Wei
ACKNOWLEDGMENTS
First of all, I would like to thank my mentor, Dr. Ruisheng Liu, for your support,
training and encouragement all the time. Thanks for guiding me into the field of renal
physiology and providing me the opportunity to foster my career in medical research. I
would also like to thank my committee members, Dr. Kay-Pong Yip, Dr. Javier Cuevas,
Dr. Byeong Cha and Dr. Alfredo Peguero-Rivera for their advice and support through
these years. I would also like to offer my thanks to my external examiner Dr. William
Welch, for accepting to chair my defense and for his constructive criticism and
comments on my dissertation.
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TABLE OF CONTENTS
List of Figures .................................................................................................................. iii
Chapter One: Introduction ............................................................................................... 1 Ischemia reperfusion injury induced acute kidney injury ....................................... 1
Renal hemodynamics in acute kidney injury .............................................. 4 Warm and cold renal ischemia ................................................................... 9
Renal autoregulation and tubuloglomerular feedback .............................. 10 Renal infarction ................................................................................................... 15
Renal infarction and hypertension ........................................................... 16
Renal infarction and chronic kidney disease ............................................ 17 Hypothesis .......................................................................................................... 19
General Hypothesis ................................................................................. 19
Chapter Two: Ischemia Reperfusion Injury Induced Acute Kidney Injury ...................... 22
Project I: the role of intratubular pressure during the ischemic phase in acute kidney injury .................................................................................................. 22
Abstract .................................................................................................... 22
Introduction .............................................................................................. 23
Methods ................................................................................................... 25
Results ..................................................................................................... 28 Discussion ............................................................................................... 36
Project II: the role of tubuloglomerular feedback in ischemia reperfusion injury induced acute kidney injury ........................................................................... 40
Introduction .............................................................................................. 40
Methods ................................................................................................... 42 Results ..................................................................................................... 47
Discussion ............................................................................................... 58 Project III: the role of renal temperature during the ischemic phase in acute
kidney injury .................................................................................................. 64
Abstract .................................................................................................... 64
Introduction .............................................................................................. 65 Methods ................................................................................................... 66 Results ..................................................................................................... 70
Discussion ............................................................................................... 75
Chapter Three: Renal Infarction Induced Chronic Kidney Disease ............................... 78 Project I: renal infarction and hypertension ........................................................ 78
Abstract .................................................................................................... 78 Introduction .............................................................................................. 79
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Methods ................................................................................................... 80
Results ..................................................................................................... 84 Discussion ............................................................................................... 96
Project II: renal infarction and chronic kidney disease ...................................... 100 Introduction ............................................................................................ 100 Methods ................................................................................................. 101 Results ................................................................................................... 106 Discussion ............................................................................................. 114
Chapter Four: Discussion ............................................................................................ 120
The role of intratubular pressure in acute kidney injury .................................... 120 The role of renal temperature in acute kidney injury ......................................... 124 The role of tubuloglomerular feedback in acute kidney injury ........................... 126
Renal infarction and hypertension .................................................................... 131 Renal infarction and chronic kidney disease .................................................... 135
Summary .......................................................................................................... 141
References .................................................................................................................. 142
Appendix: List of Abbreviation ..................................................................................... 171
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List of Figures
Figure 1. Ischemia renal disease. ................................................................................. 20
Figure 2. Proximal tubular pressure. ............................................................................. 29
Figure 3. Kidney injury markers after renal ischemia..................................................... 30
Figure 4. Histology. ....................................................................................................... 32
Figure 5. Proximal tubular pressure. ............................................................................. 33
Figure 6. Kidney injury markers after renal ischemia..................................................... 34
Figure 7. Histology. ....................................................................................................... 35
Figure 8. NOS1β protein level. ...................................................................................... 48
Figure 9. TGF response in vivo. .................................................................................... 48
Figure 10. GFR after IRI. ............................................................................................... 49
Figure 11. Plasma creatinine after IRI. .......................................................................... 50
Figure 12. NOS1 activity. .............................................................................................. 51
Figure 13. TGF response in response to acute tubular pH changes. ............................ 52
Figure 14. Urine pH. ...................................................................................................... 53
Figure 15. The effect of bicarbonate on NOS1β expression.......................................... 54
Figure 16. TGF response in vivo after high bicarbonate intake. .................................... 55
Figure 17. GFR in conscious mice after high bicarbonate intake. ................................. 55
Figure 18. NOS1β expression after high bicarbonate intake in IRI mice. ...................... 56
Figure 19. GFR in conscious mice after high bicarbonate intake in IRI mice. ............... 57
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Figure 20. Plasma creatinine after high bicarbonate intake in IRI mice. ........................ 58
Figure 21. Typical images of the cold ischemia procedure. ........................................... 68
Figure 22. Renal injury markers after cold IRI. .............................................................. 71
Figure 23. GFR in conscious mice. ............................................................................... 72
Figure 24. Histology. ..................................................................................................... 74
Figure 25. Images for renal artery. ................................................................................ 83
Figure 26. Vascular architecture of the kidney. ............................................................. 87
Figure 27. MAP after inducing renal infarction. ............................................................. 89
Figure 28. HR after inducing renal infarction. ................................................................ 90
Figure 29. PRC after inducing renal infarction. .............................................................. 90
Figure 30. Inflammatory factors mRNA level. ................................................................ 92
Figure 31. Plasma inflammatory factors. ....................................................................... 93
Figure 32. Kidney weight. .............................................................................................. 94
Figure 33. Histology. ..................................................................................................... 95
Figure 34. Vascular architecture of the kidney. ........................................................... 109
Figure 35. Plasma creatinine and GFR after surgery. ................................................. 110
Figure 36. MAP after surgery. ..................................................................................... 111
Figure 37. Inflammatory factors mRNA level. .............................................................. 112
Figure 38. Light microscopy and transmission electron microscopy ........................... 113
1
Chapter One: Introduction
Ischemia reperfusion injury induced acute kidney injury
Acute kidney injury (AKI) has been described as a rapid loss of renal function by
sudden and sustained decrease of glomerular filtration rate (GFR) with retention of
nitrogenous waste products (Bauerle et al. 2011, Mehta et al. 2007, Thadhani et al.
1996). AKI occurs in approximately 20% of hospital admissions and is increasingly
common in critically ill patients (Liangos et al. 2006, Uchino et al. 2005, Uchino et al.
2006). AKI is responsible for approximately 2 million deaths annually worldwide and
costs approximately $10 billion annually in the United States (Ali et al. 2007, Liangos et
al. 2006, Murugan & Kellum 2011, Uchino et al. 2005, Uchino et al. 2006). The classic
teaching regarding patients who survive an episode of AKI, in particular acute tubular
necrosis, was that those patients achieved full or nearly full recovery (FINKENSTAEDT
& Merrill 1956, Kjellstrand et al. 1981, LOWE 1952). AKI can cause end-stage renal
disease directly, and increase the risk of developing incident chronic kidney disease
(CKD) and worsening of underlying CKD. In addition, severity, duration, and frequency
of AKI appear to be important predictors of poor patient outcomes (Amdur et al. 2009,
Chawla et al. 2011, Chawla & Kimmel 2012, Garg et al. 2003, Ishani et al. 2009, Lo et
al. 2009, Wald et al. 2009). Patients with AKI are also more likely to experience
systemic morbidity, longer hospital length of stay, functional impairment, eventual
development of end stage kidney failure, and increased long-term mortality (Bihorac et
2
al. 2009, Coca et al. 2009, Hsu et al. 2009, Johansen et al. 2010, Levy et al. 1996, Liss
et al. 2006, Parikh et al. 2008, Wald et al. 2009). Even patients with transient AKI
experience worse long-term outcomes (Goldenberg et al. 2009, Hobson et al. 2009,
Welten et al. 2007). Even though, biomarkers of tubule injury, such as kidney injury
molecule–1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL), both being
present in the urine of animals and patients with AKI, have been found to be useful
noninvasive biomarkers of kidney injury (Bonventre et al. 2010, Han et al. 2002, Mishra
et al. 2005, Vaidya et al. 2008a, Vaidya et al. 2008b, Vaidya et al. 2010); however, at
present, no specific therapy to prevent or treat AKI or its complications exists (Chertow
et al. 2005, Faubel et al. 2012, Okusa et al. 2012).
Ischemia reperfusion injury (IRI) induced AKI is typically divided into initiation,
extension, maintenance and recovery phases. Severe injuries of thick ascending limb
(TAL) in the outer medulla and proximal tubular in renal cortex are uniform features in
various forms of AKI (Heyman et al. 1988, Lieberthal & Nigam 1998, Oken 1975,
Regner et al. 2009). Tubular obstruction with epithelial debris and backleak are
proposed to contribute to the initial phase for the reduction of GFR (Devarajan 2006,
Eltzschig & Eckle 2011, Glodowski & Wagener 2015).
Ischemia and reperfusion are unavoidable steps during kidney procurement and
transplantation. Transplanted organs experience several episodes of IRI, which can be
caused by both warm and cold ischemia. Warm ischemia occurs after cardiac death in
donors, or during the harvesting operation on living donors, and during anastomosis of
the graft. Cold ischemia occurs during allograft kidney storage in organ preservation
solutions (Homer-Vanniasinkam et al. 1997, Ploeg et al. 1988, Southard et al. 1985),
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which was invented in 1960’s. The invention of preservation solutions is the most
seminal step to avoid IRI, allowing for safe renal preservation exceeding 24 hours
(Collins et al. 1969, Wahlberg et al. 1987) : it remains the mainstay of preservation for
kidney allografts worldwide(O'Callaghan et al. 2012, Opelz & Dohler 2007).
Post-transplant renal injury has been studied extensively (Gulec et al. 2006, Land
1999, Massberg & Messmer 1998, Racusen et al. 1999, Sis et al. 2010). Prolonged
ischemia increases the occurrence of delayed graft function and worsens long-term
graft survival (Boom et al. 2000). IRI is one of the most important nonspecific factor
affecting both delayed and late allograft dysfunction (Halloran et al. 1988, Jassem &
Heaton 2004, Womer et al. 2000). RIFLE (risk, injury, failure, loss, end stage renal
disease) and acute kidney injury network criteria are the generally accepted definitions
of an AKI episode in native kidneys (Bellomo et al. 2004, Mehta et al. 2007), which are
largely based on the degrees of plasma creatinine elevation from baseline. There are no
official recommendations regarding specific diagnostic criteria for AKI in the kidney
transplant, primarily due to the difficulties in determination of the baseline creatinine
levels in graft recipients (Cavaille-Coll et al. 2013, Cooper & Wiseman 2013,
Kosieradzki & Rowinski 2008). The limited published studies of transplant AKI
epidemiology and outcomes have adopted the criteria for native kidneys (Mehrotra et al.
2012, Nakamura et al. 2012). IRI-induced AKI has been found crucial for transplanted
kidney survival. Recently AKI and graft survival of renal transplant recipients has been
analyzed in a large retrospective cohort study included 27,232 patients. Patients who
developed AKI had an increased risk of graft loss and an increased risk of mortality
(Mehrotra et al. 2012). IRI has been proposed to activate the immune system and
4
facilitate T cell interactions that possible leads to rejection (Daemen et al. 2002,
Pratschke et al. 2001), even though IRI is independent of the immunologic background
(Dragun et al. 2000).
If blood is returned to ischemic tissue before a critical time, organ function is
quickly restored (Dawidson et al. 1991). However, if ischemia lasts a longer time,
irreversible damage and even graft failure would occur. At present it is not possible to
prevent AKI during transplantation. The worldwide organ shortage has stimulated the
interest in research to minimize IRI. Post-transplant renal failure has been studied
extensively (Cavaille-Coll et al. 2013). Numerous clinical trials and analyses have been
conducted, various drug products have been tested in clinical trials in the peri-transplant
period. A few have demonstrated early improvements in graft function (Goggins et al.
2003, Rabl et al. 1993, Shilliday & Sherif 2004), but none have shown an improvement
in late graft function (Finn 1990, Fontana et al. 2005, Goggins et al. 2003, Hladunewich
et al. 2003, Lachance & Barry 1985, Martinez et al. 2010, Michael et al. 1989, Noel et
al. 1997, Rabl et al. 1993, Salmela et al. 1999, Shilliday & Sherif 2004).
Renal hemodynamics in acute kidney injury
In both humans and experimental animals, an intense and persistent renal
vasoconstriction that reduces overall kidney blood flow and GFR has long been
considered a hallmark of AKI (Lameire et al. 2005, Mason et al. 1977). It has been
reported that vascular response increased to vasoconstrictive agents, and decreased to
vasodilatory agents in arterioles taken from post-ischemic kidneys when compared with
those taken from normal kidneys (Conger & Weil 1995, Oken 1975, Oken 1984). Even
5
though direct data are still missing, tubuloglomerular feedback (TGF) responsiveness
has been postulated to contribute to the enhanced preglomerular vascular resistance
and reduction in GFR in AKI (Thurau & Boylan 1976). The commonly used AKI animal
models, which involve clamping renal arteries or pedicles, closely mimics the
mechanisms and futures of kidney transplantation (Cavaille-Coll et al. 2013).
Decreased GFR in AKI is generally believed to be the consequence of reduced
renal blood flow (RBF), but in AKI patients, RBF was found to have only a limited or no
detectable association with GFR, in a literature review of articles published from 1944-
2008 (Prowle et al. 2009, Prowle et al. 2010). The clinical trials utilizing vasodilators in
the AKI have largely failed, which included dopamine (Denton et al. 1996), fenoldopam
mesylate (Tumlin et al. 2005), atrial natriuretic peptide (Allgren et al. 1997), brain-type
natriuretic peptide (Mentzer, Jr. et al. 2007) and insulin growth factor-1 (Hirschberg et
al. 1999). Therefore, the pathophysiological mechanisms for IRI are complicated and
regulated by multiple factors that have not been fully clarified.
Severe injuries of the proximal tubular in renal cortex and TAL in the outer
medulla is a uniform feature in various forms of AKI while the glomeruli are
morphologically normal (Heyman et al. 1988, Lieberthal & Nigam 1998, Oken 1975,
Regner et al. 2009). Tubular injury can also lead to a decrease in GFR by tubular
obstruction with epithelial debris and backleak. However, normal pressures have been
observed in several tubules, making it unlikely for tubular obstruction to be the singularly
important mediator of reduction in GFR after ischemic injury (Conger et al. 1984).
Arendshorst et al. (Arendshorst et al. 1975) studied severe AKI after 1 h of unilateral
renal ischemia. They found that in the early stages (1-3 h) after ischemia, the pressures
6
in the proximal and distal tubules were elevated, and there was histological evidence of
tubular obstruction. Tubular obstruction appeared to be the predominant factor in the
decline in filtration rate at this stage. However, at a later time-point (22-26 h), tubular
pressures were normal, but preglomerular vasoconstriction was marked and this was
thought to contribute to the persistent decline in filtration rate. In addition, tubular
obstruction does not appear to be a significant pathogenic mechanism in AKI in
humans, in which only about 10% decrease in GFR could be explained by tubular
backleak (Blantz & Singh 2011, Braam et al. 1993, Myers et al. 1979, Schrier et al.
2004). A single nephron model of nephrotoxic tubular injury was established by
microperfusion of uranyl nitrate into the early proximal tubule (Peterson et al. 1989).
Within minutes of intratubular administration of uranyl nitrate, a 16-30% reduction in
single nephron glomerular filtration rate (SNGFR) was observed. The local mechanism
such as TGF could be invoked to explain the immediate decline in SNGFR. Similar
findings have been observed in the uranyl nitrate model of AKI by another group
(Flamenbaum et al. 1974).
Renal microvasculature plays a major role in maintaining hemodynamics and
tubular functions. Loss of peritubular capillaries has been found to correlate with CKD in
both animal models (Basile 2007, Kang et al. 2001, Kang et al. 2002, Ohashi et al.
2002, Yuan et al. 2003) and patients (Bohle et al. 1996, Kaukinen et al. 2009,
Lindenmeyer et al. 2007). In transplanted kidneys, fewer peritubular capillaries are
associated with the development of interstitial fibrosis and graft dysfunction (Cooper &
Wiseman 2013, Ishii et al. 2005, Snoeijs et al. 2011), which is a major cause of renal
graft loss (Nankivell et al. 2003, Perico et al. 2004). A recent clinical study found that
7
peritubular capillary loss occurs during the first 3 months after renal transplantation,
which associates with increased interstitial fibrosis and tubular atrophy and predicts
reduced graft function (Steegh et al. 2011). The densities of peritubular capillaries have
also been found to decrease with aging in animals (Kang et al. 2002). Kidneys
transplanted from older donors have shorter half-life graft survival times (de Fijter et al.
2001, Snoeijs et al. 2008), suggesting that fewer peritubular capillaries or impaired
angiogenesis may contribute to the decreased graft function. Microvasculature injuries
enhance vasoactive factors such as Ang II, adenosine and renal sympathetic nerves
and inhibit vaso-dilatation factors such as nitric oxide (NO), prostaglandin and
dopamine, thereby promoting vascular constriction and enhancing local ischemia
(Johnson & Schreiner 1997, Sanchez-Lozada et al. 2003). Therefore, peritubular
capillary injury can potentially cause local hypoxia, chronic ischemia and interstitial
inflammation, which promotes further interstitial and capillary injury, leading to interstitial
fibrosis (Aird 2003, Eddy 2000, Granger & Kubes 1994, Imhof & Dunon 1995, Kang et
al. 2002, Keller et al. 2003, Nangaku 2004, Robson et al. 1995, Sanchez-Lozada et al.
2003).
Elevations of glomerular capillary pressure have been considered a major risk
factor and play an essential role in development of CKD and diabetic nephropathy
(Anderson & Brenner 1986, Anderson & Vora 1995, Bohle et al. 1996, Dunn et al. 1986,
Johnson & Schreiner 1997, Sanchez-Lozada et al. 2003, Zatz et al. 1986). Glomerular
capillary pressure has also been measured in chronic rejection animal models (Junaid
et al. 1994, Kingma et al. 1993, Ziai et al. 2000). In transplanted kidneys with chronic
rejection, the capillary pressure has been found to be significant higher compared with
8
animals without rejection. Other studies evaluated the nephron mass and graft function.
They found that nephron mass is a major determinant for intraglomerular capillary
pressure and graft function of the transplanted kidneys (Mackenzie et al. 1994,
Mackenzie et al. 1995). Even though glomerular capillary pressure cannot be measured
directly in human kidneys, the results from several clinical observations demonstrate a
possible connection between chronic rejection and glomerular hypertension, by
evaluation of potential risk factors that could potentially raise glomerular pressure, such
as nephron mass, body weight, GFR and protein diet (Barrientos et al. 1994, Brenner et
al. 1992, Feldman et al. 1996, Pirsch et al. 1995, Salahudeen 2003, Terasaki et al.
1994). These data from both animal experiments and clinical observations support the
hypothesis that glomerular capillary pressure is a risk factor for chronic rejection and
graft function.
Few studies have evaluated the relationship between pressure and tubular cell
injury. Increasing intra-tubular pressure and tubular stretch upregulates transforming
growth factor β-1 (TGFβ-1), which is considered to contribute to tubulointerstitial
inflammation and fibrosis in the unilateral ureteral obstruction model (Quinlan et al.
2008, Sato et al. 2003). Cyclical stretch on renal epithelial cells has been reported to
induce apoptosis by activating JNK/SAPK and p38 SAPK-2 pathways (Nguyen et al.
2006). In addition, renal vein occlusion results in more severe tubular injury and higher
levels of plasma creatinine than renal arterial occlusion in C57BL/6J mice (Li et al.
2012). In patients with chronic heart failure, venous congestion was found to be an
important determinant of kidney function as measured by GFR (Damman et al. 2007).
9
These studies also suggest that higher tubular pressure might contribute to kidney
injury. However, the role of intra-tubular pressure in the development of AKI is unknown.
Warm and cold renal ischemia
IRI-induced AKI, which has been extensively studied, closely mimics IRI in
kidney transplantation in humans (Cavaille-Coll et al. 2013), even though no single
animal model duplicates human disease. Warm ischemia models achieved graded
responses by varying experimental conditions such as temperature, duration of
clamping and site of clamping, such as bilateral, unilateral, artery, vein or pedicle
clamping (Burne-Taney et al. 2003, Chen et al. 2007, Jang & Rabb 2015, Khajuria et al.
2014, Kinsey et al. 2008, Malek & Nematbakhsh 2015).
The invention of organ preservation solutions has enormously protected graft
damage induced by cold ischemia and made it possible to safely preserve renal graft for
over 24 hours (Collins et al. 1969, Homer-Vanniasinkam et al. 1997, Ploeg et al. 1988,
Southard et al. 1985, Wahlberg et al. 1987). However, transplanted kidneys usually
undergo both cold and warm ischemia. Even though cold ischemia has been studied
extensively for a long time, all of the studies that specifically focused on cold ischemia
have been carried out in vitro and ex vivo models. There is no available in vivo model to
examine IRI induced only by cold ischemia (Ishitsuka et al. 2013, O'Callaghan et al.
2012, Salahudeen 2004, Sorensen et al. 2011, Turkmen et al. 2011).
10
Renal autoregulation and tubuloglomerular feedback
Renal autoregulation is an intra-renal mechanism that maintains RBF and GFR
relatively constant independent of systemic blood pressure over a range about 80-180
mmHg. Renal autoregulation is a vital mechanism that maintains normal kidney
clearance function and protects the kidney from elevation in arterial pressure, which
causes kidney injury. The two highly coordinated mechanisms that largely mediate the
renal autoregulation are myogenic and tubuloglomerular feedback (TGF) responses,
which regulate preglomerular vasotone primarily of the afferent arteriole (Af-Art) (Burke
et al. 2014, Carlstrom et al. 2015, Hall 2015). The myogenic response of the Af-Art
responds rapidly, usually occurring within 1-2 secs following changes in arterial
pressure. In contrast, TGF acts more slowly: usually the response delay is 10-30 secs
following changes in arterial pressure (Abu-Amarah et al. 2005, Chon et al. 1993,
Cupples et al. 1996, Daniels & Arendshorst 1990, Just 2007, Pires et al. 2002).
The myogenic response is an intrinsic property of the vascular smooth muscle
cells in small arteries that constrict in response to elevations in transmural pressure.
The myogenic response varies greatly among different vascular beds. The renal
vasculature has the fastest and most complete response compare to other organs
(Carlstrom et al. 2015). Af-Arts are believed to be the major resistant vessels in kidneys
and therefore expected to largely mediate the myogenic response in the kidney
(Carmines 2010).
The existence of the TGF system was predicted in the 1930s by Goormaghtigh
(Goormaghtigh N. 1937) and Zimmerman (Zimmerman 1933). Major advances in our
understanding of the TGF system have been made using in vivo renal micropuncture
11
and in vitro microperfusion of isolated juxtaglomerular apparatus (JGA) (Ito & Carretero
1991, Liu et al. 2002, Moore et al. 1979, Navar 1978, Navar et al. 1996, Ploth et al.
1979, Schnermann et al. 1976a, Schnermann & Briggs 1999, Thurau & Schnermann
1965, Wilcox & Welch 1996). TGF response operates within each nephron principally
through the JGA and renders the single nephron GFR inversely dependent on the
tubular flow rate past the macula densa. The macula densa is a group of specialized
epithelial cells located at the distal segment of the TAL; it serves as a sensor of luminal
NaCl concentration. Increases of NaCl delivery to the macula densa promotes release
of adenosine and/or ATP, which constricts Af-Art and decreases SNGFR: a process
called the TGF response. It establishes a negative feedback by which a change in
tubular flow to the macula densa induces a reciprocal change in SNGFR, thus
preventing acute fluctuations of flow and NaCl delivery in the distal nephron (Briggs &
Schnermann 1986, Ollerstam et al. 1997, Ren et al. 2000b, Schnermann & Briggs 1992,
Welch et al. 2000, Welch & Wilcox 1990). TGF is regulated by various factors including
Ang II (Ren et al. 2002c, Welch & Wilcox 1990), adenosine (Ren et al. 2002a, Sun et al.
2001), arachidonic acid metabolites (Kurtz et al. 1986, Ren et al. 2000a), ATP (Inscho
et al. 1992, Ren et al. 2004), atrial natriuretic factor (Huang & Cogan 1987), superoxide
(Liu et al. 2004b, Ren et al. 2002b, Welch et al. 2000) and NO (Ito et al. 1993, Liu et al.
2004b, Schnermann 1998, Welch & Wilcox 1997). NO is synthesized by 3 isoforms of
NOS: Neuronal NOS (nNOS or NOS1), inducible NOS (iNOS or NOS2), and endothelial
NOS (eNOS or NOS3), all of which are expressed in the kidney. While NOS2 is
calcium-insensitive isoform, NOS1 and NOS3 are constitutive enzymes and sensitive to
calcium (Lamattina et al. 2003). NOS1 is a predominant isoform expressed in macula
12
densa cells (Mundel et al. 1992, Wilcox et al. 1992). NO generated by the macula densa
inhibits TGF response (Ito et al. 1993, Liu et al. 2004b, Schnermann 1998, Welch &
Wilcox 1997). We have recently published several studies that significantly advance the
understanding of these mechanisms. We showed that macula densa cells in the kidney
express α, β, and γ splice variants of neuronal nitric oxide synthase (NOS1) (Lu et al.
2010). NOS1β is the primary splice variant and contributes to most of the NO
generation by the macula densa in rodents. Mice with NOS1 deleted specifically from
the macula densa develop salt-sensitive hypertension, associated with enhanced TGF
responsiveness and attenuation of increases of GFR.
Renal autoregulation can be analyzed by different in vivo and in vitro methods: 1)
RBF measurement and analysis: RBF of whole kidney or in different regions of the
kidney is measured while renal arterial pressure is adjusted to evaluate autoregulatory
response. Renal arterial pressure is adjusted to step reduction by constriction of the
suprarenal aorta or increase by collusion of the mesenteric and/or celiac arteries
(Arendshorst 1979, Mattson et al. 1993, Roman et al. 1988). Renal autoregulation can
also be studied by dynamic analysis of RBF by measuring time and frequency domains,
which can separate myogenic and TGF response components (Cupples & Loutzenhiser
1998, Daniels et al. 1990, Holstein-Rathlou & Marsh 1994, Holstein-Rathlou et al. 1991,
Just 2007); 2) TGF and myogenic response measurement: TGF response can be
measured in isolated perfused JGAs in vitro (Ito et al. 1989, Kirk et al. 1985) and with
micropuncture in vivo (Bell et al. 1984, Schnermann et al. 1976b). Myogenic response
can be measure in isolated perfused Af-Arts (Edwards 1983, Ito et al. 1989), in the
13
juxtamedullary glomerular preparations (Casellas & Navar 1984, Moore & Casellas
1990) and in the hydronephrotic kidney (Hayashi et al. 1989).
Urine pH in healthy individuals fluctuates over the entire physiological range of
values from 4.5 -8.5 (Bilobrov et al. 1990). Urine pH in each individual demonstrates
marked variations throughout the day, but with patterns. A nocturnal peak in urine pH
was usually seen around 22:00. Throughout the night, urine pH decreased to a nadir
around 06:00 before rising again in the morning (Ayres et al. 1977, Barnett & Blume
1938, Cameron et al. 2012, ELLIOT et al. 1959, Jones 1843, Kanabrocki et al. 1988,
MILLS & STANBURY 1951, STANBURY & THOMSON 1951). Diet can markedly
affect acid-base status. Dietary acid load has been found to be associated with serum
bicarbonate levels and urine pH (Gunn et al. 2013, Ikizler et al. 2015, Remer & Manz
1995). The European Prospective Investigation into Cancer and Nutrition study
analyzed 22,034 men and women and compared casual urine samples with dietary acid
load. They found that dietary acid-base load was significantly related to urine pH.
Casual urine pH provides a simple tangible measure of dietary acid load (Welch et al.
2008). A recent study reported that urine pH is sensitive to reflect dietary acid-base load
and reach a steady state in about 3 days (Kanbara et al. 2012).
Current guidelines for CKD patients recommend alkali treatment with sodium
citrate or sodium bicarbonate for those with serum total carbon dioxide (CO2) less than
22 mM. Indeed, the correlation between low serum bicarbonate level and progressive
decline in estimated GFR (eGFR) has been studied extensively in patients with CKD.
Alkali therapy reduces kidney injury and slows nephropathy progression in patients with
CKD (de Brito-Ashurst et al. 2009, Dobre et al. 2015, Eustace et al. 2004, Goraya et al.
14
2012, Kovesdy et al. 2009, Mahajan et al. 2010, Navaneethan et al. 2011, Phisitkul et
al. 2010, Raphael et al. 2011, Shah et al. 2009, Susantitaphong et al. 2012), but the
mechanism is not clear.
Diet can markedly affect acid-base status and it significantly influences CKD and
its progression. In the National Health and Nutrition Examination Survey, 11,957 adults
were analyzed and found that high serum anion gap values and decreased serum total
CO2 concentration were associated with increased mortality in the general population
and with a decline in eGFR in patients with CKD (Abramowitz et al. 2012). In a separate
study, the correlation between dietary acid load and eGFR were assessed in 12,293 US
adults. High dietary acid load was found to be associated with albuminuria and low
eGFR (Banerjee et al. 2014). A cross-sectional study of the African American Study of
Kidney Disease and Hypertension (AASK) cohort indicate that a higher acid load and
lower total serum CO2 concentration (reflects serum bicarbonate concentration)
correlate with more severe CKD and readily decline of eGFR (Scialla et al. 2011).
Oral bicarbonate supplementation slows CKD progression in humans (de Brito-
Ashurst et al. 2009). Recent two intervention studies, one in patients with early CKD
without metabolic acidosis (Goraya et al. 2012), and the other in patients with advanced
CKD and metabolic acidosis (Goraya et al. 2013), suggest that a diet rich in fruits and
vegetables can improve renal function and slow the progression of CKD. In patients at
risk of CKD, alkali-generating diets that are rich in fruits and vegetables reduce the risk
of developing CKD (Chang et al. 2013, Dunkler et al. 2013, Lin et al. 2011, Nettleton et
al. 2008, Scialla et al. 2011). Oral bicarbonate supplementation has been found to delay
the decline of GFR in CKD patients (Dobre et al. 2015, Eustace et al. 2004, Kovesdy et
15
al. 2009, Navaneethan et al. 2011, Raphael et al. 2011, Shah et al. 2009,
Susantitaphong et al. 2012), but the mechanism is not clear. Recently, the relationship
between serum bicarbonate and GFR has been analyzed in people with normal renal
functions. Lower serum bicarbonate concentrations are found to be associated
independently with rapid decline of eGFR in a study of 5422 adults; in a Multi-Ethnic
Study of Atherosclerosis of 5810 participants (Driver et al. 2014), and in a Health,
Aging, and Body Composition study (Goldenstein et al. 2014). The baseline eGFR of
most or all of the participants in these studies is above 60 mL/min/1.73 m2.
Dietary acid load has also been found an important determinant of kidney injury
in rat models of CKD with and without metabolic acidosis (Khanna et al. 2004, Mahajan
et al. 2010, Nath et al. 1985, Phisitkul et al. 2010, Wesson et al. 2006, Wesson &
Simoni 2010).
In summary, most of the available data suggest that alkali intervention by diets
rich in fruits and vegetables or oral bicarbonate supplementation improve renal function
and slow decline in eGFR in normal population and patients with CKD with and without
metabolic acidosis. Urine pH reflects dietary acid-base load and plasma bicarbonate
levels.
Renal infarction
Renal infarction, characterized with an interruption of blood supply to a portion of
or the whole kidney, could result in irreversible kidney damage and subsequent necrosis
of renal parenchyma along with a decline in kidney function (Bourgault et al. 2013,
Eltawansy et al. 2014). The incidence of renal infarction was reported at approximate
16
0.003 to 0.007% in previous clinical studies (Domanovits et al. 1999, Huang et al. 2007,
Korzets et al. 2002, Paris et al. 2006). However, due to a lack of specific clinical
manifestation, renal infarction is frequently underdiagnosed and its incidence is largely
underestimated. According to the results of a retrospective postmortem study, 205 renal
infarction cases were diagnosed in a total of 14411 autopsies, indicating an actual
incidence could be around 1.4%.
Renal infarction and hypertension
The epidemiology of renal infarction is complex and has not been completely
understood. Various risk factors, including arteriosclerosis, atrial fibrillation (Antopolsky
et al. 2012a, Frost et al. 2001, Saeed 2012), hypercoagulation (Antopolsky et al. 2012a,
Saeed 2012) and endocarditis (Antopolsky et al. 2012a, Lumerman et al. 1999, Wong et
al. 1984) are involved with renal infarction. Renal thromboembolism originated from the
heart or aorta is recognized as the primary cause of renal infarction (Eltawansy et al.
2014, Korzets et al. 2002, Saeed 2012). Despite of a few case reports with normal
blood pressure (Alamir et al. 1997, Javaid et al. 2009), hypertension is commonly
observed in renal infarction. Based on the outcomes of a retrospective cohort study,
hypertension was identified in 48% of the renal infarction patients (Bourgault et al.
2013). Another retrospective clinical study also indicated that renal infarction resulted in
acute or aggravated hypertension in previous normotensive or hypertensive patients.
The renal infarction-induced hypertension was usually ameliorated following surgery or
renal artery angioplasty (Paris et al. 2006). However, the pathophysiological changes in
17
renal infarction and the mechanisms of renal infarction induced hypertension have not
been fully understood. In addition, mouse models of partial renal infarction are lacking.
Renal infarction and chronic kidney disease
CKD is characterized by a progressive loss in kidney function and could
eventually develop into end stage renal disease (ESRD). CKD is a major health issue in
the United States with increasing morbidity and significant costs. Based on the National
Kidney Foundation Kidney Disease Outcome Quality Initiative clinical practice
guidelines for CKD, the prevalence of CKD in the U.S. is approximate 13.1% and the
estimated population of ESRD is over 0.7 million by 2015 (Coresh et al. 2007,
Gilbertson et al. 2005). The risk factors for CKD are diverse and the epidemiology is
usually multiplicate. Currently, diabetes mellitus and high blood pressure have been
recognized as the two primary causes of CKD (Islam et al. 2009, Levey et al. 2010).
Despite of extensive investigation, the pathophysiological mechanisms of CKD have not
been completed elucidated. A better understanding of CKD might provide potential
therapeutic targets and advance the clinical strategies to prevent or attenuate its
progression.
Various animal models have been developed to study CKD. Five-sixths renal
mass reduction, produced by uninephrectomy plus 2/3 renal parenchyma reduction on
the contralateral kidney, is a broadly used model in rat to mimics the CKD secondary to
reduced nephron number, in which kidney function gradually decrease accompanied
with progressive proteinuria and glomerulosclerosis (Purkerson et al. 1976, Shimamura
& Morrison 1975, Yang et al. 2010). Moreover, compared with renal mass reduction
18
through surgical excision of both upper and lower poles, the approach via ligation in two
of the three renal artery branches resulted in higher blood pressure and more severe
renal injury. Unlikely to the model of rat, the development of progressive CKD following
5/6 nephrectomy in mice is highly strain-dependent. C57BL/6 mouse, the most
commonly used laboratory rodent, is relatively blood pressure and renal injury resistant
to five-sixths nephrectomy by uninephrectomy combined with the resection of 2/3
contralateral kidney. Addition of angiotensin II administration is the only strategy so far
to overcome the resistance of CKD development in 5/6 nephrectomy model of C57BL/6
mouse (Leelahavanichkul et al. 2010).
Five-sixths nephrectomy, produced by uninephrectomy and surgical removal of
2/3 contralateral kidney, is a well-established and widely used CKD model in various
kinds of laboratory animals, such as dog (Bourgoignie et al. 1987, COBURN et al. 1965,
Robertson et al. 1986), rabbit (Eddy et al. 1986, Kilicarslan et al. 2003) and rat (Griffin et
al. 1994, Laouari et al. 1997, Shimamura & Morrison 1975) with the progressive
reduction in kidney function and the development of renal injury, which mimics the
feature of CKD in human. However, it has been recognized that the susceptibility to
develop CKD following 5/6 nephrectomy is strain or genetic background dependent in
mouse. A recent study by Leelahavanichkul et al. showed that progressive CKD, as
indicated by the gradual decrease in GFR, the reciprocal changes in blood urea
nitrogen and serum creatinine, severe albuminuria and mesangial expansion in
glomeruli, was developed by 4 weeks in CD-1 mice and by 12 weeks in 129S3 mice
after 5/6 nephrectomy in which the upper and lower poles of the left kidney were
resected and the whole right kidney was removed. In addition, conscious blood
19
pressure measured by radiotelemetry was significantly elevated in both CD-1 mice and
129S3 mice following 5/6 nephrectomy. In contrast, C57BL/6 mouse model with 5/6
nephrectomy exhibited normal blood pressure and was relatively resistant to renal injury
in the remnant kidney (Leelahavanichkul et al. 2010). C57BL/6 mouse is the most
popular laboratory rodent with its advantage of stability in physiological characteristics
and heredity. Furthermore, C57BL/6 is first strain of mouse that had its genome
completely sequenced and the most broadly utilized strain as background in the
generation of genetically modified mouse. Thus, to develop animal model in C57BL/6
mouse is of greater significance. Besides nephrectomy, other approaches, such as
ligation of kidney poles (Perez-Ruiz et al. 2006) and ligation of renal artery branches
(Griffin et al. 1994, Yang et al. 2010), have also been reported to achieve a subtotal
renal mass reduction in rats. Moreover, compared with the approach surgical excision,
5/6 renal mass reduction, which was produced by uninephrectomy and 2/3 renal
parenchyma infarction via ligation in two of the three renal artery branches on
contralateral kidney, induced higher blood pressure and more severe renal injury in
Sprague-Dawley rats (Griffin et al. 1994).
