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Download date: 06 Sep 2018

 

RENAL PERFUSION AND OXYGENATION DURING ACUTE KIDNEY INJURY

Uğur Aksu

ISBN: 978-94-91715-08-2

Printing by: NetzoDruk Groningen bv

Cover: Resuscitation rain to kidneyCover design: İlker Traş

Copyright © Uğur AKSU, Amsterdam, 2015. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means mechanically, by photocopy, by recording, or otherwise, without the prior written permission of the holder of copyright.

 

Renal perfusion and oxygenation during acute kidney injury

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de Rector Magnificus

prof. dr. D.C. van den Boom

ten overstaan van een door het College voor Promoties ingestelde commissie,

in het openbaar te verdedigen in de Agnietenkapel

op dinsdag 17 november 2015 om 12.00 uur

door Uğur Aksu geboren te Üsküdar, Turkije

 

Promotiecommissie

Promotor: prof. dr. ir. C. Ince Universiteit van Amsterdam

Copromotor: dr. R. Bezemer Universiteit van Amsterdam

Overige leden: prof. dr. F. Toraman Acibadem University prof. dr. J.H. Ravesloot Universiteit van Amsterdam prof. dr. S. Florquin Universiteit van Amsterdam prof. dr. E.T. van Bavel Universiteit van Amsterdam dr. E.G. Mik Erasmus Universiteit Rotterdam dr. C.T.P. Krediet Academisch Medisch Centrum

Faculteit der Geneeskunde

 

 

“As far as we can discern, the sole purpose of human existence is to kindle a light in the darkness of mere being”

Carl Gustav Jung

In memory of my dear father…

 

 

Contents • General Introduction: The pathogenesis of acute kidney injury and the toxic triangle

of oxygen, reactive oxygen species and nitric oxide 7

• Outline of the thesis 19

• Chapter 1: Balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic shock and the effects on renal oxygenation, oxidative stress, and

inflammation 23

• Chapter 2: The acute effects of acetate-balanced colloid and crystalloid resuscitation

on renal oxygenation in a rat model of hemorrhagic shock 45

• Chapter 3: Acute effects of balanced versus unbalanced colloid resuscitation on renal

macrocirculatory and microcirculatory perfusion during endotoxemic shock 67

• Chapter 4: Effect of tempol on redox homeostasis and stress tolerance in mimetically

aged Drosophila 83

• Chapter 5: Scavenging ROS in the acute phase of renal I/R injury also protects

kidney oxygenation and NO levels 101

• Summary and conclusions 117

• Samenvatting en conclusies 121

• References list 125

• Acknowledgments 143

• Curriculum vitae and portfolio 145

• List of publications 149

 

  7  

GENERAL INTRODUCTION

THE PATHOGENESIS OF ACUTE KIDNEY INJURY AND THE TOXIC

TRIANGLE OF OXYGEN, REACTIVE OXYGEN SPECIES AND NITRIC OXIDE

Aksu U1,3, Demirci C3, Ince C1,2

1Department of Translational Physiology, Academic Medical Center, University of

Amsterdam, Amsterdam The Netherlands 2Department of Intensive Care, Erasmus MC University Hospital Rotterdam, Rotterdam,

The Netherlands 3Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey

Published in: Contrib Nephrol. 2011; 174:119-28. Review.

  8  

Abstract

Despite the identification of several of the cellular mechanisms thought to underlie the

development of acute kidney injury (AKI), the pathophysiology of AKI is still poorly

understood. It is clear, however, that instead of a single mechanism being responsible for its

etiology, AKI is associated with an entire orchestra of failing cellular mechanisms. Renal

microcirculation is the physiological compartment where these mechanisms come together

and exert their integrated deleterious action. Therefore, the study of renal microcirculation

and the identification of the determinants of its function in models of AKI can be expected to

provide insight into the pathogenesis and resolution of AKI. A major determinant of adequate

organ function is the adequate oxygen (O2) supply at the microcirculatory level and utilization

at mitochondrial levels for ATP production needed for performing organ function. The highly

complex architecture of the renal microvasculature, the need to meet a high energy demand

and the borderline hypoxemic nature of the kidney makes it an organ that is highly vulnerable

to injury. Under normal, steady-state conditions, the oxygen supply to the renal tissues is well

regulated and utilized not only for mitochondrial production of ATP (mainly for Na+

reabsorption), but also for the production of nitric oxide and the reactive oxygen species

needed for physiological control of renal function. Under pathological conditions, such as

inflammation, shock or sepsis, however, the renal microcirculation becomes compromised,

which results in a disruption of the homeostasis of nitric oxide, reactive oxygen species, and

oxygen supply and utilization. This imbalance results in these compounds exerting pathogenic

effects, such as hypoxemia and oxidative stress, resulting in further deterioration of renal

microcirculatory function. Our hypothesis is that this sequence of events underlies the

development of AKI and that integrated therapeutic modalities targeting these pathogenic

mechanisms will be effective therapeutic strategies in the clinical environment.

  9  

Introduction

Despite the advances made in unravelling the pathogenesis and improving the treatment of

acute kidney injury (AKI), current therapeutic modalities have been ineffective for adequate

treatment. Consequently, AKI remains a condition with a poor prognosis in hospitals today.

Important factors leading to AKI are renal ischemia and hypoxia that can occur as a result of

kidney transplantation, treatment of suprarenal aneurysms, cardiac surgery, renal artery

reconstructions, contrast agent-induced nephropathy, cardiac arrest, sepsis, and shock

[Lameire et al., 2005]. AKI is associated with higher early and late mortality rates [Lameire et

al., 2005] and with higher costs, especially related to the demand for retransplantation and/or

hemodialysis. Among critical care patients who have AKI and survive, up to 30% will require

long-term dialysis [Bagshaw, 2006].

Understanding of the acute kidney injury

AKI, classified according to the RIFLE criteria, is characterized by the sudden loss of

glomerular filtration rate (GFR). Current views concerning the pathophysiology of AKI

implicate a reduction in renal blood flow and consequent renal ischemia as the cause of

depressed GFR which in turn causes disturbances in fluid, electrolyte, and acid-base balances

[Kellum, 2008]. Inflammatory processes can be triggered by ischemic insults and lead to

increased expression of adhesion molecules and impaired tubular sodium reabsorption due to

intraluminal debris from tubular cells. Endothelial injury directly affects afferent arterioles

and causes endothelin release and further vasoconstriction [Abuelo, 2007], which together

results in renal microcirculatory dysfunction.

Measurements of biomarkers in blood and/or urine have recently been developed for the

diagnosis of AKI at an early stage, which can, potentially, be used to prevent progression.

Hence early warnings (i.e., before GFR falls) are important in determining the therapeutic

strategies. According to AKI definition, serum creatinine and/or blood urea nitrogen increase.

However, traditional blood and urine biomarkers (such as the fractional excretion of sodium)

are nonspecific and not sensitive. New biomarkers have been discovered by using advanced

molecular techniques. These biomarkers have been assessed primarily after a specific insult,

such as cardiac surgery, kidney transplantation, contrast administration and sepsis. Currently,

some urine biomarkers, such as neutrophil gelatinase lipocalin (NGAL), cystatin C, kidney

injury molecule, interleukin-18 have been tested for early diagnosis of AKI and was discussed

to be AKI-spesific biomarker [Coca et al., 2008; Geuss et al., 2011]. Despite these findings in

  10  

the diagnosis of AKI, however, the pathophysiology and progress of AKI to renal failure in

the respect of molecular basis remain poorly understood [Wan et al., 2008].

Shock, fluid therapy and acute kidney injury

Currently, hemorrhagic and septic shock is one of the main contributors to the development of

AKI and one of the leading causes of death in intensive care [Kellum, 2008]. Shock

constitutes a major hit to renal function as it induces a massive increase in inflammatory

mediators and activated leukocytes, which together cause severe microcirculatory dysfunction

and disruption of oxygen homeostasis that leads to oxidative stress and hypoxemia [Legrand

et al., 2008]. In the early stages of sepsis, impairment of the renal microcirculation is a key

complication potentially leading to AKI through hypoxia-induced tubular epithelial cell injury

and acute tubular necrosis.

Current treatment strategies for hemorrhagic and septic shock involve rapid and aggressive

fluid resuscitation to restore blood pressure and tissue perfusion prior to blood transfusion.

Fluid resuscitation is a cornerstone of the treatment of sepsis because it is considered crucial

for the preservation of adequate intravascular volume and the maintenance of blood pressure

[Vincent and Gerlach, 2004]. Such fluid therapy is expected to promote microvascular

perfusion and thereby renal oxygenation. However, the extent to which fluid therapy is

effective in promoting renal oxygenation has recently been questioned [Legrand et al., 2010].

Fluid resuscitation can have severe deleterious effects on the microcirculation [Boldt and

Ince, 2010] and hemodilution in a range of therapeutic scenarios have been found to lead to

renal failure [Habib et al., 2005]. Excessive fluid administration in sepsis has been found to be

associated with renal failure [Payen et al., 2008], although restrictions in fluid use can lead to

hypovolemia. Therefore, determining the optimal fluid volume to administer during sepsis to

deal with hypovolemia remains a source of controversy [Boldt and Ince, 2010]. In addition,

the type of fluid that yields the best renal outcome when used for resuscitation in sepsis is also

currently a source of uncertainty. This controversy not only includes the use of crystalloid

versus colloid solutions, but also encompasses the use of balanced versus unbalanced colloid

solutions. Because most colloid preparations are saline-based, liberal fluid resuscitation

regimes may lead to non-physiologically high sodium and chloride concentrations and may be

associated with the development of (hyperchloremic) metabolic acidosis that can affect

inflammatory and coagulation homeostasis, thereby contributing to the deterioration of renal

function. This insight has led to development of modern hydroxyl-ethylated starch (HES)

  11  

preparations, based on balanced plasma-adapted crystalloid solutions, and to the notion of

developing a totally balanced fluid resuscitation concept, including balanced crystalloids and

balanced colloids. Studies have also found that HES solutions have anti-inflammatory

properties [Hoffmann et al., 2002]. In contrast, however, various investigations have

identified potential adverse effects of HES solutions on renal function [Winkelmayer et al.,

2003, Schortgen, et al., 2001, Cittanova et al., 1996].

Despite much literature showing its deleterious effects, 0.9% NaCl is widely used as a

resuscitation solution in emergency departments and intensive care units today. However, it is

known that crystalloids have a poor plasma expanding effect since they rapidly leave the

intravascular space. Compared to colloids in this respect, about three times more volume of

crystalloids needs to be given to reach a similar systemic hemodynamic endpoint [Dubin et

al., 2010]. Therefore, colloid fluids are more effective as plasma expanders because less is

needed. However, colloids have also been shown to have adverse affects on coagulation

pathways and are often dissolved in high chloride solutions (e.g. 0.9% NaCl). Excessive

chloride levels can have deleterious effects on renal function. For example, intrarenal

administration of a chloride solution provokes renal vasoconstriction and reduces GFR

[Wilcox, 1983]. Solutions containing high amounts of chloride can cause hyperchloremic

acidosis, while solutions with buffers, such as acetate, and more physiological concentrations

of strong ions, do not. Proinflammatory mechanisms involving acidosis have been elaborately

described elsewhere [Kellum et al., 2004].

Relation between oxygen, reactive oxygen species and nitric oxide

In addition to the vasodilatory effect of NO, when it is produced by endothelial nitric oxide

synthase (NOS), NO is thought to prevent vascular dysfunction by inhibiting platelet

aggregation and preventing leukocyte activation and infiltration via endogenous anti-

inflammatory properties. The depletion of suitable cofactors of the NO-producing enzyme

(e.g. BH4), as occurs during reperfusion injury and sepsis, can enhance the production of

reactive oxygen species (ROS) by uncoupling endothelial NOS [Rabelink and Zonneveld,

2006]. Excessive NO produced by cells (occurring, for example, as a result of inflammation

from inducible NOS activation) can inhibit mitochondrial respiration by competing with

oxygen mitochondrial cytochrome oxidase in a dose-dependent manner [Cooper and Giulivi,

2007]. Thus, the production and utilization of oxygen, NO, and ROS are intrinsically

dependent on each other and a proper balance is required for ensuring adequate renal function.

