Acute Kidney Injury and Chronic Kidney Disease

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Acute Kidney Injury and Chronic Kidney DiseaseJin WeiUniversity of South Florida, [email protected]

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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.

i

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

ii

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

iii

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

iv

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),

3

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.


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



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


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



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.


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







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



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


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).


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

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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.

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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

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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).


86


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.

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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.

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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.

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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).


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

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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

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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.


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

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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.

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

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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

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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).

108


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.

110

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.

112

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

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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.




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).

125

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


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



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.


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







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


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.




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


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

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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

Acute Kidney Injury and Chronic Kidney Disease

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