Hypothesis
General Hypothesis
Renal ischemia is related with different kinds of kidney diseases. Temporary
ischemia in kidney could lead to renal ischemia reperfusion induced AKI. The
pathophysiological mechanisms of AKI are complicated and are not well elucidated. We
20
would like to determine the role of various factors including intratubal pressure, renal
temperature and renal pH in ischemia reperfusion induced AKI. Permanent ischemia in
kidney could result in renal infarction. We sought to examine whether permanent partial
renal ischemia promotes the development of CKD in the remnant kidney tissue and
investigate the potential mechanisms (Figure 1).
Hypothesis I: Increased intratubular pressure of the proximal tubule during the
ischemic phase exacerbates renal ischemia reperfusion injury and promotes the
development of AKI.
Renal ischemia is related with different kinds of kidney diseases. Temporary ischemia in kidney could lead to renal ischemia reperfusion induced AKI. We would like to determine the role of various factors including intratubal pressure, renal temperature and renal pH in ischemia reperfusion induced AKI. Permanent ischemia in kidney could result in renal infarction. We sought to examine whether permanent partial renal ischemia promotes the development of CKD in the remnant kidney tissue and investigate the potential mechanisms.
Figure 1. Ischemia renal disease.
21
Hypothesis II: Renal temperature during the ischemic phase affects the
pathophysiological process of IRI induced AKI
Hypothesis III: 1) Renal pH modulates TGF response by regulating NOS1β in the
macula densa; 2) Renal IRI downregulates NOS1β in the macula densa and enhances
TGF response, which promotes the development of AKI; 3) Bicarbonate treatment
protected against IRI-induced AKI by upregulating NOS1β in the macula densa.
Hypothesis IV: Partial renal infarction by ligating either the upper or lower branch
of the renal artery in the left kidney while the right kidney remained intact is able to
induce hypertension in C57BL/6 mouse.
Hypothesis V: Five sixth renal mass reduction by uninephrectomy and upper
renal artery branch ligation on the contralateral kidney increases blood pressure and
promotes CKD progression in C57BL/6 mouse.
22
Chapter Two: Ischemia Reperfusion Injury Induced Acute Kidney Injury
Project I: the role of intratubular pressure during the ischemic phase in acute
kidney injury
Abstract
Acute kidney injury (AKI) induced by clamping of renal vein or pedicle is more
severe than clamping of artery, but the mechanism has not been clarified. In present
study, we tested our hypothesis that increased proximal tubular pressure (Pt) during the
ischemic phase exacerbates kidney injury and promotes the development of AKI. We
induced AKI by bilateral clamping of renal arteries, pedicles or veins for 18 min at 37°
C, respectively. Pt during the ischemic phase was measured with micropuncture. We
found that higher Pt was associated with more severe AKI. To determine the role of
Pt during the ischemic phase on the development of AKI, we adjusted the Pt by altering
renal artery pressure. We induced AKI by bilateral clamping of renal veins, and the
Pt was changed by adjusting the renal artery pressure during the ischemic phase by
constriction of aorta and mesenteric artery. When we decreased renal artery pressure
from 85±5 to 65±8 mmHg, Pt decreased from 53.3±2.7 to 44.7±2.0 mmHg. Plasma
Copyright © 2016 by American Journal of Physiology - Renal Physiology
23
creatinine decreased from 2.48±0.23 to 1.91±0.21 mg/dl at 24 hours after renal
ischemia. When we raised renal artery pressure to 103±7 mmHg, Pt increased to
67.2±5.1 mmHg. Plasma creatinine elevated to 3.17±0.14 mg/dl 24 hours after renal
ischemia. Changes in KIM-1, NGAL and histology were in the similar pattern as plasma
creatinine. In summary, we found that higher Pt during the ischemic phase promoted the
development of AKI, while lower Pt protected from kidney injury. Pt may be a potential
target for treatment of AKI.
Introduction
Acute kidney injury (AKI) is a syndrome characterized by an abrupt reduction in
kidney function (Abuelo 2007, Thadhani et al. 1996), resulting in failure to maintain fluid,
electrolyte and acid-base homeostasis and retention of nitrogenous waste products
(Bauerle et al. 2011, Mehta et al. 2007). AKI occurs in about 5% of all hospital
admissions and is responsible for approximately 2 million deaths annually worldwide
(Murugan & Kellum 2011). AKI increases the risk of development of CKD (Chawla et al.
2011, Chawla & Kimmel 2012), exacerbates preexisting CKD, and can evolve into end-
stage renal disease (ESRD) (Wald et al. 2009). Patients who survive an episode of AKI
have poorer long-term outcomes with increased mortality and extensive morbidity (Ali et
al. 2007). Even though AKI is extensively studied, unfortunately, there is no approved
therapy to prevent or treat AKI (Jo et al. 2007). Therefore, further understanding of the
pathophysiological mechanism is crucial to develop therapeutic approaches for AKI.
Renal ischemia reperfusion is a common cause of AKI (Bonventre & Yang 2011,
Liano & Pascual 1996, Munshi et al. 2011). Following ischemia reperfusion, tubular
24
epithelial cells undergo serious damage, such as apical brush border disruption,
swelling, detachment from the basement membrane, and even death with acute tubular
necrosis, resulting in rapid loss of kidney function (Bonventre & Yang 2011, Devarajan
2006, Munshi et al. 2011). Several factors, such as hypoxia induced depletion
(Andreucci et al. 2003, Leemans et al. 2005, Ma et al. 2014), the imbalance between
superoxide (Masztalerz et al. 2006) and nitric oxide (Chatterjee et al. 2002, Tripatara et
al. 2007, Walker et al. 2000), and the inflammatory response have been demonstrated
to play important roles in renal ischemia reperfusion injury (Patschan et al. 2012,
Thurman 2007, Tripatara et al. 2007). However, the pathophysiological mechanisms of
AKI are complicated and are not well elucidated. This is especially true regarding the
role of hemodynamic alterations and changes in mechanical force in the development of
AKI.
The commonly used AKI models induced by occlusions of renal blood flow are
typically accomplished by clamping of the renal artery, pedicle (artery and vein) or vein.
Previous studies have reported that renal vein or pedicle clamping produced more severe
AKI than renal artery clamping alone, but the mechanism remains to be determined (Li et
al. 2012, Liano & Pascual 1996, Orvieto et al. 2007). In the present study, we tested our
hypothesis that increased intratubular pressure of the proximal tubule (Pt) during the
ischemic phase exacerbates kidney injury and promotes the development of AKI. We first
measured Pt during the ischemic phase while clamping the arteries, pedicles or veins,
respectively and found a positive correlation between the Pt and the severity of the AKI.
To determine whether the Pt during the ischemic phase is a causal factor for the kidney
injury, we adjusted the Pt by altering the renal artery pressure during clamping of the renal
25
veins. We found that increasing the Pt during the ischemic phase worsens AKI while
lowering the Pt protects renal function.
Methods
Animals and experimental protocols: C57BL/6 mice (male, 13-15 weeks old)
were purchased from Jackson Laboratory and housed in the University of South Florida
Animal Facility. All protocols were approved by the Institutional Animal Care and Use
Committee (IACUC) at the University of South Florida, College of Medicine.
Protocol I: AKI was induced by the occlusion of renal blood flow for 18 min in the
following three groups: 1) bilateral clamping of the renal arteries (RA group); 2) bilateral
clamping of the renal pedicles (artery and vein) (RP group); and 3) bilateral clamping of
the renal veins (RV group). The sham operated group underwent the same surgical
procedures and time courses as the experimental groups except without occlusion of the
renal blood flow.
The mice were anesthetized with pentobarbital (50 mg/kg, I.P.) and placed on a
temperature-controlled operating table (03-02; Vestavia Scientific, AL, USA). The body
temperature was monitored and controlled at 36.8-37.2 ° C during surgery with a
temperature control unit. Both kidneys were exposed through a single abdominal incision.
The Pt was measured as described below. The bilateral renal arteries, pedicles or veins
were carefully dissected and clamped with silver clips (FE690K; AESCULAP INC, PA,
USA) for 18 min.
26
In separate survival experiments, the Pt was not measured. After 18 min of
clamping, the clips were removed, the wounds were sutured, and the animals were
allowed to recover for 24 hours to evaluate the renal injury.
Protocol II: Before clamping the renal veins, the renal artery pressure was adjusted
as described below. Then AKI was induced with bilateral clamping of the renal veins for
18 min as described in Protocol I. The animals were divided into following three groups:
1) normal renal artery pressurerenal artery pressure (NP group); 2) low renal artery
pressure (LP group) and 3) high renal artery pressurerenal artery pressure (HP group).
NP group: The renal artery pressure was not adjusted in this group of mice. The
mean arterial pressure (MAP) was measured with a femoral artery catheter.
LP group: The aorta above the renal arteries was partially ligated to lower the renal
artery pressure during the ischemic phase. The abdominal aorta above the renal arteries
was then carefully separated from vena cava, and a 30-gauge needle was placed along
the side of the isolated aorta segment. After a 6-0 suture was tied around the aorta and
the needle, the needle was carefully removed from the ligature. The MAP was measured
at the femoral artery.
HP group: Both the superior mesenteric artery and the aorta below the renal
arteries were partially ligated by using a 30-gauge needle before clamping the renal veins
to raise the renal artery pressure during the ischemic phase. This technique was similar
the to steps described in the LP group. The MAP was measured at the carotid artery.
In the non-survival experiments, the Pt was measured as described below after
adjusting the renal artery pressure in the NP, LP and HP groups. In the survival
experiments, the Pt was not measured. The clips and ligature were removed after 18 min
27
of ischemia, and the wound was closed. The mice were allowed to recover for 24 hours
to evaluate the renal injury.
The proximal tubular pressure (Pt) during the ischemic phase was measured
using micropuncture as we previously described (Fu et al. 2012, Lu et al. 2015). The
Mice were anesthetized with pentobarbital (50 mg/kg, I.P.). A tracheostomy was
performed to facilitate breathing. The femoral artery or carotid artery were catheterized
for blood pressure measurements. The femoral vein was catheterized for infusion of
saline with 1% bovine serum albumin (1 ml/hr/100g body weight). Following an
abdominal incision, the left kidney was exposed and immobilized in a kidney holder cup
(03-12; Vestavia Scientific, AL, USA). The kidney orientation was positioned so that the
superficial tubules could be clearly visualized under the microscope (SZX16; Olympus,
Tokyo, Japan). A long superficial proximal tubule was punctured by a micropipette,
which connected with a micropressure system (Model 900A; World Precision
Instruments, FL, USA). The Pt was measured and recorded during the ischemic phase
while clamping the renal veins.
The mice were anesthetized again with isoflurane 24 hours after renal ischemia
for measuring kidney injury markers. The blood samples (100 µl) were collected through
the retro-orbital venous sinus and centrifuged at 8000 rpm for 5 minutes at 4˚C. Plasma
samples (50 µl/each) were obtained for creatinine, Kidney injury molecule-1 (KIM-1) and
Neutrophil gelatinase-associated lipocalin (NGAL) measurements. The creatinine
concentration was measured by HPLC at the O’Brien Center Core of the University of
Alabama at Birmingham. The KIM-1 was measured with a Mouse KIM-1 Immunoassay
Quantikine ELISA KIT and the NGAL was measured with a Mouse NGAL Immunoassay
28
Quantikine ELISA KIT (R&D System, MN, USA), following the manufacturer’s
instructions.
The kidneys were harvested and fixed in 4% paraformaldehyde solution 24 hours
after AKI. Fixed kidney tissues were embedded in paraffin, and 4 µm kidney tissue
slices were cut and stained with Periodic Acid Schiff (PAS). Ten randomly chosen fields
were captured under 200x magnification from the cortex, corticomedullary region
(CMR), outer medulla (OM) and inner medullar (IM), respectively. The percentage of
necrotic tubules and obstructed tubules by casts in each image was quantified as
reported (Muroya et al. 2015, Pegues et al. 2013). All morphometric analyses were
performed in a blinded manner.
The effects of interest were tested using a Student’s paired t-tests, or two-factor
analysis of variance (ANOVA) with repeated measures when appropriate. Data were
presented as mean ± SEM. The changes were considered to be significant if the p-
value was <0.05.
Results
Protocol I: The Pt was measured for 2 min before occlusion of the renal blood
flow and during 18 minutes of ischemia in the RA, RP and RV groups. The Pt at
baseline was about 10 mmHg, increased to 38.4±3.6 mmHg in the RP group (P<0.01
vs. RA group), to 53.3±2.7 mmHg in the RV group (P<0.01 vs. RP group), and
decreased to 3.2±2.1 mmHg in the RA group at the end of 18 min of ischemia. The Pt in
the sham group was constant at about 10 mmHg throughout the experiment (Figure 2A)
29
(P<0.05 vs. the RA, RP and RV groups). During the ischemic phase, the MAP was not
significantly different between groups (Figure 2B).
Tubular pressure in proximal tubule were measured using micropuncture. A) The difference of proximal tubular pressure during 18 min ischemic phase while clamping renal arteries (RA), renal pedicles (RP) and renal veins (RV). B) The mean arterial pressure during the ischemic phase in all groups of protocol I. (n=10) *P<0.01 vs Sham group; #P<0.01 vs RA group; &P<0.01 vs RP group. C) Proximal tubular pressure during 18 min ischemic phase while ligating renal pedicles. (n=3).
To exclude the possibility of incomplete occlusion of the renal blood flow by
clamping, we tightly ligated the renal pedicles with 6-0 suture and then measured the Pt
during the ischemic phase. The Pt increased from 9.2±1.4 to 35.7±3.3 mmHg over 18 min
(Figure 2C).
Figure 2. Proximal tubular pressure.
30
Figure 3. Kidney injury markers after renal ischemia.
Plasma creatinine (A), plasma KIM-1 (B) and plasma NGAL (C) were measured and compared 24 hours after renal ischemia while clamping renal arteries (RA), renal pedicles (RP) and renal veins (RV). (n=5) *P<0.01 vs Sham group; #P<0.05 vs RA group; &P<0.05 vs RP group.
The plasma creatinine, KIM-1, and NGAL were measured 24 hours after renal
ischemia. The plasma creatinine was substantially increased in all three AKI groups
compared with the sham group 24 hours after renal ischemia (P<0.01 vs. sham group).
The RV group was the highest (2.48±0.23 mg/dl) (P<0.01 vs. RP group), followed by the
RP group (1.43±0.19 mg/dl) (P<0.01 vs. RA group) and the RA group (0.45±0.07 mg/dl)
31
(Figure 3A). The plasma KIM-1 and NGAL had similar patterns as the creatinine (Figure
3B and 3C).
The histology of the kidney slices stained by PAS at 24 hours after IR was
examined among the different groups of animals. The RV mice showed the most severe
tubular injury with the largest incidence of tubular obstruction by casts and tubular
necrosis (P<0.01 vs. RP and RA groups, Figure 4A), followed by the RP mice (P<0.05
vs. RA group) and the RA mice (Figure 4B and 4C).
Protocol II: The MAP was 85±5 mmHg in the NP group. It decreased to 65±8
mmHg after partially ligating the aorta above the renal arteries in the LP group and
increased to 103±7 mmHg after partially ligating both the superior mesenteric artery and
the aorta below the renal arteries in HP group. To determine whether the Pt was altered
by adjusting the renal artery pressure during the ischemic phase, we measured the Pt
during 18 minutes of ischemia in the NP, LP and HP groups. Clamping of the renal
veins increased the Pt to 53.3±2.7 mmHg in the NP group at the end of 18 min of
ischemia (Figure 5A). When the renal artery pressure decreased in the LP group, the Pt
decreased to 44.7±2.0 mmHg (P<0.01 vs. NP group). When the renal artery pressure
increased in the HP group, the Pt increased to 67.2±5.1 mmHg at the end of 18 min of
ischemia (Figure 5B) (P<0.01 vs. NP group). To determine whether the Pt during the
ischemic phase had any effect on renal injury, we measured kidney injury markers 24
hours after renal ischemia. The plasma creatinine was 2.48±0.23 mg/dl in the NP group;
it decreased to 1.91±0.21 mg/dl in the LP group (P<0.05 vs. NP); and increased to
32
3.17±0.14 mg/dl in the HP group (Figure 6A) (P<0.05 vs. NP). The plasma KIM-1 and
NGAL were similar in pattern as the plasma creatinine (Figure 6B and 6C).
Figure 4. Histology.
Histology by PAS staining showed slices of cortex, corticomedullary region (CMR), outer medulla (OM) and inner medulla (IM) (A). Kidney injury was quantitatively measured by percentage of tubular necrosis (B) and obstructed tubules by casts (C) in the cortex, CMR, OM and IM in AKI groups by clamping renal arteries (RA), renal pedicles (RP) and renal veins (RV). (n=5) #P<0.05 vs RA group; &P<0.05 vs RP group. Quantitative analysis was performed on ten randomly chosen fields in each kidney tissue slice.
33
Tubular pressure in proximal tubule were measured using micropuncture. A) Image of the proximal tubular pressure while clamping renal vein with normal renal artery pressure (NP), low renal artery pressure (LP) and high renal artery pressure (HP) in protocol II. B) The difference of tubular pressure in proximal tubules during 18 min ischemic phase in NP group, LP group and HP group. (n=7) *P< 0.01 vs NP group.
Figure 5. Proximal tubular pressure.
34
Figure 7A shows the representative histology images from different groups of
mice. The HP group exhibited the most severe tubular injury with the larger incidence of
tubular obstruction by casts and tubular necrosis (P<0.01 vs. NP group), while the LP
group showed the least tubular injury among the 3 groups of mice (Figure 7B and 7C)
(P<0.05 vs. NP group).
Plasma creatinine (A), plasma KIM-1 (B) and plasma NGAL (C) were measured and compared 24 hours after renal ischemia while clamping renal veins with normal renal artery pressure(NP), low renal artery pressure(LP) and high renal artery pressure(HP). (n=6) *P<0.05 vs NP group.
Figure 6. Kidney injury markers after renal ischemia.
35
Histology by PAS staining showed slices of cortex, corticomedullary region (CMR), outer medulla (OM) and inner medulla (IM) (A). Kidney injury was quantitatively measured by percentage of tubular necrosis (B) and obstructed tubules by casts (C) in the cortex, CMR, OM and IM in AKI groups by clamping renal veins with low renal artery pressure(LP), normal renal artery pressure(NP) and high renal artery pressure(HP). (n=6) *P<0.05 vs LP group and HP group. Quantitative analysis was performed on ten randomly chosen fields in each kidney tissue slice.
Figure 7. Histology.
36
Discussion
In the present study, we found a positive association between the Pt during the
ischemic phase and the severity of AKI in three widely-used models by clamping of the
renal arteries, pedicles or veins. To determine the role of Pt during the ischemic phase of
the development of AKI, we adjusted Pt by altering the renal artery pressure while
clamping the renal veins. We found that increasing the Pt during the ischemic phase
exacerbates AKI while lowering the Pt protects against kidney injury.
Tubular biomechanical forces are involved in the regulation of renal tubular
function and associated with epithelial cell damage. In obstructive uropathy, increased
intratubular pressure and tubular stretch upregulate TGFβ-1, contributing to
tubulointerstitial inflammation and fibrosis (Quinlan et al. 2008, Sato et al. 2003). Also,
cyclical stretch on renal epithelial cells induces apoptosis by activating JNK/SAPK and
p38 SAPK-2 pathways (Nguyen et al. 2006). However, little is known about the role of
tubular biomechanical forces in the development of AKI. Renal vein or pedicle clamping
induced more severe AKI than renal artery clamping, but the mechanisms are elusive
(Liano & Pascual 1996, Orvieto et al. 2007). We examined whether tubular pressure
during the ischemic phase plays any significant role in the development of kidney injury.
AKI was induced with 18 min occlusion of the renal blood flow at 37 ° C in C57BL/6 mice.
First, we measured the Pt during the ischemic phase in three AKI models. We found that
clamping of the renal arteries significantly lowered the Pt below the pre-ischemia basal
level. Occlusion of the renal arteries created cessation of glomerular filtration, resulting in
a decrease of the Pt. On the contrary, the Pt was significantly elevated to approximate 50
mmHg by clamping the renal veins. The renal venous occlusion lead to kidney
37
congestion, which increased the glomerular hydrostatic pressure, consequently resulting
in an increase of the Pt. Surprisingly, clamping of the renal pedicles also significantly
increased the Pt to approximately 35 mmHg. We confirmed this finding by tightly ligating
the renal pedicles with a 6-0 suture to exclude the possibility of incomplete occlusion of
the renal blood flow. Suture ligation of the renal pedicles raised the Pt to a similar level as
clamping of the renal pedicles. Thus, this phenotype was not due to the incomplete
occlusion of the renal artery. However, we do not know the exact mechanism for this
phenomenon, which might result from decreased tubular reabsorption, tubular obstruction
and continued glomerular filtration induced by residual pressure after clamping of the
pedicles. To our knowledge, the present study is the first to examine the Pt during the
ischemic phase. Several previous studies measured the Pt after reperfusion. Arendshorst
et al. examined the Pt after occlusion of the renal artery in rats for 60 min in an elegant
study (Arendshorst et al. 1975). They found that at 1-3 hours after reperfusion, the
proximal tubules were filled with fluid and dilated; the Pt elevated to about 30 mmHg with
great heterogeneity (Arendshorst et al. 1975). The Pt measured in the other studies were
also found to be higher than baseline 1-3 days after IRI- or uranyl nitrate-induced AKI
(Conger et al. 1984, Eisenbach & Steinhausen 1973, Flamenbaum et al. 1974, Tanner &
Sophasan 1976). The heterogeneity in the Pt observed in these studies may be due to
tubular obstructions (Conger et al. 1984, Eisenbach & Steinhausen 1973, Flamenbaum
et al. 1974, Tanner & Sophasan 1976).
Although we used the method of Isotope Dilution LC-MSMS to measure creatinine
in mouse plasma to reduce the background and increase specificity (Takahashi et al.
2007), we realized the limitation of creatinine as an injury maker in mice, since 50% of
38
the creatinine is eliminated via secretion in the mouse kidney tubules (Eisner et al. 2010,
Vallon et al. 2012). Therefore, we evaluated kidney injury combined with changes in KIM-
1, NGAL, and histology in these three AKI models. We found that at 24 hours after renal
ischemia, clamping of the renal veins induced the most severe AKI while clamping of the
renal arteries induced the least renal injury. These results were consistent with the results
from previous studies (Liano & Pascual 1996, Orvieto et al. 2007). Therefore, the Pt during
the ischemic phase in these three models was positively associated with the severity of
AKI.
Acute reduction in renal blood flow in various clinical situations, such as in
hypovolemic shock, some cardiac and abdominal surgeries, cardiac arrest, acute left
heart failure, and kidney transplantation, etc. is often caused by renal arterial hypo-
perfusion. In such case, the renal artery clamping model should better mimic these clinical
conditions. Clamping of the renal vein is more consistent with clinical situations of vein
thrombosis, chronic right heart failure, or severe liver cirrhosis. Even though occlusion of
both the renal artery and vein is not a typical clinical scenario, clamping of the renal
pedicle is the most common choice for the rodent AKI models, probably due to the
technical challenge of isolating murine renal arteries. In consideration of the significant
difference in renal injury in different models, comparison and interpretation of these
results should be conducted cautiously.
To determine the significance of Pt during the ischemic phase of the development
of AKI, we modulated the Pt by adjusting the renal artery pressure while clamping the
renal veins. To adjust the renal artery pressure, we constricted the aorta and mesenteric
artery, rather than clamp them to avoid ischemic injury of tissues supplied by the aorta
39
and mesenteric artery. Constriction of the aorta above the renal arteries lowered the renal
artery pressure, which led to a decrease in the Pt compared with the normal renal artery
pressure group. On the contrary, constriction of the aorta and the superior mesenteric
artery raised the renal artery pressure, which resulted in an increase in the Pt. The kidney
injury markers were compared at 24 hours after renal ischemia. Mice with a lower Pt in
the ischemic phase exhibited less renal injury than mice with a normal Pt, while mice with
an elevated Pt showed more severe renal injury. Therefore, these results suggested that
Pt during the ischemic phase contributes to the development of AKI. A lower Pt in the
ischemic phase protects against the development of AKI, while a higher Pt aggravates
the progression of AKI. Under physiological conditions, renal autoregulation maintains the
renal blood flow and GFR relatively constant, despite fluctuations in the arterial blood
pressure over a range of about 80-160 mmHg. This buffers the transmission of
alterations in the arterial blood pressure into the renal tubules (Carlstrom et al. 2015,
Loutzenhiser et al. 2006). However, ischemia may impair this auto-regulatory capacity,
resulting in the loss of independent stability in tubular dynamics, thereby permitting the
arterial pressure transmitted to the intra-glomerulus and tubules (Adams et al. 1980, Guan
et al. 2006).
In summary, we found that the severity of AKI from clamping of the renal arteries,
veins or pedicles is positively associated with the Pt during the ischemic phase.
Furthermore, we demonstrated that elevations of the Pt during the ischemic phase by
increasing the renal artery pressure promotes the development of AKI, while decreasing
the Pt protects renal function. These findings not only provide novel insight into the
40
mechanisms of AKI but also suggest that modulation of the Pt might be a potential
approach for the treatment of AKI.
Project II: the role of tubuloglomerular feedback in ischemia reperfusion injury
induced acute kidney injury
Introduction
Urine pH in healthy individuals fluctuates over the entire physiological range of
values from 4.5 -8.5 (Bilobrov et al. 1990). Urine pH in each individual demonstrates
marked variations throughout the day, but with patterns. A nocturnal peak in urine pH
was usually seen around 22:00. Throughout the night, urine pH decreased to a nadir
around 06:00 before rising again in the morning (Ayres et al. 1977, Barnett & Blume
1938, Cameron et al. 2012, ELLIOT et al. 1959, Jones 1843, Kanabrocki et al. 1988,
MILLS & STANBURY 1951, STANBURY & THOMSON 1951). Diet can markedly
affect acid-base status. Dietary acid load has been found to be associated with serum
bicarbonate levels and urine pH (Gunn et al. 2013, Ikizler et al. 2015, Remer & Manz
1995). The European Prospective Investigation into Cancer and Nutrition study
analyzed 22,034 men and women and compared casual urine samples with dietary acid
load. They found that dietary acid-base load was significantly related to urine pH.
Casual urine pH provides a simple tangible measure of dietary acid load (Welch et al.
2008). A recent study reported that urine pH is sensitive to reflect dietary acid-base load
and reach a steady state in about 3 days (Kanbara et al. 2012).
41
Ischemia and reperfusion are natural steps during kidney procurement and
transplantation. Transplanted organs experience several episodes of IRI, which can be
caused by both warm and cold ischemia. Warm ischemia occurs after cardiac death in
donors, or during harvesting operation from living donors, and during anastomosis of
the graft. Cold ischemia occurs during allograft kidney storage in organ preservation
solutions (Homer-Vanniasinkam et al. 1997, O'Callaghan et al. 2012, Opelz & Dohler
2007, Ploeg et al. 1988, Southard et al. 1985). Post-transplant renal injury has been
studied extensively (Gulec et al. 2006, Land 1999, Massberg & Messmer 1998,
Racusen et al. 1999, Sis et al. 2010). Prolonged ischemia increases the occurrence of
delayed graft function and worsens long-term graft survival (Boom et al. 2000). IRI is
one of the most important nonspecific factor affecting both delayed and late allograft
dysfunction (Halloran et al. 1988, Jassem & Heaton 2004, Womer et al. 2000). IRI-
induces AKI has been found crucial for transplanted kidney survival. Recently AKI and
graft survival of renal transplant recipients has been analyzed in a large retrospective
cohort study included 27,232 patients. Patients who developed AKI had an increased
risk of graft loss and an increased risk of mortality (Mehrotra et al. 2012).
In the present study, we aimed to determine 1) the TGF responsiveness change
and the alteration of NOS1β activity and expression in the macula densa in IRI-induced
AKI; 2) the effect of renal pH on TGF response and the activity and expression of
NOS1β in the macula densa; and 3) the effect of bicarbonate treatment on the
development of IRI-induced AKI.
42
Methods
Protocol I: The C57BL/6 mice were induced IRI and then measured 1)
expression level of NOS1β; 2) TGF response in vivo with micropuncture; 3) GFR in
conscious mice; and 4) plasma creatinine. Protocol II: The C57BL/6J mice were divided
into three groups treated with either 0.5% NaCl, 0.72% NaHCO3 (same mEq of Na+ as
in NaCl) or 0.46% NH4Cl (same mEq of Cl- as in NaCl) for two weeks. The treated mice
were measured 1) urine pH; 2) expression level of NOS1β; 3) TGF response in vivo with
micropuncture; and 4) GFR in conscious mice.
To investigate the role of bicarbonate-induced tubular pH alterations on TGF
responsiveness, we performed experiments and measured TGF response both in vitro
and in vivo in response to acute tubular pH changes.
To determine the role of pH alterations on NOS1 activity, we measured NOS1α
and NOS1β activity in response to pH changes.
Protocol III: Both C57BL/6J mice and macula densa specific NOS1 knockout mice (MD-
NOS1KO mice) were induced IRI and then divided into two groups fed a diet containing
either 0.4% NaCl or 0.4 g/kg NaCl plus 4.3 g/kg NaHCO3 (same Na+ as in 0.4% NaCl
diet) for 7 days. The mice were measured 1) expression level of NOS1β; 2) GFR in
conscious mice; and 3) plasma creatinine.
C57BL/6 mice were anesthetized with pentobarbital (50 mg/kg, I.P.) and placed
on a temperature-controlled operating table (03-02; Vestavia Scientific, AL, USA). The
body temperature was monitored and controlled at 36.8-37.2 ° C during surgery with the
temperature control unit. Both kidneys were exposed through a single abdominal
incision. Bilateral renal arteries were carefully dissected and clamped by the silver clips
43
(FE690K; AESCULAP INC, PA, USA) for 18 min. After 18 min of clamping, the clips
were removed, the wounds were sutured, and the animals were allowed to recover for
24 hours to evaluate renal injury. Sham operated animals without clamping renal
arteries will be used as control (n=10/group).
After kidney IRI, we measured 1) expression level of NOS1β and NO generation
in the macula densa; 2) TGF response in vivo with micropuncture and in vitro in isolated
perfused JGAs; 3) GFR in conscious mice; and 4) plasma creatinine and histology.
GFR was measured by the plasma FITC-sinistrin clearance with a single bolus
injection in conscious mice before cold IRI and 7 days after cold IRI as we previous
described with modifications (Schreiber et al. 2012, Wang et al. 2016). Briefly, mice
were injected with FITC-sinistrin solution (5.6 mg/100 g BW) through the retro-orbital
venous sinus. Blood (<10 µl ) was collected into heparinized capillary tubes through the
tail vein at 3, 7, 10, 15, 35, 55, and 75 minutes after sinistrin injection. The blood
samples were centrifuged at 8000 rpm for 5 minutes at 4˚C. Plasma (1 µl) was collected
from each sample. FITC-sinistrin concentration of the plasma was measured using a
plate reader (Cytation3; BioTek, VT, USA) with 485-nm excitation and 538-nm emission.
GFR was calculated with a GraphPad Prism 6 (GraphPad Software).
Similar method for micropuncture was used as we previously described.(Fu et al.
2012, Fu et al. 2013) Briefly, mice were anesthetized with inactin (80 mg/ml, I.P.) and
ketamine (50 mg/ml, I.M.). A tracheostoma was placed to facilitate breathing and
femoral artery was catheterized for blood pressure measurement. The femoral vein was
catheterized for infusion of saline with 1% BSA (1 ml/hr/100g body weight). Following an
abdominal incision, the left kidney was exposed and immobilized in a kidney holder cup.
44
The kidney orientation was positioned so that superficial tubules could be clearly
visualized under the microscope (SZX16, Olympus, Tokyo, Japan). Tubular flow of a
selected proximal tubule with multiple visible loops was obstructed with a grease block.
Stop-flow pressure (Psf) in proximal tubule upstream of the grease block was measured
with servo-nulling method (Model 900A; World Precision Instruments, FL, USA).
Micropipette for pressure measurement with a tip diameter at 2-5 µm was filled with 2 M
KCl colored with 1% fast green. Proximal tubule distal to the grease block was perfused
with artificial tubular fluid (ATF, containing 4 NaHCO3, 5 KCl, 2 CaCl2, 7 urea, 2 MgCl2,
128 NaCl, and 1% fast green in mM; pH 7.4). Psf was measured when tubular perfusion
rate was set at 0 nl /min and 40 nl/min for 3-5 min. The change of Psf (ΔPsf) was used as
an index of TGF response mediated vasoconstriction in afferent arteriole (Fu et al.
2012).
TGF in vivo measured in isolated double-perfused JGA as described reports (Liu
et al. 2004b, Zhang et al. 2013). The mice were anesthetized with inhaled isoflurane
and the kidneys were removed and sliced. Kidney slices were placed in ice cold DMEM
containing 5% BSA and dissected under a stereomicroscope (model SMZ 1500; Nikon).
An intact JGA was microdissected under a stereomicroscope. The dissected JGA was
transferred to a temperature controlled chamber mounted on an inverted microscope.
The Af-Art was cannulated with glass micropipette and perfused with dulbecco’s
modified eagle’s medium-low glucose (DMEM), and intraluminal pressure was
maintained at 60 mmHg throughout the experiment. The end of tubule was cannulated
with another glass micropipette and perfused with the macula densa solution containing
either 80 NaCl (high NaCl) or 10 NaCl (low NaCl). The pH of the solution was 7.4. Once
45
the JGA was double perfused and temperature in bath solution was gradually increased
to 37°C for 30 min, we induced a TGF response by increasing tubular perfusate NaCl
concentration from 10 to 80 mM by measuring maximal constriction of the Af-Art. Time
control was performed using the same protocol and time course except without tubular
NaCl concentration (n=10/group).
Rabbits (It is difficult to do double perfusion in mice.) were anesthetized with
ketamine (50 mg/kg, I.M.) and kidneys were removed and sliced. Kidney slices were
placed in ice cold DMEM containing 5% bovine serum albumin. A single superficial
intact glomerulus was microdissected together with adherent tubular segments
consisting of the TAL, macula densa, and early distal tubule under a stereomicroscope
(SMZ1500, Nikon, Yuko, Japan) as described previously (Liu et al. 2004a). The
dissected sample was transferred to a temperature controlled chamber mounted on an
inverted microscope (Axiovert 100TV, ZEISS, NY, USA). The glomerulus was hold with
glass pipette. The TAL was cannulated and perfused with another glass pipette with a
macula densa solution containing: 10 HEPES, 1.0 CaCO3, 0.5 K2HPO4, 4.0 KHCO3, 1.2
MgSO4, 5.5 glucose, 0.5 Na acetate, 0.5 Na lactate, and 10 NaCl in mM; pH 7.4. For
NO measurement, the macula densa was loaded with a fluorescent NO probe 4-amino-
5-methylamino-2', 7’-difluorofluorescein diacetate (DAF-2 DA; 10 μM plus 0.1% pluronic
acid) from the tubular lumen for 30 min, then washed for 10 min with macula densa
solution. DAF-2 was excited at 490 nm with a xenon light, and the emitted fluorescence
was recorded at wavelengths of 510 to 550 nm. The rate of increase in fluorescent
intensity of DAF-2 was used to determine NO generation by the macula densa as we
previously described.(Fu et al. 2012, Wang et al. 2015) We measured TGF-induced NO
46
generation by the macula densa when we switched the tubular perfusate from 10 to 80
mM NaCl. Then we switched the tubular solution back to 10 mM NaCl , added NOS1
inhibitor 7-NI (10-4M) to tubular perfusate and perfused for 20 min. TGF-induced NO
generation was measured again after we increased tubular NaCl to 80 mM in the
presence of 7-NI. Time control was performed using the same protocol and time course
except without 7-NI.
Protein was extracted from renal cortex. Protein extracts (50 μg/lane) were
subjected to 12 % SDS-PAGE separation. After blocking for 30 min at room
temperature with 5% skim milk, the membranes were incubated overnight at 4 °C with
NOS1 antibody (mouse polyclonal, IgG; 1:3000; BD Biosciences, CA, USA). After
incubation of the membranes with horseradish peroxidase-conjugated secondary
antibody (goat anti-mouse,IgG;1:300,000;Bio-R, CA, USA), the antibody-reactive bands
were revealed by enhanced chemiluminescence detection on Hyperfilm (Amersham
Pharmacia Biotech, Piscataway, NJ) and band density was determined using the public
image program.
The mice were placed in metabolic cages for 24 hours. Urine will be collected for
measurement of pH.
The mice were anesthetized again with isoflurane 24 hours and 7 days after cold
IRI. The blood samples (100 µl/each) were collected through the retro-orbital venous
sinus and centrifuged at 8000 rpm for 5 minutes at 4˚C. The plasma (50 µl/each) was
obtained for creatinine measurement. Creatinine concentration was measured by HPLC
at the O’Brien Center Core of the University of Alabama at Birmingham.
47
To determine the role of pH alterations on NOS1 activity, NOS1α and NOS1β
activity were measured with different pH condition (5.6, 6, 6.5, 7, 7.4, 7.9 and 8.3) by
using Nitric Oxide Synthase (NOS) Activity Assay Kit (Biovision, Zurich, Switzerland)
following the manufacturer’s instruction. The NOS1α and NOS1β protein sample were
extracted through cell transfection. Vectors of NOS1α and NOS1β were kindly provided
by Dr. Dieter Saur from Technical University of Munich in Germany (Saur et al. 2002).