  12  

This delicate homeostasis is pathogenically altered during inflammation and hypoxemia, and

leads to oxidative stress and tissue damage. Oxidative stress is an imbalance between oxidants

and antioxidants that favors oxidants and causes a disruption of redox signaling and control,

leading to damage of cellular molecular structures [Clanton, 2007]. Oxygen radicals can be

released after the reduction of oxygen, and the outcome is cell injury and dysfunction. ROS is

a common term that is used for both oxygen radicals (O2– and OH–) and nonradical (H2O2,

HOCl, O3) compounds. Another commonly used term is ‘oxidant’. O2– and H2O2 can function

as both oxidizing and reducing agents.

Under normal circumstances, ROS are released at low concentrations and are neutralized by

endogenous antioxidant compounds, which can be both enzymatic, such as superoxide

dismutase, catalase and glutathione peroxidase, and nonenzymatic, such as glutathione and

vitamins C and E. Both high and low levels of oxygen promote oxidative stress, making the

need for keeping levels of tissue oxygen tensions at physiological levels imperative [Clanton,

2007].

Several studies have promoted the idea that targeting the ROS associated with cellular injury

in acute or chronic kidney disease may aid the design of future therapeutic approaches.

Oxidative stress commonly results in the degeneration of cells via apoptotic pathways.

Apoptotic-induced oxidative stress in conjunction with processes of mitochondrial

dysfunction forms the corner stone of triggered mechanisms in nephropathic conditions. The

dependency of ROS activity on oxygen availability was recently shown in a model of

oxidative stress in spontaneously hypertensive rats in which a loss of bioactive NO by high

ROS production was found to interfere with normal oxygen usage in the kidney. In addition, it

was shown that superoxide produced by NADPH oxidase was inhibited when oxygen tensions

dropped below 20 mmHg [Adler and Huang, 2004].

The main sources of ROS in the microcirculation are mitochondria, NADPH oxidase, NO

synthase and xanthine oxidase. Moreover, cytochrome P450 and cyclooxygenase are capable

of producing O2–. In mitochondria, there is continuous production of ROS during cellular

respiration. A percentage of the oxygen used in mitochondria is reduced to superoxide. This

process occurs by blockade of the electron transfer chain at the flavin mononucleotide group

of complex I or at the ubiquinone site of complex III. This free radical generation is under the

  13  

control of the endogenous antioxidant defense system, and Mn-superoxide dismutase in

mitochondria converts the superoxide to H2O2. Superoxide generates much of its biological

effects by scavenging the NO produced by three isoforms of NOS, each expressed in the

kidney: neuronal NOS, inducible NOS, and endothelial NOS. In their pathogenic action, ROS

mostly cause their deleterious effects by inducing lipid peroxidation, activation of apoptotic

pathways, alteration of intracellular calcium concentrations, and inducement of adhesion

molecule expression. Oxidative stress can also increase mitochondrial membrane

permeability, resulting in loss of mitochondrial NAD+ residues and subsequent radical

generation [Maiese and Chong, 2003]. In addition to their pathogenic action, superoxide and

NO are involved in normal kidney and vascular functions. Both may mediate the maintenance

of vascular tone (especially in the afferent arterioles) and tubular function. Angiotensin II,

mediated by superoxide together with NO, is also responsible for maintaining afferent

arteriolar tone in perfused isolated mouse afferent arterioles. Renal oxygen consumption, in

contrast, has been found to be increased by l-NG-monomethyl-arginine, a nonselective NOS

inhibitor, and S-methyl-l-thiocitrulline, a selective neuronal NOS inhibitor [Deng et al.,

2005], which emphasizes the roles of the different isoforms of NOS in modulating oxygen

utilization. These and the above-mentioned studies illustrate the complicated interdependency

between oxygen and NO species; their homeostasis becomes severely disrupted during

conditions of renal inflammation and ischemia-reperfusion (I/R), resulting in oxidative stress

and loss of renal function.

Inflammation and ischemia can severely disrupt the balance between oxygen transport and

utilization, reactive oxygen and NO metabolism, resulting in oxidative stress and regional

hypoxemia that lead to renal failure. Sepsis and reperfusion injury are the most severe

manifestations of such an inflammatory insult attacking microcirculatory function at all

levels; they can result in a viscous, self-perpetuating spiral of pathogenic events that lead to

renal failure. In this sequence of events, activated leukocytes and inflammatory mediators

disrupt the homeostasis of renal oxygenation, which leads to functional deterioration. This

model of the pathogenesis of AKI can be seen in Figure 1. In the view of the progress of AKI,

therapeutic compounds need to correct all the elements of this pathology (e.g. inflammation,

microcirculatory oxygenation, NO and oxidative stress) in an integrated manner to effectively

alter the course of AKI. We performed empirical studies on compounds that have multiple

correcting effects on these pathogenic mechanisms (e.g. dexamethasone, l-NIL, iloprost, and

  14  

APC;), and we found them successful in reverting AKI in rat models [Johannes et al., 2009;

Legrand et al., 2009; Johannes et al., 2009].

Conclusion

It is clear that physiological function of the kidney relies on a delicate balance between

oxygen transport and utilization, reactive oxygen and NO metabolism, and that this balance

affects the renal microcirculation and is essential for renal function. The question now is

whether the approach based on this integrative model provides a strategic therapeutic

rationale for the treatment of AKI in experimental scenarios.

Fig.1. An integrated model of the pathogenesis of AKI. Inflammation-induced leukocyte-endothelium interactions lead to a distortion of the homeostatic balance between O2, NO and ROS. This imbalance perpetuates the distorted leukocyte endothelium interaction, and a spiral of pathogenic events will follow. It is hypothesized that, taken together, the imbalances will fuel microcirculatory dysfunction which will lead to AKI and ultimately renal failure.

Acknowledgements The author is grateful to Rick Bezemer who drew Figure 1.

  15  

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  19  

OUTLINE OF THE THESIS

  20  

OUTLINE OF THE THESIS

The current study presented in this thesis was conducted at Department of Translational

Physiology of the Academic Medical Center of the University of Amsterdam. As most fluid

preparations are saline based, liberal fluid resuscitation regimens might lead to non-

physiologically high sodium and chloride concentrations and may be associated with the

development of (hyperchloremic) metabolic acidosis, which could affect inflammatory and

coagulation homeostasis and thereby deteriorated organ function. The ultimate aim of this

thesis is to test if fluid resuscitation regimens, besides antioxidant therapies, effect on kidney

oxygenation and redox homeostasis by using different rat models of kidney injury.

Current treatment strategies for hemorrhagic shock involve rapid and aggressive fluid

resuscitation to restore blood pressure and tissue perfusion prior to blood transfusion. In

Chapter 1, we aimed to test the hypothesis that balanced crystalloid resuscitation would be

better for the kidney than unbalanced crystalloid resuscitation in a rat hemorrhagic shock

model. For integrative investigation of the effects of hemorrhage and fluid resuscitation on the

complex interrelation between oxygen and reactive oxygen species, we measured renal

microvascular oxygen tension and plasma levels of malondialdehyde (oxidative stress

marker). Furthermore, glycocalyx degradation was assessed by measuring plasma levels of

hyaluronan (glycocalyx compartment) and plasma levels of TNF-alpha and interleukin-6 were

measured as markers of inflammation.

The use of conventional crystalloid solutions as initial resuscitation fluids is still implemented

in emergency departments even though it is known that crystalloid solutions have poor plasma

expander capacities and just 20% of the given volume remains contained in the intravascular

space. In Chapter 2, we aimed to investigate the acute effects of acetate-balanced colloid and

crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. To this

end, we examined the effects of resuscitation with different fluids: (1) 0.9% NaCl; (2)

acetated Ringer’s solution; (3) 6% HES with a molecular weight of 130 kDa and molar

substitution of 0.4 (HES 130/0.4) in 0.9% NaCl solution; and (4) 6% HES with a molecular

weight of 130 kDa and molar substitution of 0.42 (HES 130/0.42) in acetate-balanced

Ringer’s solution. We hypothesized that acetated solutions would have superior resuscitation

capacities compared to the other solutions with respect to improving renal oxygenation after

  21  

severe hemorrhage. In this line; we measured renal oxygen consumption and cortical-

medullary oxygenation in a rat model of hemorrhagic shock.

The aim of Chapter 3 was therefore to investigate the acute effects of balanced versus

unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in

a rat model of lipopolysaccharide-induced endotoxemia. We tested the hypothesis that

balanced colloid resuscitation would be better for the kidney than unbalanced colloid

resuscitation. The acute effects of two clinically applied resuscitation regimens were

investigated: 6% 130/0.4 HES in NaCl as an unbalanced colloid solution and 6% 130/0.4 HES

in Ringer’s acetate solution as a balanced colloid solution. Renal perfusion was assessed at the

macrocirculatory level using Doppler ultrasound on the renal artery and at the

microcirculatory level using laser speckle imaging on the renal cortex.

According to free radical theory, reactive oxygen species (ROS) may cause oxidative injury to

living organisms through their lifetime. Prolonged oxidative stress may cause functional

cellular decline and various age-related disorders in humans and experimental animals. The

aim of Chapter 4 was to test the hypothesis whether tempol, a tempol scavenger, restores

impaired redox homeostasis and increases stress tolerance in a mimetic aging model of

Drosophila. To this end, we investigated the extent of oxidative stress and, specifically,

oxidative protein damage in mimetically aged flies following tempol administration.

Therefore, oxidative stress parameters and, sialic acid (SA) as cell surface glycocprotein

levels were determined.

It is well known that reactive oxygen species are fundamentally implicated as primary culprits

in the pathophysiology of renal I/R injury and consequent acute kidney injury. The excess

generation of reactive oxygen species and decreases in antioxidant defenses are known to

contribute to I/R injury. In a series of recent reviews, we have described that our hypothesis

that a disturbed balance between oxygen, nitric oxide, and reactive oxygen species might form

an important component of the pathogenesis of I/R-induced acute kidney injury. In Chapter

5, we aimed to test whether the proven protective effects of tempol are indeed associated with

improved renal oxygenation and nitric oxide levels in a short-term rat model of renal I/R.

Therefore, kidney oxygenation and consumption were determined beside nitric oxide levels of

kidney tissues in a rat model of renal I/R.  

  22  

  23  

CHAPTER 1

BALANCED VS UNBALANCED CRYSTALLOID RESUSCITATION IN A NEAR-

FATAL MODEL OF HEMORRHAGIC SHOCK AND THE EFFECTS ON RENAL

OXYGENATION, OXIDATIVE STRESS, AND INFLAMMATION

Aksu U1,2, Bezemer R1, Yavuz B3, Kandil A2, Demirci C2, Ince C1

1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, The Netherlands

2Department of Biology, Faculty of Science, University of Istanbul, 3Department of Biochemistry, Cerrahpasa Medical School, University of Istanbul, Istanbul,

Turkey

Published in: Resuscitation. 2012 Jun;83(6):767-73.

  24  

Chapter 1

Balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic

shock and the effects on renal oxygenation, oxidative stress, and inflammation

Running title: Crystalloid resuscitation in hemorrhagic shock

Abstract

Background: The aim of the present study was to test the hypothesis that balanced crystalloid

resuscitation would be better for the kidney than unbalanced crystalloid resuscitation in a rat

hemorrhagic shock model.Methods: Male Wistar rats were randomly assigned to four groups

(n = 6/group): (1) time control; (2) hemorrhagic shock control; (3) hemorrhagic shock

followed by unbalanced crystalloid resuscitation (0.9% NaCl); and (4) hemorrhagic shock

followed by acetate and gluconate-balanced crystalloid resuscitation (Plasma Lyte). We tested

the solutions for their effects on renal hemodynamics and microvascular oxygenation,

strongion difference, systemic and renal markers of inflammation and oxidative stress

including glycocalyx degradation as well as their effects on renal function. Results: The main

findings of our study were that: (1) both the balanced and unbalanced crystalloid solutions

successfully restored the blood pressure, but renal blood flow was only recovered by the

balanced solution although this did not lead to improved renal microvascular oxygenation; (2)

while unbalanced crystalloid resuscitation induced hyperchloremia and worsened metabolic

acidosis in hemorrhaged rats, balanced crystalloid resuscitation prevented hyperchloremia,

restored the acid–base balance, and preserved the anion gap and strong ion difference in these

animals; (3) in addition balanced crystalloid resuscitation significantly improved renal oxygen

consumption (increased VO2, decreased EFNa+); and (4) however neither balanced nor

unbalanced crystalloid resuscitation could normalize systemic inflammation or oxidative

stress. Functional immunohistochemistry biomarkers showed improvement in L-FABP in

favor of balanced solutions in comparison to the hemorrhagic group although no such benefit

was seen for renal tubular injury (measured by NGAL) by giving either unbalanced or

balanced solutions. Conclusions: Although balanced crystalloid resuscitation seems superior

to balanced crystalloid resuscitation in protecting the kidney after hemorrhagic shock and is

certainly better than not applying fluid resuscitation, these solutions were not able to correct

systemic inflammation or oxidative stress associated with hemorrhagic shock.

  25  

Introduction

Hemorrhagic shock is one of the leading causes of death among trauma patients. Current

treatment strategies for hemorrhagic shock involve rapid and aggressive fluid resuscitation to

restore blood pressure and tissue perfusion prior to blood transfusion.

However, even when systemic vital parameters are restored, tissue oxygenation in some

organs such as the kidney remains inadequate due to disturbed (micro) vascular regulatory

mechanisms as a result of hemorrhage-induced coagulation and inflammation [Legrand et al.,

2010; Cai et al., 2009]. In this line, it has been shown that an important step during

microvascular inflammation is the loss of the endothelial cell glycocalyx [Pries and Kuebler,

2006; Taylor and Gallo, 2006; Vink and Duling, 1996; Rubio-Gayosso et al., 2006;

Nieuwdorp et al., 2006]. This loss of the glycocalyx significantly affects capillary wall

permeability, coagulation, and leukocyte adhesion and thereby disturbs microvascular

function [Constantinescu et al., 2003]. Additionally, dysfunctional nitric oxide metabolism

during oxidative stress has been proposed to cause inefficient utilization of oxygen for sodium

reabsorption [Welch et al., 2001]. Hence, there is a complex interrelation between oxygen,

nitric oxide, and reactive oxygen species in the tissues [Freeman et al., 1982; Demoncheaux et

al., 2005].

In rat models of hemorrhagic shock, it has been shown that excessive saline (0.9% NaCl)

administration for resuscitation can lead to metabolic acidosis, which has been associated with

microvascular dysfunction and consequent tissue hypoxia in sensitive organ systems such as

the kidney. Hyperchloremia, in addition, has been shown to depress renal blood flow and

glomerular filtration rate and has been shown to disturb nitric oxide production and

utilization. Therefore, using balanced crystalloids to avoid acid–base disturbances and

hyperchloremia could be beneficial. The type of buffer that should be used for balancing

fluids, however, is still subject of debate [Parekh, 2002; Pedoto et al., 1999; Wilcox,1983;

Dorje et al., 2000; Zander, 2002]. Attempts to prevent metabolic acidosis include partial

replacement of chloride by rapidly metabolized anions such as l-lactate, acetate, and

gluconate. The aim of the present study was to test the hypothesis that balanced crystalloid

resuscitation would be better for the kidney than unbalanced crystalloid resuscitation in a rat

hemorrhagic shock model. For integrative investigation of the effects of hemorrhage and fluid

resuscitation on the complex interrelation between oxygen and reactive oxygen species, we

measured renal microvascular oxygen tension and plasma levels of malondialdehyde

  26  

(oxidative stress marker). Furthermore, glycocalyx degradation was assessed by measuring

plasma levels of hyaluronan (glycocalyx compartment) and plasma levels of TNF-α and

interleukin 6 were measured as markers of inflammation.

Materials and methods

Animals

All experiments in this study were reviewed and approved by the institutional animal

experimentation committee of the Academic Medical Center of the University of Amsterdam.

Care and handling of the animals were in accordance with the guidelines for Institutional and

Animal Care and Use Committees. Experiments were performed on 24 male Wistar rats

(Harlan, the Netherlands) with body weight of 335 ± 15 g.

Surgical preparation

The rats were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg

ketamine (Nimatek®; Eurovet, Bladel, the Netherlands), 0.5 mg/kg medetomidine (Domitor;

Pfizer, New York, NY), and 0.05 mg/kg atropine-sulfate (Centrafarm, Etten-Leur, the

Netherlands). After tracheotomy the animals were mechanically ventilated with a FiO2 of 0.4.

Body temperature was maintained at 37 ± 0.5 "C during the entire experiment by external

warming. Ventilator settings were adjusted to maintain end-tidal PCO2 between 30 and 35

mmHg and arterial PCO2 between 35 and 40 mmHg.

For drug and fluid administration and hemodynamic monitoring, vessels were cannulated with

polyethylene catheters (outer diameter = 0.9 mm; Braun, Melsungen, Germany). A catheter in

the right carotid artery was connected to a pressure transducer to monitor arterial blood

pressure and heart rate. The right jugular vein was cannulated for continuous infusion of

Ringer Lactate (Baxter, Utrecht, the Netherlands) at a rate of 15 ml/kg/h and maintenance of

anesthesia. The right femoral artery was cannulated for blood shedding and the right femoral

vein for fluid resuscitation. The left kidney was exposed, decapsulated, and immobilized in a

Lucite kidney cup (K. Effenberger, Pfaffingen, Germany) via a ~4 cm incision in the left

flank. Decapsulating the kidney allowed low-noise phosphorimetric measurements of renal

microvascular oxygenation (see below). Renal vessels were carefully separated under

preservation of nerves and adrenal gland. A perivascular ultrasonic transient time flow probe

was placed around the left renal artery (type 0.7 RB; Transonic Systems Inc., Ithaca, NY,

USA) and connected to a flow meter (T206; Transonic Systems Inc.) to continuously measure

  27  

renal blood flow (RBF). The left urethra was isolated, ligated, and cannulated with a

polyethylene catheter for urine collection. A small piece of aluminum foil was placed on the

dorsal site of the renal vein to prevent contribution of underlying tissue to the

phosphorescence signal in the venous PO2 measurement (as described below).

After the surgical protocol, one optical fiber was placed 1 mm above the decapsulated kidney

and another optical fiber was placed 1 mm above the renal vein to measure renal

microvascular and venous oxygenation, respectively, using phosphorimetry. Oxyphor G2 (a

two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin, Oxygen

Enterprises Ltd., Philadelphia, PA, USA) was subsequently infused (i.e. 6 mg/kg i.v. over 5

min), followed by 30 min stabilization time. The surgical field was covered with a humidified

gauze compress throughout the entire experiment to prevent drying of the exposed tissue. A

short description of the phosphorimetric method is given below and an extensive description

of the technology can be found elsewhere [Johannes et al., 2006].

Experimental protocol

After stabilization, the animals were bled through the left femoral artery catheter at a rate of 1

ml/min using a syringe pump (Harvard 33 syringe pump; Harvard Apparatus, South Natick,

MA) till reaching a MAP of 30 mmHg. Coagulation of the shed blood was prevented by

adding 200 UI of heparin in the syringe. This MAP was maintained for 1 h by re-infusing or

withdrawing blood. At the end of this phase, the animals were randomized into: (1) balanced

fluid resuscitation with Plasma Lyte (Lyte) (n = 6) (Na+ 140 mmol L−1, Cl− 98 mmol L−1, K+

5 mmol L−1, Mg2+ 1.5 mmol L−1, acetate 27 mmol L−1, gluconate 23 mmol L−1; Plasma Lyte

148®, Baxter, Valencia, Spain); and (2) unbalanced fluid resuscitation for 90 min with saline

(NaCl) (n = 6) (0.9% NaCl). Resuscitation continued until a target MAP of 80 mmHg was

reached or for 90 min. Additionally, time control (Control) and hemorrhagic shock control

(HS) experiments were performed (n = 6 per group). The experiments were terminated by

infusion of 1 ml of 3 M potassium chloride (KCl) after which the kidney was removed and

weighed.

Hemodynamic, blood gas parameters, lactate, and acid base balance

Mean arterial pressure (MAP), heart rate (HR), and renal blood flow (RBF) were measured

continuously. Arterial blood samples (0.5 ml) were taken from the femoral artery at three time

points: (1) baseline (BL, t = 0 min); (2) hemorrhagic shock (HS, t = 60 min); and (3) 90 min

  28  

after starting resuscitation (RS, t = 150 min). The blood samples were replaced by the same

volume of balanced crystalloid (Plasma Lyte). The samples were used to determine of blood

gas parameters (ABL505 Blood Gas Analyzer; Radiometer, Copenhagen, Denmark), the

hemoglobin concentration, and the hemoglobin oxygen saturation. Renal oxygen delivery was

calculated as: DO2ren (ml/min) = RBF × arterial oxygen content (1.31 × hemoglobin × SaO2) +

(0.003 × PaO2), where SaO2 is arterial oxygen saturation and PaO2 is arterial partial pressure

of oxygen.

Renal oxygen consumption was calculated as: VO2ren (ml/min/g) = RBF × (CaO2 − CvO2),

where renal venous oxygen content (CvO2) was calculated as (1.31 × hemoglobin × SrvO2) +

(0.003 × PrvO2). An estimation of the renal vascular resistance (RVR) was made as: RVR

(dynes s cm−5) = (MAP/RBF) × 100.

Plasma lactate levels were measured by an enzymatic colorimetric method using the Roche

Modular P800 automatic analyzer (Roche Diagnostics) from samples taken at t = 0 min

(baseline) and t = 150 min (end of experiment). The anion gap (AG) was calculated as the

sum of sodium and potassium concentrations minus the sum of bicarbonate and chloride

concentrations. For calculation of strong ion difference (SID), the sum of chloride and lactate

concentrations was subtracted from the sum of sodium and potassium concentrations.

Renal function and plasma osmolality

Creatinine clearance (Clearcrea (ml/min)) was assessed as an index of the glomerular filtration

rate. Calculation of the clearance was done using: Clearcrea = (Ucrea × V)/Pcrea, where Ucrea is

the concentration of creatinine in urine, V is the urine volume per unit time and Pcrea is the

concentration of creatinine in plasma. Additionally, excretion fraction of Na+ [EFNa (%)] was

calculated to use as a marker of tubular function as: EFNa+ = (UNa × Pcrea)/(PNa+ × Ucrea) × 100,

where UNa is Na+ concentration in urine and PNa is the Na+ concentration in plasma. Clearcrea

and EFNa+ were determined at t = 0 min and t = 150 min. Furthermore, the renal energy

efficiency for sodium transport (VO2ren/TNa+ ) was assessed using the ratio of the total amount

of VO2ren over the total amount of sodium reabsorbed (TNa+ , mmol/min). The osmolality of

the plasma and urine were determined using the freezing point method using an osmotic

pressure meter (Osmostation, OM-6050; Arkray) from a sample taken at the end of the

experiment.

  29  

Renal microvascular oxygenation and renal venous PO2

Renal microvascular PO2 (µPO2) and renal venous PO2 (PrvO2) were measured by oxygen

dependent quenching of phosphorescence lifetimes of the systemically infused albumin

targeted (and therefore circulation confined) phosphorescent dye Oxyphor G2 [Vinogradov et

al., 2002]. A linear relationship between reciprocal phosphorescence lifetime and oxygen

tension (given by the Stern–Volmer relation) allows quantitative measurement of µPO2.

Oxyphor G2 (a two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin)

has two excitation peaks (λexcitation1 = 440 nm, λexcitation2 = 632 nm) and one emission peak

(λemission = 800 nm) [ Dunphy et al., 2002].These optical properties allow (near) simultaneous

lifetime measurements in microcirculation of the kidney cortex (CµPO2) and the outer

medulla (MµPO2) due to different optical penetration depths of the two excitation

wavelengths [Johannes et al., 2006]. For the measurement of renal venous PO2 (PrvO2), a

mono-wavelength (λexcitation = 632 nm) phosphorimeter was used [Mik et al., 2008].

Oxidative stress and inflammation markers

Determination of malondialdehyde (MDA) levels was used to quantify the lipid peroxidation

in tissues and plasmas. Tissues were homogenized in 5 mM (cold) Na-phosphate buffer. The

homogenates were centrifuged at 12,000 × g for 15 min at 4 oC and supernatants were used

for MDA determination. The level of lipid peroxides was expressed as micromoles of MDA

per miligram of protein (Bradford assay). Plasma levels of TNF-α and IL-6 were measured by

ELISA as markers of systemic inflammation.

Glycocalyx degradation

Hyaluronan is the main component of endothelial glycocalyx, and alterations in its

concentration can be attributed to glycocalyx volume loss [Nieuwdorp et al., 2006]. Plasma

hyaluronan concentrations were determined using the Corgenix hyaluronic acid test kit

(Corgenix Inc., Westminster, Colo) that is based on an enzyme-linked hyaluronic acid binding

protein assay.