The pH value of the protein sample was adjusted by adding either HCl or NaOH and
measured by using orion micro pH electrode (Thermo Scientific, MA, USA).
Results
Protocol I: To determine whether renal IRI decreases expression of NOS1β in the
macula densa, we compared NOS1β expression in renal cortex between IRI and sham
operated mice. In renal cortex, NOS1β level decreased by 84±6% in IRI mice compared
with sham operated mice (n=6, p<0.01 vs sham, Figure 8A and 8B).
To determine whether renal IRI enhance TGF response in vivo, TGF response
was measured right after and 7 days after IRI. Right after IRI, Psf was from 32.2±4.5 to
31.4±3.8 mmHg when perfusion rate was increased stepwise from 0 to 40 nL/min (n=4).
TGF response which was measured by maximal change of Psf was 0.8±0.6 mmHg. In
separate experiments, we measured TGF response in the mice 7 days after IRI and
found TGF increased to 7.2±0.8 mmHg (n=5, p<0.01 vs right after IRI). TGF response in
sham operated mice was 4.7±0.5 mmHg right after operation (n=3) and 4.1 ±0.3 mmHg
7 days after sham operation (n=3, p<0.01 vs IRI, Figure 9).
48
NOS1β expression in renal cortex were measured and compared between IRI and sham operated mice. A) Representative Image of NOS1β Expression after IRI (n=8); B) NOS1β level decreased by 84±6% in IRI mice compared with sham operated mice.*p<0.01 vs sham.
TGF response was measured right after and 7 days after IRI. TGF response was 0.8±0.6 mmHg right after IRI and increased to 7.2±0.8 mmHg 7 days after IRI,. #p<0.01 vs right after IRI; *p<0.01 vs Sham.
Sham IRI
TG
F R
es
po
ns
e i
n v
ivo
(m
mH
g)
0
2
4
6
8
10
Right after surgery
7 days after surgery
*
#
Figure 8. NOS1β protein level.
Figure 9. TGF response in vivo.
49
GFR in conscious mice were measured before IRI at baseline and 7 days after IRI. GFR in the sham operated mice had no significant decrease 7 days after surgery. However, the GFR decreased by 21.6±1.9% in IRI mice compared with the sham operated mice. *p<0.01 vs Sham.
To determine whether renal IRI decrease GFR, we measured GFR in conscious
mice before IRI at baseline and 7 days after IRI. Baseline GFR was similar between IRI
and sham operated mice. GFR in the sham operated mice had no significant decrease 7
days after surgery. However, the GFR decreased by 21.6±1.9% in IRI mice compared
with the sham operated mice (n=6, p<0.01 vs Sham, Figure 10). Plasma creatinine was
measured 24 hours and 7 days after IRI to evaluate kidney injury. At 24 hours after IRI,
the plasma creatinine was 0.08±0.03 mg/dl in sham operated mice and increased to
0.45±0.07 mg/dl in IRI mice. At 7 days after cold IRI, the plasma creatinine was still
significantly higher in IRI mice compared with sham operated mice (n=6, p<0.01 vs Sham,
Figure 11).
Baseline 7 days
GF
R (
µl/
min
)
0
50
100
150
200
250
300IRI
Sham
*
Figure 10. GFR after IRI.
50
Plasma creatinine was measured 24 hours and 7 days after IRI. *p<0.01 vs Sham.
Protocol II: To determine whether changes of pH alter NOS1 activity in renal
cortex, NOS1α and NOS1β activity were measured with different pH value (5.6, 6, 6.5,
7, 7.4, 7.9 and 8.3), separately. NOS1β activity was 0.3865±0.0477 mU/mg when pH
value was 7.4. It gradually decreased in response to the decrease of pH value.
However, NOS1β activity reached its peak value (0.4492±0.0199 mU/mg) when pH was
7.9. NOS1α activity was 0.3912±0.0268 mU/mg when pH value was 7.4 and increased
to 0.4666±0.0368 mU/mg when pH value decreased to 6.5 (Figure 12).
We measured TGF response in vitro in isolated and double perfused JGA. When
we switched the tubular perfusate from 10 to 80 mM NaCl solution with a pH adjusted to
7.4, the Af-Art constricted from 16.1±0.7 to 12.5±0.5 µm µm. The difference in diameter
between 80 and 10 mM NaCl is a TGF response, which was 3.6 ± 0.3 µm. Then we
24 hours 7 days
Pla
sm
a c
rea
tin
ine
(m
g/d
l)
0.0
0.1
0.2
0.3
0.4
0.5
0.6IRI
Sham*
*
Figure 11. Plasma creatinine after IRI.
51
switched the tubular solution back to 10 mM NaCl for 15 min and pH of tubular perfusate
was adjusted to 8.4. When we increased tubular NaCl to 80 mM, the Af-Art constricted
from 17.3±0.6 µm to 14.5± 0.7 µm. TGF was reduced by 2.8 ± 0.2 µm (p<0.05, n=5,
Figure 13A), indicating that TGF was blunted when pH at the macula densa increased.
NOS1α and NOS1β activity with different pH value.
We measured TGF response in vivo with micropuncture. When we increased
tubular pH from 5.5 to 8.0, ΔPsf was decreased from 12.4 ± 1.5 at pH 5.5 to 9.9 ± 1.8mmHg
at pH 8.0 (n=9 tubules/4 rats, p<0.05, Figure 13B). These data demonstrated that
increases of tubular pH blunted TGF response both in vitro and in vivo. To determine
whether the effect of tubular pH-induced TGF inhibition is mediated by activation of
nNOS, we added in LNMA to the tubular perfusate to inhibit NOS and measured TGF
response. ΔPsf was 11.1 ± 1.4 mmHg at pH 5.5 and 13.0± 1.6 mmHg at pH 8.0,
respectively (n=7 tubules/4 rats, Figure 13C). LNMA eliminated the pH-induced TGF
5.6 6 6.5 7 7.4 7.9 8.3
NO
S1
ac
tiv
ity (
mU
/mg
)
0.1
0.2
0.3
0.4
0.5
0.6NOS1a
NOS1b
Figure 12. NOS1 activity.
52
inhibition when increasing tubular pH from 5.5 to 8.0. These data indicated that the
increases of tubular pH blunted TGF response mediated by enhancing NO generation.
A) Increase of pH at the macula densa blunts TGF isolated double-perfused JGA. *p<0.05 vs pH 7.4. B) Increase of tubular pH blunts TGF response in vivo. *p<0.05 vs pH 5.5. C) LNMA Eliminated the pH Induced TGF Inhibition when Increasing Tubular pH.
Figure 13. TGF response in response to acute tubular pH changes.
53
To determine whether high bicarbonate intake increase urine pH, we measured
pH in urine collected with metabolic cages. Urine pH was significantly increased to
7.9±0.3 in NaHCO3 treated group and decreased to 6.5±0.2 in NH4Cl treated group,
compared with the NaCl treated group (7.1±0.3) (n=5, p<0.05, Figure 14).
To determine whether high bicarbonate intake increase renal NOS1β expression,
the expression of NOS1β in renal cortex was measured and compared in NaHCO3,
NH4Cl and NaCl treated mice. The NOS1β protein was significantly increased with a
more than 5 folds higher in NaHCO3 treated group than that in NaCl treated group. On
the contrary, NOS1β level was significantly decreased in NH4Cl group with about 1/3
value of that in NaCl group (n=4, p<0.05, Figure 15A and 15B).
NaCl NaHCO3 NH4Cl
Uri
ne
pH
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5*
*
Figure 14. Urine pH.
54
The protein level of NOS1β was compared in NaHCO3 treated, NaCl treated and NH4Cl treated mice. The NOS1β protein was significantly increased with a more than 5 folds higher in NaHCO3 treated group than that in NaCl treated group. On the contrary, NOS1β level was significantly decreased in NH4Cl group with about 1/3 value of that in NaCl group. *p<0.01 vs NaCl group.
To determine whether high bicarbonate intake inhibited TGF response, TGF
response was measured and compared in NaHCO3, NH4Cl and NaCl treated mice. TGF
was significantly blunted to 3.1±0.5 mmHg in NaHCO3 group but significantly enhanced
to 7.4±0.8 mmHg in NH4Cl group, compared with NaCl group (4.4±0.7 mmHg) (n=4
mice/10 tubules, p<0.05 vs NaCl group, Figure 16).
Figure 15. The effect of bicarbonate on NOS1β expression.
55
TGF was measured and compared in NaHCO3 treated, NaCl treated and NH4Cl treated mice. *p<0.05 vs NaCl group.
To determine whether increased bicarbonate intake raise GFR, GFR was
measured and compared in NaHCO3, NH4Cl and NaCl treated mice. There was no
significant difference among three groups, which was 237.6±13.7 μl/min in NaHCO3
treated mice, 229.5±17.2 μl/min in NH4Cl treated mice and 241±12.8 μl/min in NaCl
treated mice (n=5, Figure 17).
GFR was measured and compared in NaHCO3, NH4Cl and NaCl treated mice.
NaHCO3 NaCl NH4Cl
TG
F r
es
po
ns
e i
n v
ivo
(m
mH
g)
0
2
4
6
8
10
*
*
NaHCO3 NaCl NH4Cl
GF
R (
µl/
min
)
0
50
100
150
200
250
300
Figure 16. TGF response in vivo after high bicarbonate intake.
Figure 17. GFR in conscious mice after high bicarbonate intake.
56
Protocol III: To determine whether bicarbonate prevent IRI-induced decrease in
NOS1β expression, the expression of NOS1β in renal cortex was measured and
compared 7 days after IRI between NaHCO3 and NaCl treated mice. In renal cortex,
NOS1β protein abundance was 3.6±0.16 times higher in NaHCO3 treated mice than
that in NaCl treated mice (n=5, p<0.05 vs NaCl group, Figure 18A and 18B).
A) Representative Image of NOS1β Expression after High Bicarbonate Intake in IRI Mice. B) The protein level of NOS1β was compared between NaCl and NaHCO3 treated mice after IRI. *p< 0.01 vs sham.
To determine whether bicarbonate prevent IRI-induced decrease of GFR, GFR
was measured and compared before IRI at baseline and 7 days after IRI between
NaHCO3 and NaCl treated mice. Baseline GFR was similar between NaHCO3 and NaCl
Figure 18. NOS1β expression after high bicarbonate intake in IRI mice.
57
treated mice. At 7 days after IRI, GFR increased by 15.8±2.2% in NaHCO3 treated mice
compared with NaCl treated mice (n=6, p<0.05 vs NaCl group).
To further determine the role of NOS1β in the alteration of GFR by bicarbonate,
we measured GFR before IRI at baseline and 7 days after IRI in MD-NOS1KO mice. The
difference of GFR between NaCl treated (172.5±9.3 μl/min) and NaHCO3 treated mice
(186.2±10.2 μl/min) was attenuated (n=4, Figure 19).
GFR was measured and compared before IRI at baseline and 7 days after IRI between NaHCO3 and NaCl treated mice. The difference of GFR between NaCl treated (172.5±9.3 μl/min) and NaHCO3 treated mice (186.2±10.2 μl/min) was attenuated. *p< 0.05 vs baseline; *p< 0.05 vs NaCl.
Plasma creatinine was measured 7 days after IRI to evaluate kidney injury. At 7
days after cold IRI, the plasma creatinine was 0.37±0.03 in NaCl treated WT mice and
decreased to 0.28±0.04 in NaHCO3 treated WT mice (n=6, p<0.05 vs NaCl). However,
the decrease of plasma creatinine in NaHCO3 treated MD-NOS1KO mice was limited
compared with NaCl treated MD-NOS1KO mice (n=4, Figure 20).
Baseline 7 days
GF
R (
µl/
min
)
0
50
100
150
200
250
300
WT+NaCl
WT+NaHCO3
MD-NOS1KO+NaCl
MD-NOS1KO+NaHCO3
*
*
**
#
Figure 19. GFR in conscious mice after high bicarbonate intake in IRI mice.
58
Plasma creatinine was measured and compared 7 days after IRI between NaHCO3 and NaCl treated mice. The decrease of plasma creatinine in NaHCO3 treated MD-NOS1KO mice was limited compared with NaCl treated MD-NOS1KO mice. *p< 0.05 vs NaCl.
Discussion
In the present study, we examined 1) the effect of renal pH on TGF response and
the activity and expression of NOS1β in the macula densa; 2) the TGF responsiveness
change and the alteration of NOS1β activity and expression in the macula densa in IRI-
induced AKI; and 3) the effect of bicarbonate treatment on the development of IRI-
induced AKI. We found that 1) renal pH modulates TGF response by regulating NOS1β
in the macula densa; 2) renal IRI downregulates NOS1β in the macula densa and
enhances TGF response, which promotes the development of AKI; and 3) bicarbonate
treatment protected against IRI-induced AKI by upregulating NOS1β in the macula
densa.
In both humans and experimental animals, reductions in GFR have been
attributed to persistent vasoconstriction. An intense and persistent renal
WT MD-NOS1KO
Pla
sm
a c
rea
tin
ine
(m
g/d
l)
0.0
0.1
0.2
0.3
0.4
0.5
0.6NaCl
NaHCO3
*
Figure 20. Plasma creatinine after high bicarbonate intake in IRI mice.
59
vasoconstriction that reduces overall kidney blood flow and GFR has long been
considered a hallmark of AKI (Lameire et al. 2005, Mason et al. 1977). In fact, ischemic
AKI has been referred to as “vasomotor nephropathy”. A severe injury of the proximal
tubular in renal cortex and TAL in the outer medulla is a uniform feature in various forms
of AKI while the glomeruli are morphologically normal (Heyman et al. 1988, Lieberthal &
Nigam 1998, Oken 1975, Regner et al. 2009). Tubular injury can also lead to a
decrease in GFR by tubular obstruction with epithelial debris and backleak. However,
normal pressures have been observed in several tubules, making it unlikely for tubular
obstruction to be the singularly important mediator of reduction in GFR after ischemic
injury (Conger et al. 1984). Arendshorst et al.(Arendshorst et al. 1975) studied severe
AKI after 1 h of unilateral renal ischemia. They found that in the early stages (1-3 h)
after ischemia, the pressures in the proximal and distal tubules were elevated, and there
was histological evidence of tubular obstruction. Tubular obstruction appeared to be the
predominant factor in the decline in filtration rate at this stage. However, at a later time-
point (22-26 h), tubular pressures were normal, but preglomerular vasoconstriction was
marked and this was thought to contribute to the persistent decline in filtration rate. In
addition, tubular obstruction does not appear to be a significant pathogenic mechanism
in AKI in humans, in which only about 10% decrease in GFR could be explained by
tubular backleak (Blantz & Singh 2011, Braam et al. 1993, Myers et al. 1979, Schrier et
al. 2004). A single nephron model of nephrotoxic tubular injury was established by
microperfusion of uranyl nitrate into the early proximal tubule (Peterson et al. 1989).
Within minutes of intratubular administration of uranyl nitrate, a 16-30% reduction in
SNGFR was observed. The local mechanism such as TGF could be invoked to explain
60
the immediate decline in SNGFR. Similar findings have been observed in the uranyl
nitrate model of AKI by another group (Flamenbaum et al. 1974). Hence, an increase in
preglomerular resistance appears to be consistent in nearly all forms of AKI and TGF
has been postulated to dictate the enhanced preglomerular vascular resistance and
reduction in GFR in AKI (Thurau & Boylan 1976). In present study, we measured TGF in
vivo with micropuncture right after and 7days after following 18 min clamping of renal
arteries. The results indicate that renal ischemia impairs TGF response early after IRI,
but enhances TGF response later after IRI.
As an enzyme to catalyze the generation of NO and citrulline from L-arginine,
NOS1 is affected by pH. However, the optimal pH for NOS1 is controversial in previous
studies. The maximal efficiency of NOS1 was measured at a pH 6.7 with the brain
tissue (Chen et al. 1997). However, the most favorable pH for NOS1 in the kidney tissue
was indicated to be around 8.0 (Liu et al. 2004a, Wang et al. 2003a). There are three
splice variants of NOS1, α, β, and γ. NOS1α is the primary isoform in brain but NOS1β
is the majority in kidney. Hence, in present study, we generated the distinct NOS1α and
NOS1β by their expression vectors and measured the activity under conditions with
graded pH environment. The results indicated that the most favable pH for NOS1α was
6.5 and the optimium for NOS1β was 7.9, which is consistant of the previous studies
and explains the controversy.
In previous studies, we reported that the NO generation was enhanced by the
macula densa during TGF response (Liu et al. 2004a). The activation of nNOS is
mediated by elevation of intracellular pH in the macula densa via NHE (Liu et al.
2004a). Microperfusion studies showed when luminal pH was increased, there was a
61
linear correlation between changes in intracellular pH and extracellular pH in macula
densa, and intracellular pH of the macula densa at high NaCl was always higher than at
low NaCl (Liu et al. 2008). Also, in cultured tubular cells decreasing pH in culture
medium with different concentration of NaHCO3 accordingly reduced intracellular pH
(Yaqoob et al. 1996). These studies indicate changing luminal pH can be used to alter
intracellular pH. In our study, it is very likely that modulation of luminal pH took effect by
altering intracellular pH. During TGF response NO concentration by the macula densa
increased significantly (Liu et al. 2002). The inhibition of NO generation by either NOS
blockers (Welch & Wilcox 1997) or specific nNOS inhibitors (Ren et al. 2000b, Welch et
al. 2000) enhances TGF responsiveness both in vivo and in vitro. We previously found
that raising intracellular pH of the macula densa from 7.3 to 7.8 significantly increased
NO concentration in microdissected and perfused macula densa and 7-Nitroindazole
blocked the increase of NO induced by elevated pH (Liu et al. 2004a). In present study,
we found tubular pH directly regulate TGF responsiveness at the macula densa in vivo
with micropuncture and in vitro with microperfusion. Increases of pH in tubules blunt
TGF response. NOS inhibition eliminated this effect, indicating NO mediated the
attenuation of TGF induced by elevation of tubular pH.
Current guidelines for CKD patients recommend alkali treatment with sodium
citrate or sodium bicarbonate for those with serum total CO2 less than 22 mM (2000).
Indeed, the correlation between low serum bicarbonate level and progressive decline in
eGFR has been studied extensively in patients with CKD. Alkali therapy reduces kidney
injury and slows nephropathy progression in patients with CKD (de Brito-Ashurst et al.
2009, Dobre et al. 2015, Eustace et al. 2004, Goraya et al. 2012, Kovesdy et al. 2009,
62
Mahajan et al. 2010, Navaneethan et al. 2011, Phisitkul et al. 2010, Raphael et al. 2011,
Shah et al. 2009, Susantitaphong et al. 2012), but the mechanism is not clear. Diet can
markedly affect acid-base status and it significantly influences CKD and its
progression. In the National Health and Nutrition Examination Survey, 11,957 adults
were analyzed and found that high serum anion gap values and decreased serum total
CO2 concentration were associated with increased mortality in the general population
and with a decline in eGFR in patients with CKD (Abramowitz et al. 2012). In a separate
study, the correlation between dietary acid load and eGFR were assessed in 12,293 US
adults. High dietary acid load was found to be associated with albuminuria and low
eGFR (Banerjee et al. 2014). A cross-sectional study of the African American Study of
Kidney Disease and Hypertension cohort indicate that a higher acid load and lower total
serum CO2 concentration (reflects serum bicarbonate concentration) correlate with more
severe CKD and readily decline of eGFR (Scialla et al. 2011). Oral bicarbonate
supplementation slows CKD progression in humans (de Brito-Ashurst et al.
2009). Recent two intervention studies, one in patients with early CKD without metabolic
acidosis (Goraya et al. 2012), and the other in patients with advanced CKD and
metabolic acidosis (Goraya et al. 2013), suggest that a diet rich in fruits and vegetables
can improve renal function and slow the progression of CKD. In patients at risk of CKD,
alkali-generating diets that are rich in fruits and vegetables reduce the risk of developing
CKD (Chang et al. 2013, Dunkler et al. 2013, Lin et al. 2011, Nettleton et al. 2008,
Scialla et al. 2011). Oral bicarbonate supplementation has been found to delay the
decline of GFR in CKD patients (Dobre et al. 2015, Eustace et al. 2004, Kovesdy et al.
2009, Navaneethan et al. 2011, Raphael et al. 2011, Shah et al. 2009, Susantitaphong
63
et al. 2012), but the mechanism is not clear. Recently, the relationship between serum
bicarbonate and GFR has been analyzed in people with normal renal functions. Lower
serum bicarbonate concentrations are found to be associated independently with rapid
decline of eGFR in a study of 5422 adults; in a Multi-Ethnic Study of Atherosclerosis of
5810 participants (Driver et al. 2014), and in a Health, Aging, and Body Composition
study (Goldenstein et al. 2014). The baseline eGFR of most or all of the participants in
these studies is above 60 mL/min/1.73 m2. Dietary acid load has also been found an
important determinant of kidney injury in rat models of CKD with and without metabolic
acidosis (Khanna et al. 2004, Mahajan et al. 2010, Nath et al. 1985, Phisitkul et al.
2010, Wesson et al. 2006, Wesson & Simoni 2010). In present study, we measured
GFR and plasma creatinine 7 days after IRI in bicarbonate treated C57BL/6J and MD-
NOS1KO mice. We found NaHCO3 treatment can limit the decrease of GFR and the
increase of plasma creatinine in C57BL/6J mice, but this limitation was attenuated in
MD-NOS1KO mice. The results indicate upregulation of NOS1β in the macula densa by
a high bicarbonate intake could be a novel mechanism, but also a potential target for
prevention and treatment for IRI-induced AKI.
In conclusion, we found 1) renal pH modulates TGF response by regulating
NOS1β in the macula densa; 2) renal IRI downregulates NOS1β in the macula densa
and enhances TGF response, which promotes the development of AKI; and 3)
bicarbonate treatment protected against IRI-induced AKI by upregulating NOS1β in the
macula densa.
64
Project III: the role of renal temperature during the ischemic phase in acute
kidney injury
Abstract
Ischemia and reperfusion are natural steps during kidney transplantation, and IRI
is considered one of the most important nonspecific factors affecting allograft
dysfunction. Transplanted organs experience several episodes of ischemia, in which
cold ischemia occurs during allograft storage in preservation solutions.
Even though cold ischemia has been studied extensively, all of the studies have
been carried out in vitro and ex vivo models. There is no in vivo model available to
examine renal IRI induced solely by cold ischemia.
In the present study, we developed an in vivo mouse model to study renal IRI
induced exclusively by cold ischemia through clamping the renal pedicle for 1 to 5
hours. During the ischemic phase, blood was flushed from the kidney with cold saline
through a small opening on the renal vein. The kidney was kept cold in a kidney cup
with circulating cooled saline, while the body temperature was maintained at 37℃ during
the experiment. The level of kidney injury was evaluated by plasma creatinine, KIM-1,
NAGL, GFR, and histology.
Plasma creatinine was significantly increased from 0.15±0.04 mg/dl in the sham
group to 1.14±0.21 and 2.65±0.14 mg/dl in 4 and 5-hours ischemia groups at 24 hours
after cold IRI. The plasma creatinine in mice with ischemic time <3 hours demonstrated
no significant increase compared with sham mice. Changes in KIM-1, NAGL, GFR and
histology were similar to plasma creatinine.
65
In summary, we developed and characterized a novel in vivo IRI-induced AKI
mouse model exclusively produced by cold ischemia.
Introduction
Renal transplantation is the best replacement therapy for patients with end-stage
kidney disease. Not only is kidney transplantation a life-saving procedure, but it also
improves the patient’s quality of life (Hricik et al. 2001, Kaplan & Meier-Kriesche 2002,
Murray et al. 1984, Wolfe et al. 1999).
Ischemia reperfusion injury (IRI) has remained one of the most serious pitfalls of
the kidney transplantation procedure. IRI-induced acute AKI can crucially affect the
transplanted kidney’s survival and is considered one of the most important nonspecific
factors affecting both early and late allograft dysfunction (Nehus & Devarajan 2012).
Ischemia and reperfusion are natural steps during kidney procurement and
transplantation. Transplanted organs experience several episodes of IRI, which can be
caused by both warm and cold ischemia. Warm ischemia occurs after cardiac death in
cadaveric donors, or during harvesting operations from living donors, and during
anastomosis of the graft. Cold ischemia occurs during allograft kidney storage in organ
preservation solutions (Southard et al. 1985), which was invented in 1960’s and is the
most seminal step to avoid IRI, allowing for safe renal preservation exceeding 24 hours
(Collins et al. 1969). It remains the mainstay of preservation for kidney allografts
worldwide (O'Callaghan et al. 2012, Opelz & Dohler 2007).
Even though cold ischemia has been studied extensively for a long time, all of
the studies have been carried out in vitro and ex vivo models (O'Callaghan et al. 2012).
66
There is no in vivo model available to examine IRI induced solely by cold ischemia. In
the present study, we developed a novel in vivo model to study renal IRI induced
exclusively by cold ischemia.
Methods
C57BL/6 mice were anesthetized with pentobarbital (50 mg/ml, IP) and their body
temperature was maintained at 36.8-37.2˚C during surgery with a temperature-
controlled operating table (03-02; Vestavia Scientific, AL, USA). A midline abdominal
incision was performed. The right pedicle was ligated, and the right kidney was
removed. The left kidney, aorta and inferior vena cava were exposed (Rong et al. 2012).
Then, the left kidney was placed in a kidney cup and incubated with cool saline (Figure
21A and 21B), while the temperature was maintained at 4±0.5°C by circulating sterilized
saline solution from a saline reservoir on ice, driven with a low flow peristaltic pump
(P720; Instech Laboratories, PA, USA). Four 5 mm microvascular mini clips (FE690K;
AESCULAP INC, PA, USA) were used to prevent bleeding and induce ischemia (Figure
21C) in the following steps: 1) the inferior vena cava and the aorta below the renal
arteries were clamped together (# 1 clip), and then the aorta above the renal arteries
was clamped (# 2 clip); 2) the left renal vein near the vena cava was clamped (# 3 clip),
and a small opening was cut on the left renal vein between clip #3 and the left kidney; 3)
the aorta between the left renal artery and clip #1 was infused with 1-1.5 mL of 4°C
0.9% sterilized saline using a 30G needle until the solution from the opening on the
renal vein cleared and the color of the left kidney became white (Figure 20B); 4) the left
renal pedicle between the opening and the left kidney was clamped (# 4 clip). Then the
67
small opening was sutured with 10-0 suture (BV75-4; ETHICON, GA, USA); 5) three
clips (# 1, 3 and 2 clips) on the inferior vena cava, left renal vein and aorta were
removed, sequentially. The ischemic durations were 1, 2, 3, 4 and 5 hours, respectively.
The time of ischemia was starting from clamping the aorta with clip #2. The clip on the
renal pedicle (# 4 clip) and the kidney cup were removed at the end of the respective
ischemic times. Blood flow was restored. The wounds were sutured, and the mice were
recovered in an incubator until fully awake. The sham–operated mice underwent
identical surgical procedures, and the left kidney was incubated with 4±0.5°C sterilized
saline for 5 hours without ischemia induction as a control group. Twenty-four hours after
cold IRI, some of the mice in different groups were anesthetized again with isoflurane to
collect blood samples and kidney tissues as described below. Other mice in different
groups were kept for 7 days to measure glomerular filtration rate (GFR).
The mice were anesthetized again with isoflurane 24 hours and 7 days after cold
IRI for measuring kidney injury markers. The blood samples (100 µl/each) were
collected through the retro-orbital venous sinus and centrifuged at 8000 rpm for 5
minutes at 4˚C. The plasma (50 µl/each) was obtained for creatinine, KIM-1, and NGAL
measurements. Creatinine concentration was measured by HPLC at the O’Brien Center
Core of the University of Alabama at Birmingham. KIM-1 was measured with a Mouse
KIM-1 Immunoassay Quantikine ELISA KIT, and NGAL was measured with a Mouse
NGAL Immunoassay Quantikine ELISA KIT (R&D System, MN, USA), following the
manufacturer’s instructions.
68
A) The left kidney in a kidney cup and incubated with cool saline. B) The left kidney during the ischemic phase. C) Usage image of clips.
Figure 21. Typical images of the cold ischemia procedure.
69
GFR was measured by the plasma FITC-sinistrin clearance with a single bolus
injection in conscious mice before cold IRI and 7 days after cold IRI as we previous
described with modifications (Schreiber et al. 2012, Wang et al. 2016). Briefly, mice
were injected with FITC-sinistrin solution (5.6 mg/100 g BW) through the retro-orbital
venous sinus. Blood (<10 µl ) was collected into heparinized capillary tubes through the
tail vein at 3, 7, 10, 15, 35, 55, and 75 minutes after sinistrin injection. The blood
samples were centrifuged at 8000 rpm for 5 minutes at 4˚C. Plasma (1 µl) was collected
from each sample. FITC-sinistrin concentration of the plasma was measured using a
plate reader (Cytation3; BioTek, VT, USA) with 485-nm excitation and 538-nm emission.
GFR was calculated with a GraphPad Prism 6 (GraphPad Software).
The kidneys were harvested and fixed in 4% paraformaldehyde solution 24 hours
after cold IRI. Fixed kidney tissues were embedded in paraffin, and 4 µm kidney tissue
slices were cut and stained with PAS. Ten randomly chosen fields were captured under
400x magnification from the cortex, CMR, OM and IM respectively. The percentage of
either necrotic tubules or obstructed tubules by casts in each image was quantified as
reported (Muroya et al. 2015, Pegues et al. 2013). All morphometric analyses were
performed in a blinded manner.
The effects of interest were tested using a Student’s paired t-tests, or two-factor
analysis of variance (ANOVA) with repeated measures when appropriate. Data were
presented as a mean ± standard error of the mean. The changes were considered to be
significant if the p-value was less than 0.05.
70
Results
Plasma creatinine was measured 24 hours and 7 days after cold IRI to evaluate
kidney injury. At 24 hours after cold IRI, the plasma creatinine had no significant
increase in mice with 1 and 2 hours cold ischemia, which were 0.15±0.04 and 0.16±0.05
mg/dl, respectively, compared with sham group (0.09±0.04 mg/dl). The plasma
creatinine increased to 0.34±0.06 mg/dl in mice with 3 hours cold ischemia (P<0.01 vs.
Sham group, 1 and 2 hours groups), to 1.14±0.21 mg/dl in mice with 4 hours cold
ischemia (P<0.01 vs Sham group, 1, 2, and 3 hours groups) and 2.65±0.14 mg/dl in
mice with 5 hours cold ischemia (P<0.01 vs other 5 groups). At 7 days after cold IRI, the
plasma creatinine was still significantly higher in mice with 3 and 4 hours cold ischemia
compared with the other 3 groups (Figure 22A) (P<0.01 vs Sham group, 1 and 2 hours
groups). The mice with 5 hours cold ischemia all died within 2-3 days after cold IRI. The
plasma creatinine had no significant difference among sham, 1 and 2 hours groups.
Plasma KIM-1 and NGAL were measured 24 hours after cold IRI. The plasma
KIM-1 and NGAL had no significant changes in mice with 1 and 2 hours of cold
ischemia compared with the sham-operated animals, but significantly and stepwise
increased in the mice with 3, 4, and 5 hours cold ischemia (Figure 22B and 22C)
(P<0.05 vs Sham group, 1 and 2 hours groups).
71
Plasma creatinine, KIM-1, and NGAL were measured and compared after cold ischemia. A) At 24 hours after cold IRI, the plasma creatinine were highest in the mice with 5 hours cold ischemia, followed by the mice with 4 and 3 hours cold ischemia, and lowest in the mice with 1 and 2 hours cold ischemia. At 7 days after cold IRI, the plasma creatinine was still significantly higher in mice with 3 and 4 hours cold ischemia compared with the other 3 groups (n=6/group). *P<0.01 vs Sham group, 1 and 2 hours groups; #P <0.05 vs 3 hours group; &P<0.01 vs 4 hours group. B) The plasma KIM-1 significantly and stepwise increased in the mice with 3, 4, and 5 hours cold ischemia compared with the mice with 1 and 2 hours cold ischemia (n=5/group). *P<0.01 vs Sham group, 1 and 2 hours groups; #P <0.01 vs 3 hours group; &P<0.05 vs 4 hours group. C) The plasma NGAL was also higher in mice with 3, 4 and 5 hours cold ischemia compared with the mice with 1 and 2 hours cold ischemia (n=5/group).*P<0.05 vs Sham group, 1 and 2 hours groups; #P<0.05 vs 3 hours group; &P<0.01 vs 4 hours group.
Figure 22. Renal injury markers after cold IRI.
72
GFR was measured in conscious mice before cold IRI at baseline and 7 days
after cold IRI. Baseline GFR was similar in all groups of mice. GFR in the sham group
was decreased by about 30% due to uninephrectomy. At 7 days after cold IRI, the GFR
had no significant difference in mice with 1 and 2 hours cold ischemia (161.7±12 and
163.4±8.4µl/min, respectively) compared with the sham group (175.2±15.1 µl/min). The
GFR decreased by 24.1±5.1% in mice with 3 hours cold ischemia and by 42.0±7.4% in
mice with 4 hours cold ischemia compared with the sham group (Figure 23) (P<0.05 vs
Sham group).
GFR were measured in conscious mice before IRI at baseline as well as 7 days after IRI in the mice with cold ischemia. GFR had no significant difference in mice with 1 and 2 hours cold ischemia compared with the sham group. GFR decreased by 24.1±5.1% in mice with 3 hours cold ischemia and by 42.0±7.4% in mice with 4 hours cold ischemia compared with the sham group. (n=6/group) *P<0.05 vs Sham group, 1 and 2 hours groups; #P<0.05 vs 3 hours group.
Baseline 7 days
GF
R (
µl/
min
)
0
50
100
150
200
250
300 Sham
1 h
2 h
3 h
4 h
*
*#
Figure 23. GFR in conscious mice.
73
Figure 24. Histology (continued on next page).
Cortex CMR OM IM
Sham
1 h
2 h
3 h
4 h
5 h
A
74
Histology by PAS staining showed slices of the cortex, corticomedullary region (CMR), outer medulla (OM) and inner medulla (IM) (A). Kidney injury was quantitatively measured by percentage of tubular necrosis (B) and obstructed tubules by casts (C) in the cortex, CMR, OM and IM in all cold ischemia groups. (n=4/group) *P<0.01 vs 1 and 2 hours group; #P<0.05 vs 3 hours group; &P<0.01 vs 4 hours group. Quantitative analysis was performed on ten randomly chosen fields in each kidney tissue slice.
A comparison of histologic appearance of PAS-stained kidney sections 24 hours
after different cold ischemic time length is presented in Figure 24A. The kidneys with no
more than 2 hours cold ischemia exhibited only moderate tubular injury, such as
epithelial cell swelling and intratubular debris. As the cold ischemic time prolonged, the
degree of tubular injury was significantly increased with marked diffusely obstructed
tubules by casts (especially in OM and IM) and necrotic tubules (particulary in CMR and
OM). The mice with 5 hours cold ischemia showed the most severe tubular injury with
the largest incidence of tubular obstruction by casts and tubular necrosis (P<0.05 vs 1,
2 3 and 4 hours groups), followed by the mice with 4 hours cold ischemia and the mice
with 3 hours cold ischemia (Figure 24B and 24C) (P<0.05 vs 1 and 2 hours groups).
Figure 24. Histology.
75
Discussion
In the present study, we develop and characterize a novel in vivo IRI-induced AKI
model that is exclusively induced by cold ischemia, which provides a new tool to
investigate renal IRI in kidney transplantation.
In this model, the blood in the kidney was first flushed out with cold saline and
then the kidney was maintained in a kidney cup with circulating cold saline. IRI was
induced by clamping of the renal pedicle. The body temperature was maintained at
37°C during the experiment. After releasing the clip on the renal pedicle, the kidney cup
was removed and the kidney was reperfused by blood at body temperature. This
approach mimics the cold ischemia and kidney transplantation surgical procedures but
avoids warm ischemia.
Although UW solution is the primary choice for cold storage of deceased donor
kidneys in clinical transplantation (Belzer et al. 1992, Muhlbacher et al. 1999, Southard
et al. 1990), various kinds of perfusion and preservation solutions, including normal
saline (Martins 2006, Rong et al. 2012, Tian et al. 2010), phosphate buffered saline
(Lutz et al. 2007), Ringer’s solution (Wang et al. 2003b) and UW solution (Tse et al.
2014) are used in mouse kidney transplantation. While the effects of different solutions
in mouse kidney transplants have not been compared, it has been considered that the
preservation solutions with sticky properties may result in obstructions of the
microcirculation in mice (Tse et al. 2014). Hence, we perfused and preserved the kidney
with cold saline during the ischemic stage in this model, as most recent studies did in
mouse kidney transplants (Rong et al. 2012, Tian et al. 2010, Tse et al. 2013).