Immunohistochemical analysis

Kidney tissues were fixed in 4% formalin and embedded in paraffin. Kidney sections (5 µm)

were deparaffinized with xylene and rehydrated with decreasing percentages of ethanol and

finally with water. Antigen retrieval was accomplished by microwaving slides in citrate buffer

  30  

(pH 6.0) (Thermo Scientific, AP-9003-500) for 10 min. Slides were left to cool for 20 min at

room temperature and then rinsed with distilled water. Surroundings of the sections were

marked with a PAP pen. The endogenous peroxidase activity was blocked with 3% H2O2 for

10 min at room temperature and later rinsed with distilled water and PBS. Blocking reagent

(LabVision, TA-125-UB) was applied to each slide followed by 5 min incubation at room

temperature in a humid chamber. Kidney sections were incubated for overnight at 4 oC with

Lipocalin 2 antibody (NGAL) (abcam 41105) and polyclonal antibody to rat L-FABP (Hycult

Biotect HP8010). Antibodies were diluted in a large volume of UltrAb Diluent (Thermo

Scientific, TA-125-UD). The sections were washed in PBS three times for 5 min each time

and then incubated for 30 min at room temperature with biotinylated goat anti-rabbit

antibodies (LabVision, TP-125-BN). After slides were washed in PBS, the streptavidin

peroxidase label reagent (LabVision, TS-125-HR) was applied for 30 min at room

temperature in a humid chamber. The colored product was developed by incubation with

AEC. The slides were counterstained with Mayer’s hematoxylin (LabVision, TA- 125-MH)

and mounted in vision mount (LabVision, TA-060-UG) after being washed in distilled water.

Both the intensity and the distribution of specific L-FABP and NGAL staining were scored.

For each sample, a histological score (HSCORE) value was derived by summing the

percentages of cells that stained at each intensity multiplied by the weighted intensity of the

staining [HSCORE = S Pi (i + 1), where i is the intensity score and Pi is the corresponding

percentage of the cells] [Senturk et al., 1996].

Statistical analysis

Values are reported as mean ± SEM. The decay curves of phosphorescence intensity were

analyzed using software programmed in Labview 6.1 (National Instruments, Austin, TX,

USA). Statistical analysis was performed using GraphPad Prism version 4.0 for Windows

(GraphPad Software, San Diego, CA, USA). Two-way ANOVA for repeated measurements

with a Bonferroni post hoc test was used for comparisons; a p-value of <0.05 was considered

statistically significant.

Results

Fluid resuscitation and plasma osmolality

For restoration of the MAP from 30 mmHg to 80 mmHg, 56.8 ± 3.8 ml of saline was required

and only 30.4 ± 4.7 ml of Plasma Lyte was required (p < 0.01 vs HS + NaCl group). The

plasma osmolality at the end of resuscitation time point was 237 ± 4 mOsm/kg in the control

  31  

group, 253 ± 2 mOsm/kg in the HS group (p < 0.01 vs control), 244 ± 2 mOsm/kg in the HS +

NaCl group (p < 0.01 vs HS), and 247 ± 2 mOsm/kg in HS + Lyte group.

Plasma ions

Anion gap values, the negative strong ion difference, and plasma ion levels are presented in

Table 1. Hemorrhagic shock did not affect the anion gap, the SID, and the sodium and

chloride levels. Hemorrhagic shock did induce metabolic acidosis as reflected by a decreased

pH (p < 0.05 vs control) and plasma HCO3− level (p < 0.01 vs control). The metabolic

acidosis could be restored by Plasma Lyte resuscitation, but not by NaCl resuscitation. NaCl

resuscitation, moreover, increased the plasma chloride levels (p < 0.001 vs control), which

were decreased by Plasma Lyte resuscitation (p < 0.05 vs HS). Sodium concentration was

similar in all groups. The NaCl resuscitation group had the lowest anion gap (p < 0.01) and

negative SID was also significantly lower in the NaCl group compared to the other groups

(p < 0.05). The highest value of negative SID was found in the HS + Lyte group.

Systemic and renal hemodynamics

Systemic and renal hemodynamics are presented in Table 2. Baseline values were found

similar in each group. Hemorrhage caused marked effects on hemodynamics without

significant differences among groups (p > 0.05). In all groups, MAP and RBF decreased

during hemorrhage but RVR and HR did not change. During resuscitation, MAP was

increased in all groups and the target MAP of 80 mmHg was successfully achieved and

maintained throughout resuscitation in all groups. Resuscitation did not affect HR (p > 0.05).

During hemorrhagic shock, RBF dropped ∼74% (p < 0.01) without differences among groups.

The most effective fluid to restore RBF was the Plasma-Lyte preparation. None of the fluids

significantly affected RVR during resuscitation

  32  

Table 1. Anion gap, negative strong ion difference and pH values and plasma ion levels at baseline (t= 0 min) and at the end of resuscitation (t = 150 min). Cp< 0.05 vs control, Hp<0.05 vs HS, Np< 0.05 vs HS+NaCl.

Baseline Resuscitation Anion gap

           Control 20.8 ± 0.5 22.3 ± 3.2 HS 18.6 ± 2.5 30.2 ± 1.6 HS+NaCl 16.3 ± 1.2 0.6 ± 4.0C,H HS+Lyte 22.6 ± 3.3 29.8 ± 6.0 Strong ion difference (negative)

     Control 39.7 ± 1.8 34.3 ± 6.0 HS 37.1 ± 2.6 26.2 ± 3.1 HS+NaCl 36.0 ± 0.8 12.6 ± 5.0C HS+Lyte 39.2 ± 2.6 47.6 ± 5.3H,N pH

           Control 7.38 ± 0.01 7.38 ± 0.01 HS 7.37 ± 0.01 7.18 ± 0.00C HS+NaCl 7.39 ± 0.03 7.18 ± 0.00C HS+Lyte 7.38 ± 0.03 7.36 ± 0.04H,N HCO3

-(mmol/l)          Control 20.9 ± 1.9 20.6 ± 2.2

HS 20.3 ± 0.4 6.9 ± 1.0C HS+NaCl 21.7 ± 0.7 15.2 ± 1.4 HS+Lyte 21.0 ± 0.6 21.3 ± 0.9H Cl- (mmol/l)

           Control 107.7   ± 3.4   110.7 ± 2.6  HS 106.4   ± 2.8   113.8 ± 1.9  HS+NaCl 109.2   ± 1.1   133.8 ± 4.0C,H  HS+Lyte 105.5   ± 3.2   95.3 ± 5.6H,N  Na+ (mmol/l)

           Control 144.7   ± 0.3   146.0 ± 1.5  HS 140.8   ± 1.9   144.2 ± 1.0  HS+NaCl 142.8   ± 1.1   145.2 ± 0.7  HS+Lyte 143.5   ± 2.1   141.5 ± 0.5  

  33  

Table 2. Hemodynamic parameters at baseline (t=0 min), during shock (t=60 min) and resuscitation (t=150 min). Cp < 0.05 vs. control, Hp < 0.05 vs. HS

Baseline Shock Resuscitation Mean arterial pressure (mmHg)

               Control 103 ± 2 102 ± 1 102 ± 3 HS 102 ± 2 34 ± 1 C 35 ± 1C HS+NaCl 99 ± 4 35 ± 2 C 76 ± 6 C,H HS+Lyte 100 ± 5 34 ± 3 C 75 ± 6 C,H Heart rate (bpm)

                 Control 245 ± 6 253 ± 3 254 ± 11 HS 229 ± 7 256 ± 6 247 ± 12 HS+NaCl 226 ± 8 237 ± 16 247 ± 13 HS+Lyte 260 ± 13 260 ± 15 281 ± 12 Renal blood flow (ml/min)

                 Control 5.1 ± 0.7 5.9 ± 0.8 6 ± 0.9 HS 5.1 ± 0.2 1.4 ± 0.1 C 1.2 ± 0.1 C HS+NaCl 5.2 ± 0.8 1.8 ± 0.3 C 3.3 ± 1.1 HS+Lyte 5.4 ± 0.2 1.7 ± 0.2 C 3.9 ± 0.2 Renal vascular resistance (dyn s cm-5)

           Control 1435 ± 114 1505 ± 164 1299 ± 171 HS 1584 ± 47 1927 ± 260 2518 ± 465 HS+NaCl 1393 ± 170 1704 ± 235 3117 ± 1443 HS+Lyte 1916 ± 15 1937 ± 195 1516 ± 106

Renal oxygenation and function

Renal oxygenation and function parameters are presented in Figs. 1 and 2, respectively. DO2,

VO2, CµPO2, and MµPO2 all decreased during the hemorrhage. CµPO2 and MµPO2 could not

be improved by fluid resuscitation using NaCl or Plasma Lyte. Both NaCl and Plasma Lyte

resuscitation increased DO2 and VO2 although the increase in VO2 was more pronounced in

the HS + Lyte group. VO2/TNa+ was increased during hemorrhage and could not be improved

by fluid resuscitation (0.68 ± 0.20 in the control group vs 1.67 ± 0.62 and 1.69 ± 0.32 in the

HS + NaCl and HS + Lyte groups, respectively, p < 0.05). Creatinine clearance was decreased

in all groups compared to the control group. NaCl resuscitation significantly increased the

EFNa+ group (p < 0.05 vs all groups) (Fig. 2).

  34  

Fig.1. Renal oxygenation at baseline (t=0 min) and during shock (t=60 min) and resuscitation (t=150 min). µPO2: microvascular oxygen tension; DO2: renal oxygen delivery; VO2: renal oxygen consumption. Cp< 0.05 vs control group, Hp< 0.05 vs HS group.

Fig.2. Renal function parameters at baseline (t=0 min) and the end of resuscitation (t=150min).Cp<0.05 vs controlgroup, Np<0.05 vs HS+NaCl group.

  35  

Oxidative stress and inflammation

All types of resuscitation decreased the plasma lactate levels. Additionally, lactate levels in

resuscitated groups were similar with control group (Table 3). The plasma MDA levels in the

resuscitated groups were lower than in the control group, reflecting the dilution of plasma by

resuscitation. Additionally, tissue MDA levels in all groups were higher than in the control

group (p < 0.05) (Table 3). This was accompanied by increase in plasma levels of hyaluronan,

an endothelial glycocalyx component (p < 0.05 vs control group) (Table 3). Plasma cytokines

levels TNF-α and IL-6 increased during shock (p < 0.01 vs control) and resuscitation did not

restore these parameters (Table 3).

Immunohistochemical analysis

Lipocalin 2 (NGAL) and L-FABP reactivity were both increased during hemorrhagic shock

(p < 0.01). Fluid resuscitation could only slightly reduce the NGAL levels (p > 0.05 in the HS

+ Lyte group and p < 0.05 in the HS + NaCl group). NaCl resuscitation could not correct the

L-FABP levels while Plasma Lyte resuscitation could significantly reduce the L-FABP levels

(p < 0.01; Fig. 3).

  36  

Table 3. Plasma and tissue biochemistry parameters. Cp < 0.05 vs. control, Hp < 0.05 vs.HS Resuscitation Plasma lactate (mmol/l)

   Control 2.8 ± 0.2 HS 11.2 ± 1.8 C HS+NaCl 3.3 ± 0.7 H HS+Lyte 3.6 ± 0.5 H Plasma NO (µmol)

     Control 76.9 ± 14.1 HS 81.2 ± 18.5 HS+NaCl 147.9 ± 23.5 HS+Lyte 102.2 ± 21.6 Tissue NO /protein content (µmol/g)

   Control 1.2 ± 0.1 HS 1.9 ± 0.2 HS+NaCl 1.6 ± 0.6 HS+Lyte 0.7 ± 0.3 Plasma TNF-α (pg/ml)

   Control 68 ± 13 HS 291 ± 16.26 C HS+NaCl 368 ± 47.99 C HS+Lyte 247 ± 48.7 C Plasma IL-6 (pg/ml)

     Control 170 ± 25 HS 14132 ± 4347 C HS+NaCl 21258 ± 5312 C HS+Lyte 12808 ± 6146 C Plasma MDA (µmol/l)

     Control 20.7 ± 2 HS 21.6 ± 3.1 HS+NaCl 15.9 ± 1.6 HS+Lyte 16.6 ± 1.8 Tissue MDA/protein content (umol/g)

   Control 1.7 ± 0.2 HS 3.4 ± 0.6 C HS+NaCl 3.5 ± 0.6 C HS+Lyte 3.1 ± 0.3 C Plasma hyaluronan (ng/ml)

   Control 7.9 ± 2.7 HS 158.1 ± 20.8 C HS+NaCl 97.1 ± 13.4 C HS+Lyte 152.7 ± 56.9 C

  37  

Fig.3. Renal tissue NGAL and L-FABP levels at the end of resuscitation (t=150 min). Cp<0.05 vs control group, Hp<0.05 vs HS group.

Discussion

In the present study, an acetate and gluconate-balanced crystalloid solution was tested for its

effects on the plasma ion levels and acid–base balance; renal oxygenation, oxidative stres

status, glycocalyx integrity; and systemic cytokine levels in a rat model of hemorrhagic shock.

The main findings of our study were that: (1) both the balanced and unbalanced crystalloid

solutions successfully restored the blood pressure, but renal blood flow was only recovered by

the balanced solution although this did not lead to improved renal oxygenation; (2) less

balanced fluid was required to restore blood pressure; (3) while unbalanced crystalloid

resuscitation induced hyperchloremia and worsened metabolic acidosis in hemorrhaged rats,

balanced crystalloid resuscitation prevented hyperchloremia, restored the acid–base balance,

and preserved the anion gap and strong ion difference in these animals; (4) neither balanced

nor unbalanced crystalloid resuscitation could normalize systemic inflammation (TNF-α and

IL-6); (5) only balanced crystalloid resuscitation significantly reduced renal oxidative stres as

reflected by reduced L-FABP reactivity, but none of the fluids could restore the increased

NGAL, MDA, and hyaluronan levels; and (6) balanced crystalloid resuscitation significantly

improved renal oxygen consumption (increased VO2, decreased EFNa+ ), but none of the fluids

was able to restore creatinine clearance rate in this shortterm protocol.

The results of our study show that renal microcirculatory hypoxia occurs during hemorrhage

and remains after crystalloid resuscitation. In addition, this appears to be relatively

independent from systemic and renal macrohemodynamics, but arises from intrarenal

mechanisms that may be associated with hypoxia in the microcirculation. A mechanism

  38  

potentially responsible for the disassociation between macro- and microcirculatory parameters

could be the inadequate resolution of inflammatory activation by just improving systemic

hemodynamics [Ulloa and Tracey, 2005]. Indeed, crystalloid resuscitation was unable to

prevent systemic inflammation. Combined with a suboptimal fluid composition, this possibly

leads to disturbed plasma ion levels (e.g., hyperchloremia), reduced oxygen carrying capacity,

and consequent microvascular dysfunction and hypoxia.

As renal dysfunction is a common complication following major hemorrhage and fluid

resuscitation, there is a continuing research on the efficacy of fluid resuscitation strategies to

protect the kidney. However, the type of fluid that should be used for resuscitation to yield the

best renal outcome remains controversial today and the composition of resuscitation fluids is

still subject of debate. Buffers such as acetate, gluconate, and lactate are commonly used in

resuscitation fluids. These buffers are converted to bicarbonate in the liver, raising the pH of

the solution toward normal blood pH of 7.4 [Stewart 1983; Richards et al., 1982].

Furthermore, recent investigations have shown that unbalanced solutions containing high

amounts of chloride (e.g., 0.9% NaCl) might cause hyperchloremic acidosis [Scheingraber et

al., 1999; McFarlane and Lee, 1994; Prough and Bidani, 1999] whereas solutions with buffers

and more physiological concentrations of strong ions do not [Kellum et al., 1998; Kellum,

1998]. As demonstrated by the present work, unbalanced crystalloid resuscitation leads to

hyperchloremia since a reduction in the strong ion difference is required to match the diluted

non-volatile weak acid. In this line, the decrease in pH as observed following NaCl

resuscitation in the present study appears to have been caused by chloride loading.

Resuscitation with the balanced crystalloid preparation, in contrast, completely restored

metabolic acidosis and improved renal blood flow. Clinical studies have also shown that

balanced solutions alter the acid–base status significantly less compared to saline solutions

[Khajavi et al., 2008]. The role of chloride in modulating vasoconstrictor responses to

vasoactive agents has previously been investigated in isolated rat kidneys [Quilley et al.,

1993]. Hyperchloremic fluids induced intrarenal vasoconstriction as indicated by increased

RVR and decreased RBF and glomerular filtration rate [Pedoto et al., 1999; Wilcox, 1983;

Naylor and Forsyth, 1986]. This is in agreement with the findings of increased RVR and

decreased microcirculatory perfusion in the present study. Whether these perturbations are

caused by the high levels of chloride directly or from the consequent disturbances in the acid–

base balance remains unknown.

  39  

Acidemia might affect a variety of vasoregulatory mechanisms. First, acidemia increases

endogenous catecholamine release, which induces the release of both pro and anti-

inflammatory cytokines [Le Tulzo et al., 1997; Liskaser et al., 2000] and nitric oxide [Haque

et al., 2003; Celotto et al., 2008]. Moreover, studies have shown that fluid resuscitation

following hemorrhage can exacerbate the systemic inflammatory responses which may be

even more harmful than the initial hemorrhage [Le Tulzo et al., 1997]. Jensen et al. have

shown that as a result of acid loading, macrophages increase their tumor necrosis factor

secretion [Jensen et al., 1990]. Increased levels of tumor necrosis factor might have influenced

microvascular perfusion either by the direct vasoactive properties of these molecules, which

could have contributed to the worsening of shock, or via direct tissue injury.

To test the effects of fluid resuscitation on systemic inflammation and oxidative stress in our

model, we measured plasma TNF-α and IL-6 levels as markers of systemic inflammation,

MDA and L-FABP as markers of oxidative stress, hyaluronan levels as markers of glycocalyx

degradation, and NGAL as a marker of renal tubular injury. We found that neither balanced

nor unbalanced crystalloid resuscitation could restore systemic inflammation, oxidative stress,

glycocalyx, or renal injury. The balanced crystalloid resuscitation, however, could

significantly reduce L-FABP reactivity. Nonetheless, our results suggest that even balanced

crystalloid resuscitation fails to prevent harmful systemic inflammatory responses and

oxidative stress following hemorrhagic shock.

In this study we found that hemorrhage severely disturbed the renal oxygen

consumption/sodium reabsortion balance and that this could not be restored by resuscitation

with balanced or unbalanced crystalloid solutions. Under normal physiological conditions,

there is a positive correlation between renal oxygen consumption (VO2) and tubular sodium

reabsorption and glomerular filtration rate [Kiil, 1977; Adler and Huang 2002; Lassen and

Thaysen, 1961]. Excessive nitric oxide (NO) production by cells as a result of inducible NO

synthase activation consequent to inflammation can inhibit mitochondrial respiration by

competing with oxygen mitochondrial cytochrome oxidase [Terada et al., 1992; Cooper and

Giulivi, 2007]. Thus, the production and utilization of oxygen, NO, and reactive oxygen

species are directly dependent on each other and together determine the adequacy of renal

function. In this line, renal oxygen consumption has been found to be increased by

nonselective NOS inhibition and selective neuronal NOS inhibition [Deng et al., 2005], which

demonstrates that different isoforms of NOS are involved in modulating renal oxygen

  40  

utilization. Hence, there is a complex interdependency between oxygen, reactive oxygen

species, and NO; their homeostasis becomes severely altered during conditions of renal

inflammation and ischemia/reperfusion, resulting in oxidative stress and loss of renal

function.

Conclusions

While unbalanced crystalloid resuscitation induces hyperchloremia and worsens metabolic

acidosis in hemorrhaged rats, balanced crystalloid resuscitation prevents hyperchloremia,

restores the acid–base balance, and preserves the anion gap and strong ion difference in these

animals. Balanced crystalloid resuscitation prevents renal hypoperfusion better than

unbalanced crystalloid resuscitation. However, although the balanced preparation improves

some parameters, it does not improve oxidative stress and systemic inflammation.

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  44  

  45  

Chapter 2

THE ACUTE EFFECTS OF ACETATE-BALANCED COLLOID AND

CRYSTALLOID RESUSCITATION ON RENAL OXYGENATION IN A RAT

MODEL OF HEMORRHAGIC SHOCK.

Almac E1,2, Aksu U1,3, Bezemer R1, Jong W1, Kandil A3, Yuruk K1, Demirci-Tansel C3,

Ince C1

1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

2Department of Anesthesiology, St. Antonius Hospital Nieuwegein, Nieuwegein, The Netherlands

3Department of Biology, Faculty of Science, University of Istanbul, Istanbul, Turkey

Published in: Resuscitation. 2012 Sep;83(9):1166-72.

  46  

Chapter 2

The acute effects of acetate-balanced colloid and crystalloid resuscitation on renal

oxygenation in a rat model of hemorrhagic shock

Running title: Colloid vs crystalloid resuscitation in hemorrhagic shock

Abstract

Introduction: Fluid resuscitation therapy is the initial step of treatment for hemorrhagic shock.

In the present study we aimed to investigate the acute effects of acetate-balanced colloid and

crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. We

hypothesized that acetate-balanced solutions would be superior in correcting impaired renal

perfusion and oxygenation after severe hemorrhage compared to unbalanced solutions.

Methods: In anesthetized, mechanically ventilated rats, hemorrhagic shock was induced by

withdrawing blood from the femoral artery until mean arterial pressure (MAP) was reduced to

30 mmHg. One hour later, animals were resuscitated with either hydroxyethyl starch (HES,

130/0.42 kDa) dissolved in saline (HES-NaCl; n = 6) or a acetate-balanced Ringer’s solution

(HES-RA; n = 6), as well as with acetated Ringer’s solution (RA; n = 6) or 0.9% NaCl alone

(NaCl; n = 6) until a target MAP of 80 mmHg was reached. Oxygen tension in the renal

cortex (CµPO2), outer medulla (MµPO2), and renal vein were measured using

phosphorimetry. Results: Hemorrhagic shock (MAP = 30 mmHg) significantly decreased

renal oxygenation and oxygen consumption. Restoring the MAP to 80 mmHg required 24.8 ±

1.7 ml of NaCl, 21.7 ± 1.4 ml of RA, 5.9 ± 0.5 ml of HES-NaCl (p < 0.05 vs. NaCl and RA),

and 6.0 ± 0.4 ml of HES-RA (p < 0.05 vs. NaCl and RA). NaCl, RA, and HES-NaCl

resuscitation led to hyperchloremic acidosis, while HES-RA resuscitation did not. Only HES-

RA resuscitation could restore renal blood flow back to ∼85% of baseline level (from 1.9 ±

0.1 ml/min during shock to 5.1 ml ± 0.2 ml/min 60min after HES-RA resuscitation) which

was associated with an improved renal oxygenation (CµPO2 increased from 24 ± 2 mmHg

during shock to 50 ± 2 mmHg 60 min after HES-RA resuscitation) albeit not to baseline level.

At the end of the protocol, creatinine clearance was decreased in all groups with no

differences between the different resuscitation groups. Conclusion: While resuscitation with

the NaCl and RA (crystalloid solutions) and the HES-NaCl (unbalanced colloid solution) led

to hyperchloremic acidosis, resuscitation with the HES-RA (acetate-balanced colloid solution)

  47  

did not. The HES-RA was furthermore the only fluid restoring renal blood flow back to ∼85%

of baseline level and most prominently improved renal microvascular oxygenation.

  48  

Introduction

Hemorrhagic shock is the major cause of mortality after major trauma and aggressive fluid

resuscitation is often the initial step to restore the circulating intravascular volume to prevent

organ hypoperfusion, organ failure, and eventually death [Coimbra et al., 2006; Liu et al.,

2003]. Acute renal failure (ARF) is a serious complication contributing to the high mortality

in these patients [Morris et al., 1991]. The use of conventional crystalloid solutions (e.g.,

isotonic saline) as initial resuscitation fluids is still implemented in emergency departments

even though it is known that crystalloid solutions have poor plasma expander capacities and

just 20% of the given volume remains contained in the intravascular space [Svensen and

Hahn, 1997]. Hence, to restore perfusion, large volumes of crystalloid solutions are required.

Additionally, hyperchloremic acidosis is a known risk in patients treated with isotonic saline.

Hyperchloremia is suggested to cause afferent renal artery vasoconstriction in animal models,

possibly leading to kidney dysfunction [Wilcox, 1983; Bullivant et al., 1989; Wilcox and

Peart, 1987]. Hydroxyethyl starch (HES) solutions have been used clinically as a colloid

solution, and have been shown to have superior plasma expanding capacities compared to

traditional crystalloid solutions [Haisch et al., 2001]. However, these HES solutions, in turn,

have been suggested to have adverse effects on systemic coagulation properties and are

potentially harmful for the kidney [Warren and Durieux, 1997]. Consequently, new HES

solutions (mean molecular weight: 130 kDa, degree of substitution: 0.4; HES 130/0.4) have

been developed and have been shown to improve microvascular perfusion and reduce

macromolecular leakage [Boldt et al., 1996; Nohe et al., 2005; Hoffmann et al., 2002].