76
The severity of renal injury induced by ischemia is associated with the ischemic
times (Bonventre & Yang 2011, Munshi et al. 2011, Wei & Dong 2012). As indicated by
the levels of plasma creatinine, KIM-1, NGAL, and histology, the mice with 5 hours of
cold renal ischemia induced severe AKI and died with 2-3 days after cold IRI. The mice
with less than 2 hours of cold ischemia had no significant kidney injury. Thus, in this
model, the severity of renal injury could be easily controlled by adjusting the duration of
cold ischemia in the kidney. The plasma creatinine level and GFR at 7 days after
surgery indicated that the mice made almost a full recovery of renal function from
moderate AKI induced by less than 3 hours cold ischemia. This is consistent with the
clinical situation in deceased-donor kidney transplant in which the prolonged cold
ischemia time is associated with a higher risk of delayed graft function and a lower graft
survival rate (Kayler et al. 2011, Perez Valdivia et al. 2011, Sert et al. 2014). It has been
well-documented that the corticomedullary region, comprising the S3 segment of the
proximal tubule and thick ascending limb, is most susceptible in IRI (Bonventre & Yang
2011, Devarajan 2006). The histology of this cold renal IRI model showed the same
pattern that the most severe tubular injury was observed in the CMR with the largest
percentage of necrotic tubules. However, in this model, the inner medulla exhibited
diffuse tubular obstruction by casts, which is inconsistent with warm renal IRI models,
suggesting that the pathophysiological changes and mechanisms of IRI induced by cold
ischemia might be distinguished from those caused by warm ischemia (Bonventre &
Yang 2011, Devarajan 2006).
This innovative in vivo renal ischemia model offers the potential to investigate the
distinctive pathophysiological changes in cold ischemia reperfusion induced AKI beyond
77
the traditional AKI models induced by warm renal ischemia reperfusion. In addition, by
using this new model, it becomes possible to investigate the differences and similarities
in signal pathway and mechanisms of AKI induced by warm or cold ischemia. The
availability of this model in the mouse further advances the capacity for investigation of
mechanisms with the utilization of genetically modified mice. Moreover, by changing the
perfusion and preservation solutions, the effect upon the cold storage of any potential
therapeutic agent or novel organ preservation solution in kidney transplant can be
assessed in this model.
In summary, we developed and characterized a novel in vivo IRI mouse model in
which the renal IRI is exclusively induced by cold ischemia. The cold ischemia phase in
this model to a large extent mimics the kidney storage in deceased-donor kidney
transplants. Therefore with our sophisticated surgical procedure, this new design
provides an additional tool for investigating the pathophysiological changes and the
underlying mechanisms in renal IRI induced by cold ischemia, as well as in studying the
preservation solutions.
78
Chapter Three: Renal Infarction Induced Chronic Kidney Disease
Project I: renal infarction and hypertension
Abstract
Hypertension is frequently observed in patients with renal infarction. The
pathophysiological mechanism for the development of hypertension in renal infarction
has not been elucidated. In the present study, we developed an animal model of
hypertension by ligation of a renal artery branch to induce renal infarction to test our
hypothesis that partial renal infarction induces hypertension mediated by inflammation.
The renal artery in C57BL/6 mice typically bifurcates into two branches before
reaching the renal hilum. We labeled the vascular architecture originated from each
renal artery branch with lectin with confocal microscope.
Partial renal infarction was performed by ligating either upper (LU) or lower (LL)
branch of the left renal artery while the right kidney remained intact. MAP quickly
increased within 3 days after renal infarction, which was from 95.8±6.1 to 120.4±4.6
mmHg in UL group and from 97.5±5.6 to 114.3±5.6 mmHg in LL group 4 weeks after
renal infarction. Plasma renin concentration (PRC) and heart rate (HR) were elevated
and returned to baseline within 7 days after renal infarction. Inflammatory markers of
TNFα and IL-6 in infarcted kidney and plasma significantly increased. Moreover,
79
resection of the infarction zone 2 weeks after renal infarction normalized the blood
pressure and the pro-inflammatory cytokines.
In conclusion, partial renal infarction with the ligation of a renal artery branche in
C57BL/6 mice induced hypertension, which was associated with initially elevated renin
and sympathetic nerve activity and correlated with chronically enhanced inflammatory
response that is dependent on the infarcted region.
Introduction
Renal infarction, characterized with an interruption of blood supply to a portion of
or the whole kidney, could result in irreversible kidney damage and subsequent necrosis
of renal parenchyma along with a decline in kidney function (Bourgault et al. 2013,
Eltawansy et al. 2014). The incidence of renal infarction was reported at approximate
0.003 to 0.007% in previous clinical studies (Domanovits et al. 1999, Huang et al. 2007,
Korzets et al. 2002, Paris et al. 2006). However, due to a lack of specific clinical
manifestation, renal infarction is frequently underdiagnosed and its incidence is largely
underestimated. According to the results of a retrospective postmortem study, 205 renal
infarction cases were diagnosed in a total of 14411 autopsies, indicating an actual
incidence could be around 1.4%.
The epidemiology of renal infarction is complex and has not been completely
understood. Various risk factors, including arteriosclerosis, atrial fibrillation (Antopolsky
et al. 2012a, Frost et al. 2001, Saeed 2012), hypercoagulation (Antopolsky et al. 2012a,
Saeed 2012) and endocarditis (Antopolsky et al. 2012a, Lumerman et al. 1999, Wong et
al. 1984) are involved with renal infarction. Renal thromboembolism originated from the
80
heart or aorta is recognized as the primary cause of renal infarction (Eltawansy et al.
2014, Korzets et al. 2002, Saeed 2012). Despite of a few case reports with normal
blood pressure (Alamir et al. 1997, Javaid et al. 2009), hypertension is commonly
observed in renal infarction. Based on the outcomes of a retrospective cohort study,
hypertension was identified in 48% of the renal infarction patients (Bourgault et al.
2013). Another retrospective clinical study also indicated that renal infarction resulted in
acute or aggravated hypertension in previous normotensive or hypertensive patients.
The renal infarction-induced hypertension was usually ameliorated following surgery or
renal artery angioplasty (Paris et al. 2006).
However, the pathophysiological changes in renal infarction and the mechanisms
of renal infarction induced hypertension have not been fully understood. In addition,
mouse models of partial renal infarction are lacking. Therefore, in present study, we
created a partial renal infarction model in C57BL/6 mice by ligating either the upper or
lower branch of the renal artery in the left kidney while the right kidney remained intact
to determine whether partial renal infarction induces hypertension and investigate the
potential underlying mechanisms.
Methods
Dylight 488 and Dylight 594 labeled tomato lectin (DL-1174 and DL-1177; Vector
Laboratories, CA, USA) were used to mark vasculature from two branches of the renal
artery. The mice were anesthetized with pentobarbital (50 mg/mL, IP). The left kidney,
aorta and inferior vena were exposed after making an abdominal incision. Two
branches of the left renal artery were carefully separated from the left renal vein. The
81
following steps were performed to mark vasculature: 1) the vena cava and the aorta
below the left renal artery were clamped by one microvascular mini clip (FE690K;
AESCULAP INC, PA, USA), and then the aorta above the left renal arteries was
clamped by another clip; 2) an opening was cut on the left renal vein, and the blood was
flushed out from the left kidney by injecting 1-1.5 mL 0.9% saline through the aorta; 3)
the upper branch of the renal artery was clamped, 1 mL Dylight 488 labeled tomato
lectin solution was injected to the kidney through the aorta; 4) 1 mL 0.9% saline was
injected to the kidney again to flush out Dylight 488 labeled tomato lectin solution; 5) the
lower branch of the renal artery was clamped after removing the clip on the upper
branch, 1 mL Dylight 594 labeled tomato lectin solution was then injected to the kidney
through the aorta; 6) clip on the lower branch was removed and 1-1.5 mL 10% buffered
formalin (Sigma, MO, USA) was injected to the kidney through the aorta to fix the
kidney. The left kidney was removed immediately from the mouse and fixed in 10%
buffered formalin. 100um kidney slices were then sectioned with vibratome (HM 650V;
Thermo Fisher SCIENTIFIC, MA, USA). Fixed kidney were examined and the 3D
reconstruction of the kidney were got under a multiphoton laser scanning microscope
(FV1000 MPE; Olympus Corporation, Tokyo, Japan).
MAP and HR were continuously measured with a telemetry system in conscious
mice as described previously (Song et al. 2015, Zhang et al. 2014). Mice were
anesthetized with inhaled isoflurane. The left carotid artery was exposed by a small
incision in the middle of the neck. The pressure catheter was implanted in the left
carotid artery and the telemetric device was placed subcutaneously in the right ventral
flank of the mice. MAP and HR were measured and recorded for 10 seconds every 2
82
min for 4 hours from 1:00 PM to 5:00 PM starting from 7 days after the transmitter
implantation.
The mice were divided into four groups: 1) upper branch ligation group (UL); 2)
lower branch ligation group (LL); 3) upper renal resection group (UL+R); and 4) lower
renal resection group (LL+R). Basal MAP and HR were recorded for three days, partial
renal infarction was then performed in UL and LL groups by ligating either upper or
lower branch of renal artery in the left kidney while the right kidney remained intact.
Briefly, the mice were anesthetized with pentobarbital (50 mg/mL, IP). A midline
abdominal incision was performed to expose the left kidney. The two branches of renal
artery in the left kidney were carefully separated from the renal vein (Figure 24). Either
upper or lower branch of renal artery (Figure 25) was ligated using a 7–0 braided silk
suture (07C1500160; Teleflex Medical OEM, IL, USA). The wound was sutured and the
mouse was kept in the recovery cage for 24 hours. In UL+R and LL+R groups, partial
renal infarction was performed as described before and the infarction zone was
resected 14 days after inducing renal infarction. MAP and HR were measured and
recorded for 4 weeks in all groups. Four weeks after inducing partial renal infarction, all
animals were anesthetized again with inhaled isoflurane. Left kidneys were harvest for
inflammatory factors levels measurement and histology.
We collected about 20 µL blood sample from the tail vein in 50 µL heparinized
micro-hematocrit capillary tube (22-362-566; Fisher, NH, USA) 24 hours and once a
week after inducing renal infarction. Plasma was got by 5 minutes centrifugation (8000
rpm, 4˚C). All of plasma samples were stored at -80°C before renin assay. PRC was
measured by using angiotensin I (Ang I) enzyme immunoassay (EIA) kit as we reported
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previously (S-1188; Bachem, CA, USA). Briefly, two microliter plasma sample was thaw
at room temperature and added into 28 µL EIA buffer to make 15 fold dilution. The
diluted plasma (10 µL) was incubated for 20 min with 0.4 μmol/L porcine
angiotensinogen (AGT) (SCP0021; Sigma, MO, USA), 50 mmol/L sodium acetate (pH
6.5), 2 mmol/L AEBSF (A8456; Sigma, MO, USA), 5 mmol/L EDTA (pH 8) and 1 mmol/L
8-hydoxoyquinoline (252565; Sigma, MO, USA) in an Eppendorf tube. The total volume
of this master mix was 50 µL. The amount of Ang I was measured using an EIA kit. PRC
was expressed as the amount of Ang I generated per hour per microliter of plasma.
Representative image for two branches of renal artery.
Total RNAs were extracted from the left kidney. The RNAs were digested with
RNase-free DNase (Promega, WI, USA) to avoid the genomic contamination. Reverse
transcription was performed to synthesize cDNA templates. The primers set for TNFα,
IL-6 and β-actin genes were designed using Prime3Plus (TNFα- F: 5’-
ATGAGAAGTTCCCAAATGGC-3’, TNFα-R: 5’- CTCCACTTGGTGGTTTGCTA-3’; IL-6-
F: 5’-CTCTGGGAAATCGTGGAAAT-3’, IL-6-R: 5’- CCAGTTTGGTAGCATCCATC-3’;
and β-actin-F: 5’-GTCCCTCACCCTCCCAAAAG-3’, β-actin-R: 5’-
GCTGCCTCAACACCTC-AACCC-3’). Real-time PCR analysis was performed using iQ
Figure 25. Images for renal artery.
84
SYBR Green Supermix (Bio-Rad, CA, USA) and CFX96 Real-Time Detection System
(Bio-Rad, CA, USA) according to the manufacturer's protocol. The comparative Ct
method (2–ΔΔCt) was used to analyze data and calculate the relative mRNA expression
level after PCR program.
The plasma levels of IL-6 and TNF-α were measured by using enzyme linked
immunosorbent assay (ELISA) kit (Abcam, Cambridge, MA). ELISA were performed as
described in the manufacturers.
The kidneys were harvested at 4 weeks after inducing renal infarction and fixed
in 4% paraformaldehyde solution for 24 hours. The kidney tissue was embedded in
paraffin and 2 µm kidney tissue slices were cut and stained with Masson trichrome at
USF health diagnostic laboratory (Sugimoto et al. 2007, Zeisberg et al. 2003). The renal
injury was evaluated by morphometric analysis of interstitial fibrosis and tubular crysts.
Results
To characterize the vascular architecture originated from two renal artery
branches, tomato lectin with different conjugated fluorophores was perfused via each
branch respectively. The fluorescence images of the whole kidney slice showed a clear
border between green and red fluorescence signals. The approximate 2/3 upper portion
of the kidney slice only displayed the green florescence while red fluorescence signal
exclusively existed in the lower part (Figure 26A). The fluorescence images with higher
magnification showed the detailed vascular architecture in the border zone. Some
85
peritubular capillaries exhibited both green and red florescence, however, no glomeruli
showed both green and red fluorescence (Figure 26B and Figure 26C).
Figure 26. Vascular architecture of the kidney (continued on next page).
Figure 26. Vascular architecture of the kidney (continued on next page).
86
Figure 26. Vascular architecture of the kidney (continued on next page).
87
The fluorescence images of the whole kidney slice showed a clear border between green and red signals (A). In the border zone, either red or green fluorescence signal displayed alone in each specific glomerulus (B and C). Blue signal: DAPI; Red signal: Dylight 594 labeled tomato lectin; Green signal: Dylight 488 labeled tomato lectin.
Figure 26. Vascular architecture of the kidney.
88
To determine whether partial renal infarction alters blood pressure, we measured
MAP with the telemetry system in the mice after inducing renal infarction. Baseline MAP
was similar in UL, LL and sham groups. MAP increased by 42.9±3.7 % (from 95.8±6.1
to 136.9±6.2 mmHg) in UL group and by 35.6±4.3 % (from 97.5±5.6 to132.3±3.1
mmHg) in LL group in 2 to 3 days after inducing renal infarction and gradually declined
until 6 to 7 days after inducing renal infarction. Four weeks after inducing renal
infarction, MAP increased by 23.8±2.5% to 120.4±4.6 mmHg in UL group (p<0.01 vs
baseline) and by 19.0±1.1% to 114.3±5.6 mmHg in LL group (p<0.01 vs baseline)
compared with baseline (Figure 27A). In sham group, MAP was constant during the
experiment.
To determine whether the infarction zone has any effect on the blood pressure,
we removed the infarction zone 14 days after inducing renal infarction. MAP in UL+R
and LL+R groups followed the same trend as that in UL and LL groups before removing
the infarction zone. MAP raised by 23.8±2.6% in UL+R group and by 24.1±1.9% in
LL+R group. However, the MAP slowly returned to baseline both in UL+R and LL+R
groups in 5 to 7 days after removing the infarction zone (Figure 27B).
To determine whether renal infarction has any effect on sympathetic nerve
activity, HR were recorded with the telemetry system. Baseline heart rate in UL group
(526±15 bpm) was similar to that in LL group (538±14 bpm). The HR increased to
732±21 bpm in UL group and 687±17 bpm in LL group in 2 to 3 days after inducing
renal infarction (p<0.01 vs baseline, Figure 28). The HR gradually returned to the basal
89
value in 5 to 6 days after inducing renal infarction. HR in sham group was stable during
experiment.
MAP were measured in all of groups. A) The change of MAP after inducing renal infarction in UL and LL groups. (n=8) *p< 0.01 vs baseline, #p< 0.01 vs sham. B) The change of MAP after inducing renal infarction and resecting infarction zone in UL+R and LL+R groups. (n=5) *p< 0.01 vs baseline. UL: upper branch ligation; LL: lower branch ligation; UL+R: upper branch resection; LL+R: lower branch resection.
Figure 27. MAP after inducing renal infarction.
90
The HR increased to the peak in 2 to 3 days after inducing renal infarction and gradually returned to the basal value in 5 to 6 days after inducing renal infarction in UL and LL groups. HR in sham group was stable after surgery and had no difference with baseline. (n=8) *p< 0.01 vs baseline. UL: upper branch ligation; LL: lower branch ligation.
In UL and LL groups, the PRC increased about five folds 24 hours after inducing renal infarction compared with baseline and returned to basal value in 1 week after inducing renal infarction. (n=7) *p< 0.01 vs baseline. UL: upper branch ligation; LL: lower branch ligation.
Days-5 0 5 10 15 20 25 30
HR
(b
pm
)
400
500
600
700
800
UL
LL
Sham
*
Days-5 0 5 10 15 20 25 30
Pla
sm
a r
en
in c
on
ce
ntr
ati
on
(m
g/d
l)
0
100
200
300
400
500
UL
LL*
Figure 28. HR after inducing renal infarction.
Figure 29. PRC after inducing renal infarction.
91
To determine whether PRC has any effect on the increase of blood pressure, we
measured PRC after inducing renal infarction in UL and LL groups. PRC increased
about five fold 24 hours after inducing renal infarction both in UL and LL groups
compared with baseline (p<0.01 vs baseline, Figure 29). PRC returned to basal value
in 1 week after inducing renal infarction.
To determine whether renal infarction induces inflammation, we measured
mRNA and protein levels of TNFα and IL-6 in left kidney homogeneous at 4 weeks after
inducing renal infarction. In UL and LL groups, TNFα mRNA level increased about 4
folds (p<0.01 vs. sham) compared with sham group (Figure 30A). IL-6 mRNA level also
increased about 3 folds (p<0.01 vs. sham) compared with sham group (Figure 30B). In
UL+R and LL+R groups, TNFα and IL-6 mRNA levels had no significant increase
compared with sham group.
Plasma TNFα and IL-6 levels were measured after inducing renal infarction.
Baseline plasma TNFα was similar in all groups. Plasma TNFα was about 9 folds higher
in UL, LL, UL+R and LL+R groups (p<0.01 vs. sham) compared with sham group at 24
h after inducing renal infarction, and still 5 folds higher (p<0.01 vs. sham) at 14 days
after inducing renal infarction. At 28 days after inducing renal infarction, plasma TNFα
was 60.69±16.25 pg/ml in UL group and 82.62±19.40 pg/ml in LL group (p<0.01 vs.
sham), which was significant higher than sham group (9.51±4.47 pg/ml), but it had no
significant increase in UL+R and LL+R groups (Figure 31A). The difference of plasma
IL-6 in all groups was the same as plasma TNFα at 24 h and 14 days after inducing
renal infarction. At 28 days after inducing renal infarction, plasma IL-6 was also
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significant higher in UL and LL groups (96.71±14.07 and 71.79±17.43 pg/ml) compared
with sham group (p<0.01 vs. sham), but it had no significant change in UL+R and LL+R
groups (Figure 31B).
The mRNA levels of TNFα and IL-6 in left kidney homogeneous were measured and compared among UL, LL, UL+R, LL+R and sham groups 4 weeks after inducing renal infarction. A) TNFα mRNA level increased about 4 folds both in UL and LL groups compared with sham group. (n=5) *p<0.01 vs sham. B) IL-6 mRNA level increased about 3 folds both in UL and LL groups compared with sham group. (n=5) *p<0.05 vs sham. UL: upper branch ligation; LL: lower branch ligation; UL+R: upper branch resection; LL+R: lower branch resection.
Figure 30. Inflammatory factors mRNA level.
93
Plasma TNFα (A) and IL-6 levels (B) were measured and compared among sham, UL, UL+R, LL and LL+R groups after inducing renal infarction. At 28 days after inducing renal infarction, plasma TNFα and IL-6 was significant higher than sham group, but it had no significant increase in UL+R and LL+R groups (n=7) *p<0.01 vs sham. UL: upper branch ligation; LL: lower branch ligation; UL+R: upper branch resection; LL+R: lower branch resection.
Figure 31. Plasma inflammatory factors.
94
Kidney weight were measured at 4 weeks after inducing renal infarction. The
right kidney weight had no significant difference among all groups. However, the left
kidney weight were lower in UL and LL groups than sham group (p<0.05 vs. sham). The
left kidney weight in UL+R and LL+R groups were also lower than sham group (p<0.01
vs. sham), but had no significant difference compared with UL and LL groups (Figure
32A and 32B).
Kidney images (A) and kidney weight (B) at 28 days after inducing renal infarction. (UL and LL groups: n=8; UL+R and LL+R groups: n=5) *p<0.05 vs sham. UL: upper branch ligation; LL: lower branch ligation; UL+R: upper branch resection; LL+R: lower branch resection.
Figure 32. Kidney weight.
95
At 4 weeks after inducing renal infarction, interstitial fibrosis was observed by
collagen accumulation in widened interstitial space and the tubular cysts was observed
by enlargement of tubular lumen (Figure 33).
At 28 days after inducing renal infarction, interstitial fibrosis was observed by collagen accumulation in widened interstitial space (yellow triangle) and the tubular cysts was observed by enlargement of tubular lumen (black triangle).
Figure 33. Histology.
96
Discussion
In the present study, we developed a partial renal infarction model in C57BL/6
mouse by ligating one of the two renal artery branches in the left kidney with an intact
contralateral kidney. MAP significantly raised in mice with either branch ligation. PRC
and HR were increased. Inflammatory response was enhanced. Resection of the
infarction zone normalized blood pressure.
In human, the renal artery is typically divided into five segmental arteries near the
renal hilum with one posterior and four anterior (el-Galley & Keane 2000, Urban et al.
2001). Accessory renal artery is a common vascular variant in human and exists in
approximately one-third of the population (Spring et al. 1979, Urban et al. 2001).
Moreover, the anatomy of renal artery and even its branching have been well
characterized in various kinds of laboratory animals, like dog (Marques-Sampaio et al.
2007), pig (Evan et al. 1996, Sampaio et al. 1998), rabbit and rat (Fuller & Huelke 1973,
Yoldas & Dayan 2014). However, the morphology of the renal artery in mouse, in
particularly, the most used laboratory rodent C57BL/6 mouse was not documented
before. On the basis of our observation in current study, accessory renal artery is rare in
C57BL/6 mouse; the left renal artery is bifurcate into two branches at approximate 5 to
10 mm before entering parenchyma; the diameter of upper branch is generally about
twice of the lower, suggesting the renal blood supply via the upper branch is larger than
that through the lower. Accordingly, ligation of upper branch causes ischemia of nearly
2/3 kidney in the upper portion, while occlusion of the lower branch results in
approximate 1/3 renal parenchymal ischemia in the lower part of the kidney. Moreover,
as the branching site of either adrenal artery or ureteral artery from the stem of renal
97
artery is prior to the bifurcation point of the renal artery branches, ligation in either
branch of renal artery would not effect on the blood supply to the adrenal grand or
ureter.
To characterize the vascular architecture originated from the two renal artery
branches, tomato lectin with different conjugated fluorophores was perfused via either
upper or lower branch respectively to distinguish the vasculature from each other.
Tomato lectin, a kind of subunit glycoprotein from Lycopersicon esculentum is utilized in
the visualization of vasculature on account of its capacity of binding to glycocalyx of
vascular endothelium. The fluorescence images of the whole kidney slice showed a
clear border between green and red signals which indicated that the vasculature
distributed in the upper portion of renal parenchyma had its origin from the upper renal
artery branch; on the contrary, the rest renal vasculature existed in the lower part of the
kidney was originated solely from the other renal artery branch. In the border zone,
even though some peritubular capillaries exhibited both green and red color, for each
specific glomerulus, either red or green fluorescence signal displayed only, indicating no
overlapping in blood supply for glomeruli between the two branches.
To determine the changes in blood pressure after partial renal infarction, we
measured the MAP with the telemetry system. Blood pressure raised rapidly and
peaked at 2 to 3 days after surgery, then gradually declined but kept at an elevated
level throughout the remainder experiment course. Even though the upper renal artery
branch supplies more blood flow to a larger portion of the renal parenchyma compared
with the lower branch, there is no significant difference in MAP between the renal
infarction models with the ligation in either upper or lower branch. While no studies in
98
mouse models, Griffin et al. reported that renal mass reduction in Sprague-Dawley rats
by unilateral ligation in one or two of the three renal artery branches plus
uninephrectomy of contralateral kidney resulted in a significant increase in systolic
blood pressure with the peak at 3 to 5 days after surgery (Griffin et al. 1994). In addition,
previous studies by Fekete et al. (Fekete et al. 1975) and Tarjan et al. (Tarjan & Fekete
1975) demonstrated that unilateral total kidney ligation in rats induced hypertension.
Inflammation has been demonstrated to play an essential role in the
development of hypertension (Das 2006, Guzik et al. 2007, Majid et al. 2015). Patients
with renal infarction exhibit enhanced inflammatory response (Antopolsky et al. 2012a,
Bourgault et al. 2013, Korzets et al. 2002). Hence, in regard to the underlying
mechanisms contributing to the blood pressure outcomes after renal infarction in the
present study, we examined the level of pro-inflammatory cytokines, TNF-α and IL-6, in
both partially infarcted kidney and plasma. We found that renal infarction not only
resulted in local inflammation but also lead to upgraded systemic inflammatory status.
Furthermore, resection of the infarction zone normalized the blood pressure and
significantly reduced the pro-inflammatory cytokines level. These results consist with the
clinical outcomes of renal infarction patients that nephrectomy of the infarction zone
ameliorated hypertension (Paris et al. 2006). In addition, our finding is also consistent
with the previous studies on renal mass reduction, in which compared with the approach
with excision of both kidney poles, the reduction of renal parenchyma mass in Sprague-
Dawley rats by ligating two of the three artery branches induced higher blood pressure
with an enhanced renal inflammation (Griffin et al. 1994, Yang et al. 2010).
99
Besides inflammation, renal infarction induced hypertension in clinical patients is
also associated upregulated renin angiotensin system (Candel-Pau et al. 2008, Paris et
al. 2006). Similarly, current study also demonstrated that ligation in renal artery branch
initially induced upregulation in plasma renin. However, it returned to normal within a
week and stayed at a normal level, but the blood pressure maintained at the similar high
level. Renin is generated and released from granular cells in JGA and its secretion is
controlled by renal baroreceptor, sodium chloride delivery at macula densa and
sympathetic nervous system (Kurtz 2011, Peti-Peterdi & Harris 2010, Vander 1967).
The fluorescence images of intrarenal vascular architecture indicated that the blood
supply of each specific glomerulus was solely from only one of the two renal artery
branches without any overlapping. Thus, ligation of either branch of renal artery would
not induce partial ischemia in JGAs, which could promote renin release like in 2K1C
model. Therefore, the increased level of plasma renin might be partially released from
the JGAs in the infarction area before they turn to ischemic necrosis. On the other hand,
the heart rate, which represents the intensity of sympathetic nerve activity (Petretta et
al. 1997, Robinson et al. 1966), was significantly increased after renal infarction and
gradually decline to basaline along with the change in plasma renin. These results
indicate that the enhanced sympathetic activity may contribute to the increased plasma
renin concentration.
In summary, we characterized the intrarenal vascular architecture originated from
each renal artery branch in mouse kidney. Ligation in one of the renal artery branches in
C57BL/6 mice induced hypertension, which was associated with the initially upregulated
sympathetic nerve activity and increased plasma renin concentration and correlated
100
with chronically enhanced inflammatory response. Furthermore, in renal infarction
induced hypertensive mice, resection of the infarction zone normalized hypertension
along with a downregulation of pro-inflammatory cytokines. We conclude that renal
infarction induces hypertension mediated by enhanced inflammatory response
dependent on the infarcted tissue.
Project II: renal infarction and chronic kidney disease
Introduction
CKD is characterized by a progressive loss in kidney function and could
eventually develop into ESRD. CKD is a major health issue in the United States with
increasing morbidity and significant costs. Based on the National Kidney Foundation
Kidney Disease Outcome Quality Initiative clinical practice guidelines for CKD, the
prevalence of CKD in the U.S. is approximate 13.1% and the estimated population of
ESRD is over 0.7 million by 2015 (Coresh et al. 2007, Gilbertson et al. 2005). The risk
factors for CKD are diverse and the epidemiology is usually multiplicate. Currently,
diabetes mellitus and high blood pressure have been recognized as the two primary
causes of CKD (Islam et al. 2009, Levey et al. 2010). Despite of extensive investigation,
the pathophysiological mechanisms of CKD have not been completed elucidated. A
better understanding of CKD might provide potential therapeutic targets and advance
the clinical strategies to prevent or attenuate its progression.
Various animal models have been developed to study CKD. Five-sixths renal
mass reduction, produced by uninephrectomy combined with 2/3 parenchyma reduction
101
on the contralateral kidney, is a broadly used model to mimics the CKD secondary to
reduced nephron number (Purkerson et al. 1976, Shimamura & Morrison 1975, Yang et
al. 2010). Whereas, the development of CKD following 5/6 nephrectomy, achieved by
uninephrectomy and surgical excision of 2/3 contralateral kidney, is highly strain-
dependent in mice. C57BL/6 mouse, the most commonly used strain, is relatively
resistant to 5/6 nephrectomy induced CKD (Leelahavanichkul et al. 2010). On the other
hand, compared with 2/3 renal mass reduction through surgical excision of kidney
poles, the approach via ligation in two of the three renal artery branches resulted in
higher blood pressure and more severe renal injury in 5/6 renal mass reduction models
of rats (Griffin et al. 1994). However, a combination of uninephrectomy and renal artery
branch ligation on contralateral kidney to achieve the total 5/6 renal mass reduction has
nerve been reported before in mice because of the technical difficulty in isolation of
renal artery branches. Thus, in the present study, we aimed to develop a 5/6 renal mass
reduction model by uninephrectomy combined with a ligation of upper renal artery
branch on the contralateral kidney in C57BL/6 mouse and determine whether this model
is able to increase blood pressure and promote the progression of CKD in C57BL/6
mouse (Kren & Hostetter 1999).
Methods
Dylight 488 and Dylight 594 labeled tomato lectin (DL-1174 and DL-1177; Vector
Laboratories, CA, USA) were used to mark vasculature from two branches of the renal
artery. The mice were anesthetized with pentobarbital (50 mg/mL, IP). The left kidney,
aorta and inferior vena were exposed after making an abdominal incision. Two
102
branches of the left renal artery were carefully separated from the left renal vein. The
following steps were performed to mark vasculature: 1) the vena cava and the aorta
below the left renal artery were clamped by one microvascular mini clip (FE690K;
AESCULAP INC, PA, USA), and then the aorta above the left renal arteries was
clamped by another clip; 2) an opening was cut on the left renal vein, and the blood was
flushed out from the left kidney by injecting 1-1.5 mL 0.9% saline through the aorta; 3)
the upper branch of the renal artery was clamped, 1 mL Dylight 488 labeled tomato
lectin solution was injected to the kidney through the aorta; 4) 1 mL 0.9% saline was
injected to the kidney again to flush out Dylight 488 labeled tomato lectin solution; 5) the
lower branch of the renal artery was clamped after removing the clip on the upper
branch, 1 mL Dylight 594 labeled tomato lectin solution was then injected to the kidney
through the aorta; 6) clip on the lower branch was removed and 1-1.5 mL 10% buffered
formalin (Sigma, MO, USA) was injected to the kidney through the aorta to fix the
kidney. The left kidney was removed immediately from the mouse and fixed in 10%
buffered formalin. 100 µm kidney slices were then sectioned with vibratome (HM 650V;
Thermo Fisher SCIENTIFIC, MA, USA). Fixed kidney were examined and the 3D
reconstruction of the kidney were got under a multiphoton laser scanning microscope
(FV1000 MPE; Olympus Corporation, Tokyo, Japan).
The mice were anesthetized again with isoflurane 24 hours after renal ischemia.
The blood samples (100 µl) were collected through the retro-orbital venous sinus and
centrifuged at 8000 rpm for 5 minutes at 4˚C. The plasma (50 µl/each) was obtained for
creatinine, KIM-1 and NGAL measurements. Creatinine concentration was measured by
HPLC at the O’Brien Center Core of the University of Alabama at Birmingham.
103
MAP were continuously measured with a telemetry system in conscious mice as
described previously (Song et al. 2015, Zhang et al. 2014). Mice were anesthetized with
inhaled isoflurane. The left carotid artery was exposed by a small incision in the middle
of the neck. The pressure catheter was implanted in the left carotid artery and the
telemetric device was placed subcutaneously in the right ventral flank of the mice. MAP
were measured and recorded every 1 min for 2 hours from 2:00 PM to 4:00 PM
beginning 7 days after the catheter implantation.
The mice were divided into upper branch ligation group (UX+Ligation) and upper
renal resection group (UX+Resection). In UX+Ligation group, partial renal infarction was
performed by ligating upper branch of renal artery in the left kidney while removing the
right kidney. Briefly, the mice were anesthetized with pentobarbital (50 mg/mL, IP) and a
midline abdominal incision was made to expose two kidneys. After removing the right
kidney, the two branches of renal artery in the left kidney were exposed and upper
branch of renal artery was ligated using a 7–0 braided silk suture (07C1500160;
Teleflex Medical OEM, IL, USA). The wound was sutured and the mouse was kept in
the recovery cage for 24 hours. In UX+Resection group, the ligation zone was resected
right after ligating upper branch. MAP were measured and recorded for 3 months in all
groups. The mice in UX group underwent uninephrectomy of right kidney without
ligation or resection of left kidney. At the end of experiments, all of mice were
anesthetized again with inhaled isoflurane and left kidneys were harvest for
inflammatory factors levels measurement and kidney injury evaluation.
The mice were anesthetized every two weeks with isoflurane 24 hours after
surgery. The blood samples (40 µl) were collected through the retro-orbital venous sinus
104
and centrifuged at 8000 rpm for 5 minutes at 4˚C. The plasma (25 µl/each) was
obtained for creatinine measurement. Creatinine concentration was measured by HPLC
at the O’Brien Center Core of the University of Alabama at Birmingham.
GFR was measured by the plasma FITC-sinistrin clearance with a single bolus
injection in conscious mice every two weeks after surgery as we previous described
with modifications (Schreiber et al. 2012, Wang et al. 2016). Briefly, mice were injected
with FITC-sinistrin solution (5.6 mg/100 g BW) through the retro-orbital venous sinus.
Blood (<10 µl ) was collected into heparinized capillary tubes through the tail vein at 3,
7, 10, 15, 35, 55, and 75 minutes after sinistrin injection. The blood samples were
centrifuged at 8000 rpm for 5 minutes at 4˚C. Plasma (1 µl) was collected from each
sample. FITC-sinistrin concentration of the plasma was measured using a plate reader
(Cytation3; BioTek, VT, USA) with 485-nm excitation and 538-nm emission. GFR was
calculated with a GraphPad Prism 6 (GraphPad Software).
Mice were placed in metabolic cages 24 h to collect urine every month after
surgery. To calculate urine ACR, urine albumin concentration was measured by using
mouse albumin ELISA kit (ab108792; Abcam, Cambridge, MA) and urine creatinine
concentration was measured by HPLC at the O’Brien Center Core of the University of
Alabama at Birmingham.
We collected about 20 µL blood sample from the tail vein in 50 µL heparinized
micro-hematocrit capillary tube (22-362-566; Fisher, NH, USA) 24 hours and once a
week after inducing renal infarction. Plasma was got by 5 minutes centrifugation (8000
rpm, 4˚C). All of plasma samples were stored at -80°C before renin assay. PRC was
measured by using angiotensin I (Ang I) enzyme immunoassay (EIA) kit as we reported
105
previously (S-1188; Bachem, CA, USA). Briefly, two microliter plasma sample was thaw
at room temperature and added into 28 µL EIA buffer to make 15 fold dilution. The
diluted plasma (10 µL) was incubated for 20 min with 0.4 μmol/L porcine
angiotensinogen (AGT) (SCP0021; Sigma, MO, USA), 50 mmol/L sodium acetate (pH
6.5), 2 mmol/L AEBSF (A8456; Sigma, MO, USA), 5 mmol/L EDTA (pH 8) and 1 mmol/L
8-hydoxoyquinoline (252565; Sigma, MO, USA) in an Eppendorf tube. The total volume
of this master mix was 50 µL. The amount of Ang I was measured using an EIA kit. PRC
was expressed as the amount of Ang I generated per hour per microliter of plasma.
Total RNAs were extracted from the left and right kidneys, respectively. The
RNAs were digested with RNase-free DNase (Promega, WI, USA) to avoid the genomic
contamination. Reverse transcription was performed to synthesize cDNA templates.
The primers set for TNFα, IL-6 and β-actin genes were designed using Prime3Plus
(TNFα- F: 5’- ATGAGAAGTTCCCAAATGGC-3’, TNFα-R: 5’-
CTCCACTTGGTGGTTTGCTA-3’; IL-6-F: 5’-CTCTGGGAAATCGTGGAAAT-3’, IL-6-R:
5’- CCAGTTTGGTAGCATCCATC-3’; and β-actin-F: 5’-GTCCCTCACCCTCCCAAAAG-
3’, β-actin-R: 5’-GCTGCCTCAACACCTC-AACCC-3’). Real-time PCR analysis was
performed using iQ SYBR Green Supermix (Bio-Rad, CA, USA) and CFX96 Real-Time
Detection System (Bio-Rad, CA, USA) according to the manufacturer's protocol. The
comparative Ct method (2–ΔΔCt) was used to analyze data and calculate the relative
mRNA expression level after PCR program.
The kidneys were harvested and fixed in 4% paraformaldehyde solution 12
weeks after ligation. Fixed kidney tissues were embedded in paraffin, and 2 µm kidney
tissue slices were cut and stained with PAS. Ten randomly chosen fields were captured
106
under 400x magnification and the degree of glomerular injury was quantified from the
percentage of mesangial expansion as reported (Tervaert et al. 2010).