Although effective in restoring systemic hemodynamic parameters, aggressive (i.e., large

volume) fluid resuscitation introduces non-physiologic levels of plasma ions which depress

microvascular function and organ perfusion [Legrand et al., 2010]. The kidney is especially

susceptible for this type of injury due to its complex microvascular structure and high oxygen

dependency [Evans et al., 2008]. Over the past few years, research has therefore been focused

on balancing fluids to optimally match physiological conditions and thereby prevent

microvascular dysfunction and organ hypoperfusion [Spahn et al., 2007; Stern, 2001; Horstick

et al., 2002]. Balanced fluids are suggested to have a more physiological electrolyte

composition than conventional saline-based fluids.

In the present study we aimed to investigate the acute effects of acetate-balanced colloid and

crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. To this

  49  

end, we examined the effects of resuscitation with different fluids: (1) 0.9% NaCl; (2)

acetated Ringer’s solution; (3) 6% HES with a molecular weight of 130 kDa and molar

substitution of 0.4 (HES 130/0.4) in 0.9% NaCl solution (HES-NaCl); and (4) 6% HES with a

molecular weight of 130 kDa and molar substitution of 0.42 (HES 130/0.42) in acetate-

balanced Ringer’s solution (HES-RA). We hypothesized that acetated solutions would have

superior resuscitation capacities compared to the other solutions with respect to improving

renal oxygenation after severe hemorrhage.

Materials and methods

Animals

All experiments in this study were approved by the institutional Animal Experimentation

Committee of the Academic Medical Center of the University of Amsterdam. Care and

handling of the animals were in accordance with the guidelines for Institutional and Animal

Care and Use Committees. Experiments were performed on 30 Sprague-Dawley rats (Harlan,

the Netherlands) with mean ± SD body weight of 350 ± 20 g.

Surgical preparation

The rats were anesthetized with an intraperitoneal injection of a mixture of 100 mg/kg

ketamine (Nimatek®; Eurovet, Bladel, the Netherlands), 0.5 mg/kg medetomidine (Domitor;

Pfizer, New York, NY), and 0.05 mg/kg atropine-sulfate (Centrafarm, Etten-Leur, the

Netherlands). After tracheotomy, the animals were mechanically ventilated with an FiO2 of

0.4. Body temperature was maintained at 37 ± 0.5 ◦C during the entire experiment by external

warming. The ventilator settings were adjusted to maintain end-tidal PCO2 between 30 and 35

mmHg and arterial PCO2 between 35 and 40 mmHg. Vessels were cannulated with

polyethylene catheters (outer diameter = 0.9 mm; Braun, Melsungen, Germany) for drug and

fluid administration and hemodynamic monitoring. A catheter in the right carotid artery was

connected to a pressure transducer to monitor mean arterial blood pressure (MAP) and heart

rate. The right jugular vein was cannulated for continuous infusion of Ringer Lactate (Baxter,

Utrecht, the Netherlands) at a rate of 15 ml/kg/h. The right femoral artery was cannulated for

blood shedding and the right femoral vein for fluid resuscitation. The left kidney was

exposed, decapsulated, and immobilized in a Lucite kidney cup (K. Effenberger, Pfaffingen,

Germany) via a 4 cm incision in the left flank. Renal vessels were carefully separated under

preservation of nerves and adrenal gland. A perivascular ultrasonic transient time flow probe

was placed around the left renal artery (type 0.7 RB; Transonic Systems Inc., Ithaca, NY,

  50  

USA) and connected to a flow meter (T206; Transonic Systems Inc.) to continuously measure

renal blood flow (RBF). An estimation of the renal vascular resistance (RVR) was made as

RVR [dynes s cm−5] = (MAP/RBF) × 100. The left ureter was isolated, ligated and cannulated

with a polyethylene catheter for urine collection. The surgical field was covered with a

humidified gauze compress throughout the entire experiment to prevent drying of the exposed

tissue.

After the surgical protocol (approximately 60 min) one optical fiber was placed 1 mm above

the decapsulated kidney and another optical fiber 1 mm above the renal vein to measure

oxygenation using a phosphorescence lifetime technique. A small piece of aluminum foil was

placed on the dorsal site of the renal vein to prevent contribution of underlying tissue to the

phosphorescence signal in the venous PO2 measurement. Oxyphor G2 (a two-layer glutamate

dendrimer of tetra-(4-carboxy-phenyl) benzoporphyrin; Oxygen Enterprises Ltd.,

Philadelphia, PA, USA) was subsequently infused (6 mg/kg IV over 5 min) followed by a 30

min stabilization period. A short description of the phosphorescence quenching method is

given below and a more detailed description of the technology has been previously described

[Johannes et al., 2006].

Experimental protocol

After stabilization, the animals in experimental groups were bled by the left femoral artery

catheter at a rate of 1 ml/min using a syringe pump (Harvard 33 syringe pump; Harvard

Apparatus, South Natick, MA) until a MAP of 30 mmHg was reached which was maintained

for 1 h by re-infusing or withdrawing blood. Coagulation of the shed blood was prevented by

adding 200 UI of heparin in the syringe.

At the end of the hemorrhage phase, the animals were randomized into 5 groups for

resuscitation until a target MAP of 80 mmHg was reached with: (1) 0.9% NaCl (NaCl; Na+

154 mmol l−1, Cl− 154 mmol l−1; pH 5.5; n = 6); (2) Ringer’s Acetate (RA; Na+ 130 mmol l−1,

Cl− 112 mmol l−1, K+ 5.4 mmol l−1, Ca+2 0.9 mmol l−1, Mg+2 1.0 mmol l−1, acetate− 27 mmol

l−1; pH = 5.0–7.0; n = 6); (3) 6% HES with a molecular weight of 130 kDa and molar

substitution of 0.4 (HES 130/0.4) in 0.9% NaCl solution (HES-NaCl; Voluven®, Fresenius

Kabi, Bad Homburg, Germany; n = 6); or (4) 6% HES with a molecular weight of 130 kDa

and molar substitution of 0.42 (HES 130/0.42) in acetate-balanced Ringer’s solution (HES-

  51  

RA; Plasma Volume®, Baxter, Germany; n = 6). In addition, sham operated control

experiments were performed (n = 6).

The experiments were terminated by infusion of 1 ml of 3 M potassium chloride (KCl).

Blood gas parameters

Arterial blood samples (0.5 ml) were taken from the femoral artery at time points: (1) baseline

(BL, t = 0 min); (2) after hemorrhagic shock (HS, t = 60 min); (3) 15 min after starting

resuscitation (R15, t = 75 min), and (4) at the end of the protocol (R60, t = 120 min).

The blood samples were replaced by the same volume of test solution. The samples were used

to determine blood gas parameters (ABL505 blood gas analyzer; Radiometer, Copenhagen,

Denmark), hemoglobin concentration, and hemoglobin oxygen saturation (OSM 3,

Radiometer).

Renal microvascular and venous oxygenation

Microvascular oxygen tension in the renal cortex (CµPO2), outer medulla (MµPO2), and renal

venous oxygen tension (PrvO2) were measured by oxygen-dependent quenching of

phosphorescence lifetimes of the systemically infused albumin-targeted (and therefore

circulation-confined) phosphorescent dye Oxyphor G2 [Johannes et al., 2006; Mik et al.,

2004; Vinogradov et al., 2002; Dunphy et al., 2002]. Oxyphor G2 (a two-layer glutamate

dendrimer of tetra- (4-carboxy-phenyl) benzoporphyrin) has two excitation peaks (λexcitation1 =

440 nm, λexcitation2 = 632 nm) and one emission peak (λemission = 800 nm) [Dunphy et al.,

2002]. These optical properties allow (near) simultaneous lifetime measurements in

microcirculation of the kidney cortex and the outer medulla due to different optical

penetration depths of the excitation light [Johannes et al., 2006]. For the measurement of renal

venous PO2 (PrvO2), a mono-wavelength phosphorimeter was used [Mik et al., 2008]. Oxygen

measurements based on phosphorescence lifetime techniques rely on the principle that

phosphorescence can be quenched by energy transfer to oxygen resulting in shortening of the

phosphorescence lifetime. A linear relationship between reciprocal phosphorescence lifetime

and oxygen tension (given by the Stern–Volmer relation) allows quantitative measurement of

PO2. Details of the technique have previously been published [Johannes et al., 2006].

Renal oxygen delivery and consumption

  52  

Arterial oxygen content (AOC) was calculated by (1.31 ×hemoglobin × SaO2) + (0.003 ×

PaO2), where SaO2 is arterial oxygen saturation and PaO2 is arterial partial pressure of oxygen.

Renal venous oxygen (RVOC) content was calculated as (1.31 × hemoglobin × SrvO2) +

(0.003 × PrvO2), where SrvO2 is venous oxygen saturation and PrvO2 is renal vein partial

pressure of oxygen. Renal oxygen delivery was calculated as DO2 (ml/min) = RBF × AOC.

Renal oxygen consumption is calculated as VO2ren (ml/min/g) = RBF × (AOC − RVOC). The

renal oxygen extraction ratio was calculated as O2ERren (%) = VO2ren/DO2 × 100.

Assessment of kidney function

Creatinine clearance (Clearcrea, [ml/min]) was assessed as an index of the glomerular filtration

rate. Calculation of the clearance was done using the standard formula: Clearcrea = (Ucrea ×

V)/Pcrea, where Ucrea is the concentration of creatinine in urine, V is the urine volume per unit

time and Pcrea is the concentration of creatinine in plasma.

Furthermore, all urine samples were analyzed for sodium (Na+) concentration. The renal

energy efficiency for sodium transport (VO2ren/TNa+ ) was assessed using the ratio of the total

amount of VO2ren over the total amount of sodium reabsorbed (TNa+ , [mmol/min]).

Statistical analysis

Values are reported as the mean ± SEM. The decay curves of phosphorescence intensity were

analyzed using software programed in Labview 6.1 (National Instruments, Austin, TX, USA).

Statistical analysis was performed using GraphPad Prism version 4.0 for Windows (GraphPad

Software, San Diego, CA, USA). Twoway ANOVA with a Bonferroni post hoc test was used

and a p-value of <0.05 was considered statistically significant.

Results

Fluid and electrolyte balance

The amount of fluids given during resuscitation and the plasma chloride and sodium levels

and plasma pH are presented in Table 1. Restoring the MAP from 30 mmHg (shock) to 80

mmHg required 24.8 ± 1.7 ml of NaCl, 21.7 ± 1.4 ml of RA, 5.9 ± 0.5 ml of HES-NaCl (p <

0.05 vs. NaCl and RA), and 6.0 ± 0.4 ml of HES-RA (p < 0.05 vs. NaCl and RA). Plasma

chloride levels were significantly increased (p < 0.05 vs. time control) after NaCl (119.6 ± 6.1

mmol l−1), RA (110.2 ± 1.7 mmol l−1), and HES-NaCl (112.4 ± 3.5 mmol l−1) resuscitation,

  53  

but not after HES-RA (106.0 ± 3.5 mmol l−1) resuscitation. Similarly, plasma pH was

significantly decreased (p < 0.05 vs. time control) after NaCl (7.10 ± 0.03), RA (7.15 ± 0.01),

and HESNaCl (7.20 ± 0.02) resuscitation, but not after HES-RA (7.26 ± 0.02) resuscitation.

Hence, NaCl, RA, and HES-NaCl resuscitation led to hyperchloremic acidosis, while HES-

RA resuscitation did not.

Table 1: Amount of resuscitation fluid required to increase the mean arterial pressure from 30 to 80 mmHg and the plasma sodium (Na+ ) and chloride (Cl−) concentrations and plasma pH at baseline (BL) and after 60 min of resuscitation (R60). Tp<0.05 vs. time control, Np<0.05 vs. 0.9% NaCl, Rp<0.05 vs. Ringer’s Acetate.