The kidneys were harvested to evaluate kidney injury under transmission
electron microscope 3 months after surgery. Briefly, with an incision to the right atrium
of the heart, 1M phosphate buffered saline (PBS) solution with 0.8 Units/ml heparin was
perfused via the left ventricle to flush the blood as previously described (3). Small tissue
pieces (approximately 1 mm3) of renal cortex were cut and fixed in 2% glutaraldehyde
in 0.1 M sodium cacodylate buffer (pH 7.4) overnight at room temperature. The tissue
pieces were rinsed with 0.1 M sodium cacodylate buffer (pH 7.4) and treated with 1%
osmium tetroxide for 2 h. Then the tissue pieces were dehydrated with a series of
graded ethanol, sequentially infiltrated with 2:1, 1:1, 1:2 mixtures of acetone and resin,
and embedded in 100% resin. Thin sections (approximately 90-100 nm) were cut and
examined with transmission electron microscope (JEM-1400Plus) at USF Health Lisa
Muma Weitz Laboratory. The thickness of glomerular basement membrane (GBM) was
defined by the shortest distance between endothelial plasma membrane and podocyte
foot processes. The mean GBM thickness of each glomerulus was calculated as
described previously. The podocyte foot process was detected and the average width of
foot process in each glomerulus was determined as described previously.
Results
To characterize the vascular architecture originated from two renal artery
branches, tomato lectin with different conjugated fluorophores was perfused via each
branch respectively. The fluorescence images of the whole kidney slice showed a clear
107
border between green and red fluorescence signals. The approximate 2/3 upper portion
of the kidney slice only displayed the green florescence while red fluorescence signal
exclusively existed in the lower part (Figure 34A). The fluorescence images with higher
magnification showed the detailed vascular architecture in the border zone. Some
peritubular capillaries exhibited both green and red florescence, however, no glomeruli
showed both green and red fluorescence (Figure 34B).
Kidney function of these mice was evaluated by measuring plasma creatinine
concentration and GFR after surgery. The plasma creatinine increased from 0.11±0.02
mg/dl to 0.21±0.03 mg/dl in UX+Ligation group and from 0.12±0.03 mg/dl to 0.18±0.02
mg/dl in UX+Resection group two weeks after surgery, but did not change in UX group.
The plasma creatinine further increased to 0.31±0.05 mg/dl in UX+Ligation group in 12
weeks after surgery (p<0.05 vs. baseline), but remained stable in UX+Resection group
(Figure 35A). Baseline GFR was similar in all groups of mice. GFR in UX group was
decreased by about 30% due to uninephrectomy. GFR decreased by 50.3±2.2% from
249.5±12.7 to 118.4±11.9 µl/min in UX+Ligation group and 41.5±4.4% from 221.5±9.1
to 129.5±15.6 µl/min in UX+Resection group two weeks after surgery. Twelve weeks
after surgery, the GFR further decreased to 82.9±19.2 µl/min in UX+Ligation group
(p<0.01 vs. baseline), but had no decrease in UX+Resection group (Figure 35B).
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Figure 34. Vascular architecture of the kidney (continued on next page).
109
The fluorescence images of the whole kidney slice showed a clear border between green and red signals (A). In the border zone, either red or green fluorescence signal displayed alone in each specific glomerulus (B). Blue signal: DAPI; Red signal: Dylight 594 labeled tomato lectin; Green signal: Dylight 488 labeled tomato lectin.
Figure 34. Vascular architecture of the kidney.
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We measured MAP with the telemetry system in the mice after surgery. Baseline
MAP was similar in all three groups. MAP increased by 29.1±4.4 % from 97.8±5.3 to
126.2±7.8 mmHg in UX+Ligation group in 2 weeks after surgery and further increase to
137.64±13.97 mmHg in 12 weeks after surgery (p<0.01 vs. baseline). In contrast, MAP
in UX+Resection group was only increased by 14.2±2.6 % from 93.5±3.5 to 106.7±6.5
mmHg in 2 weeks after surgery, and then stabilized. MAP in UX group was constant
during the experiment (Figure 36).
A) AT 12 weeks after surgery, plasma creatinine was significant higher in UX+Ligation group compared with UX and UX+Resectin groups (n=8; p<0.01 vs. UX and UX+Resection groups). B) GFR was significant lower in UX+Ligation group compared with UX and UX+Resectin groups (n=8; p<0.05 vs. UX and UX+Resection groups).
Figure 35. Plasma creatinine and GFR after surgery.
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We measured mRNA and protein levels of TNFα and IL-6 at 12 weeks after
surgery. In UX+Ligation group, TNFα mRNA level increased 3.8±0.24 folds (p<0.01 vs.
UX) compared with UX group (Figure 37A) and IL-6 mRNA level also increased
4.1±0.81 folds (p<0.01 vs. UX) compared with UX group (Figure 37B). In UX+Resection
group, TNFα and IL-6 mRNA levels had no significant increase compared with UX
group.
MAP after surgery were measured and compared among UX, UX+Ligation and UX+Resection groups. At 12 weeks after surgery, MAP increased by 40.6±6.2 % in UX+Ligation group, but had no significant increase in UX and UX+Resection groups. (n=5; p<0.01 vs. UX and UX+Resection groups).
Baseline 2 4 6 8 10 12
MA
P (
mm
Hg
)
80
100
120
140
160UX
UX+Resection
UX+Ligation
**
**
**
Figure 36. MAP after surgery.
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The mRNA levels of TNFα and IL-6 were measured and compared among UX, UX+Ligation and UX+Resection groups 12 weeks after surgery. A) TNFα mRNA level increased 3.8±0.24 folds compared with UX group. (n=6) *p<0.01 vs UX and UX+Resection. B) IL-6 mRNA level increased 4.1±0.81 folds compared with UX group. (n=6) *p<0.05 vs UX and UX+Resection.
Figure 38A showed the representative histology images from different groups of
mice. The UX+Ligation group exhibited the more severe glomerular injury (p<0.05 vs. UX
group) compared with UX group (Figure 38C). Widened foot process width and GBM
thickness were observed in UX+Ligation group (p<0.05 vs. UX group) compared with UX
group (Figure 38B, 38D and 38E).
Figure 37. Inflammatory factors mRNA level.
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A) The representative histology images from different groups of mice. B) Transmission electron microscopic images from different groups of mice. C) The UX+Ligation group exhibited the more severe glomerular injury compared with UX group (n=6) *p<0.05 vs UX and UX+Resection. D and E) Widened foot process width and GBM thickness were observed in UX+Ligation group compared with UX group (n=5) *p<0.05 vs UX and UX+Resection.
Figure 38. Light microscopy and transmission electron microscopy
114
Discussion
In the present study, we compared the blood pressure and renal injury in two 5/6
renal mass reduction models of C57BL/6 mouse achieved via different approaches. We
found that 5/6 renal mass reduction by uninephrectomy plus 2/3 renal infarction rather
than 2/3 renal ablation on the contralateral kidney increased blood pressure and
promoted the development of CKD in C57BL/6 mouse. The divergence in blood
pressure and CKD progression between these two models might be mediated by the
stimulated renal and systemic inflammatory response due to the remnant infarcted
tissue.
Five-sixths nephrectomy, produced by uninephrectomy and surgical removal of
2/3 contralateral kidney, is a well-established and widely used CKD model in various
kinds of laboratory animals, such as dog (Bourgoignie et al. 1987, COBURN et al. 1965,
Robertson et al. 1986), rabbit (Eddy et al. 1986, Kilicarslan et al. 2003) and rat (Griffin et
al. 1994, Laouari et al. 1997, Shimamura & Morrison 1975) with the progressive
reduction in kidney function and the development of renal injury, which mimics the
feature of CKD in human. However, it has been recognized that the susceptibility to
develop CKD following 5/6 nephrectomy is strain or genetic background dependent in
mouse. A recent study by Leelahavanichkul et al. showed that progressive CKD, as
indicated by the gradual decrease in GFR, the reciprocal changes in blood urea
nitrogen and serum creatinine, severe albuminuria and mesangial expansion in
glomeruli, was developed by 4 weeks in CD-1 mice and by 12 weeks in 129S3 mice
after 5/6 nephrectomy in which the upper and lower poles of the left kidney were
resected and the whole right kidney was removed. In addition, conscious blood
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pressure measured by radiotelemetry was significantly elevated in both CD-1 mice and
129S3 mice following 5/6 nephrectomy. In contrast, C57BL/6 mouse model with 5/6
nephrectomy exhibited normal blood pressure and was resistant to progressive CKD.
Besides 5/6 nephrectomy, several other approaches have also been reported to
achieve a total 5/6 renal mass reduction in rats (Fleck et al. 2006, van et al. 2013, Yang
et al. 2010).
Moreover, Along with the common procedure uninephrectomy, approximate 2/3
renal mass reduction on contralateral kidney by ligating two of the three renal artery
branches induced higher blood pressure and more severe renal injury, compared with
the approach by surgical excision of kidney poles. On the basis of these studies in
Sprague Dawley (SD) rats, in the present study, we sought to determine whether
subtotal renal mass reduction by renal artery branch ligation rather than ablative surgery
in C57BL/6 mouse is able to result in similar outcomes in SD rats. Our results
demonstrated that renal mass reduction by uninephrectomy combined with upper
branch of renal artery ligation on contralateral kidney induced permanent hypertension
and the development of CKD in C57BL/6 mice. In contrast, the mouse model with
surgical excision of the infarction region exhibited normal blood pressure and resistance
to injury in the remnant kidney, as previous reported 5/6 nephrectomy model with
resection of kidney poles in C57BL/6 mouse (Leelahavanichkul et al. 2010).
The mechanism underlying the divergence in blood pressure response and the
susceptibility to CKD between these two remnant kidney models with different
approaches remains to be determined.
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Inflammation has been recognized to promote the development of hypertension
(Harrison et al. 2011). The elevated C-reactive protein (CRP) level (Bourgault et al.
2013, Guasti et al. 2011) and the increase in white blood cells count (Antopolsky et al.
2012b, Bourgault et al. 2013, Lessman et al. 1978) in blood test indicated an
upregulated systemic inflammatory status in renal infarction patients. Moreover, the
administration of cyclophosphamide, an immune-suppressor, prevented the blood
pressure response to partial renal infarct in SD rats, which further suggests the critical
role of inflammation contributing to the hypertension in renal infarction (Norman, Jr. et
al. 1988). In the present study, we examined the level of pro-inflammatory cytokines,
TNF-α and IL-6, in the remnant kidneys from these two different models with reduced
renal mass. We found that the remnant kidney with upper branch of renal artery ligation
exhibited upgraded inflammation; in contrast, the remnant kidney with surgical excision
of the infarction region had normal level of pro-inflammatory cytokines. These results
are consist with the blood pressure changes in these two mouse models with different
approaches, indicating the immunological reaction against the infarcted tissue in the
remnant kidney might be associated with the permanent hypertension in response to
renal mass reduction by uninephrectomy plus a ligation in upper branch of renal artery
on contralateral kidney.
Furthermore, Ang II administration could induce hypertension in C57BL/6 mice
with 5/6 nephrectomy and overcome the resistance to CKD. The antagonist of Ang II
type I receptor could decrease the blood pressure and blunt the progression of 5/6
nephrectomy induced CKD in CD-1 mice. It has also been indicated that the
upregulation in renin angiotensin aldosterone system (RAAS) is involved with the acute
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increased blood pressure in renal infarction patients (Arakawa et al. 1970, Candel-Pau
et al. 2008, Paris et al. 2006). However, our results demonstrated that the plasma renin
in 5/6 renal mass reduction mouse model with ligation in upper branch of renal artery
kept stable at normal range, which indicates that the hypertension in this model was
independent with RAAS. This finding is consistent with the previous studies on renal
infarction in rats. Norman et al.’s study demonstrated that despite of a transient increase
in plasma renin activity measured at 1 week after a ligation in two out of three renal
artery branches in SD rats, the plasma renin activity declined to normal level by 4 weeks
after renal infarction while hypertension was maintained (Norman, Jr. et al. 1988).
Munich-Wista rats with 5/6 renal mass reduction exhibited even lower plasma renin
concentration than sham-operated rats at 4 weeks after renal infarction (Anderson et al.
1986). Besides the plasma renin, Pupilli et al.’s study also demonstrated that even
though the intrarenal renin concentration in the infarcted tissue was higher than the
remnant kidney parenchyma, it was comparable with that in the kidney from sham-
operated rats (Pupilli et al. 1992). Moreover, the fluorescence images of intrarenal
vascular architecture showed that the blood supply for each specific glomerulus was
solely originated from only one of the two renal artery branches without any overlapping.
Thus, ligation of the upper branch of renal artery would not induce partial ischemia in
JGAs in remnant kidney, which is consistent with the normal plasma renin level in this
model. Thus, it has been suggested that blood pressure played a critical role in the
development of CKD in mouse model with 5/6 nephrectomy; the susceptibility to CKD in
different strains correlated with their blood pressure changes after renal mass reduction
(Leelahavanichkul et al. 2010).
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Hypertension has been defined as one of the leading causes of CKD and to
accelerate the progression of CKD (Chanda & Fenves 2009, Metcalfe 2007, Ritz et al.
1993). High systemic blood pressure transmitted into glomerular capillary increases the
mechanical forces in glomerulus. This mechanical insult in glomerulus upregulates the
cytokines, such as fibronectin (Gruden et al. 2000), transforming growth factor-beta1
(Gruden et al. 2000), enhances the accumulation of mesangial extracellular matrix
(Riser et al. 1992), stimulates glomerular monocyte infiltration via an NF-kB and
monocyte chemoattractant protein-1 mediated pathway (Gruden et al. 2005, Ha et al.
2002), which promotes the pathogenesis of the glomerular injury. In the present study,
significant glomerular injury, as indicated by the progressive proteinuria and
glomerulosclerosis was observed in the remnant kidney with upper branch of renal
artery ligation but not in the kidney with surgical excision of the infarction region, which
was consistent with the divergence of blood pressure in these two reduced renal mass
models with different approaches, suggesting the critical role of blood pressure in the
development of CKD in the C57BL/6 mouse model with renal mass reduction.
Upregulated systemic Inflammation has been recognized to involve in the
progression of CKD (Gupta et al. 2012, Silverstein 2009). A recent prospective cohort
study of CKD patients demonstrated that plasma levels of pro-inflammatory biomarkers
including IL-6 and TNF-alpha were strongly inversely correlated with kidney function, as
assessed by eGFR and serum cystatin C, and positively associated with glomerular
injury, as evaluated with albuminuria or urine albumin to creatinine ratio (Gupta et al.
2012). It has been known that inflammation stimulates oxidative stress (Cachofeiro et al.
2008, Reuter et al. 2010, Shah et al. 2007), vasoconstriction (Linden et al. 2008,
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Remuzzi et al. 2005, Vianna et al. 2011), leukocyte adhesion (Betjes 2013, Hill et al.
1994, Kato et al. 2008), mesangial cell proliferation (Lopez-Novoa et al. 2011, Migliorini
et al. 2013, Schlondorff & Banas 2009), and tubulointerstitial fibrosis (Hodgkins &
Schnaper 2012, Ke et al. 2016), which promotes the progression of CKD. Moreover, it
has been reported that downregulation of inflammation slowed the progression of CKD.
Quiroz et al.’s study demonstrated that administration of melatonin, a potent anti-
inflammatory hormone produced in pineal gland, significantly attenuated the decline in
kidney function as indicated by plasma creatinine level and ameliorated the
deterioration of renal injury, as assessed with proteinuria and glomerulosclerosis, in SD
rats with 5/6 renal mass reduction (Quiroz et al. 2008). In addition, Clopidogrel, an
inhibitor of adenosine diphosphate receptor on platelet cell membrance, was reported to
attenuate the early progression of CKD in Wistar rats by downregulating the systemic
inflammation (Tu et al. 2008). Therefore, in the present study, besides blood pressure,
the inflammatory status in the remnant kidney might also contribute to the divergence in
CKD in these two C57BL/6 mouse models with different approaches.
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Chapter Four: Discussion
The role of intratubular pressure in acute kidney injury
In the present study, we found a positive association between the Pt during the
ischemic phase and the severity of AKI in three widely-used models by clamping of the
renal arteries, pedicles or veins. To determine the role of Pt during the ischemic phase of
the development of AKI, we adjusted Pt by altering the renal artery pressure while
clamping the renal veins. We found that increasing the Pt during the ischemic phase
exacerbates AKI while lowering the Pt protects against kidney injury.
Tubular biomechanical forces are involved in the regulation of renal tubular
function and associated with epithelial cell damage. In obstructive uropathy, increased
intratubular pressure and tubular stretch upregulate TGFβ-1, contributing to
tubulointerstitial inflammation and fibrosis (Sato et al. 2003). Also, cyclical stretch on renal
epithelial cells induces apoptosis by activating JNK/SAPK and p38 SAPK-2 pathways
(Nguyen et al. 2006). However, little is known about the role of tubular biomechanical
forces in the development of AKI. Renal vein or pedicle clamping induced more severe
AKI than renal artery clamping, but the mechanisms are elusive (Liano & Pascual 1996,
Orvieto et al. 2007). We examined whether tubular pressure during the ischemic phase
plays any significant role in the development of kidney injury. AKI was induced with 18
min occlusion of the renal blood flow at 37 ° C in C57BL/6 mice. First, we measured the
Pt during the ischemic phase in three AKI models. We found that clamping of the renal
121
arteries significantly lowered the Pt below the pre-ischemia basal level. Occlusion of the
renal arteries created cessation of glomerular filtration, resulting in a decrease of the Pt.
On the contrary, the Pt was significantly elevated to approximate 50 mmHg by clamping
the renal veins. The renal venous occlusion lead to kidney congestion, which increased
the glomerular hydrostatic pressure, consequently resulting in an increase of the Pt.
Surprisingly, clamping of the renal pedicles also significantly increased the Pt to
approximately 35 mmHg. We confirmed this finding by tightly ligating the renal pedicles
with a 6-0 suture to exclude the possibility of incomplete occlusion of the renal blood flow.
Suture ligation of the renal pedicles raised the Pt to a similar level as clamping of the renal
pedicles. Thus, this phenotype was not due to the incomplete occlusion of the renal artery.
However, we do not know the exact mechanism for this phenomenon, which might result
from decreased tubular reabsorption, tubular obstruction and continued glomerular
filtration induced by residual pressure after clamping of the pedicles. To our knowledge,
the present study is the first to examine the Pt during the ischemic phase. Several
previous studies measured the Pt after reperfusion. Arendshorst et al. examined the Pt
after occlusion of the renal artery in rats for 60 min in an elegant study (Arendshorst et al.
1975). They found that at 1-3 hours after reperfusion, the proximal tubules were filled with
fluid and dilated; the Pt elevated to about 30 mmHg with great heterogeneity (Arendshorst
et al. 1975). The Pt measured in the other studies were also found to be higher than
baseline 1-3 days after IRI- or uranyl nitrate-induced AKI (Conger et al. 1984, Eisenbach
& Steinhausen 1973, Flamenbaum et al. 1974, Tanner & Sophasan 1976). The
heterogeneity in the Pt observed in these studies may be due to tubular obstructions
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(Conger et al. 1984, Eisenbach & Steinhausen 1973, Flamenbaum et al. 1974, Tanner &
Sophasan 1976).
Although we used the method of Isotope Dilution LC-MSMS to measure creatinine
in mouse plasma to reduce the background and increase specificity (Takahashi et al.
2007), we realized the limitation of creatinine as an injury maker in mice, since 50% of
the creatinine is eliminated via secretion in the mouse kidney tubules (Eisner et al. 2010,
Vallon et al. 2012). Therefore, we evaluated kidney injury combined with changes in KIM-
1, NGAL, and histology in these three AKI models. We found that at 24 hours after renal
ischemia, clamping of the renal veins induced the most severe AKI while clamping of the
renal arteries induced the least renal injury. These results were consistent with the results
from previous studies (Liano & Pascual 1996, Orvieto et al. 2007). Therefore, the Pt during
the ischemic phase in these three models was positively associated with the severity of
AKI.
Acute reduction in renal blood flow in various clinical situations, such as in
hypovolemic shock, some cardiac and abdominal surgeries, cardiac arrest, acute left
heart failure, and kidney transplantation, etc. is often caused by renal arterial hypo-
perfusion. In such case, the renal artery clamping model should better mimic these clinical
conditions. Clamping of the renal vein is more consistent with clinical situations of vein
thrombosis, chronic right heart failure, or severe liver cirrhosis. Even though occlusion of
both the renal artery and vein is not a typical clinical scenario, clamping of the renal
pedicle is the most common choice for the rodent AKI models, probably due to the
technical challenge of isolating murine renal arteries. In consideration of the significant
123
difference in renal injury in different models, comparison and interpretation of these
results should be conducted cautiously.
To determine the significance of Pt during the ischemic phase of the development
of AKI, we modulated the Pt by adjusting the renal artery pressure while clamping the
renal veins. To adjust the renal artery pressure, we constricted the aorta and mesenteric
artery, rather than clamp them to avoid ischemic injury of tissues supplied by the aorta
and mesenteric artery. Constriction of the aorta above the renal arteries lowered the renal
artery pressure, which led to a decrease in the Pt compared with the normal renal artery
pressure group. On the contrary, constriction of the aorta and the superior mesenteric
artery raised the renal artery pressure, which resulted in an increase in the Pt. The kidney
injury markers were compared at 24 hours after renal ischemia. Mice with a lower Pt in
the ischemic phase exhibited less renal injury than mice with a normal Pt, while mice with
an elevated Pt showed more severe renal injury. Therefore, these results suggested that
Pt during the ischemic phase contributes to the development of AKI. A lower Pt in the
ischemic phase protects against the development of AKI, while a higher Pt aggravates
the progression of AKI. Under physiological conditions, renal autoregulation maintains the
renal blood flow and GFR relatively constant, despite fluctuations in the arterial blood
pressure over a range of about 80-160 mmHg. This buffers the transmission of
alterations in the arterial blood pressure into the renal tubules (Carlstrom et al. 2015,
Loutzenhiser et al. 2006). However, ischemia may impair this auto-regulatory capacity,
resulting in the loss of independent stability in tubular dynamics, thereby permitting the
arterial pressure transmitted to the intra-glomerulus and tubules (Adams et al. 1980, Guan
et al. 2006).
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The role of renal temperature in acute kidney injury
In the present study, we develop and characterize a novel in vivo IRI-induced AKI
model that is exclusively induced by cold ischemia, which provides a new tool to
investigate renal IRI in kidney transplantation.
In this model, the blood in the kidney was first flushed out with cold saline and
then the kidney was maintained in a kidney cup with circulating cold saline. IRI was
induced by clamping of the renal pedicle. The body temperature was maintained at
37°C during the experiment. After releasing the clip on the renal pedicle, the kidney cup
was removed and the kidney was reperfused by blood at body temperature. This
approach mimics the cold ischemia and kidney transplantation surgical procedures but
avoids warm ischemia.
Although UW solution is the primary choice for cold storage of deceased donor
kidneys in clinical transplantation (Belzer et al. 1992, Muhlbacher et al. 1999, Southard
et al. 1990), various kinds of perfusion and preservation solutions, including normal
saline (Martins 2006, Rong et al. 2012, Tian et al. 2010), phosphate buffered saline
(Lutz et al. 2007), Ringer’s solution (Wang et al. 2003b) and UW solution (Tse et al.
2014) are used in mouse kidney transplantation. While the effects of different solutions
in mouse kidney transplants have not been compared, it has been considered that the
preservation solutions with sticky properties may result in obstructions of the
microcirculation in mice (Tse et al. 2014). Hence, we perfused and preserved the kidney
with cold saline during the ischemic stage in this model, as most recent studies did in
mouse kidney transplants (Rong et al. 2012, Tian et al. 2010, Tse et al. 2013).
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The severity of renal injury induced by ischemia is associated with the ischemic
times (Bonventre & Yang 2011, Munshi et al. 2011, Wei & Dong 2012). As indicated by
the levels of plasma creatinine, KIM-1, NGAL, and histology, the mice with 5 hours of
cold renal ischemia induced severe AKI and died with 2-3 days after cold IRI. The mice
with less than 2 hours of cold ischemia had no significant kidney injury. Thus, in this
model, the severity of renal injury could be easily controlled by adjusting the duration of
cold ischemia in the kidney. The plasma creatinine level and GFR at 7 days after
surgery indicated that the mice made almost a full recovery of renal function from
moderate AKI induced by less than 3 hours cold ischemia. This is consistent with the
clinical situation in deceased-donor kidney transplant in which the prolonged cold
ischemia time is associated with a higher risk of delayed graft function and a lower graft
survival rate (Kayler et al. 2011, Perez Valdivia et al. 2011, Sert et al. 2014). It has been
well-documented that the corticomedullary region, comprising the S3 segment of the
proximal tubule and thick ascending limb, is most susceptible in IRI (Bonventre & Yang
2011, Devarajan 2006). The histology of this cold renal IRI model showed the same
pattern that the most severe tubular injury was observed in the CMR with the largest
percentage of necrotic tubules. However, in this model, the inner medulla exhibited
diffuse tubular obstruction by casts, which is inconsistent with warm renal IRI models,
suggesting that the pathophysiological changes and mechanisms of IRI induced by cold
ischemia might be distinguished from those caused by warm ischemia (Bonventre &
Yang 2011, Devarajan 2006).
This innovative in vivo renal ischemia model offers the potential to investigate the
distinctive pathophysiological changes in cold ischemia reperfusion induced AKI beyond
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the traditional AKI models induced by warm renal ischemia reperfusion. In addition, by
using this new model, it becomes possible to investigate the differences and similarities
in signal pathway and mechanisms of AKI induced by warm or cold ischemia. The
availability of this model in the mouse further advances the capacity for investigation of
mechanisms with the utilization of genetically modified mice. Moreover, by changing the
perfusion and preservation solutions, the effect upon the cold storage of any potential
therapeutic agent or novel organ preservation solution in kidney transplant can be
assessed in this model.
The role of tubuloglomerular feedback in acute kidney injury
In the present study, we examined 1) the effect of renal pH on TGF response and
the activity and expression of NOS1β in the macula densa; 2) the TGF responsiveness
change and the alteration of NOS1β activity and expression in the macula densa in IRI-
induced AKI; and 3) the effect of bicarbonate treatment on the development of IRI-
induced AKI. We found that 1) renal pH modulates TGF response by regulating NOS1β
in the macula densa; 2) renal IRI downregulates NOS1β in the macula densa and
enhances TGF response, which promotes the development of AKI; and 3) bicarbonate
treatment protected against IRI-induced AKI by upregulating NOS1β in the macula
densa.
In both humans and experimental animals, reductions in GFR have been
attributed to persistent vasoconstriction. An intense and persistent renal
vasoconstriction that reduces overall kidney blood flow and GFR has long been
considered a hallmark of AKI (Lameire et al. 2005, Mason et al. 1977). In fact, ischemic
127
AKI has been referred to as “vasomotor nephropathy”. A severe injury of the proximal
tubular in renal cortex and TAL in the outer medulla is a uniform feature in various forms
of AKI while the glomeruli are morphologically normal (Heyman et al. 1988, Lieberthal &
Nigam 1998, Oken 1975, Regner et al. 2009). Tubular injury can also lead to a
decrease in GFR by tubular obstruction with epithelial debris and backleak. However,
normal pressures have been observed in several tubules, making it unlikely for tubular
obstruction to be the singularly important mediator of reduction in GFR after ischemic
injury (Conger et al. 1984). Arendshorst et al.(Arendshorst et al. 1975) studied severe
AKI after 1 h of unilateral renal ischemia. They found that in the early stages (1-3 h)
after ischemia, the pressures in the proximal and distal tubules were elevated, and there
was histological evidence of tubular obstruction. Tubular obstruction appeared to be the
predominant factor in the decline in filtration rate at this stage. However, at a later time-
point (22-26 h), tubular pressures were normal, but preglomerular vasoconstriction was
marked and this was thought to contribute to the persistent decline in filtration rate. In
addition, tubular obstruction does not appear to be a significant pathogenic mechanism
in AKI in humans, in which only about 10% decrease in GFR could be explained by
tubular backleak (Blantz & Singh 2011, Braam et al. 1993, Myers et al. 1979, Schrier et
al. 2004). A single nephron model of nephrotoxic tubular injury was established by
microperfusion of uranyl nitrate into the early proximal tubule (Peterson et al. 1989).
Within minutes of intratubular administration of uranyl nitrate, a 16-30% reduction in
SNGFR was observed. The local mechanism such as TGF could be invoked to explain
the immediate decline in SNGFR. Similar findings have been observed in the uranyl
nitrate model of AKI by another group (Flamenbaum et al. 1974). Hence, an increase in
128
preglomerular resistance appears to be consistent in nearly all forms of AKI and TGF
has been postulated to dictate the enhanced preglomerular vascular resistance and
reduction in GFR in AKI (Thurau & Boylan 1976). In present study, we measured TGF in
vivo with micropuncture right after and 7days after following 18 min clamping of renal
arteries. The results indicate that renal ischemia impairs TGF response early after IRI,
but enhances TGF response later after IRI.
As an enzyme to catalyze the generation of NO and citrulline from L-arginine,
NOS1 is affected by pH. However, the optimal pH for NOS1 is controversial in previous
studies. The maximal efficiency of NOS1 was measured at a pH 6.7 with the brain
tissue (Chen et al. 1997). However, the most favorable pH for NOS1 in the kidney tissue
was indicated to be around 8.0 (Liu et al. 2004a, Wang et al. 2003a). There are three
splice variants of NOS1, α, β, and γ. NOS1α is the primary isoform in brain but NOS1β
is the majority in kidney. Hence, in present study, we generated the distinct NOS1α and
NOS1β by their expression vectors and measured the activity under conditions with
graded pH environment. The results indicated that the most favable pH for NOS1α was
6.5 and the optimium for NOS1β was 7.9, which is consistant of the previous studies
and explains the controversy.
In previous studies, we reported that the NO generation by the macula densa
enhances during TGF response (Liu et al. 2004a). The activation of nNOS is mediated
by elevation of intracellular pH in the macula densa via NHE (Liu et al. 2004a).
Microperfusion studies showed when luminal pH was increased, there was a linear
correlation between changes in intracellular pH and extracellular pH in macula densa,
and intracellular pH of the macula densa at high NaCl was always higher than at low
129
NaCl (Liu et al. 2008). Also, in cultured tubular cells decreasing pH in culture medium
with different concentration of NaHCO3 accordingly reduced intracellular pH (Yaqoob et
al. 1996). These studies indicate changing luminal pH can be used to alter intracellular
pH. In our study, it is very likely that modulation of luminal pH took effect by altering
intracellular pH. During TGF response NO concentration by the macula densa
increased significantly (Liu et al. 2002). The inhibition of NO generation by either NOS
blockers (Welch & Wilcox 1997) or specific nNOS inhibitors (Ren et al. 2000b, Welch et
al. 2000) enhances TGF responsiveness both in vivo and in vitro. We previously found
that raising intracellular pH of the macula densa from 7.3 to 7.8 significantly increased
NO concentration in microdissected and perfused macula densa and 7-Nitroindazole
blocked the increase of NO induced by elevated pH (Liu et al. 2004a). In present study,
we found tubular pH directly regulate TGF responsiveness at the macula densa in vivo
with micropuncture and in vitro with microperfusion. Increases of pH in tubules blunt
TGF response. NOS inhibition eliminated this effect, indicating NO mediated the
attenuation of TGF induced by elevation of tubular pH.
Current guidelines for CKD patients recommend alkali treatment with sodium
citrate or sodium bicarbonate for those with serum total CO2 less than 22 mM (2000).
Indeed, the correlation between low serum bicarbonate level and progressive decline in
eGFR has been studied extensively in patients with CKD. Alkali therapy reduces kidney
injury and slows nephropathy progression in patients with CKD (de Brito-Ashurst et al.
2009, Dobre et al. 2015, Eustace et al. 2004, Goraya et al. 2012, Kovesdy et al. 2009,
Mahajan et al. 2010, Navaneethan et al. 2011, Phisitkul et al. 2010, Raphael et al. 2011,
Shah et al. 2009, Susantitaphong et al. 2012), but the mechanism is not clear. Diet can
130
markedly affect acid-base status and it significantly influences CKD and its
progression. In the National Health and Nutrition Examination Survey, 11,957 adults
were analyzed and found that high serum anion gap values and decreased serum total
CO2 concentration were associated with increased mortality in the general population
and with a decline in eGFR in patients with CKD (Abramowitz et al. 2012). In a separate
study, the correlation between dietary acid load and eGFR were assessed in 12,293 US
adults. High dietary acid load was found to be associated with albuminuria and low
eGFR (Banerjee et al. 2014). A cross-sectional study of the African American Study of
Kidney Disease and Hypertension cohort indicate that a higher acid load and lower total
serum CO2 concentration (reflects serum bicarbonate concentration) correlate with more
severe CKD and readily decline of eGFR (Scialla et al. 2011). Oral bicarbonate
supplementation slows CKD progression in humans (de Brito-Ashurst et al.
2009). Recent two intervention studies, one in patients with early CKD without metabolic
acidosis (Goraya et al. 2012), and the other in patients with advanced CKD and
metabolic acidosis (Goraya et al. 2013), suggest that a diet rich in fruits and vegetables
can improve renal function and slow the progression of CKD. In patients at risk of CKD,
alkali-generating diets that are rich in fruits and vegetables reduce the risk of developing
CKD (Chang et al. 2013, Dunkler et al. 2013, Lin et al. 2011, Nettleton et al. 2008,
Scialla et al. 2011). Oral bicarbonate supplementation has been found to delay the
decline of GFR in CKD patients (Dobre et al. 2015, Eustace et al. 2004, Kovesdy et al.
2009, Navaneethan et al. 2011, Raphael et al. 2011, Shah et al. 2009, Susantitaphong
et al. 2012), but the mechanism is not clear. Recently, the relationship between serum
bicarbonate and GFR has been analyzed in people with normal renal functions. Lower
131
serum bicarbonate concentrations are found to be associated independently with rapid
decline of eGFR in a study of 5422 adults; in a Multi-Ethnic Study of Atherosclerosis of
5810 participants (Driver et al. 2014), and in a Health, Aging, and Body Composition
study (Goldenstein et al. 2014). The baseline eGFR of most or all of the participants in
these studies is above 60 mL/min/1.73 m2. Dietary acid load has also been found an
important determinant of kidney injury in rat models of CKD with and without metabolic
acidosis (Khanna et al. 2004, Mahajan et al. 2010, Nath et al. 1985, Phisitkul et al.
2010, Wesson et al. 2006, Wesson & Simoni 2010). In present study, we measured
GFR and plasma creatinine 7 days after IRI in bicarbonate treated C57BL/6J and MD-
NOS1KO mice. We found NaHCO3 treatment can limit the decrease of GFR and the
increase of plasma creatinine in C57BL/6J mice, but this limitation was attenuated in
MD-NOS1KO mice. The results indicate upregulation of NOS1β in the macula densa by
a high bicarbonate intake could be a novel mechanism, but also a potential target for
prevention and treatment for IRI-induced AKI.
Renal infarction and hypertension
In the present study, we developed a partial renal infarction model in C57BL/6
mouse by ligating one of the two renal artery branches in the left kidney with an intact
contralateral kidney. MAP significantly raised in mice with either branch ligation. PRC
and HR were increased. Inflammatory response was enhanced. Resection of the
infarction zone normalized blood pressure.
In human, the renal artery is typically divided into five segmental arteries near the
renal hilum with one posterior and four anterior (el-Galley & Keane 2000, Urban et al.
132
2001). Accessory renal artery is a common vascular variant in human and exists in
approximately one-third of the population (Spring et al. 1979, Urban et al. 2001).
Moreover, the anatomy of renal artery and even its branching have been well
characterized in various kinds of laboratory animals, like dog (Marques-Sampaio et al.
2007), pig (Evan et al. 1996, Sampaio et al. 1998), rabbit and rat (Fuller & Huelke 1973,
Yoldas & Dayan 2014). However, the morphology of the renal artery in mouse, in
particularly, the most used laboratory rodent C57BL/6 mouse was not documented
before. On the basis of our observation in current study, accessory renal artery is rare in
C57BL/6 mouse; the left renal artery is bifurcate into two branches at approximate 5 to
10 mm before entering parenchyma; the diameter of upper branch is generally about
twice of the lower, suggesting the renal blood supply via the upper branch is larger than
that through the lower. Accordingly, ligation of upper branch causes ischemia of nearly
2/3 kidney in the upper portion, while occlusion of the lower branch results in
approximate 1/3 renal parenchymal ischemia in the lower part of the kidney. Moreover,
as the branching site of either adrenal artery or ureteral artery from the stem of renal
artery is prior to the bifurcation point of the renal artery branches, ligation in either
branch of renal artery would not effect on the blood supply to the adrenal grand or
ureter.
To characterize the vascular architecture originated from the two renal artery
branches, tomato lectin with different conjugated fluorophores was perfused via either
upper or lower branch respectively to distinguish the vasculature from each other.
Tomato lectin, a kind of subunit glycoprotein from Lycopersicon esculentum is utilized in
the visualization of vasculature on account of its capacity of binding to glycocalyx of
133
vascular endothelium. The fluorescence images of the whole kidney slice showed a
clear border between green and red signals which indicated that the vasculature
distributed in the upper portion of renal parenchyma had its origin from the upper renal
artery branch; on the contrary, the rest renal vasculature existed in the lower part of the
kidney was originated solely from the other renal artery branch. In the border zone,
even though some peritubular capillaries exhibited both green and red color, for each
specific glomerulus, either red or green fluorescence signal displayed only, indicating no
overlapping in blood supply for glomeruli between the two branches.
To determine the changes in blood pressure after partial renal infarction, we
measured the MAP with the telemetry system. Blood pressure raised rapidly and
peaked at 2 to 3 days after surgery, then gradually declined but kept at an elevated
level throughout the remainder experiment course. Even though the upper renal artery
branch supplies more blood flow to a larger portion of the renal parenchyma compared
with the lower branch, there is no significant difference in MAP between the renal
infarction models with the ligation in either upper or lower branch. While no studies in
mouse models, Griffin et al. reported that renal mass reduction in Sprague-Dawley rats
by unilateral ligation in one or two of the three renal artery branches plus
uninephrectomy of contralateral kidney resulted in a significant increase in systolic
blood pressure with the peak at 3 to 5 days after surgery (Griffin et al. 1994). In addition,
previous studies by Fekete et al. (Fekete et al. 1975) and Tarjan et al. (Tarjan & Fekete
1975) demonstrated that unilateral total kidney ligation in rats induced hypertension.
Inflammation has been demonstrated to play an essential role in the
development of hypertension (Das 2006, Guzik et al. 2007, Majid et al. 2015). Patients
134
with renal infarction exhibit enhanced inflammatory response (Antopolsky et al. 2012a,
Bourgault et al. 2013, Korzets et al. 2002). Hence, in regard to the underlying
mechanisms contributing to the blood pressure outcomes after renal infarction in the
present study, we examined the level of pro-inflammatory cytokines, TNF-α and IL-6, in
both partially infarcted kidney and plasma. We found that renal infarction not only
resulted in local inflammation but also lead to upgraded systemic inflammatory status.
Furthermore, resection of the infarction zone normalized the blood pressure and
significantly reduced the pro-inflammatory cytokines level. These results consist with the
clinical outcomes of renal infarction patients that nephrectomy of the infarction zone
ameliorated hypertension (Paris et al. 2006). In addition, our finding is also consistent
with the previous studies on renal mass reduction, in which compared with the approach
with excision of both kidney poles, the reduction of renal parenchyma mass in Sprague-
Dawley rats by ligating two of the three artery branches induced higher blood pressure
with an enhanced renal inflammation (Griffin et al. 1994).
Besides inflammation, renal infarction induced hypertension in clinical patients is
also associated upregulated renin angiotensin system (Arakawa et al. 1970, Candel-
Pau et al. 2008, Paris et al. 2006). Similarly, current study also demonstrated that
ligation in renal artery branch initially induced upregulation in plasma renin. However, it
returned to normal within a week and stayed at a normal level, but the blood pressure
maintained at the similar high level. Renin is generated and released from granular cells
in JGA and its secretion is controlled by renal baroreceptor, sodium chloride delivery at
macula densa and sympathetic nervous system (Kurtz 2011, Peti-Peterdi & Harris 2010,
Vander 1967). The fluorescence images of intrarenal vascular architecture indicated
135
that the blood supply of each specific glomerulus was solely from only one of the two
renal artery branches without any overlapping. Thus, ligation of either branch of renal
artery would not induce partial ischemia in JGAs, which could promote renin release like
in 2K1C model. Therefore, the increased level of plasma renin might be partially
released from the JGAs in the infarction area before they turn to ischemic necrosis. On
the other hand, the heart rate, which represents the intensity of sympathetic nerve
activity (Petretta et al. 1997, Robinson et al. 1966), was significantly increased after
renal infarction and gradually decline to basaline along with the change in plasma renin.
These results indicate that the enhanced sympathetic activity may contribute to the
increased plasma renin concentration.
Renal infarction and chronic kidney disease
In the present study, we compared the blood pressure and renal injury in two 5/6
renal mass reduction models of C57BL/6 mouse achieved via different approaches. We
found that 5/6 renal mass reduction by uninephrectomy plus 2/3 renal infarction rather
than 2/3 renal ablation on the contralateral kidney increased blood pressure and
promoted the development of CKD in C57BL/6 mouse. The divergence in blood
pressure and CKD progression between these two models might be mediated by the
stimulated renal and systemic inflammatory response due to the remnant infarcted
tissue.
Five-sixths nephrectomy, produced by uninephrectomy and surgical removal of
2/3 contralateral kidney, is a well-established and widely used CKD model in various
kinds of laboratory animals, such as dog (Bourgoignie et al. 1987, COBURN et al. 1965,
136
Robertson et al. 1986), rabbit (Eddy et al. 1986, Kilicarslan et al. 2003) and rat (Laouari
et al. 1997, Shimamura & Morrison 1975) with the progressive reduction in kidney
function and the development of renal injury, which mimics the feature of CKD in
human. However, it has been recognized that the susceptibility to develop CKD
following 5/6 nephrectomy is strain or genetic background dependent in mouse. A
recent study by Leelahavanichkul et al. showed that progressive CKD, as indicated by
the gradual decrease in GFR, the reciprocal changes in blood urea nitrogen and serum
creatinine, severe albuminuria and mesangial expansion in glomeruli, was developed by
4 weeks in CD-1 mice and by 12 weeks in 129S3 mice after 5/6 nephrectomy in which
the upper and lower poles of the left kidney were resected and the whole right kidney
was removed. In addition, conscious blood pressure measured by radiotelemetry was
significantly elevated in both CD-1 mice and 129S3 mice following 5/6 nephrectomy. In
contrast, C57BL/6 mouse model with 5/6 nephrectomy exhibited normal blood pressure
and was resistant to progressive CKD. Besides 5/6 nephrectomy, several other
approaches have also been reported to achieve a total 5/6 renal mass reduction in rats
(Fleck et al. 2006, van et al. 2013, Yang et al. 2010).
Moreover, Along with the common procedure uninephrectomy, approximate 2/3
renal mass reduction on contralateral kidney by ligating two of the three renal artery
branches induced higher blood pressure and more severe renal injury, compared with
the approach by surgical excision of kidney poles. On the basis of these studies in
Sprague Dawley (SD) rats, in the present study, we sought to determine whether
subtotal renal mass reduction by renal artery branch ligation rather than ablative surgery
in C57BL/6 mouse is able to result in similar outcomes in SD rats. Our results
137
demonstrated that renal mass reduction by uninephrectomy combined with upper
branch of renal artery ligation on contralateral kidney induced permanent hypertension
and the development of CKD in C57BL/6 mice. In contrast, the mouse model with
surgical excision of the infarction region exhibited normal blood pressure and resistance
to injury in the remnant kidney, as previous reported 5/6 nephrectomy model with
resection of kidney poles in C57BL/6 mouse (Leelahavanichkul et al. 2010).
The mechanism underlying the divergence in blood pressure response and the
susceptibility to CKD between these two remnant kidney models with different
approaches remains to be determined.
Inflammation has been recognized to promote the development of hypertension
(Harrison et al. 2011). The elevated C-reactive protein (CRP) level (Bourgault et al.
2013, Guasti et al. 2011) and the increase in white blood cells count (Antopolsky et al.
2012b, Bourgault et al. 2013, Lessman et al. 1978) in blood test indicated a upregulated
systemic inflammatory status in renal infarction patients. Moreover, the administration of
cyclophosphamide, an immune-suppressor, prevented the blood pressure response to
partial renal infarct in SD rats, which further suggests the critical role of inflammation
contributing to the hypertension in renal infarction (Norman, Jr. et al. 1988). In the
present study, we examined the level of pro-inflammatory cytokines, TNF-α and IL-6, in
the remnant kidneys from these two different models with reduced renal mass. We
found that the remnant kidney with upper branch of renal artery ligation exhibited
upgraded inflammation; in contrast, the remnant kidney with surgical excision of the
infarction region had normal level of pro-inflammatory cytokines. These results are
consist with the blood pressure changes in these two mouse models with different
138
approaches, indicating the immunological reaction against the infarcted tissue in the
remnant kidney might be associated with the permanent hypertension in response to
renal mass reduction by uninephrectomy plus a ligation in upper branch of renal artery
on contralateral kidney.
Furthermore, Ang II administration could induce hypertension in C57BL/6 mice
with 5/6 nephrectomy and overcome the resistance to CKD. The antagonist of Ang II
type I receptor could decrease the blood pressure and blunt the progression of 5/6
nephrectomy induced CKD in CD-1 mice. It has also been indicated that the
upregulation in renin angiotensin aldosterone system (RAAS) is involved with the acute
increased blood pressure in renal infarction patients (Arakawa et al. 1970, Candel-Pau
et al. 2008). However, our results demonstrated that the plasma renin in 5/6 renal mass
reduction mouse model with ligation in upper branch of renal artery kept stable at
normal range, which indicates that the hypertension in this model was independent with
RAAS. This finding is consistent with the previous studies on renal infarction in rats.
Norman et al.’s study demonstrated that despite of a transient increase in plasma renin
activity measured at 1 week after a ligation in two out of three renal artery branches in
SD rats, the plasma renin activity declined to normal level by 4 weeks after renal
infarction while hypertension was maintained (Norman, Jr. et al. 1988). Munich-Wista
rats with 5/6 renal mass reduction exhibited even lower plasma renin concentration than
sham-operated rats at 4 weeks after renal infarction (Anderson et al. 1986). Besides the
plasma renin, Pupilli et al.’s study also demonstrated that even though the intrarenal
renin concentration in the infarcted tissue was higher than the remnant kidney
parenchyma, it was comparable with that in the kidney from sham-operated rats (Pupilli
139
et al. 1992). Moreover, the fluorescence images of intrarenal vascular architecture
showed that the blood supply for each specific glomerulus was solely originated from
only one of the two renal artery branches without any overlapping. Thus, ligation of the
upper branch of renal artery would not induce partial ischemia in JGAs in remnant
kidney, which is consistent with the normal plasma renin level in this model. Thus, it has
been suggested that blood pressure played a critical role in the development of CKD in
mouse model with 5/6 nephrectomy; the susceptibility to CKD in different strains
correlated with their blood pressure changes after renal mass reduction
(Leelahavanichkul et al. 2010).
Hypertension has been defined as one of the leading causes of CKD [32, 33, 34]
and to accelerate the progression of CKD (Chanda & Fenves 2009, Metcalfe 2007, Ritz
et al. 1993). High systemic blood pressure transmitted into glomerular capillary
increases the mechanical forces in glomerulus. This mechanical insult in glomerulus
upregulates the cytokines, such as fibronectin (Gruden et al. 2000), transforming growth
factor-beta1 (Gruden et al. 2000), enhances the accumulation of mesangial extracellular
matrix (Riser et al. 1992), stimulates glomerular monocyte infiltration via an NF-kB and
monocyte chemoattractant protein-1 mediated pathway (Gruden et al. 2005, Ha et al.
2002), which promotes the pathogenesis of the glomerular injury. In the present study,
significant glomerular injury, as indicated by the progressive proteinuria and
glomerulosclerosis was observed in the remnant kidney with upper branch of renal
artery ligation but not in the kidney with surgical excision of the infarction region, which
was consistent with the divergence of blood pressure in these two reduced renal mass
140
models with different approaches, suggesting the critical role of blood pressure in the
development of CKD in the C57BL/6 mouse model with renal mass reduction.
Upregulated systemic Inflammation has been recognized to involve in the
progression of CKD (Gupta et al. 2012, Silverstein 2009). A recent prospective cohort
study of CKD patients demonstrated that plasma levels of pro-inflammatory biomarkers
including IL-6 and TNF-alpha were strongly inversely correlated with kidney function, as
assessed by eGFR and serum cystatin C, and positively associated with glomerular
injury, as evaluated with albuminuria or urine albumin to creatinine ratio (Gupta et al.
2012). It has been known that inflammation stimulates oxidative stress (Cachofeiro et al.
2008, Reuter et al. 2010, Shah et al. 2007), vasoconstriction (Linden et al. 2008,
Remuzzi et al. 2005, Vianna et al. 2011), leukocyte adhesion (Betjes 2013, Hill et al.
1994, Kato et al. 2008), mesangial cell proliferation (Lopez-Novoa et al. 2011, Migliorini
et al. 2013, Schlondorff & Banas 2009), and tubulointerstitial fibrosis (Hodgkins &
Schnaper 2012, Ke et al. 2016), which promotes the progression of CKD. Moreover, it
has been reported that downregulation of inflammation slowed the progression of CKD.
Quiroz et al.’s study demonstrated that administration of melatonin, a potent anti-
inflammatory hormone produced in pineal gland, significantly attenuated the decline in
kidney function as indicated by plasma creatinine level and ameliorated the
deterioration of renal injury, as assessed with proteinuria and glomerulosclerosis, in SD
rats with 5/6 renal mass reduction (Quiroz et al. 2008). In addition, Clopidogrel, an
inhibitor of adenosine diphosphate receptor on platelet cell membrance, was reported to
attenuate the early progression of CKD in Wistar rats by downregulating the systemic
inflammation (Tu et al. 2008). Therefore, in the present study, besides blood pressure,
141
the inflammatory status in the remnant kidney might also contribute to the divergence in
CKD in these two C57BL/6 mouse models with different approaches.
Summary
In the present study, we investigated the pathophysiological mechanisms of renal
ischemia related kidney diseases. Temporary ischemia could lead to renal ischemia
reperfusion induced AKI. We determined the role of various factors including intratubal
pressure, renal temperature and renal pH in ischemia reperfusion induced AKI and we
found that 1) Increased intratubular pressure of the proximal tubule during the ischemic
phase exacerbates renal ischemia reperfusion injury and promotes the development of
AKI; 2) the pathophysiological changes and the underlying mechanisms in renal IRI
induced by cold ischemia is different with that induced by warm ischemia; 3)
bicarbonate treatment elevate renal pH and protected against IRI-induced AKI by
upregulating NOS1β in the macula densa. Permanent ischemia in kidney could result in
renal infarction. We examined the effect of permanent renal ischemia on the
development of CKD in the remnant kidney tissue and we found that permanent
ischemia induced renal infarction increases blood pressure and promotes CKD
progression.
142
References
2000. Clinical practice guidelines for nutrition in chronic renal failure. K/DOQI, National Kidney Foundation. Am J Kidney Dis. 35, S1-140.
Abramowitz, M. K., Hostetter, T. H. & Melamed, M. L. 2012. The serum anion gap is altered in early kidney disease and associates with mortality. Kidney Int. 82, 701-709.
Abu-Amarah, I., Bidani, A. K., Hacioglu, R., Williamson, G. A. & Griffin, K. A. 2005. Differential effects of salt on renal hemodynamics and potential pressure transmission in stroke-prone and stroke-resistant spontaneously hypertensive rats. Am J Physiol Renal Physiol 289, F305-F313.
Abuelo, J. G. 2007. Normotensive ischemic acute renal failure. N.Engl.J.Med. 357, 797-805.
Adams, P. L., Adams, F. F., Bell, P. D. & Navar, L. G. 1980. Impaired renal blood flow autoregulation in ischemic acute renal failure. Kidney Int. 18, 68-76.
Aird, W. C. 2003. The role of the endothelium in severe sepsis and multiple organ dysfunction syndrome. Blood 101, 3765-3777.
Alamir, A., Middendorf, D. F., Baker, P., Nahman, N. S., Jr., Fontaine, A. B. & Hebert, L. A. 1997. Renal artery dissection causing renal infarction in otherwise healthy men. Am.J.Kidney Dis. 30, 851-855.
Ali, T., Khan, I., Simpson, W., Prescott, G., Townend, J., Smith, W. & Macleod, A. 2007. Incidence and outcomes in acute kidney injury: a comprehensive population-based study. J.Am.Soc.Nephrol. 18, 1292-1298.
Allgren, R. L., Marbury, T. C., Rahman, S. N., Weisberg, L. S., Fenves, A. Z., Lafayette, R. A., Sweet, R. M., Genter, F. C., Kurnik, B. R., Conger, J. D. & Sayegh, M. H. 1997. Anaritide in acute tubular necrosis. Auriculin Anaritide Acute Renal Failure Study Group. N.Engl.J.Med. 336, 828-834.
Amdur, R. L., Chawla, L. S., Amodeo, S., Kimmel, P. L. & Palant, C. E. 2009. Outcomes following diagnosis of acute renal failure in U.S. veterans: focus on acute tubular necrosis. Kidney Int. 76, 1089-1097.
Anderson, S. & Brenner, B. M. 1986. The role of intraglomerular pressure in the initiation and progression of renal disease. J.Hypertens.[Suppl] 4, S236-S238.
Anderson, S., Rennke, H. G. & Brenner, B. M. 1986. Therapeutic advantage of converting enzyme inhibitors in arresting progressive renal disease associated with systemic hypertension in the rat. J.Clin.Invest 77, 1993-2000.
Anderson, S. & Vora, J. P. 1995. Current concepts of renal hemodynamics in diabetes. J Diabetes Complications 9, 304-307.
143
Andreucci, M., Michael, A., Kramers, C., Park, K. M., Chen, A., Matthaeus, T., Alessandrini, A., Haq, S., Force, T. & Bonventre, J. V. 2003. Renal ischemia/reperfusion and ATP depletion/repletion in LLC-PK(1) cells result in phosphorylation of FKHR and FKHRL1. Kidney Int. 64, 1189-1198.
Antopolsky, M., Simanovsky, N., Stalnikowicz, R., Salameh, S. & Hiller, N. 2012a. Renal infarction in the ED: 10-year experience and review of the literature. Am.J.Emerg.Med. 30, 1055-1060.
Antopolsky, M., Simanovsky, N., Stalnikowicz, R., Salameh, S. & Hiller, N. 2012b. Renal infarction in the ED: 10-year experience and review of the literature. Am.J.Emerg.Med. 30, 1055-1060.
Arakawa, K., Torii, S., Naito, S., Minohara, A., Uemura, N. & Nakamura, M. 1970. Plasma renin activity as a more specific diagnostic aid for renal infarction. Arch.Intern.Med. 125, 830-834.
Arendshorst, W. J. 1979. Autoregulation of renal blood flow in spontaneously hypertensive rats. Circ.Res. 44, 344-349.
Arendshorst, W. J., Finn, W. F. & Gottschalk, C. W. 1975. Pathogenesis of acute renal failure following temporary ischemia in the rat. Circ.Res. 37, 558-568.
Ayres, J. W., Weidler, D. J., MacKichan, J. & Wagner, J. G. 1977. Circadian rhythm of urinary pH in man with and without chronic antacid administration. Eur.J Clin.Pharmacol. 12, 415-420.
Banerjee, T., Crews, D. C., Wesson, D. E., Tilea, A., Saran, R., Rios, B. N., Williams, D. E. & Powe, N. R. 2014. Dietary acid load and chronic kidney disease among adults in the United States. BMC.Nephrol 15, 137.
Barnett, G. D. & Blume, F. E. 1938. ALKALINE TIDES. J Clin.Invest 17, 159-165.
Barrientos, A., Portoles, J., Herrero, J. A., Torralbo, A., Prats, D., Gutierrez-Millet, V. & Blanco, J. 1994. Glomerular hyperfiltration as a nonimmunologic mechanism of progression of chronic renal rejection. Transplantation 57, 753-756.
Basile, D. P. 2007. The endothelial cell in ischemic acute kidney injury: implications for acute and chronic function. Kidney Int. 72, 151-156.
Bauerle, J. D., Grenz, A., Kim, J. H., Lee, H. T. & Eltzschig, H. K. 2011. Adenosine generation and signaling during acute kidney injury. J Am.Soc.Nephrol. 22, 14-20.
Bell, P. D., Reddington, M., Ploth, D. & Navar, L. G. 1984. Tubuloglomerular feedback-mediated decreases in glomerular pressure in Munich-Wistar rats. Am.J.Physiol. 247, F877-F880.
Bellomo, R., Ronco, C., Kellum, J. A., Mehta, R. L. & Palevsky, P. 2004. Acute renal failure - definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. Crit Care 8, R204-R212.
Belzer, F. O., D'Alessandro, A. M., Hoffmann, R. M., Knechtle, S. J., Reed, A., Pirsch, J. D., Kalayoglu, M. & Sollinger, H. W. 1992. The use of UW solution in clinical transplantation. A 4-year experience. Ann.Surg. 215, 579-583.
144
Betjes, M. G. 2013. Immune cell dysfunction and inflammation in end-stage renal disease. Nat.Rev.Nephrol. 9, 255-265.
Bihorac, A., Yavas, S., Subbiah, S., Hobson, C. E., Schold, J. D., Gabrielli, A., Layon, A. J. & Segal, M. S. 2009. Long-term risk of mortality and acute kidney injury during hospitalization after major surgery. Ann.Surg. 249, 851-858.
Bilobrov, V. M., Chugaj, A. V. & Bessarabov, V. I. 1990. Urine pH variation dynamics in healthy individuals and stone formers. Urol.Int. 45, 326-331.
Blantz, R. C. & Singh, P. 2011. Analysis of the prerenal contributions to acute kidney injury. Contrib.Nephrol. 174, 4-11.
Bohle, A., Mackensen-Haen, S. & Wehrmann, M. 1996. Significance of postglomerular capillaries in the pathogenesis of chronic renal failure. Kidney Blood Press Res. 19, 191-195.
Bonventre, J. V., Vaidya, V. S., Schmouder, R., Feig, P. & Dieterle, F. 2010. Next-generation biomarkers for detecting kidney toxicity. Nat.Biotechnol. 28, 436-440.
Bonventre, J. V. & Yang, L. 2011. Cellular pathophysiology of ischemic acute kidney injury. J.Clin.Invest 121, 4210-4221.
Boom, H., Mallat, M. J., de Fijter, J. W., Zwinderman, A. H. & Paul, L. C. 2000. Delayed graft function influences renal function, but not survival. Kidney Int. 58, 859-866.
Bourgault, M., Grimbert, P., Verret, C., Pourrat, J., Herody, M., Halimi, J. M., Karras, A., Amoura, Z., Jourde-Chiche, N., Izzedine, H., Francois, H., Boffa, J. J., Hummel, A., Bernadet-Monrozies, P., Fouque, D., Canoui-Poitrine, F. et al. 2013. Acute renal infarction: a case series. Clin.J.Am.Soc.Nephrol. 8, 392-398.
Bourgoignie, J. J., Gavellas, G., Martinez, E. & Pardo, V. 1987. Glomerular function and morphology after renal mass reduction in dogs. J.Lab Clin.Med. 109, 380-388.
Braam, B., Mitchell, K. D., Koomans, H. A. & Navar, L. G. 1993. Relevance of the tubuloglomerular feedback mechanism in pathophysiology. J.Am.Soc.Nephrol. 4, 1257-1274.
Brenner, B. M., Cohen, R. A. & Milford, E. L. 1992. In renal transplantation, one size may not fit all. J.Am.Soc.Nephrol. 3, 162-169.
Briggs, J. P. & Schnermann, J. 1986. Macula densa control of renin secretion and glomerular vascular tone: evidence for common cellular mechanisms. Renal Physiol.(Basel) 9, 193-203.
Burke, M., Pabbidi, M. R., Farley, J. & Roman, R. J. 2014. Molecular mechanisms of renal blood flow autoregulation. Curr.Vasc.Pharmacol. 12, 845-858.
Burne-Taney, M. J., Kofler, J., Yokota, N., Weisfeldt, M., Traystman, R. J. & Rabb, H. 2003. Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury. Am.J.Physiol Renal Physiol 285, F87-F94.
145
Cachofeiro, V., Goicochea, M., de Vinuesa, S. G., Oubina, P., Lahera, V. & Luno, J. 2008. Oxidative stress and inflammation, a link between chronic kidney disease and cardiovascular disease. Kidney Int.Suppl, S4-S9.
Cameron, M., Maalouf, N. M., Poindexter, J., Adams-Huet, B., Sakhaee, K. & Moe, O. W. 2012. The diurnal variation in urine acidification differs between normal individuals and uric acid stone formers. Kidney Int. 81, 1123-1130.
Candel-Pau, J., Castilla-Fernandez, Y., Madrid-Aris, A., Vilalta-Cases, R., Lara-Moctezuma, L. E., Garcia-Pena, P., Perez, M. & Nieto-Rey, J. L. 2008. Hypertension and segmental renal infarction in children: apropos of two cases. Pediatr.Nephrol. 23, 841-845.
Carlstrom, M., Wilcox, C. S. & Arendshorst, W. J. 2015. Renal autoregulation in health and disease. Physiol Rev. 95, 405-511.
Carmines, P. K. 2010. The renal vascular response to diabetes. Curr.Opin.Nephrol.Hypertens. 19, 85-90.
Casellas, D. & Navar, L. G. 1984. In vitro perfusion of juxtamedullary nephrons in rats. Am.J.Physiol. 246, F349-F358.
Cavaille-Coll, M., Bala, S., Velidedeoglu, E., Hernandez, A., Archdeacon, P., Gonzalez, G., Neuland, C., Meyer, J. & Albrecht, R. 2013. Summary of FDA workshop on ischemia reperfusion injury in kidney transplantation. Am.J.Transplant. 13, 1134-1148.
Chanda, R. & Fenves, A. Z. 2009. Hypertension in patients with chronic kidney disease. Curr.Hypertens.Rep. 11, 329-336.
Chang, A., Van, H. L., Jacobs, D. R., Jr., Liu, K., Muntner, P., Newsome, B., Shoham, D. A., Durazo-Arvizu, R., Bibbins-Domingo, K., Reis, J. & Kramer, H. 2013. Lifestyle-related factors, obesity, and incident microalbuminuria: the CARDIA (Coronary Artery Risk Development in Young Adults) study. Am J Kidney Dis. 62, 267-275.
Chatterjee, P. K., Patel, N. S., Kvale, E. O., Cuzzocrea, S., Brown, P. A., Stewart, K. N., Mota-Filipe, H. & Thiemermann, C. 2002. Inhibition of inducible nitric oxide synthase reduces renal ischemia/reperfusion injury. Kidney Int. 61, 862-871.
Chawla, L. S., Amdur, R. L., Amodeo, S., Kimmel, P. L. & Palant, C. E. 2011. The severity of acute kidney injury predicts progression to chronic kidney disease. Kidney Int. 79, 1361-1369.
Chawla, L. S. & Kimmel, P. L. 2012. Acute kidney injury and chronic kidney disease: an integrated clinical syndrome. Kidney Int.
Chen, D., Hurst, W. J., Ding, J. M., Faiman, L. E., Mayer, B. & Gillette, M. U. 1997. Localization and characterization of nitric oxide synthase in the rat suprachiasmatic nucleus: evidence for a nitrergic plexus in the biological clock. J.Neurochem. 68, 855-861.
Chen, G., Bridenbaugh, E. A., Akintola, A. D., Catania, J. M., Vaidya, V. S., Bonventre, J. V., Dearman, A. C., Sampson, H. W., Zawieja, D. C., Burghardt, R. C. & Parrish, A. R. 2007. Increased susceptibility of
146
aging kidney to ischemic injury: identification of candidate genes changed during aging, but corrected by caloric restriction. Am.J.Physiol Renal Physiol 293, F1272-F1281.
Chertow, G. M., Burdick, E., Honour, M., Bonventre, J. V. & Bates, D. W. 2005. Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J Am.Soc.Nephrol. 16, 3365-3370.
Chon, K. H., Chen, Y. M., Holstein-Rathlou, N. H., Marsh, D. J. & Marmarelis, V. Z. 1993. On the efficacy of linear system analysis of renal autoregulation in rats. IEEE Trans.Biomed.Eng 40, 8-20.
COBURN, J. W., GONICK, H. C., RUBINI, M. E. & KLEEMAN, C. R. 1965. STUDIES OF EXPERIMENTAL RENAL FAILURE IN DOGS. I. EFFECT OF 5-6 NEPHRECTOMY ON CONCENTRATING AND DILUTING CAPACITY OF RESIDUAL NEPHRONS. J.Clin.Invest 44, 603-614.
Coca, S. G., Yusuf, B., Shlipak, M. G., Garg, A. X. & Parikh, C. R. 2009. Long-term risk of mortality and other adverse outcomes after acute kidney injury: a systematic review and meta-analysis. Am.J Kidney Dis. 53, 961-973.
Collins, G. M., Bravo-Shugarman, M. & Terasaki, P. I. 1969. Kidney preservation for transportation. Initial perfusion and 30 hours' ice storage. Lancet (Dec. 6), 1219-1222.
Conger, J. D., Robinette, J. B. & Kelleher, S. P. 1984. Nephron heterogeneity in ischemic acute renal failure. Kidney Int. 26, 422-429.
Conger, J. D. & Weil, J. V. 1995. Abnormal vascular function following ischemia-reperfusion injury. J Investig.Med. 43, 431-442.
Cooper, J. E. & Wiseman, A. C. 2013. Acute kidney injury in kidney transplantation. Curr.Opin.Nephrol.Hypertens. 22, 698-703.
Coresh, J., Selvin, E., Stevens, L. A., Manzi, J., Kusek, J. W., Eggers, P., Van, L. F. & Levey, A. S. 2007. Prevalence of chronic kidney disease in the United States. JAMA 298, 2038-2047.
Cupples, W. A. & Loutzenhiser, R. D. 1998. Dynamic autoregulation in the in vitro perfused hydronephrotic rat kidney. Am.J.Physiol. 275, F126-F130.
Cupples, W. A., Novak, P., Novak, V. & Salevsky, F. C. 1996. Spontaneous blood pressure fluctuations and renal blood flow dynamics. Am.J.Physiol. 270, F82-F89.
Daemen, M. A., de, V. B. & Buurman, W. A. 2002. Apoptosis and inflammation in renal reperfusion injury. Transplantation 73, 1693-1700.
Damman, K., Navis, G., Smilde, T. D., Voors, A. A., van der Bij, W., van Veldhuisen, D. J. & Hillege, H. L. 2007. Decreased cardiac output, venous congestion and the association with renal impairment in patients with cardiac dysfunction. Eur.J Heart Fail. 9, 872-878.
Daniels, F. H. & Arendshorst, W. J. 1990. Tubuloglomerular feedback kinetics in spontaneously hypertensive and Wistar-Kyoto rats. Am.J.Physiol. 259, F529-F534.
147
Daniels, F. H., Arendshorst, W. J. & Roberds, R. G. 1990. Tubuloglomerular feedback and autoregulation in spontaneously hypertensive rats. Am.J.Physiol. 258, F1479-F1489.
Das, U. N. 2006. Hypertension as a low-grade systemic inflammatory condition that has its origins in the perinatal period. J.Assoc.Physicians India 54, 133-142.
Dawidson, I., Rooth, P., Lu, C., Sagalowsky, A., Diller, K., Palmer, B., Peters, P., Risser, R., Sandor, Z. & Seney, F. 1991. Verapamil improves the outcome after cadaver renal transplantation. J.Am.Soc.Nephrol. 2, 983-990.
de Brito-Ashurst, I., Varagunam, M., Raftery, M. J. & Yaqoob, M. M. 2009. Bicarbonate supplementation slows progression of CKD and improves nutritional status. J.Am.Soc.Nephrol. 20, 2075-2084.
de Fijter, J. W., Mallat, M. J., Doxiadis, I. I., Ringers, J., Rosendaal, F. R., Claas, F. H. & Paul, L. C. 2001. Increased immunogenicity and cause of graft loss of old donor kidneys. J.Am.Soc.Nephrol. 12, 1538-1546.
Denton, M. D., Chertow, G. M. & Brady, H. R. 1996. "Renal-dose" dopamine for the treatment of acute renal failure: scientific rationale, experimental studies and clinical trials. Kidney Int. 50, 4-14.
Devarajan, P. 2006. Update on mechanisms of ischemic acute kidney injury. J.Am.Soc.Nephrol. 17, 1503-1520.
Dobre, M., Rahman, M. & Hostetter, T. H. 2015. Current status of bicarbonate in CKD. J.Am.Soc.Nephrol. 26, 515-523.
Domanovits, H., Paulis, M., Nikfardjam, M., Meron, G., Kurkciyan, I., Bankier, A. A. & Laggner, A. N. 1999. Acute renal infarction. Clinical characteristics of 17 patients. Medicine (Baltimore) 78, 386-394.
Dragun, D., Hoff, U., Park, J. K., Qun, Y., Schneider, W., Luft, F. C. & Haller, H. 2000. Ischemia-reperfusion injury in renal transplantation is independent of the immunologic background. Kidney Int. 58, 2166-2177.
Driver, T. H., Shlipak, M. G., Katz, R., Goldenstein, L., Sarnak, M. J., Hoofnagle, A. N., Siscovick, D. S., Kestenbaum, B., de Boer, I. H. & Ix, J. H. 2014. Low serum bicarbonate and kidney function decline: the Multi-Ethnic Study of Atherosclerosis (MESA). Am.J.Kidney Dis. 64, 534-541.
Dunkler, D., Dehghan, M., Teo, K. K., Heinze, G., Gao, P., Kohl, M., Clase, C. M., Mann, J. F., Yusuf, S. & Oberbauer, R. 2013. Diet and kidney disease in high-risk individuals with type 2 diabetes mellitus. JAMA Intern.Med. 173, 1682-1692.
Dunn, B. R., Anderson, S. & Brenner, B. M. 1986. The hemodynamic basis of progressive renal disease. Semin.Nephrol. 6, 122-138.
Eddy, A. A. 2000. Molecular basis of renal fibrosis. Pediatr.Nephrol. 15, 290-301.
Eddy, A. A., Falk, R. J., Sibley, R. K. & Hostetter, T. H. 1986. Subtotal nephrectomy in the rabbit: a model of chronic hypercalcemia, nephrolithiasis, and obstructive nephropathy. J.Lab Clin.Med. 107, 508-516.
148
Edwards, R. M. 1983. Segmental effects of norepinephrine and angiotensin II on isolated renal microvessels. Am.J.Physiol. 244, F526-F534.
Eisenbach, G. M. & Steinhausen, M. 1973. Micropuncture studies after temporary ischemia of rat kidneys. Pflugers Arch. 343, 11-25.
Eisner, C., Faulhaber-Walter, R., Wang, Y., Leelahavanichkul, A., Yuen, P. S., Mizel, D., Star, R. A., Briggs, J. P., Levine, M. & Schnermann, J. 2010. Major contribution of tubular secretion to creatinine clearance in mice. Kidney Int. 77, 519-526.
el-Galley, R. E. & Keane, T. E. 2000. Embryology, anatomy, and surgical applications of the kidney and ureter. Surg.Clin.North Am. 80, 381-401, xiv.
ELLIOT, J. S., SHARP, R. F. & Lewis, L. 1959. Urinary pH. J Urol. 81, 339-343.
Eltawansy, S. A., Patel, S., Rao, M., Hassanien, S. & Maniar, M. 2014. Acute renal infarction presenting with acute abdominal pain secondary to newly discovered atrial fibrillation: a case report and literature review. Case.Rep.Emerg.Med. 2014, 981409.
Eltzschig, H. K. & Eckle, T. 2011. Ischemia and reperfusion--from mechanism to translation. Nat.Med. 17, 1391-1401.
Eustace, J. A., Astor, B., Muntner, P. M., Ikizler, T. A. & Coresh, J. 2004. Prevalence of acidosis and inflammation and their association with low serum albumin in chronic kidney disease. Kidney Int. 65, 1031-1040.
Evan, A. P., Connors, B. A., Lingeman, J. E., Blomgren, P. & Willis, L. R. 1996. Branching patterns of the renal artery of the pig. Anat.Rec. 246, 217-223.
Faubel, S., Chawla, L. S., Chertow, G. M., Goldstein, S. L., Jaber, B. L. & Liu, K. D. 2012. Ongoing Clinical Trials in AKI. Clin.J Am.Soc.Nephrol.
Fekete, A., Kiss, B. & Spitzar, E. 1975. Renal function in rats after unilateral ligation of renal artery. Acta Med.Acad.Sci.Hung. 32, 55-62.
Feldman, H. I., Fazio, I., Roth, D., Berlin, J. A., Brayman, K., Burns, J. E. & Grossman, R. A. 1996. Recipient body size and cadaveric renal allograft survival. J.Am.Soc.Nephrol. 7, 151-157.
FINKENSTAEDT, J. T. & Merrill, J. P. 1956. Renal function after recovery from acute renal failure. N.Engl.J Med. 254, 1023-1026.
Finn, W. F. 1990. Prevention of ischemic injury in renal transplantation. Kidney Int. 37, 171-182.
Flamenbaum, W., Huddleston, M. L., McNeil, J. S. & Hamburger, R. J. 1974. Uranyl nitrate-induced acute renal failure in the rat: micropuncture and renal hemodynamic studies. Kidney Int. 6, 408-418.
Fleck, C., Appenroth, D., Jonas, P., Koch, M., Kundt, G., Nizze, H. & Stein, G. 2006. Suitability of 5/6 nephrectomy (5/6NX) for the induction of interstitial renal fibrosis in rats--influence of sex, strain, and surgical procedure. Exp.Toxicol.Pathol. 57, 195-205.
149
Fontana, I., Germi, M. R., Beatini, M., Fontana, S., Bertocchi, M., Porcile, E., Saltalamacchia, L., Ornis, S., Ghinolfi, D., Bonifazio, L. & Valente, U. 2005. Dopamine "renal dose" versus fenoldopam mesylate to prevent ischemia-reperfusion injury in renal transplantation. Transplant.Proc. 37, 2474-2475.
Frost, L., Engholm, G., Johnsen, S., Moller, H., Henneberg, E. W. & Husted, S. 2001. Incident thromboembolism in the aorta and the renal, mesenteric, pelvic, and extremity arteries after discharge from the hospital with a diagnosis of atrial fibrillation. Arch.Intern.Med. 161, 272-276.
Fu, Y., Hall, J. E., Lu, D., Lin, L., Manning, R. D., Jr., Cheng, L., Gomez-Sanchez, C. E., Juncos, L. A. & Liu, R. 2012. Aldosterone blunts tubuloglomerular feedback by activating macula densa mineralocorticoid receptors. Hypertension 59, 599-606.