BL R60 Amount of fluid required (ml)

Time control HS control

0.9 % NaCl

24.8 ± 1.7 Ringer's Acetate

21.7 ± 1.4

HES-NaCl

5.9 ± 0.5N,R HES-RA

6.0 ± 0.4N,R

Cl- (mmol/l) Time control 104.0 ± 1.0 105.0 ± 2.0

HS control 110.4 ± 1.1 119.5 ± 3.2 0.9 % NaCl 87.2 ± 5.4 119.6 ± 6.1T Ringer's Acetate 105.6 ± 3.7 110.2 ± 1.7T HES-NaCl 97.6 ± 1.2 112.4 ± 3.5T HES-RA 102.4 ± 4.1 106.0 ± 3.5 Na+ (mmol/l)

Time control 142.0 ± 2.0 143.0 ± 2.0 HS control 148.4 ± 2.0 149.0 ± 3.1 0.9 % NaCl 141.8 ± 1.0 143.2 ± 0.8 Ringer's Acetate 142.3 ± 1.2 144.2 ± 0.5 HES-NaCl 143.2 ± 1.0 143.8 ± 0.6 HES-RA 142.5 ± 0.9 143.8 ± 0.5 pH

Time control 7.31 ± 0.10 7.30 ± 0.10 HS control 7.35 ± 0.01 7.11 ± 0.02 0.9 % NaCl 7.27 ± 0.01 7.10 ± 0.03T Ringer's Acetate 7.31 ± 0.01 7.15 ± 0.01T HES-NaCl 7.29 ± 0.03 7.20 ± 0.02T HES-RA 7.31 ± 0.01 7.26 ± 0.02

  54  

Systemic and renal hemodynamics

Systemic and renal hemodynamic variables are presented in Table 2. The baseline values

measured in each group were found to be similar (p > 0.05). In all groups, MAP, RBF

decreased during hemorrhage without significant differences between groups.

During resuscitation, MAP was consistently increased in all groups, though the target MAP of

80 mmHg was not successfully maintained after 60 min of resuscitation. In crystalloid treated

groups, NaCl and RA, MAP was lower at the end of the protocol (44 ± 4 and 48 ± 3 mmHg,

respectively) compared to in the colloid treated groups, HES-NaCl and HES RA (58 ± 5 and

52 ± 3 mmHg, respectively).

Resuscitation improved RBF in all groups starting in the early phase of resuscitation (p <

0.05). Improvement of RBF after 60 min of resuscitation was most pronounced in the HES-

RA group (5.1 ± 0.2 ml/min; 85% of baseline value) and least in 0.9% NaCl group (2.4 ± 0.5

ml/min; 42% of baseline value).

Renal oxygenation

Renal DO2, VO2, CµPO2, and MµPO2 are presented in Table 3. All these parameters

decreased during hemorrhage without significant differences between groups. At the end of

resuscitation, DO2 was improved compared to hemorrhagic shock. This increase was

significant in the RA group (0.41 ± 0.07 ml O2/min) and HES-RA group (0.39 ± 0.06 ml

O2/min) compared to HS control (p < 0.05). VO2, however, could not be increased by fluid

resuscitation (p > 0.05 vs. HS control).

Resuscitation improved CµPO2 and MµPO2 albeit not to baseline level. At R60, CµPO2 was

higher in the HES-RA group compared to other groups and significantly different comparing

to the NaCl group (p < 0.05).

  55  

Table 2: Mean arterial pressure (MAP), renal blood flow (RBF), and renal vascular resistance (RVR) at baseline (BL), during hemorrhagic shock (HS), and after 15 and 60 min of resuscitation (R15 and R60, respectively). Hp < 0.05 vs. HS control, Np < 0.05 vs. 0.9 % NaCl. BL HS R15 R60 MAP (mmHg)                      Time control 102 ± 1.0 104 ± 2.0 99 ± 3.0 105 ± 4.0 HS Control 95 ± 9.0 30 ± 2.0 30 ± 3.0 30 ± 2.0 0.9% NaCl 102 ± 2.0 31 ± 1.0 73 ± 8.0 44 ± 4.0 Ringer's Acetate 101 ± 2.0 33 ± 1.0 57 ± 2.0H 48 ± 3.0 HES-NaCl 102 ± 3.0 30 ± 1.0 67 ± 6.0 58 ± 5.0 HES-RA 101 ± 3.0 32 ± 2.0 62 ± 3.0 52 ± 3.0 RBF (ml.min-1)                      Time control 5.6 ± 0.4 5.6 ± 0.1 5.8 ± 0.6 5.5 ± 1.0 HS Control 5.4 ± 0.2 1.2 ± 0.2 1.1 ± 0.3 0.9 ± 0.2 0.9% NaCl 5.6 ± 0.3 1.4 ± 0.1 2.9 ± 0.6H 2.4 ± 0.5 Ringer's Acetate 5.6 ± 0.4 1.4 ± 0.2 3.8 ± 0.6H 3.6 ± 0.4H HES-NaCl 5.5 ± 0.2 1.7 ± 0.3 3.1 ± 0.6H 3.4 ± 0.4H HES-RA 5.9 ± 0.2 1.9 ± 0.1 4.9 ± 0.6H 5.1 ± 0.2H,N RVR (dyn.s.sec-5)                      Time control 16.4 ± 1.1 15.1 ± 1.5 17.2 ± 1.2 16.9 ± 1.5 HS Control 17.5 ± 1.8 24.5 ± 5.2 27.6 ± 5.4 37.7 ± 5.6 0.9% NaCl 18.6 ± 1.5 22.3 ± 1.8 29.3 ± 4.6 21.4 ± 3.5 Ringer's Acetate 18.5 ± 1.5 20.8 ± 1.2 15.8 ± 2.6 14.2 ± 2.0 HES-NaCl 18.6 ± 1.3 19.9 ± 3.0 21.4 ± 3.5 18.7 ± 2.7 HES-RA 17.1 ± 1.0 17.6 ± 0.9 13.1 ± 1.1 10.2 ± 0.5H,N

Renal function

Creatinine clearance and VO2/TNa+ are presented in Fig. 1. There were no differences at

baseline in creatinine clearance (not shown). During hemorrhagic shock urine production

decreased dramatically. In the HS control group, all animals suffered from anuria at the end of

the protocol. All groups had a lower creatinine clearance at the end of resuscitation (p < 0.05

vs. time control). The NaCl resuscitated group had the lowest creatinine clearance rate at R60.

The VO2/TNa+ was found to be unaffected by fluid resuscitation.

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Table 3: Renal oxygen delivery (DO2), oxygen consumption (VO2) and microvascular oxygen tension in the renal cortex (CµpO2) and medulla (MµpO2) at baseline (BL), during hemorrhagic shock (HS), and after 15 and 60 min of resuscitation (R15 and R60, respectively). Hp < 0.05 vs. HS control, Np < 0.05 vs. 0.9% NaCl.Rp < 0.05 vs. Ringer’s Acetate.

 BL HS R15 R60

DO2 (ml O2/min)                      Time control 1.30 ± 0.10 1.32 ± 0.15 1.27 ± 0.08 1.4 ± 0.01

HS control 1.42 ± 0.11 0.19 ± 0.05 0.16 ± 0.05 0.13 ± 0.05 0.9 % NaCl 1.36 ± 0.09 0.22 ± 0.01 0.33 ± 0.06 0.27 ± 0.06

Ringer's Acetate 1.24 ± 0.03 0.22 ± 0.04 0.48 ± 0.08H 0.41 ± 0.07H HES-NaCl 1.39 ± 0.04 0.29 ± 0.09 0.4 ± 0.09H 0.35 ± 0.05H

HES-RA 1.32 ± 0.08 0.2 ± 0.02 0.53 ± 0.09H 0.39 ± 0.06H VO2 (ml O2/min/g)

                     Time control 0.20 ± 0.02 0.25 ± 0.01 0.24 ± 0.01 0.28 ± 0.02 HS control 0.15 ± 0.08 0.07 ± 0.02 0.07 ± 0.02 0.06 ± 0.02

0.9 % NaCl 0.19 ± 0.06 0.07 ± 0.01 0.08 ± 0.02 0.05 ± 0.01 Ringer's Acetate 0.10 ± 0.03 0.06 ± 0.02 0.10 ± 0.04 0.07 ± 0.03

HES-NaCl 0.21 ± 0.03 0.11 ± 0.04 0.09 ± 0.03 0.07 ± 0.02 HES-RA 0.15 ± 0.02 0.07 ± 0.01 0.11 ± 0.03 0.11 ± 0.02

CµpO2 (mmHg)                        Time control 80.0 ± 2.0 78.0 ± 2.0 78.0 ±   2.0 76.0 ± 2.0

HS control 83.0 ± 2.0 28.0 ± 6.0 22.0 ±   4.0 19.0 ± 5.0 0.9 % NaCl 85.0 ± 4.1 19.7 ± 2.5 42.9 ± 2.9 33.0 ± 2.5

Ringer's Acetate 75.0 ± 6.0 21.0 ± 3.8 43.6 ± 3.1 41.0 ± 2.0 HES-NaCl 81.0 ± 7.0 27.0 ± 4.0 39.8 ± 3.5 45.0 ± 5.0

HES-RA 85.0 ± 4.0 24.1 ± 1.5 53.1 ± 3.5R 49.8 ± 2.4N MµpO2 (mmHg)

                       Time control 67.0 ± 2.0 66.0 ± 1.0 64.0 ± 1.0 64.0 ± 2.0 HS control 62.0 ± 2.0 19.0 ± 6.0 15.0 ± 2.0 14.0 ± 2.0

0.9 % NaCl 67.0 ± 3.0 9.1 ± 1.2 41.4 ± 1.6 30.4 ± 1.4 Ringer's Acetate 61.0 ± 4.5 11.2 ± 3.1 30.7 ± 2.8 29.6 ± 1.0

HES-NaCl 73.0 ± 1.3 15.6 ± 2.7 34.9 ± 2.3 29.9 ± 2.6 HES-RA 67.0 ± 3.3 22.2 ± 2.7 39.2 ± 5.8 30.5 ± 3.5

 

  57  

Fig.1. Creatinine clearance and the ratio of the renal oxygen consumption (VO2) over the total amount of sodium reabsorbed (TNa+) after 60 min of resuscitation. Tp < 0.05 vs. time control, Np < 0.05 vs. 0.9% NaCl.

Discussion

In the present study, we examined the acute effects of acetatebalanced colloid and crystalloid

resuscitation on renal oxygenation in a rat model of hemorrhagic shock. We tested the

hypothesis that acetate-balanced solutions would be superior in correcting impaired renal

perfusion and oxygenation after severe hemorrhage compared to unbalanced solutions. Our

main findings were that: (1) hemorrhagic shock was associated with acute decreases in blood

pressure, renal perfusion and oxygenation, and urine production; (2) volume replacement

therapy with balanced and unbalanced crystalloid and colloid solutions partially corrected

these parameters; and (3) the acetate-balanced colloid solution HES-RA was the only

resuscitation fluid that could restore renal blood flow back to ∼85% of baseline level which

was associated with the most prominently improved renal oxygenation.

Hemorrhagic shock is one of the major causes of acute renal failure due to decreased blood

pressure and consequent hypoperfusion of the kidney. The presence of acute renal failure

significantly increases morbidity and mortality [Lindseth et al., 1975]. The first step in the

correction of hemorrhage-induced hypotension is aggressive volume replacement therapy

[Spahn et al., 2007] which aims to increase the circulating intravascular volume, blood

pressure, and organ perfusion [Hoffmann et al., 2002; Kemming et al., 2005]. However, in

contrast to blood, resuscitation fluids have poor oxygen transporting capacity and rheological

properties. In addition, the fluids used for volume replacement therapy have been suggested to

  58  

increase inflammation and disturb homeostasis and the acid–base balance [Xiao et al., 2004;

Crimi et al., 2006; Santibanez-Gallerani et al., 2001; Yada-Langui et al., 2004]. Over time, a

variety of colloid and crystalloid solutions has been used, including isotonic saline and saline-

based colloid solutions. Although saline-based solutions have been associated with disturbed

acid–base balance due to non-physiological electrolyte composition and pH, these yet remain

the most popular solutions for volume replacement therapy in peri-operative care

[Scheingraber et al., 1999; Waters et al., 2001; Waters et al., 1999; Juca et al., 2005; Wilcox,

1983; Bullivant et al., 1989; Wilcox and Peart 1987]. With respect to the kidney, saline-based

solutions are known to be more frequently associated with hyperchloremic acidosis, due to

their high levels of chloride, resulting in renal vascular constriction and decreased renal

perfusion [Kellum, 2002; Brill et al., 2002; Williams et al., 1999]. This we have confirmed in

the present study.