Fu, Y., Lu, Y., Liu, E. Y., Zhu, X., Mahajan, G. J., Lu, D., Roman, R. J. & Liu, R. 2013. Testosterone enhances tubuloglomerular feedback by increasing superoxide production in the macula densa. Am.J.Physiol Regul.Integr.Comp Physiol 304, R726-R733.
Fuller, P. M. & Huelke, D. F. 1973. Kidney vascular supply in the rat, cat and dog. Acta Anat.(Basel) 84, 516-522.
Garg, A. X., Suri, R. S., Barrowman, N., Rehman, F., Matsell, D., Rosas-Arellano, M. P., Salvadori, M., Haynes, R. B. & Clark, W. F. 2003. Long-term renal prognosis of diarrhea-associated hemolytic uremic syndrome: a systematic review, meta-analysis, and meta-regression. JAMA 290, 1360-1370.
Gilbertson, D. T., Liu, J., Xue, J. L., Louis, T. A., Solid, C. A., Ebben, J. P. & Collins, A. J. 2005. Projecting the number of patients with end-stage renal disease in the United States to the year 2015. J.Am.Soc.Nephrol. 16, 3736-3741.
Glodowski, S. D. & Wagener, G. 2015. New insights into the mechanisms of acute kidney injury in the intensive care unit. J.Clin.Anesth. 27, 175-180.
Goggins, W. C., Pascual, M. A., Powelson, J. A., Magee, C., Tolkoff-Rubin, N., Farrell, M. L., Ko, D. S., Williams, W. W., Chandraker, A., Delmonico, F. L., Auchincloss, H. & Cosimi, A. B. 2003. A prospective, randomized, clinical trial of intraoperative versus postoperative Thymoglobulin in adult cadaveric renal transplant recipients. Transplantation 76, 798-802.
Goldenberg, I., Chonchol, M. & Guetta, V. 2009. Reversible acute kidney injury following contrast exposure and the risk of long-term mortality. Am.J Nephrol. 29, 136-144.
Goldenstein, L., Driver, T. H., Fried, L. F., Rifkin, D. E., Patel, K. V., Yenchek, R. H., Harris, T. B., Kritchevsky, S. B., Newman, A. B., Sarnak, M. J., Shlipak, M. G. & Ix, J. H. 2014. Serum bicarbonate concentrations and kidney disease progression in community-living elders: the Health, Aging, and Body Composition (Health ABC) Study. Am.J.Kidney Dis. 64, 542-549.
Goormaghtigh N. 1937. L'appareil neuro-myo-arteriel juxta-glomerulaire du rein:ses reactions en pathologie et ses rapports avec le tube urinifere. [Fre]C R Soc Biol 124, 293-296.
Goraya, N., Simoni, J., Jo, C. & Wesson, D. E. 2012. Dietary acid reduction with fruits and vegetables or bicarbonate attenuates kidney injury in patients with a moderately reduced glomerular filtration rate due to hypertensive nephropathy. Kidney Int. 81, 86-93.
150
Goraya, N., Simoni, J., Jo, C. H. & Wesson, D. E. 2013. A comparison of treating metabolic acidosis in CKD stage 4 hypertensive kidney disease with fruits and vegetables or sodium bicarbonate. Clin.J Am Soc Nephrol 8, 371-381.
Granger, D. N. & Kubes, P. 1994. The microcirculation and inflammation: modulation of leukocyte- endothelial cell adhesion. J.Leukoc.Biol. 55, 662-675.
Griffin, K. A., Picken, M. & Bidani, A. K. 1994. Method of renal mass reduction is a critical modulator of subsequent hypertension and glomerular injury. J.Am.Soc.Nephrol. 4, 2023-2031.
Gruden, G., Setti, G., Hayward, A., Sugden, D., Duggan, S., Burt, D., Buckingham, R. E., Gnudi, L. & Viberti, G. 2005. Mechanical stretch induces monocyte chemoattractant activity via an NF-kappaB-dependent monocyte chemoattractant protein-1-mediated pathway in human mesangial cells: inhibition by rosiglitazone. J.Am.Soc.Nephrol. 16, 688-696.
Gruden, G., Zonca, S., Hayward, A., Thomas, S., Maestrini, S., Gnudi, L. & Viberti, G. C. 2000. Mechanical stretch-induced fibronectin and transforming growth factor-beta1 production in human mesangial cells is p38 mitogen-activated protein kinase-dependent. Diabetes 49, 655-661.
Guan, Z., Gobe, G., Willgoss, D. & Endre, Z. H. 2006. Renal endothelial dysfunction and impaired autoregulation after ischemia-reperfusion injury result from excess nitric oxide. Am.J.Physiol Renal Physiol 291, F619-F628.
Guasti, L., Dentali, F., Castiglioni, L., Maroni, L., Marino, F., Squizzato, A., Ageno, W., Gianni, M., Gaudio, G., Grandi, A. M., Cosentino, M. & Venco, A. 2011. Neutrophils and clinical outcomes in patients with acute coronary syndromes and/or cardiac revascularisation. A systematic review on more than 34,000 subjects. Thromb.Haemost. 106, 591-599.
Gulec, B., Coskun, K., Oner, K., Aydin, A., Yigitler, C., Kozak, O., Uzar, A. & Arslan, I. 2006. Effects of perfusion solutions on kidney ischemia-reperfusion injury in pigs. Transplant.Proc. 38, 371-374.
Gunn, C. A., Weber, J. L., Coad, J. & Kruger, M. C. 2013. Increasing fruits and vegetables in midlife women: a feasibility study. Nutr.Res. 33, 543-551.
Gupta, J., Mitra, N., Kanetsky, P. A., Devaney, J., Wing, M. R., Reilly, M., Shah, V. O., Balakrishnan, V. S., Guzman, N. J., Girndt, M., Periera, B. G., Feldman, H. I., Kusek, J. W., Joffe, M. M. & Raj, D. S. 2012. Association between albuminuria, kidney function, and inflammatory biomarker profile in CKD in CRIC. Clin.J.Am.Soc.Nephrol. 7, 1938-1946.
Guzik, T. J., Hoch, N. E., Brown, K. A., McCann, L. A., Rahman, A., Dikalov, S., Goronzy, J., Weyand, C. & Harrison, D. G. 2007. Role of the T cell in the genesis of angiotensin II induced hypertension and vascular dysfunction. J.Exp.Med. 204, 2449-2460.
Ha, H., Yu, M. R., Choi, Y. J., Kitamura, M. & Lee, H. B. 2002. Role of high glucose-induced nuclear factor-kappaB activation in monocyte chemoattractant protein-1 expression by mesangial cells. J.Am.Soc.Nephrol. 13, 894-902.
Hall, J. E. 2015. Textbook of Medical Physiology. In: ed. L. Belfus, pp. 216-230. Philadelphia: Elsevier Inc.
151
Halloran, P. F., Aprile, M. A., Farewell, V., Ludwin, D., Smith, E. K., Tsai, S. Y., Bear, R. A., Cole, E. H., Fenton, S. S. & Cattran, D. C. 1988. Early function as the principal correlate of graft survival. A multivariate analysis of 200 cadaveric renal transplants treated with a protocol incorporating antilymphocyte globulin and cyclosporine. Transplantation 46, 223-228.
Han, W. K., Bailly, V., Abichandani, R., Thadhani, R. & Bonventre, J. V. 2002. Kidney Injury Molecule-1 (KIM-1): a novel biomarker for human renal proximal tubule injury. Kidney Int. 62, 237-244.
Harrison, D. G., Guzik, T. J., Lob, H. E., Madhur, M. S., Marvar, P. J., Thabet, S. R., Vinh, A. & Weyand, C. M. 2011. Inflammation, immunity, and hypertension. Hypertension 57, 132-140.
Hayashi, K., Epstein, M. & Loutzenhiser, R. 1989. Pressure-induced vasoconstriction of renal microvessels in normotensive and hypertensive rats. Studies in the isolated perfused hydronephrotic kidney. Circ.Res. 65, 1475-1484.
Heyman, S. N., Brezis, M., Reubinoff, C. A., Greenfeld, Z., Lechene, C., Epstein, F. H. & Rosen, S. 1988. Acute renal failure with selective medullary injury in the rat. J Clin.Invest 82, 401-412.
Hill, P. A., Lan, H. Y., Nikolic-Paterson, D. J. & Atkins, R. C. 1994. ICAM-1 directs migration and localization of interstitial leukocytes in experimental glomerulonephritis. Kidney Int. 45, 32-42.
Hirschberg, R., Kopple, J., Lipsett, P., Benjamin, E., Minei, J., Albertson, T., Munger, M., Metzler, M., Zaloga, G., Murray, M., Lowry, S., Conger, J., McKeown, W., O'shea, M., Baughman, R., Wood, K. et al. 1999. Multicenter clinical trial of recombinant human insulin-like growth factor I in patients with acute renal failure. Kidney Int. 55, 2423-2432.
Hladunewich, M. A., Corrigan, G., Derby, G. C., Ramaswamy, D., Kambham, N., Scandling, J. D. & Myers, B. D. 2003. A randomized, placebo-controlled trial of IGF-1 for delayed graft function: a human model to study postischemic ARF. Kidney Int. 64, 593-602.
Hobson, C. E., Yavas, S., Segal, M. S., Schold, J. D., Tribble, C. G., Layon, A. J. & Bihorac, A. 2009. Acute kidney injury is associated with increased long-term mortality after cardiothoracic surgery. Circulation 119, 2444-2453.
Hodgkins, K. S. & Schnaper, H. W. 2012. Tubulointerstitial injury and the progression of chronic kidney disease. Pediatr.Nephrol. 27, 901-909.
Holstein-Rathlou, N. H. & Marsh, D. J. 1994. Renal blood flow regulation and arterial pressure fluctuations: a case study in nonlinear dynamics. Physiol Rev. 74, 637-681.
Holstein-Rathlou, N.-H., Wagner, A. J. & Marsh, D. J. 1991. Tubuloglomerular feedback dynamics and renal blood flow autoregulation in rats. Am.J.Physiol. 260, F53-F68.
Homer-Vanniasinkam, S., Crinnion, J. N. & Gough, M. J. 1997. Post-ischaemic organ dysfunction: a review. Eur.J.Vasc.Endovasc.Surg. 14, 195-203.
Hricik, D. E., Halbert, R. J., Barr, M. L., Helderman, J. H., Matas, A. J., Pirsch, J. D., Schenkel, F. A., Siegal, B. & Ferguson, R. M. 2001. Life satisfaction in renal transplant recipients: preliminary results from the transplant learning center. Am.J.Kidney Dis. 38, 580-587.
152
Hsu, C. Y., Chertow, G. M., McCulloch, C. E., Fan, D., Ordonez, J. D. & Go, A. S. 2009. Nonrecovery of kidney function and death after acute on chronic renal failure. Clin.J Am.Soc.Nephrol. 4, 891-898.
Huang, C. C., Lo, H. C., Huang, H. H., Kao, W. F., Yen, D. H., Wang, L. M., Huang, C. I. & Lee, C. H. 2007. ED presentations of acute renal infarction. Am.J.Emerg.Med. 25, 164-169.
Huang, C. L. & Cogan, M. G. 1987. Atrial natriuretic factor inhibits maximal tubuloglomerular feedback response. Am.J.Physiol. 252, F825-F828.
Ikizler, H. O., Zelnick, L., Ruzinski, J., Curtin, L., Utzschneider, K. M., Kestenbaum, B., Himmelfarb, J. & de Boer, I. H. 2015. Dietary Acid Load Is Associated With Serum Bicarbonate but not Insulin Sensitivity in Chronic Kidney Disease. J Ren Nutr.
Imhof, B. A. & Dunon, D. 1995. Leukocyte migration and adhesion. Adv.Immunol. 58, 345-416.
Inscho, E. W., Ohishi, K. & Navar, L. G. 1992. Effects of ATP on pre- and postglomerular juxtamedullary microvasculature. Am.J.Physiol. 263, F886-F893.
Ishani, A., Xue, J. L., Himmelfarb, J., Eggers, P. W., Kimmel, P. L., Molitoris, B. A. & Collins, A. J. 2009. Acute kidney injury increases risk of ESRD among elderly. J Am.Soc.Nephrol. 20, 223-228.
Ishii, Y., Sawada, T., Kubota, K., Fuchinoue, S., Teraoka, S. & Shimizu, A. 2005. Injury and progressive loss of peritubular capillaries in the development of chronic allograft nephropathy. Kidney Int. 67, 321-332.
Ishitsuka, Y., Fukumoto, Y., Kondo, Y., Irikura, M., Kadowaki, D., Narita, Y., Hirata, S., Moriuchi, H., Maruyama, T., Hamasaki, N. & Irie, T. 2013. Comparative Effects of Phosphoenolpyruvate, a Glycolytic Intermediate, as an Organ Preservation Agent with Glucose and N-Acetylcysteine against Organ Damage during Cold Storage of Mouse Liver and Kidney. ISRN.Pharmacol. 2013, 375825.
Islam, T. M., Fox, C. S., Mann, D. & Muntner, P. 2009. Age-related associations of hypertension and diabetes mellitus with chronic kidney disease. BMC.Nephrol. 10, 17.
Ito, S., Arima, S., Juncos, L. A. & Carretero, O. A. 1993. Endothelium-derived relaxing factor/nitric oxide modulates angiotensin II action in the isolated microperfused rabbit afferent but not efferent arteriole. J.Clin.Invest. 91, 2012-2019.
Ito, S. & Carretero, O. A. 1991. Macula densa control of glomerular hemodynamics. Kidney Int Suppl 32, S83-S85.
Ito, S., Carretero, O. A., Abe, K., Beierwaltes, W. H. & Yoshinaga, K. 1989. Effect of prostanoids on renin release from rabbit afferent arterioles with and without macula densa. Kidney Int. 35, 1138-1144.
Jang, H. R. & Rabb, H. 2015. Immune cells in experimental acute kidney injury. Nat.Rev.Nephrol. 11, 88-101.
Jassem, W. & Heaton, N. D. 2004. The role of mitochondria in ischemia/reperfusion injury in organ transplantation. Kidney Int. 66, 514-517.
153
Javaid, M. M., Butt, M. A., Syed, Y. & Carr, P. 2009. Acute renal infarction: an unusual cause of abdominal pain. Ren Fail. 31, 406-408.
Jo, S. K., Rosner, M. H. & Okusa, M. D. 2007. Pharmacologic treatment of acute kidney injury: why drugs haven't worked and what is on the horizon. Clin.J.Am.Soc.Nephrol. 2, 356-365.
Johansen, K. L., Smith, M. W., Unruh, M. L., Siroka, A. M., O'Connor, T. Z. & Palevsky, P. M. 2010. Predictors of health utility among 60-day survivors of acute kidney injury in the Veterans Affairs/National Institutes of Health Acute Renal Failure Trial Network Study. Clin.J Am.Soc.Nephrol. 5, 1366-1372.
Johnson, R. J. & Schreiner, G. F. 1997. Hypothesis: the role of acquired tubulointerstitial disease in the pathogenesis of salt-dependent hypertension. Kidney Int. 52, 1169-1179.
Jones, H. B. 1843. On the Variations of the Acidity of the Urine in the State of Health on the Influence of Medicines on the Acidity of the Urine. [Abstract]. Abstracts of the Papers Communicated to the Royal Society of London 5, 825-826.
Junaid, A., Kren, S. M., Rosenberg, M. E., Nath, K. A. & Hostetter, T. H. 1994. Physiological and structural responses to chronic experimental renal allograft injury. Am.J.Physiol 267, F1102-F1106.
Just, A. 2007. Mechanisms of renal blood flow autoregulation: dynamics and contributions. Am J Physiol Regul.Integr.Comp Physiol. 292, R1-17.
Kanabrocki, E. L., Snedeker, P. W., Zieher, S. J., Raymond, R., Gordey, J., Bird, T., Sothern, R. B., Hrushesky, W. J., Marks, G., Olwin, J. H. & . 1988. Circadian characteristics of dialyzable and non-dialyzable human urinary electrolytes, trace elements and total solids. Chronobiol.Int. 5, 175-184.
Kanbara, A., Miura, Y., Hyogo, H., Chayama, K. & Seyama, I. 2012. Effect of urine pH changed by dietary intervention on uric acid clearance mechanism of pH-dependent excretion of urinary uric acid. Nutr.J 11, 39.
Kang, D. H., Anderson, S., Kim, Y. G., Mazzalli, M., Suga, S., Jefferson, J. A., Gordon, K. L., Oyama, T. T., Hughes, J., Hugo, C., Kerjaschki, D., Schreiner, G. F. & Johnson, R. J. 2001. Impaired angiogenesis in the aging kidney: vascular endothelial growth factor and thrombospondin-1 in renal disease. Am.J.Kidney Dis. 37, 601-611.
Kang, D. H., Kanellis, J., Hugo, C., Truong, L., Anderson, S., Kerjaschki, D., Schreiner, G. F. & Johnson, R. J. 2002. Role of the microvascular endothelium in progressive renal disease. J.Am.Soc.Nephrol. 13, 806-816.
Kaplan, B. & Meier-Kriesche, H. U. 2002. Death after graft loss: an important late study endpoint in kidney transplantation. Am.J.Transplant. 2, 970-974.
Kato, S., Chmielewski, M., Honda, H., Pecoits-Filho, R., Matsuo, S., Yuzawa, Y., Tranaeus, A., Stenvinkel, P. & Lindholm, B. 2008. Aspects of immune dysfunction in end-stage renal disease. Clin.J.Am.Soc.Nephrol. 3, 1526-1533.
154
Kaukinen, A., Lautenschlager, I., Helin, H., Karikoski, R. & Jalanko, H. 2009. Peritubular capillaries are rarefied in congenital nephrotic syndrome of the Finnish type. Kidney Int. 75, 1099-1108.
Kayler, L. K., Magliocca, J., Zendejas, I., Srinivas, T. R. & Schold, J. D. 2011. Impact of cold ischemia time on graft survival among ECD transplant recipients: a paired kidney analysis. Am.J.Transplant. 11, 2647-2656.
Ke, Y., Tang, H., Ye, C., Lei, C. T., Wang, Y. M., Su, H., Zhang, C. & Jiang, H. J. 2016. Role and Association of Inflammatory and Apoptotic Caspases in Renal Tubulointerstitial Fibrosis. Kidney Blood Press Res. 41, 643-653.
Keller, T. T., Mairuhu, A. T., de Kruif, M. D., Klein, S. K., Gerdes, V. E., ten, C. H., Brandjes, D. P., Levi, M. & van Gorp, E. C. 2003. Infections and endothelial cells. Cardiovasc.Res. 60, 40-48.
Khajuria, A., Tay, C., Shi, J., Zhao, H. & Ma, D. 2014. Anesthetics attenuate ischemia-reperfusion induced renal injury: effects and mechanisms. Acta Anaesthesiol.Taiwan. 52, 176-184.
Khanna, A., Simoni, J., Hacker, C., Duran, M. J. & Wesson, D. E. 2004. Increased endothelin activity mediates augmented distal nephron acidification induced by dietary protein. J Am Soc Nephrol 15, 2266-2275.
Kilicarslan, H., Yildirim, S., Bagcivan, I., Gokce, G., Sarac, B. & Sarioglu, Y. 2003. The effect of chronic renal failure on phosphodiesterase inhibitor-induced relaxation responses in rabbit cavernosal strips. Eur.J.Pharmacol. 462, 155-160.
Kingma, I., Chea, R., Davidoff, A., Benediktsson, H. & Paul, L. C. 1993. Glomerular capillary pressures in long-surviving rat renal allografts. Transplantation 56, 53-60.
Kinsey, G. R., Li, L. & Okusa, M. D. 2008. Inflammation in acute kidney injury. Nephron Exp.Nephrol. 109, e102-e107.
Kirk, K. L., Bell, P. D., Barfuss, D. W. & Ribadeneira, M. 1985. Direct visualization of the isolated and perfused macula densa. Am.J.Physiol. 248, F890-F894.
Kjellstrand, C. M., Ebben, J. & Davin, T. 1981. Time of death, recovery of renal function, development of chronic renal failure and need for chronic hemodialysis in patients with acute tubular necrosis. Trans.Am.Soc.Artif.Intern.Organs 27, 45-50.
Korzets, Z., Plotkin, E., Bernheim, J. & Zissin, R. 2002. The clinical spectrum of acute renal infarction. Isr.Med.Assoc.J. 4, 781-784.
Kosieradzki, M. & Rowinski, W. 2008. Ischemia/reperfusion injury in kidney transplantation: mechanisms and prevention. Transplant.Proc. 40, 3279-3288.
Kovesdy, C. P., Anderson, J. E. & Kalantar-Zadeh, K. 2009. Association of serum bicarbonate levels with mortality in patients with non-dialysis-dependent CKD. Nephrol.Dial.Transplant. 24, 1232-1237.
Kren, S. & Hostetter, T. H. 1999. The course of the remnant kidney model in mice. Kidney Int. 56, 333-337.
155
Kurtz, A. 2011. Renin release: sites, mechanisms, and control. Annu.Rev.Physiol 73, 377-399.
Kurtz, A., Pfeilschifter, J., Brown, C. D. & Bauer, C. 1986. NaCl transport stimulates prostaglandin release in cultured renal epithelial (MDCK) cells. Am.J.Physiol 250, C676-C681.
Lachance, S. L. & Barry, J. M. 1985. Effect of furosemide on dialysis requirement following cadaveric kidney transplantation. J.Urol. 133, 950-951.
Lamattina, L., Garcia-Mata, C., Graziano, M. & Pagnussat, G. 2003. Nitric oxide: the versatility of an extensive signal molecule. Annu.Rev.Plant Biol. 54, 109-136.
Lameire, N., Van, B. W. & Vanholder, R. 2005. Acute renal failure. Lancet 365, 417-430.
Land, W. 1999. Postischemic reperfusion injury and allograft dysfunction: is allograft rejection the result of a fateful confusion by the immune system of danger and benefit? Transplant.Proc. 31, 332-336.
Laouari, D., Friedlander, G., Burtin, M., Silve, C., Dechaux, M., Garabedian, M. & Kleinknecht, C. 1997. Subtotal nephrectomy alters tubular function: effect of phosphorus restriction. Kidney Int. 52, 1550-1560.
Leelahavanichkul, A., Yan, Q., Hu, X., Eisner, C., Huang, Y., Chen, R., Mizel, D., Zhou, H., Wright, E. C., Kopp, J. B., Schnermann, J., Yuen, P. S. & Star, R. A. 2010. Angiotensin II overcomes strain-dependent resistance of rapid CKD progression in a new remnant kidney mouse model. Kidney Int. 78, 1136-1153.
Leemans, J. C., Stokman, G., Claessen, N., Rouschop, K. M., Teske, G. J., Kirschning, C. J., Akira, S., van der Poll, T., Weening, J. J. & Florquin, S. 2005. Renal-associated TLR2 mediates ischemia/reperfusion injury in the kidney. J.Clin.Invest 115, 2894-2903.
Lessman, R. K., Johnson, S. F., COBURN, J. W. & Kaufman, J. J. 1978. Renal artery embolism: clinical features and long-term follow-up of 17 cases. Ann.Intern.Med. 89, 477-482.
Levey, A. S., Astor, B. C., Stevens, L. A. & Coresh, J. 2010. Chronic kidney disease, diabetes, and hypertension: what's in a name? Kidney Int. 78, 19-22.
Levy, E. M., Viscoli, C. M. & Horwitz, R. I. 1996. The effect of acute renal failure on mortality. A cohort analysis. JAMA 275, 1489-1494.
Li, X., Liu, M., Bedja, D., Thoburn, C., Gabrielson, K., Racusen, L. & Rabb, H. 2012. Acute renal venous obstruction is more detrimental to the kidney than arterial occlusion: implication for murine models of acute kidney injury. Am.J.Physiol Renal Physiol 302, F519-F525.
Liangos, O., Wald, R., O'Bell, J. W., Price, L., Pereira, B. J. & Jaber, B. L. 2006. Epidemiology and outcomes of acute renal failure in hospitalized patients: a national survey. Clin.J Am.Soc.Nephrol. 1, 43-51.
Liano, F. & Pascual, J. 1996. Epidemiology of acute renal failure: a prospective, multicenter, community-based study. Madrid Acute Renal Failure Study Group. Kidney Int. 50, 811-818.
156
Lieberthal, W. & Nigam, S. K. 1998. Acute renal failure. I. Relative importance of proximal vs. distal tubular injury. Am.J Physiol 275, F623-F631.
Lin, J., Fung, T. T., Hu, F. B. & Curhan, G. C. 2011. Association of dietary patterns with albuminuria and kidney function decline in older white women: a subgroup analysis from the Nurses' Health Study. Am J Kidney Dis. 57, 245-254.
Linden, E., Cai, W., He, J. C., Xue, C., Li, Z., Winston, J., Vlassara, H. & Uribarri, J. 2008. Endothelial dysfunction in patients with chronic kidney disease results from advanced glycation end products (AGE)-mediated inhibition of endothelial nitric oxide synthase through RAGE activation. Clin.J.Am.Soc.Nephrol. 3, 691-698.
Lindenmeyer, M. T., Kretzler, M., Boucherot, A., Berra, S., Yasuda, Y., Henger, A., Eichinger, F., Gaiser, S., Schmid, H., Rastaldi, M. P., Schrier, R. W., Schlondorff, D. & Cohen, C. D. 2007. Interstitial vascular rarefaction and reduced VEGF-A expression in human diabetic nephropathy. J.Am.Soc.Nephrol. 18, 1765-1776.
Liss, P., Persson, P. B., Hansell, P. & Lagerqvist, B. 2006. Renal failure in 57 925 patients undergoing coronary procedures using iso-osmolar or low-osmolar contrast media. Kidney Int. 70, 1811-1817.
Liu, R., Carretero, O. A., Ren, Y. & Garvin, J. L. 2004a. Increased intracellular pH at the macula densa activates nNOS during tubuloglomerular feedback. Kidney Int. 67, 1837-1843.
Liu, R., Carretero, O. A., Ren, Y., Wang, H. & Garvin, J. L. 2008. Intracellular pH regulates superoxide production by the macula densa. Am.J.Physiol Renal Physiol 295, F851-F856.
Liu, R., Pittner, J. & Persson, A. E. 2002. Changes of cell volume and nitric oxide concentration in macula densa cells caused by changes in luminal NaCl concentration. J Am Soc Nephrol 13, 2688-2696.
Liu, R., Ren, Y., Garvin, J. L. & Carretero, O. A. 2004b. Superoxide enhances tubuloglomerular feedback by constricting the afferent arteriole. Kidney Int. 66, 268-274.
Lo, L. J., Go, A. S., Chertow, G. M., McCulloch, C. E., Fan, D., Ordonez, J. D. & Hsu, C. Y. 2009. Dialysis-requiring acute renal failure increases the risk of progressive chronic kidney disease. Kidney Int. 76, 893-899.
Lopez-Novoa, J. M., Rodriguez-Pena, A. B., Ortiz, A., Martinez-Salgado, C. & Lopez Hernandez, F. J. 2011. Etiopathology of chronic tubular, glomerular and renovascular nephropathies: clinical implications. J.Transl.Med. 9, 13.
Loutzenhiser, R., Griffin, K., Williamson, G. & Bidani, A. 2006. Renal autoregulation: new perspectives regarding the protective and regulatory roles of the underlying mechanisms. Am.J.Physiol Regul.Integr.Comp Physiol 290, R1153-R1167.
LOWE, K. G. 1952. The late prognosis in acute tubular necrosis; an interim follow-up report on 14 patients. Lancet 1, 1086-1088.
157
Lu, D., Fu, Y., Lopez-Ruiz, A., Zhang, R., Juncos, R., Liu, H., Manning, R. D., Jr., Juncos, L. A. & Liu, R. 2010. Salt-sensitive splice variant of nNOS expressed in the macula densa cells. Am.J.Physiol Renal Physiol 298, F1465-F1471.
Lu, Y., Wei, J., Stec, D. E., Roman, R. J., Ge, Y., Cheng, L., Liu, E. Y., Zhang, J., Hansen, P. B., Fan, F., Juncos, L. A., Wang, L., Pollock, J., Huang, P. L., Fu, Y., Wang, S. et al. 2015. Macula Densa Nitric Oxide Synthase 1beta Protects against Salt-Sensitive Hypertension. J.Am.Soc.Nephrol.
Lumerman, J. H., Hom, D., Eiley, D. & Smith, A. D. 1999. Heightened suspicion and rapid evaluation with CT for early diagnosis of partial renal infarction. J.Endourol. 13, 209-214.
Lutz, J., Lu, R., Strobl, M., Huang, H., Deng, M., Wang, M., Ouyang, N. & Heemann, U. 2007. ICOS/B7RP-1 interference in mouse kidney transplantation. Transplantation 84, 223-230.
Ma, Z., Wei, Q., Dong, G., Huo, Y. & Dong, Z. 2014. DNA damage response in renal ischemia-reperfusion and ATP-depletion injury of renal tubular cells. Biochim.Biophys.Acta 1842, 1088-1096.
Mackenzie, H. S., Azuma, H., Rennke, H. G., Tilney, N. L. & Brenner, B. M. 1995. Renal mass as a determinant of late allograft outcome: insights from experimental studies in rats. Kidney Int.Suppl 52, S38-S42.
Mackenzie, H. S., Tullius, S. G., Heemann, U. W., Azuma, H., Rennke, H. G., Brenner, B. M. & Tilney, N. L. 1994. Nephron supply is a major determinant of long-term renal allograft outcome in rats. J.Clin.Invest 94, 2148-2152.
Mahajan, A., Simoni, J., Sheather, S. J., Broglio, K. R., Rajab, M. H. & Wesson, D. E. 2010. Daily oral sodium bicarbonate preserves glomerular filtration rate by slowing its decline in early hypertensive nephropathy. Kidney Int. 78, 303-309.
Majid, D. S., Prieto, M. C. & Navar, L. G. 2015. Salt-Sensitive Hypertension: Perspectives on Intrarenal Mechanisms. Curr.Hypertens.Rev. 11, 38-48.
Malek, M. & Nematbakhsh, M. 2015. Renal ischemia/reperfusion injury; from pathophysiology to treatment. J.Renal Inj.Prev. 4, 20-27.
Marques-Sampaio, B. P., Pereira-Sampaio, M. A., Henry, R. W., Favorito, L. A. & Sampaio, F. J. 2007. Dog kidney: anatomical relationships between intrarenal arteries and kidney collecting system. Anat.Rec.(Hoboken.) 290, 1017-1022.
Martinez, F., Kamar, N., Pallet, N., Lang, P., Durrbach, A., Lebranchu, Y., Adem, A., Barbier, S., Cassuto-Viguier, E., Glowaki, F., Le, M. Y., Rostaing, L., Legendre, C., Hermine, O. & Choukroun, G. 2010. High dose epoetin beta in the first weeks following renal transplantation and delayed graft function: Results of the Neo-PDGF Study. Am.J.Transplant. 10, 1695-1700.
Martins, P. N. 2006. Technique of kidney transplantation in mice with anti-reflux urinary reconstruction. Int.Braz.J.Urol. 32, 713-718.
Mason, J., Olbricht, C., Takabatake, T. & Thurau, K. 1977. The early phase of experimental acute renal failure. I. Intratubular pressure and obstruction. Pflugers Arch. 370, 155-163.
158
Massberg, S. & Messmer, K. 1998. The nature of ischemia/reperfusion injury. Transplant.Proc. 30, 4217-4223.
Masztalerz, M., Wlodarczyk, Z., Czuczejko, J., Slupski, M. & Kedziora, J. 2006. Superoxide anion as a marker of ischemia-reperfusion injury of the transplanted kidney. Transplant.Proc. 38, 46-48.
Mattson, D. L., Lu, S., Roman, R. J. & Cowley, A. W., Jr. 1993. Relationship between renal perfusion pressure and blood flow in different regions of the kidney. Am.J.Physiol. 264, R578-R583.
Mehrotra, A., Rose, C., Pannu, N., Gill, J., Tonelli, M. & Gill, J. S. 2012. Incidence and consequences of acute kidney injury in kidney transplant recipients. Am.J.Kidney Dis. 59, 558-565.
Mehta, R. L., Kellum, J. A., Shah, S. V., Molitoris, B. A., Ronco, C., Warnock, D. G. & Levin, A. 2007. Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit Care 11, R31.
Mentzer, R. M., Jr., Oz, M. C., Sladen, R. N., Graeve, A. H., Hebeler, R. F., Jr., Luber, J. M., Jr. & Smedira, N. G. 2007. Effects of perioperative nesiritide in patients with left ventricular dysfunction undergoing cardiac surgery:the NAPA Trial. J.Am.Coll.Cardiol. 49, 716-726.
Metcalfe, W. 2007. How does early chronic kidney disease progress? A background paper prepared for the UK Consensus Conference on early chronic kidney disease. Nephrol.Dial.Transplant. 22 Suppl 9, ix26-ix30.
Michael, H. J., Francos, G. C., Burke, J. F., Besarab, A., Moritz, M., Gillum, D. & Jarrell, B. 1989. A comparison of the effects of cyclosporine versus antilymphocyte globulin on delayed graft function in cadaver renal transplant recipients. Transplantation 48, 805-808.
Migliorini, A., Ebid, R., Scherbaum, C. R. & Anders, H. J. 2013. The danger control concept in kidney disease: mesangial cells. J.Nephrol. 26, 437-449.
MILLS, J. N. & STANBURY, S. W. 1951. Intrinsic diurnal rhythm in urinary electrolyte output. J Physiol 115, 18p-19p.
Mishra, J., Dent, C., Tarabishi, R., Mitsnefes, M. M., Ma, Q., Kelly, C., Ruff, S. M., Zahedi, K., Shao, M., Bean, J., Mori, K., Barasch, J. & Devarajan, P. 2005. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 365, 1231-1238.
Moore, L. C. & Casellas, D. 1990. Tubuloglomerular feedback dependence of autoregulation in rat juxtamedullary afferent arterioles. Kidney Int. 37, 1402-1408.
Moore, L. C., Schnermann, J. & Yarimizu, S. 1979. Feedback mediation of SNGFR autoregulation in hydropenic and. Am.J.Physiol. 237, F63-F74.
Muhlbacher, F., Langer, F. & Mittermayer, C. 1999. Preservation solutions for transplantation. Transplant.Proc. 31, 2069-2070.
Mundel, P., Bachmann, S., Bader, M., Fischer, A., Kummer, W., Mayer, B. & Kriz, W. 1992. Expression of nitric oxide synthase in kidney macula densa cells. Kidney Int. 42, 1017-1019.
159
Munshi, R., Hsu, C. & Himmelfarb, J. 2011. Advances in understanding ischemic acute kidney injury. BMC.Med. 9, 11.
Muroya, Y., Fan, F., Regner, K. R., Falck, J. R., Garrett, M. R., Juncos, L. A. & Roman, R. J. 2015. Deficiency in the Formation of 20-Hydroxyeicosatetraenoic Acid Enhances Renal Ischemia-Reperfusion Injury. J.Am.Soc.Nephrol. 26, 2460-2469.
Murray, J. E., Merrill, J. P., Harrison, J. H., Wilson, R. E. & Dammin, G. J. 1984. Prolonged survival of human-kidney homografts by immunosuppressive drug therapy. Ann.Plast.Surg. 12, 70-83.
Murugan, R. & Kellum, J. A. 2011. Acute kidney injury: what's the prognosis? Nat.Rev.Nephrol. 7, 209-217.
Myers, B. D., Chui, F., Hilberman, M. & Michaels, A. S. 1979. Transtubular leakage of glomerular filtrate in human acute renal failure. Am.J Physiol 237, F319-F325.
Nakamura, M., Seki, G., Iwadoh, K., Nakajima, I., Fuchinoue, S., Fujita, T. & Teraoka, S. 2012. Acute kidney injury as defined by the RIFLE criteria is a risk factor for kidney transplant graft failure. Clin.Transplant. 26, 520-528.
Nangaku, M. 2004. Mechanisms of tubulointerstitial injury in the kidney: final common pathways to end-stage renal failure. Intern.Med. 43, 9-17.
Nankivell, B. J., Borrows, R. J., Fung, C. L., O'Connell, P. J., Allen, R. D. & Chapman, J. R. 2003. The natural history of chronic allograft nephropathy. N.Engl.J.Med. 349, 2326-2333.
Nath, K. A., Hostetter, M. K. & Hostetter, T. H. 1985. Pathophysiology of chronic tubulo-interstitial disease in rats. Interactions of dietary acid load, ammonia, and complement component C3. J.Clin.Invest. 76, 667-675.
Navaneethan, S. D., Schold, J. D., Arrigain, S., Jolly, S. E., Wehbe, E., Raina, R., Simon, J. F., Srinivas, T. R., Jain, A., Schreiber, M. J., Jr. & Nally, J. V., Jr. 2011. Serum bicarbonate and mortality in stage 3 and stage 4 chronic kidney disease. Clin.J.Am.Soc.Nephrol. 6, 2395-2402.
Navar, L. G. 1978. Renal autoregulation: perspectives from whole kidney and single nephron studies. Am.J.Physiol. 234, F357-F370.
Navar, L. G., Inscho, E. W., Majid, D. S., Imig, J. D., Harrison-Bernard, L. M. & Mitchell, K. D. 1996. Paracrine regulation of the renal microcirculation. Physiol.Rev. 76, 425-536.
Nehus, E. J. & Devarajan, P. 2012. Acute kidney injury: AKI in kidney transplant recipients--here to stay. Nat.Rev.Nephrol. 8, 198-199.
Nettleton, J. A., Steffen, L. M., Palmas, W., Burke, G. L. & Jacobs, D. R., Jr. 2008. Associations between microalbuminuria and animal foods, plant foods, and dietary patterns in the Multiethnic Study of Atherosclerosis. Am J Clin.Nutr. 87, 1825-1836.
160
Nguyen, H. T., Hsieh, M. H., Gaborro, A., Tinloy, B., Phillips, C. & Adam, R. M. 2006. JNK/SAPK and p38 SAPK-2 mediate mechanical stretch-induced apoptosis via caspase-3 and -9 in NRK-52E renal epithelial cells. Nephron Exp.Nephrol 102, e49-e61.
Noel, C., Hazzan, M., Coppin, M. C., Codaccioni, M. X., Pruvot, F. R., Labalette, M. & Lelievre, G. 1997. A randomized controlled trial of pentoxifylline for the prevention of delayed graft function in cadaveric kidney graft. Clin.Transplant. 11, 169-173.
Norman, R. A., Jr., Galloway, P. G., Dzielak, D. J. & Huang, M. 1988. Mechanisms of partial renal infarct hypertension. J.Hypertens. 6, 397-403.
O'Callaghan, J. M., Knight, S. R., Morgan, R. D. & Morris, P. J. 2012. Preservation solutions for static cold storage of kidney allografts: a systematic review and meta-analysis. Am.J.Transplant. 12, 896-906.
Ohashi, R., Shimizu, A., Masuda, Y., Kitamura, H., Ishizaki, M., Sugisaki, Y. & Yamanaka, N. 2002. Peritubular capillary regression during the progression of experimental obstructive nephropathy. J.Am.Soc.Nephrol. 13, 1795-1805.
Oken, D. E. 1975. Acute renal failure (vasomotor nephropathy): micropuncture studies of the pathogenetic mechanisms. Annu.Rev.Med. 26, 307-319.
Oken, D. E. 1984. Hemodynamic basis for human acute renal failure (vasomotor nephropathy). Am.J Med. 76, 702-710.
Okusa, M. D., Molitoris, B. A., Palevsky, P. M., Chinchilli, V. M., Liu, K. D., Cheung, A. K., Weisbord, S. D., Faubel, S., Kellum, J. A., Wald, R., Chertow, G. M., Levin, A., Waikar, S. S., Murray, P. T., Parikh, C. R., Shaw, A. D. et al. 2012. Design of Clinical Trials in Acute Kidney Injury: A Report from an NIDDK Workshop--Prevention Trials. Clin.J Am.Soc.Nephrol.
Ollerstam, A., Pittner, J., Persson, A. E. G. & Thorup, C. 1997. Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase. J.Clin.Invest. 99, 2212-2218.
Opelz, G. & Dohler, B. 2007. Multicenter analysis of kidney preservation. Transplantation 83, 247-253.
Orvieto, M. A., Zorn, K. C., Mendiola, F., Lyon, M. B., Mikhail, A. A., Gofrit, O. N. & Shalhav, A. L. 2007. Recovery of renal function after complete renal hilar versus artery alone clamping during open and laparoscopic surgery. J.Urol. 177, 2371-2374.
Parikh, C. R., Coca, S. G., Wang, Y., Masoudi, F. A. & Krumholz, H. M. 2008. Long-term prognosis of acute kidney injury after acute myocardial infarction. Arch.Intern.Med. 168, 987-995.
Paris, B., Bobrie, G., Rossignol, P., Le, C. S., Chedid, A. & Plouin, P. F. 2006. Blood pressure and renal outcomes in patients with kidney infarction and hypertension. J.Hypertens. 24, 1649-1654.
Patschan, D., Patschan, S. & Muller, G. A. 2012. Inflammation and microvasculopathy in renal ischemia reperfusion injury. J.Transplant. 2012, 764154.
Pegues, M. A., McCrory, M. A., Zarjou, A. & Szalai, A. J. 2013. C-reactive protein exacerbates renal ischemia-reperfusion injury. Am.J.Physiol Renal Physiol 304, F1358-F1365.
161
Perez Valdivia, M. A., Gentil, M. A., Toro, M., Cabello, M., Rodriguez-Benot, A., Mazuecos, A., Osuna, A. & Alonso, M. 2011. Impact of cold ischemia time on initial graft function and survival rates in renal transplants from deceased donors performed in Andalusia. Transplant.Proc. 43, 2174-2176.
Perez-Ruiz, L., Ros-Lopez, S., Cardus, A., Fernandez, E. & Valdivielso, J. M. 2006. A forgotten method to induce experimental chronic renal failure in the rat by ligation of the renal parenchyma. Nephron Exp.Nephrol. 103, e126-e130.
Perico, N., Cattaneo, D., Sayegh, M. H. & Remuzzi, G. 2004. Delayed graft function in kidney transplantation. Lancet 364, 1814-1827.
Peterson, O. W., Gabbai, F. B., Myers, R. R., Mizisin, A. P. & Blantz, R. C. 1989. A single nephron model of acute tubular injury: role of tubuloglomerular feedback. Kidney Int. 36, 1037-1044.
Peti-Peterdi, J. & Harris, R. C. 2010. Macula densa sensing and signaling mechanisms of renin release. J.Am.Soc.Nephrol. 21, 1093-1096.
Petretta, M., Bonaduce, D., Scalfi, L., de, F. E., Marciano, F., Migaux, M. L., Themistoclakis, S., Ianniciello, A. & Contaldo, F. 1997. Heart rate variability as a measure of autonomic nervous system function in anorexia nervosa. Clin.Cardiol. 20, 219-224.
Phisitkul, S., Khanna, A., Simoni, J., Broglio, K., Sheather, S., Rajab, M. H. & Wesson, D. E. 2010. Amelioration of metabolic acidosis in patients with low GFR reduced kidney endothelin production and kidney injury, and better preserved GFR. Kidney Int. 77, 617-623.
Pires, S. L., Julien, C., Chapuis, B., Sassard, J. & Barres, C. 2002. Spontaneous renal blood flow autoregulation curves in conscious sinoaortic baroreceptor-denervated rats. Am.J Physiol Renal Physiol 282, F51-F58.
Pirsch, J. D., Armbrust, M. J., Knechtle, S. J., D'Alessandro, A. M., Sollinger, H. W., Heisey, D. M. & Belzer, F. O. 1995. Obesity as a risk factor following renal transplantation. Transplantation 59, 631-633.
Ploeg, R. J., Vreugdenhil, P., Goossens, D., McAnulty, J. F., Southard, J. H. & Belzer, F. O. 1988. Effect of pharmacologic agents on the function of the hypothermically preserved dog kidney during normothermic reperfusion. Surgery 103, 676-683.
Ploth, D. W., Rudolph, J., LaGrange, R. & Navar, L. G. 1979. Tubuloglomerular feedback and single nephron function after converting enzyme inhibition in the rat. J.Clin.Invest. 64, 1325-1335.
Pratschke, J., Wilhelm, M. J., Laskowski, I., Kusaka, M., Beato, F., Tullius, S. G., Neuhaus, P., Hancock, W. W. & Tilney, N. L. 2001. Influence of donor brain death on chronic rejection of renal transplants in rats. J.Am.Soc.Nephrol. 12, 2474-2481.
Prowle, J. R., Ishikawa, K., May, C. N. & Bellomo, R. 2009. Renal blood flow during acute renal failure in man. Blood Purif. 28, 216-225.
Prowle, J. R., Ishikawa, K., May, C. N. & Bellomo, R. 2010. Renal plasma flow and glomerular filtration rate during acute kidney injury in man. Ren Fail. 32, 349-355.
162
Pupilli, C., Chevalier, R. L., Carey, R. M. & Gomez, R. A. 1992. Distribution and content of renin and renin mRNA in remnant kidney of adult rat. Am.J.Physiol 263, F731-F738.
Purkerson, M. L., Hoffsten, P. E. & Klahr, S. 1976. Pathogenesis of the glomerulopathy associated with renal infarction in rats. Kidney Int. 9, 407-417.
Quinlan, M. R., Docherty, N. G., Watson, R. W. & Fitzpatrick, J. M. 2008. Exploring mechanisms involved in renal tubular sensing of mechanical stretch following ureteric obstruction. Am J Physiol Renal Physiol 295, F1-F11.
Quiroz, Y., Ferrebuz, A., Romero, F., Vaziri, N. D. & Rodriguez-Iturbe, B. 2008. Melatonin ameliorates oxidative stress, inflammation, proteinuria, and progression of renal damage in rats with renal mass reduction. Am.J.Physiol Renal Physiol 294, F336-F344.
Rabl, H., Khoschsorur, G., Colombo, T., Petritsch, P., Rauchenwald, M., Koltringer, P., Tatzber, F. & Esterbauer, H. 1993. A multivitamin infusion prevents lipid peroxidation and improves transplantation performance. Kidney Int. 43, 912-917.
Racusen, L. C., Solez, K., Colvin, R. B., Bonsib, S. M., Castro, M. C., Cavallo, T., Croker, B. P., Demetris, A. J., Drachenberg, C. B., Fogo, A. B., Furness, P., Gaber, L. W., Gibson, I. W., Glotz, D., Goldberg, J. C., Grande, J. et al. 1999. The Banff 97 working classification of renal allograft pathology. Kidney Int. 55, 713-723.
Raphael, K. L., Wei, G., Baird, B. C., Greene, T. & Beddhu, S. 2011. Higher serum bicarbonate levels within the normal range are associated with better survival and renal outcomes in African Americans. Kidney Int. 79, 356-362.
Regner, K. R., Zuk, A., Van Why, S. K., Shames, B. D., Ryan, R. P., Falck, J. R., Manthati, V. L., McMullen, M. E., Ledbetter, S. R. & Roman, R. J. 2009. Protective effect of 20-HETE analogues in experimental renal ischemia reperfusion injury. Kidney Int. 75, 511-517.
Remer, T. & Manz, F. 1995. Potential renal acid load of foods and its influence on urine pH. J.Am.Diet.Assoc. 95, 791-797.
Remuzzi, G., Perico, N., Macia, M. & Ruggenenti, P. 2005. The role of renin-angiotensin-aldosterone system in the progression of chronic kidney disease. Kidney Int.Suppl, S57-S65.
Ren, Y., Arima, S., Carretero, O. A. & Ito, S. 2002a. Possible role of adenosine in macula densa control of glomerular hemodynamics. Kidney Int. 61, 169-176.
Ren, Y., Carretero, O. A. & Garvin, J. L. 2002b. Mechanism by which superoxide potentiates tubuloglomerular feedback. Hypertension 39, 624-628.
Ren, Y., Garvin, J. L. & Carretero, O. A. 2000a. Efferent arteriole tubuloglomerular feedback in the renal nephron. Kidney Int. 59, 222-229.
Ren, Y., Garvin, J. L. & Carretero, O. A. 2000b. Role of macula densa nitric oxide and cGMP in the regulation of tubuloglomerular feedback. Kidney Int. 58, 2053-2060.
163
Ren, Y., Garvin, J. L. & Carretero, O. A. 2002c. Vasodilator action of angiotensin-(1-7) on isolated rabbit afferent arterioles. Hypertension 39, 799-802.
Ren, Y., Garvin, J. L., Liu, R. & Carretero, O. A. 2004. Role of macula densa adenosine triphosphate (ATP) in tubuloglomerular feedback. Kidney Int. 66, 1479-1485.
Reuter, S., Gupta, S. C., Chaturvedi, M. M. & Aggarwal, B. B. 2010. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic.Biol.Med. 49, 1603-1616.
Riser, B. L., Cortes, P., Zhao, X., Bernstein, J., Dumler, F. & Narins, R. G. 1992. Intraglomerular pressure and mesangial stretching stimulate extracellular matrix formation in the rat. J.Clin.Invest 90, 1932-1943.
Ritz, E., Fliser, D. & Siebels, M. 1993. Pathophysiology of hypertensive renal damage. Am.J.Hypertens. 6, 241S-244S.
Robertson, J. L., Goldschmidt, M., Kronfeld, D. S., Tomaszewski, J. E., Hill, G. S. & Bovee, K. C. 1986. Long-term renal responses to high dietary protein in dogs with 75% nephrectomy. Kidney Int. 29, 511-519.
Robinson, B. F., Epstein, S. E., Beiser, G. D. & Braunwald, E. 1966. Control of heart rate by the autonomic nervous system. Studies in man on the interrelation between baroreceptor mechanisms and exercise. Circ.Res. 19, 400-411.
Robson, S. C., Candinas, D., Hancock, W. W., Wrighton, C., Winkler, H. & Bach, F. H. 1995. Role of endothelial cells in transplantation. Int.Arch.Allergy Immunol. 106, 305-322.
Roman, R. J., Cowley, A. W., Jr., Garcia-Estan, J. & Lombard, J. H. 1988. Pressure-diuresis in volume-expanded rats: cortical and medullary hemodynamics. Hypertension 12, 168-176.
Rong, S., Lewis, A. G., Kunter, U., Haller, H. & Gueler, F. 2012. A knotless technique for kidney transplantation in the mouse. J.Transplant. 2012, 127215.
Saeed, K. 2012. Renal infarction. Int.J.Nephrol.Renovasc.Dis. 5, 119-123.
Salahudeen, A. K. 2003. Obesity and survival on dialysis. Am.J.Kidney Dis. 41, 925-932.
Salahudeen, A. K. 2004. Cold ischemic injury of transplanted kidneys: new insights from experimental studies. Am.J.Physiol Renal Physiol 287, F181-F187.
Salmela, K., Wramner, L., Ekberg, H., Hauser, I., Bentdal, O., Lins, L. E., Isoniemi, H., Backman, L., Persson, N., Neumayer, H. H., Jorgensen, P. F., Spieker, C., Hendry, B., Nicholls, A., Kirste, G. & Hasche, G. 1999. A randomized multicenter trial of the anti-ICAM-1 monoclonal antibody (enlimomab) for the prevention of acute rejection and delayed onset of graft function in cadaveric renal transplantation: a report of the European Anti-ICAM-1 Renal Transplant Study Group. Transplantation 67, 729-736.
Sampaio, F. J., Pereira-Sampaio, M. A. & Favorito, L. A. 1998. The pig kidney as an endourologic model: anatomic contribution. J.Endourol. 12, 45-50.
164
Sanchez-Lozada, L. G., Tapia, E., Johnson, R. J., Rodriguez-Iturbe, B. & Herrera-Acosta, J. 2003. Glomerular hemodynamic changes associated with arteriolar lesions and tubulointerstitial inflammation. Kidney Int.Suppl, S9-14.
Sato, M., Muragaki, Y., Saika, S., Roberts, A. B. & Ooshima, A. 2003. Targeted disruption of TGF-beta1/Smad3 signaling protects against renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction. J Clin.Invest 112, 1486-1494.
Saur, D., Neuhuber, W. L., Gengenbach, B., Huber, A., Schusdziarra, V. & Allescher, H. D. 2002. Site-specific gene expression of nNOS variants in distinct functional regions of rat gastrointestinal tract. Am.J.Physiol Gastrointest.Liver Physiol 282, G349-G358.
Schlondorff, D. & Banas, B. 2009. The mesangial cell revisited: no cell is an island. J.Am.Soc.Nephrol. 20, 1179-1187.
Schnermann, J. 1998. Juxtaglomerular cell complex in the regulation of renal salt excretion. Am.J.Physiol 274, R263-R279.
Schnermann, J. & Briggs, J. P. 1992. Function of the juxtaglomerular apparatus. Control of glomerular hemodynamics and renin secretion. In: eds. D. W. Seldin & G. Giebisch The Kidney: Physiology and Pathology, Chapt. 35, pp. 1249-1289. New York: Raven Press.
Schnermann, J. & Briggs, J. P. 1999. The macula densa is worth its salt. J.Clin.Invest. 104, 1007-1009.
Schnermann, J., Ploth, D. W. & Dahlheim, H. 1976a. Inter-relationship between autoregulation of glomerular filtration rate, tubuloglomerular feedback and juxtaglomerular renin activity in normotensive and hypertensive rats. Clin.Sci.Mol.Med. 51, 105-107.
Schnermann, J., Ploth, D. W. & Hermle, M. 1976b. Activation of tubulo-glomerular feedback by chloride transport. Pflugers.Arch. 362, 229-240.
Schreiber, A., Shulhevich, Y., Geraci, S., Hesser, J., Stsepankou, D., Neudecker, S., Koenig, S., Heinrich, R., Hoecklin, F., Pill, J., Friedemann, J., Schweda, F., Gretz, N. & Schock-Kusch, D. 2012. Transcutaneous measurement of renal function in conscious mice. Am.J.Physiol Renal Physiol 303, F783-F788.
Schrier, R. W., Wang, W., Poole, B. & Mitra, A. 2004. Acute renal failure: definitions, diagnosis, pathogenesis, and therapy. J Clin Invest. 114, 5-14.
Scialla, J. J., Appel, L. J., Astor, B. C., Miller, E. R., III, Beddhu, S., Woodward, M., Parekh, R. S. & Anderson, C. A. 2011. Estimated net endogenous acid production and serum bicarbonate in African Americans with chronic kidney disease. Clin.J Am Soc Nephrol 6, 1526-1532.
Sert, I., Colak, H., Tugmen, C., Dogan, S. M. & Karaca, C. 2014. The effect of cold ischemia time on delayed graft function and acute rejection in kidney transplantation. Saudi.J.Kidney Dis.Transpl. 25, 960-966.
Shah, S. N., Abramowitz, M., Hostetter, T. H. & Melamed, M. L. 2009. Serum bicarbonate levels and the progression of kidney disease: a cohort study. Am.J.Kidney Dis. 54, 270-277.
165
Shah, S. V., Baliga, R., Rajapurkar, M. & Fonseca, V. A. 2007. Oxidants in chronic kidney disease. J.Am.Soc.Nephrol. 18, 16-28.
Shilliday, I. R. & Sherif, M. 2004. Calcium channel blockers for preventing acute tubular necrosis in kidney transplant recipients. Cochrane.Database.Syst.Rev, CD003421.
Shimamura, T. & Morrison, A. B. 1975. A progressive glomerulosclerosis occurring in partial five-sixths nephrectomized rats. Am.J.Pathol. 79, 95-106.
Silverstein, D. M. 2009. Inflammation in chronic kidney disease: role in the progression of renal and cardiovascular disease. Pediatr.Nephrol. 24, 1445-1452.
Sis, B., Mengel, M., Haas, M., Colvin, R. B., Halloran, P. F., Racusen, L. C., Solez, K., Baldwin, W. M., III, Bracamonte, E. R., Broecker, V., Cosio, F., Demetris, A. J., Drachenberg, C., Einecke, G., Gloor, J., Glotz, D. et al. 2010. Banff '09 meeting report: antibody mediated graft deterioration and implementation of Banff working groups. Am.J.Transplant. 10, 464-471.
Snoeijs, M. G., Buurman, W. A., Christiaans, M. H., van Hooff, J. P., Goldschmeding, R., van Suylen, R. J., Peutz-Kootstra, C. J. & van Heurn, L. W. 2008. Histological assessment of preimplantation biopsies may improve selection of kidneys from old donors after cardiac death. Am.J.Transplant. 8, 1844-1851.
Snoeijs, M. G., van, B. A., Swennen, E., Haenen, G. R., Roberts, L. J., Christiaans, M. H., Peppelenbosch, A. G., Buurman, W. A. & Ernest van Heurn, L. W. 2011. Tubular epithelial injury and inflammation after ischemia and reperfusion in human kidney transplantation. Ann.Surg. 253, 598-604.
Song, J., Lu, Y., Lai, E. Y., Wei, J., Wang, L., Chandrashekar, K., Wang, S., Shen, C., Juncos, L. A. & Liu, R. 2015. Oxidative status in the macula densa modulates tubuloglomerular feedback responsiveness in angiotensin II-induced hypertension. Acta Physiol (Oxf) 213, 249-258.
Sorensen, I., Rong, S., Susnik, N., Gueler, F., Shushakova, N., Albrecht, M., Dittrich, A. M., von, V. S., Becker, J. U., Melk, A., Bohlmann, A., Reingruber, S., Petzelbauer, P., Haller, H. & Schmitt, R. 2011. Bbeta(15-42) attenuates the effect of ischemia-reperfusion injury in renal transplantation. J.Am.Soc.Nephrol. 22, 1887-1896.
Southard, J. H., Rice, M. J., Ametani, M. S. & Belzer, F. O. 1985. Effects of short-term hypothermic perfusion and cold storage on function of the isolated-perfused dog kidney. Cryobiology 22, 147-155.
Southard, J. H., van Gulik, T. M., Ametani, M. S., Vreugdenhil, P. K., Lindell, S. L., Pienaar, B. L. & Belzer, F. O. 1990. Important components of the UW solution. Transplantation 49, 251-257.
Spring, D. B., Salvatierra, O., Jr., Palubinskas, A. J., Amend, W. J., Jr., Vincenti, F. G. & Feduska, N. J. 1979. Results and significance of angiography in potential kidney donors. Radiology 133, 45-47.
STANBURY, S. W. & THOMSON, A. E. 1951. Diurnal variation in electrolyte excretion. Clin.Sci.(Lond) 10, 267-293.
166
Steegh, F. M., Gelens, M. A., Nieman, F. H., van Hooff, J. P., Cleutjens, J. P., van Suylen, R. J., Daemen, M. J., van Heurn, E. L., Christiaans, M. H. & Peutz-Kootstra, C. J. 2011. Early loss of peritubular capillaries after kidney transplantation. J.Am.Soc.Nephrol. 22, 1024-1029.
Sugimoto, H., Grahovac, G., Zeisberg, M. & Kalluri, R. 2007. Renal fibrosis and glomerulosclerosis in a new mouse model of diabetic nephropathy and its regression by bone morphogenic protein-7 and advanced glycation end product inhibitors. Diabetes 56, 1825-1833.
Sun, D., Samuelson, L. C., Yang, T., Huang, Y., Paliege, A., Saunders, T., Briggs, J. & Schnermann, J. 2001. Mediation of tubuloglomerular feedback by adenosine: evidence from mice lacking adenosine 1 receptors. Proc.Natl.Acad.Sci.USA 98, 9983-9988.
Susantitaphong, P., Sewaralthahab, K., Balk, E. M., Jaber, B. L. & Madias, N. E. 2012. Short- and long-term effects of alkali therapy in chronic kidney disease: a systematic review. Am.J.Nephrol. 35, 540-547.
Takahashi, N., Boysen, G., Li, F., Li, Y. & Swenberg, J. A. 2007. Tandem mass spectrometry measurements of creatinine in mouse plasma and urine for determining glomerular filtration rate. Kidney Int. 71, 266-271.
Tanner, G. A. & Sophasan, S. 1976. Kidney pressures after temporary renal artery occlusion in the rat. Am.J.Physiol 230, 1173-1181.
Tarjan, E. & Fekete, A. 1975. Experimental renal hypertension in rats. Int.Urol.Nephrol. 7, 149-156.
Terasaki, P. I., Koyama, H., Cecka, J. M. & Gjertson, D. W. 1994. The hyperfiltration hypothesis in human renal transplantation. Transplantation 57, 1450-1454.
Tervaert, T. W., Mooyaart, A. L., Amann, K., Cohen, A. H., Cook, H. T., Drachenberg, C. B., Ferrario, F., Fogo, A. B., Haas, M., de, H. E., Joh, K., Noel, L. H., Radhakrishnan, J., Seshan, S. V., Bajema, I. M. & Bruijn, J. A. 2010. Pathologic classification of diabetic nephropathy. J.Am.Soc.Nephrol. 21, 556-563.
Thadhani, R., Pascual, M. & Bonventre, J. V. 1996. Acute renal failure. N.Engl.J Med. 334, 1448-1460.
Thurau, K. & Boylan, J. W. 1976. Acute renal success. The unexpected logic of oliguria in acute renal failure. Am.J Med. 61, 308-315.
Thurau, K. & Schnermann, J. 1965. Die Natriumkonzentration an den Macula densa Zellen als regulierender Faktor fur das Glomerulumfiltrat (Mikropunktionsversuche). [Ger]. Klin.Wochenschr. 43, 410-413.
Thurman, J. M. 2007. Triggers of inflammation after renal ischemia/reperfusion. Clin.Immunol. 123, 7-13.
Tian, Y., Chen, J., Gaspert, A., Segerer, S., Clavien, P. A., Wuthrich, R. P. & Fehr, T. 2010. Kidney transplantation in mice using left and right kidney grafts. J.Surg.Res. 163, e91-e97.
167
Tripatara, P., Patel, N. S., Webb, A., Rathod, K., Lecomte, F. M., Mazzon, E., Cuzzocrea, S., Yaqoob, M. M., Ahluwalia, A. & Thiemermann, C. 2007. Nitrite-derived nitric oxide protects the rat kidney against ischemia/reperfusion injury in vivo: role for xanthine oxidoreductase. J.Am.Soc.Nephrol. 18, 570-580.
Tse, G. H., Hesketh, E. E., Clay, M., Borthwick, G., Hughes, J. & Marson, L. P. 2014. Mouse kidney transplantation: models of allograft rejection. J.Vis.Exp., e52163.
Tse, G. H., Hughes, J. & Marson, L. P. 2013. Systematic review of mouse kidney transplantation. Transpl.Int. 26, 1149-1160.
Tu, X., Chen, X., Xie, Y., Shi, S., Wang, J., Chen, Y. & Li, J. 2008. Anti-inflammatory renoprotective effect of clopidogrel and irbesartan in chronic renal injury. J.Am.Soc.Nephrol. 19, 77-83.
Tumlin, J. A., Finkel, K. W., Murray, P. T., Samuels, J., Cotsonis, G. & Shaw, A. D. 2005. Fenoldopam mesylate in early acute tubular necrosis: a randomized, double-blind, placebo-controlled clinical trial. Am.J.Kidney Dis. 46, 26-34.
Turkmen, K., Martin, J., Akcay, A., Nguyen, Q., Ravichandran, K., Faubel, S., Pacic, A., Ljubanovic, D., Edelstein, C. L. & Jani, A. 2011. Apoptosis and autophagy in cold preservation ischemia. Transplantation 91, 1192-1197.
Uchino, S., Bellomo, R., Goldsmith, D., Bates, S. & Ronco, C. 2006. An assessment of the RIFLE criteria for acute renal failure in hospitalized patients. Crit Care Med. 34, 1913-1917.
Uchino, S., Kellum, J. A., Bellomo, R., Doig, G. S., Morimatsu, H., Morgera, S., Schetz, M., Tan, I., Bouman, C., Macedo, E., Gibney, N., Tolwani, A. & Ronco, C. 2005. Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 294, 813-818.
Urban, B. A., Ratner, L. E. & Fishman, E. K. 2001. Three-dimensional volume-rendered CT angiography of the renal arteries and veins: normal anatomy, variants, and clinical applications. Radiographics 21, 373-386.
Vaidya, V. S., Ferguson, M. A. & Bonventre, J. V. 2008a. Biomarkers of acute kidney injury. Annu.Rev.Pharmacol.Toxicol. 48, 463-493.
Vaidya, V. S., Ozer, J. S., Dieterle, F., Collings, F. B., Ramirez, V., Troth, S., Muniappa, N., Thudium, D., Gerhold, D., Holder, D. J., Bobadilla, N. A., Marrer, E., Perentes, E., Cordier, A., Vonderscher, J., Maurer, G. et al. 2010. Kidney injury molecule-1 outperforms traditional biomarkers of kidney injury in preclinical biomarker qualification studies. Nat.Biotechnol. 28, 478-485.
Vaidya, V. S., Waikar, S. S., Ferguson, M. A., Collings, F. B., Sunderland, K., Gioules, C., Bradwin, G., Matsouaka, R., Betensky, R. A., Curhan, G. C. & Bonventre, J. V. 2008b. Urinary biomarkers for sensitive and specific detection of acute kidney injury in humans. Clin.Transl.Sci. 1, 200-208.
Vallon, V., Eraly, S. A., Rao, S. R., Gerasimova, M., Rose, M., Nagle, M., Anzai, N., Smith, T., Sharma, K., Nigam, S. K. & Rieg, T. 2012. A role for the organic anion transporter OAT3 in renal creatinine secretion in mice. Am.J.Physiol Renal Physiol 302, F1293-F1299.
168
van, K. A., Verhaar, M. C., Bongartz, L. G. & Joles, J. A. 2013. 5/6th nephrectomy in combination with high salt diet and nitric oxide synthase inhibition to induce chronic kidney disease in the Lewis rat. J.Vis.Exp., e50398.
Vander, A. J. 1967. Control of renin release. Physiol Rev. 47, 359-382.
Vianna, H. R., Soares, C. M., Tavares, M. S., Teixeira, M. M. & Silva, A. C. 2011. [Inflammation in chronic kidney disease: the role of cytokines]. J.Bras.Nefrol. 33, 351-364.
Wahlberg, J. A., Love, R., Landegaard, L., Southard, J. H. & Belzer, F. O. 1987. 72-hour preservation of the canine pancreas. Transplantation 43, 5-8.
Wald, R., Quinn, R. R., Luo, J., Li, P., Scales, D. C., Mamdani, M. M. & Ray, J. G. 2009. Chronic dialysis and death among survivors of acute kidney injury requiring dialysis. JAMA 302, 1179-1185.
Walker, L. M., Walker, P. D., Imam, S. Z., Ali, S. F. & Mayeux, P. R. 2000. Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: studies with the inducible nitric oxide synthase inhibitor L-N(6)-(1-Iminoethyl)lysine. J.Pharmacol.Exp.Ther. 295, 417-422.
Wang, H., Carretero, O. A. & Garvin, J. L. 2003a. Inhibition of apical Na+/H+ exchangers on the macula densa cells augments tubuloglomerular feedback. Hypertension 41, 688-691.
Wang, L., Shen, C., Liu, H., Wang, S., Chen, X., Roman, R. J., Juncos, L. A., Lu, Y., Wei, J., Zhang, J., Yip, K. P. & Liu, R. 2015. Shear stress blunts tubuloglomerular feedback partially mediated by primary cilia and nitric oxide at the macula densa. Am.J.Physiol Regul.Integr.Comp Physiol 309, R757-R766.
Wang, M., Bai, J., Baumann, M. & Heemann, U. 2003b. New model for simultaneous heart and kidney transplantation in mice. Microsurgery 23, 164-168.
Wang, X., Chandrashekar, K., Wang, L., Lai, E. Y., Wei, J., Zhang, G., Wang, S., Zhang, J., Juncos, L. A. & Liu, R. 2016. Inhibition of Nitric Oxide Synthase 1 Induces Salt-Sensitive Hypertension in Nitric Oxide Synthase 1alpha Knockout and Wild-Type Mice. Hypertension 67, 792-799.
Wei, Q. & Dong, Z. 2012. Mouse model of ischemic acute kidney injury: technical notes and tricks. Am.J.Physiol Renal Physiol 303, F1487-F1494.
Welch, A. A., Mulligan, A., Bingham, S. A. & Khaw, K. T. 2008. Urine pH is an indicator of dietary acid-base load, fruit and vegetables and meat intakes: results from the European Prospective Investigation into Cancer and Nutrition (EPIC)-Norfolk population study. Br.J Nutr. 99, 1335-1343.
Welch, W. J., Tojo, A. & Wilcox, C. S. 2000. Roles of NO and oxygen radicals in tubuloglomerular feedback in SHR. Am.J.Physiol.Renal Physiol. 278, F769-F776.
Welch, W. J. & Wilcox, C. S. 1990. Feedback responses during sequential inhibition of angiotensin and thromboxane. Am.J.Physiol. 258, F457-F466.
Welch, W. J. & Wilcox, C. S. 1997. Role of nitric oxide in tubuloglomerular feedback: effects of dietary salt. Clin.Exp.Pharmacol.Physiol. 24, 582-586.
169
Welten, G. M., Schouten, O., Chonchol, M., Hoeks, S. E., Feringa, H. H., Bax, J. J., Dunkelgrun, M., van Gestel, Y. R., Van Domburg, R. T. & Poldermans, D. 2007. Temporary worsening of renal function after aortic surgery is associated with higher long-term mortality. Am.J Kidney Dis. 50, 219-228.
Wesson, D. E. & Simoni, J. 2010. Acid retention during kidney failure induces endothelin and aldosterone production which lead to progressive GFR decline, a situation ameliorated by alkali diet. Kidney Int. 78, 1128-1135.
Wesson, D. E., Simoni, J. & Prabhakar, S. 2006. Endothelin-induced increased nitric oxide mediates augmented distal nephron acidification as a result of dietary protein. J Am Soc Nephrol 17, 406-413.
Wilcox, C. S. & Welch, W. J. 1996. TGF and nitric oxide: effects of salt intake and salt-sensitive hypertension. Kidney Int. 49 (Suppl 55), S-9-S-13.
Wilcox, C. S., Welch, W. J., Murad, F., Gross, S. S., Taylor, G., Levi, R. & Schmidt, H. H. W. 1992. Nitric oxide synthase in macula densa regulates glomerular capillary pressure. Proc.Natl.Acad.Sci.USA 89, 11993-11997.
Wolfe, R. A., Ashby, V. B., Milford, E. L., Ojo, A. O., Ettenger, R. E., Agodoa, L. Y., Held, P. J. & Port, F. K. 1999. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N.Engl.J.Med. 341, 1725-1730.
Womer, K. L., Vella, J. P. & Sayegh, M. H. 2000. Chronic allograft dysfunction: mechanisms and new approaches to therapy. Semin.Nephrol. 20, 126-147.
Wong, W. S., Moss, A. A., Federle, M. P., Cochran, S. T. & London, S. S. 1984. Renal infarction: CT diagnosis and correlation between CT findings and etiologies. Radiology 150, 201-205.
Yang, H. C., Zuo, Y. & Fogo, A. B. 2010. Models of chronic kidney disease. Drug Discov.Today Dis.Models. 7, 13-19.
Yaqoob, M., Edelstein, C. L., Wieder, E. D., Alkhunaizi, A. M., Gengaro, P. E., Nemenoff, R. A. & Schrier, R. W. 1996. Nitric oxide kinetics during hypoxia in proximal tubules: effects of acidosis and glycine. Kidney Int. 49, 1314-1319.
Yoldas, A. & Dayan, M. O. 2014. Morphological characteristics of renal artery and kidney in rats. ScientificWorldJournal. 2014, 468982.
Yuan, H. T., Li, X. Z., Pitera, J. E., Long, D. A. & Woolf, A. S. 2003. Peritubular capillary loss after mouse acute nephrotoxicity correlates with down-regulation of vascular endothelial growth factor-A and hypoxia-inducible factor-1 alpha. Am.J.Pathol. 163, 2289-2301.
Zatz, R., Dunn, B. R., Meyer, T. W., Anderson, S., Rennke, H. G. & Brenner, B. M. 1986. Prevention of diabetic glomerulopathy by pharmacological amelioration of glomerular capillary hypertension. J.Clin.Invest. 77, 1925-1930.
Zeisberg, M., Hanai, J., Sugimoto, H., Mammoto, T., Charytan, D., Strutz, F. & Kalluri, R. 2003. BMP-7 counteracts TGF-beta1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat.Med. 9, 964-968.
170
Zhang, J., Chandrashekar, K., Lu, Y., Duan, Y., Qu, P., Wei, J., Juncos, L. A. & Liu, R. 2014. Enhanced expression and activity of Nox2 and Nox4 in the macula densa in ANG II-induced hypertensive mice. Am.J.Physiol Renal Physiol 306, F344-F350.
Zhang, Q., Lin, L., Lu, Y., Liu, H., Duan, Y., Zhu, X., Zou, C., Manning, R. D., Jr. & Liu, R. 2013. Interaction between nitric oxide and superoxide in the macula densa in aldosterone-induced alterations of tubuloglomerular feedback. Am.J.Physiol Renal Physiol 304, F326-F332.
Ziai, F., Nagano, H., Kusaka, M., Coito, A. J., Troy, J. L., Nadeau, K. C., Rennke, H. G., Tilney, N. L., Brenner, B. M. & Mackenzie, H. S. 2000. Renal allograft protection with losartan in Fisher-->Lewis rats: hemodynamics, macrophages, and cytokines. Kidney Int. 57, 2618-2625.
Zimmerman, K. W. 1933. Ueber den Bau des Glomerulus der Saeugerniere [Ger]. Z.Microskop.Anat.Forsch. 32, 176-287.
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Appendix: List of Abbreviation
AKI Acute kidney injury
Af-Art Afferent arteriole
CKD Chronic kidney disease
CO2 Carbon dioxide
CMR Corticomedullary region
DMEM Dulbecco’s modified eagle’s medium-low glucose
DAF-2 DA 4-amino-5-7’-difluorofluorescein diacetate methylamino-2'
eGFR Estimated glomerular filtration rate
GFR Glomerular filtration rate
IRI Ischemia reperfusion injury
IM Inner medullar
JGA Juxtaglomerular apparatus
KIM-1 Kidney injury molecule–1
MAP Mean arterial pressure
MD-NOS1KO mice Macula densa specific NOS1 knockout mice
NO Nitric oxide
NGAL Neutrophil gelatinase-associated lipocalin
OM Outer medulla
Pt Proximal tubular pressure
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PAS Periodic Acid Schiff
Psf Stop-flow pressure
PRC Plasma renin concentration
PCR Polymerase chain reaction
RBF Renal blood flow
SNGFR Single nephron glomerular filtration rate
TAL Thick ascending limb
TGF Tubuloglomerular feedback
TGFβ-1 Transforming growth factor β-1