Balanced solutions, in contrast, provide an alternative with optimized physiological

composition in terms of sodium, potassium, calcium, magnesium, and chloride levels, and

their relative contributions regarding osmolality. Buffers such as acetate, gluconate, pyruvate,

and lactate can be used in resuscitation fluids and are converted to bicarbonate in liver and

raise the pH of the solution to normal blood pH (7.4). These solutions achieve a physiological

acid–base balance with either bicarbonate or metabolizable anions and reduce of the risk of

iatrogenic disruptions. In animal models of sepsis it has also been demonstrated that balanced

solutions lead to less metabolic acidosis, reduced inflammatory cytokine levels, and longer

survival compared to resuscitation with normal saline [Kellum, 2002; Kellum et al., 2006].

Infusion of solutions containing lactate, however, has multiple side effects and, aside from

those, lactate buffers require high levels of liver metabolism and oxygen consumption

[Zander, 2002].

In our model, as shown by others, hyperchloremia led to progressive renal vasoconstriction

(increased RVR and decreased RBF) and a fall in glomerular filtration rate (decreased

creatinine clearance). These phenomena have been shown to be independent of the renal

nerve system and to be related to tubular chloride reabsorption and chloride-induced renal

vasoconstriction [Wilcox, 1983]. Increased RVR and decreased creatinine clearance were

most pronounced following NaCl resuscitation and were less pronounced in the HES-RA

resuscitated group. Furthermore, HES-RA resuscitation was the only regime that could

significantly increase renal DO2. This can be explained by the composition of the different

  59  

fluids: where 0.9% NaCl has a chloride content of 154 mmol l−1, HES-RA has a chloride

content of 112 mmol l−1. It should be pointed out, however, that the improved renal

oxygenation in the HES-RA group compared to the other groups is not directly associated

with acetate-balancing, per se; rather, it is probably due to less chloride infused in HES-RA in

this MAP-targeted resuscitation protocol. Acetate itself does not correct hyperchloremic

acidosis, lactic acidosis, and does not protect renal function. However, as HES-RA

resuscitation prevented hyperchloremic acidosis, it also led to avoiding microvascular

constriction in renal cortex and medulla by which renal oxygenation was improved.

Therefore, in essence, this study provided evidence that the excess chloride in resuscitation is

toxic and disturbs both the acid–base balance and the organ function.

In this line, metabolic acidosis has been shown to be a common complication in critically ill

patients and has been shown to serve as an independent predictor of outcome [Smith et al.,

2001; Gunnerson et al., 2006]. Furthermore, restricting chloride-rich fluids in intensive care

have been shown significantly improve the acid–base status in critically ill patients [Yunos et

al., 2011]. However, although several animal studies, including the present study, suggest that

hyperchloremic metabolic acidosis leads to renal vasoconstriction and potentially to kidney

dysfunction, whether this also occurs in patients remains to be verified.

The results from our study demonstrated once more the need for larger volumes of

crystalloids to achieve similar systemic and microcirculatory goals, compared to colloids.

Blood pressure increased the first 15–20 min of resuscitation and then gradually declined even

though fluid infusion continued. Hence, the volume expansion effect of both crystalloids and

colloids were temporary. Nonetheless, significantly lower volumes of colloids were required

and the colloid solutions were also more effective in maintaining blood pressure after 1 h of

resuscitation. The low efficacy of the crystalloid solutions can be explained by the fact that

only 20% of their volume remains in the vascular lumen and 80% leaks out, leading to tissue

edema and consequent impaired tissue oxygenation.

Excessive fluid overload leads to hemodilution which eventually may impair tissue

oxygenation. In experimental studies it has been demonstrated that acute isovolemic

hemodilution is associated with increases in red blood cell aggregation which triggers

endothelium-dependent thrombogenic and pro-inflammatory responses [Morariu et al., 2006].

Animal studies have demonstrated the direct influence of hemodilution on microvascular flow

  60  

and renal oxygen supply [Johannes et al., 2007]. Johannes et al. have found that the renal

microvascular oxygenation drops at very early stages of isovolemic hemodilution. It was also

shown that the kidney is particularly vulnerable to decreases in oxygen delivery and that the

critical hematocrit associated with a decrease in microvascular oxygenation is much higher

for the kidney than for the heart or intestines [van Bommel et al., 2008]. This was underscored

by a study demonstrating an increased risk of acute kidney injury in cardiopulmonary bypass-

associated hemodilution [Habib et al., 2005]. The reasons for such a high sensitivity to

hemodilution could involve endothelial dysfunction with an inflammatory component leading

to tissue edema and increase of diffusion distance from microcirculation to the tissue cells.

Although earlier studies suggested negative effects of colloids on microcirculation, there is

increasing evidence supporting the opposite [Krieter et al., 1995]. Compared to crystalloid

solutions, colloid solutions increase plasma viscosity. Elevating plasma viscosity in extreme

hemodilution has been shown to increase microvascular flow through nitric oxide-mediated

vasodilation [Tsai et al., 2005]. Others have demonstrated the importance of sufficient blood

viscosity with respect to functional capillary density and tissue oxygenation. Hence, during

acute hemodilution as occur during aggressive fluid resuscitation, increasing plasma viscosity

by administration of colloids may be beneficial for the microcirculation [Tsai and Intaglietta,

2001]. Indeed, the administration of hyperoncotic and hyperviscous solutions has been shown

to be advantageous in hemorrhagic shock due to normalization of colloid osmotic pressure

which leads to the recovery of microcirculatory perfusion and oxygenation [Wettstein et al.,

2006]. Furthermore, Lang et al. described that colloids improved microvascular perfusion and

reduced endothelial tissue edema. In contrast, the authors showed that crystalloids leak

rapidly into the interstitium, causing endothelial tissue swelling and consequently reducing

capillary perfusion and increasing the oxygen diffusion distance [Lang et al., 2001]. The

results from the present study confirm this as microvascular oxygenation in the renal cortex

was lower in the crystalloid resuscitated groups compared to the colloid resuscitated groups.

This was most marked when comparing the unbalanced crystalloid olution (NaCl) to the

acetate-balanced colloid solution (HES-RA) and was also translated into a significantly higher

creatinine clearance rate in the HES-RA group compared to the NaCl group.

Our study has, however, some limitations which should be acknowledged. First, translation of

the findings in our animal model to clinical scenarios should be done with utmost care. Here,

we imitated major hemorrhage by withdrawing blood until mean arterial pressure was

  61  

decreased to 30 mmHg. Most trauma patients, however, suffer from multiple injuries which

may influence their inflammatory state, potentially interfering with the hemorrhageinduced

hypovolemia and subsequent treatment. Moreover, this model does not reflect the challenges

in treatment of a neurological trauma patient. Nonetheless, our model does demonstrate the

efficacy of volume replacement therapy using different types of fluids on renal perfusion and

oxygenation after severe hemorrhage. Second, the rather short follow-up period after of

hemorrhagic shock and resuscitation does not allow assessment of renal (dys)function and

injury in the long-term. Third, blood lactate and base excess levels were not monitored in the

experiments so the effects of the tested solutions on these parameters remain to be elucidated.

Conclusions

In conclusion, while resuscitation with the NaCl and RA (crystalloid solutions) and the HES-

NaCl (unbalanced colloid solution) led to hyperchloremic acidosis, resuscitation with the

HES-RA (acetatebalanced colloid solution) did not. The acetate-balanced colloid solution

HES-RA was furthermore the only fluid restoring renal blood flow back to ∼85% of baseline

level and most prominently improved renal microvascular oxygenation. However, the

longterm effects of HES-RA resuscitation on renal function warrants further study.

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  67  

Chapter 3

ACUTE EFFECTS OF BALANCED VERSUS UNBALANCED COLLOID

RESUSCITATION ON RENAL MACROCIRCULATORY AND

MICROCIRCULATORY PERFUSION DURING ENDOTOXEMIC SHOCK

Aksu U1,2, Bezemer R1, Demirci C2, Ince C1.

1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands

2Department of Biology, Faculty of Science, University of Istanbul, Istanbul, Turkey

Published in: Shock. 2012 Feb;37(2):205-9.

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Chapter 3

Acute effects of balanced versus unbalanced colloid resuscitation on renal

macrocirculatory and microcirculatory perfusion during endotoxemic shock

Running title: Balanced vs unbalanced colloid resuscitation in sepsis

This study was designed to investigate the acute effects of balanced versus unbalanced colloid

resuscitation on renal macrocirculatory and microcirculatory perfusions during

lipopolysaccharide-induced endotoxemic shock in rats. We tested the hypothesis that balanced

colloid resuscitation would be better for the kidney than unbalanced colloid resuscitation.

Shock was induced by lipopolysaccharide (10 mg/kg i.v. over 30 min). When mean arterial

pressure (MAP) was decreased to 40 mmHg, fluid resuscitation was started with either

hydroxyethyl starch (HES130/0.42) dissolved in saline (HES-NaCl) as an unbalanced colloid

solution or HES130/0.42 dissolved in Ringer’s acetate (HES-RA) as a balanced colloid

solution. Microvascular perfusion in the renal cortex was monitored using laser speckle

imaging, and in addition, systemic hemodynamics, renal artery blood flow (RBF), and plasma

ion levels were measured. Shock decreased MAP, led to anuria, and worsened all other

parameters. Hydroxyethyl starch-NaCl improved MAP (p > 0.05) but did not improve RBF (p

> 0.05), metabolic acidosis (p > 0.05), and plasma ion levels (p > 0.05). Hydroxyethyl starch-

RA improved MAP (p < 0.05), RBF (p < 0.05), and renal microvascular perfusion (p < 0.05),

but did not improve metabolic acidosis (p > 0.05) and plasma ion levels (p > 0.05). Both

HES-NaCl and HES-RA treatment could normalize creatinine clearance but not fractional

sodium excretion. In endotoxemic rats, balanced colloid (HES) resuscitation was shown to be

superior to un- balanced colloid resuscitation in terms of improvement of renal macrovascular

and microvascular perfusions. However, whether this results in improved renal function in the

long term warrants further study.

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Introduction

In the early stage of sepsis, impairment of the renal microcirculation is a key complication

potentially leading to renal failure through hypoxia-induced tubular epithelial cell injury and

acute tubular necrosis [Weinberg et al., 1991; De Backer et al., 2002; Sakr et al., 2004;

Klenzak and Himmelfarb, 2005]. Fluid resuscitation during sepsis is considered crucial for the

preservation of adequate intravascular volume and blood pressure and thereby promotion of

microvascular perfusion and renal oxygenation [Rivers et al., 2001]. It has been shown,

furthermore, that hypoxic microvascular areas might arise in the renal cortex in untreated

endotoxemia [Johannes et al., 2009].

As renal dysfunction is a key complication in intensive care units, there is a continuing

concern about the efficacy of fluid resuscitation. However, the type of fluid that should be

used for resuscitation in sepsis to yield the best renal outcome remains controversial today

[Marx, 2003]. This controversy includes not only the use of crystalloid versus colloid

solutions but also the use of balanced versus unbalanced colloid solutions. It is known that

crystalloid solutions have poor plasma expander capacities, and just 20% of the given volume

remains contained in the intravascular space [Svensen and Hahn, 1997]. Colloid solutions,

because of their high colloid osmotic pressure, are known to have superior plasma expanding

capacities compared with traditional crystalloid solutions. As most colloid preparations are

saline based, liberal fluid resuscitation regimens might lead to nonphysiologically high

sodium and chloride concentrations and may be associated with the development of

(hyperchloremic) metabolic acidosis, which could affect inflammatory and coagulation

homeostasis and thereby deteriorated organ function [Morgan, 2005; Schindler, 2004]. This

insight has led to development of modern hydroxyethyl starch (HES) preparations based on

balanced, plasma-adapted, crystalloid solutions and to the idea of developing a totally

balanced fluid resuscitation concept, including balanced crystalloids and balanced colloids

[Schindler, 2004]. However, whether these new, balanced fluids are able to improve renal

microcirculatory perfusion and renal function under septic conditions remains to be

elucidated.

The aim of this study was therefore to investigate the acute effects of balanced versus

unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in

a rat model of lipopolysaccharide (LPS)-induced endotoxemia. We tested the hypothesis that

balanced colloid resuscitation would be better for the kidney than unbalanced colloid

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resuscitation. The acute effects of two clinically applied resuscitation regimens were

investigated: 6% 130/0.4 HES in NaCl (HES-NaCl) as an unbalanced colloid solution and 6%

130/0.4 HES in Ringer’s acetate solution (HES-RA) as a balanced colloid solution. Renal

perfusion was assessed at the macrocirculatory level using Doppler ultrasound on the renal

artery and at the microcirculatory level using laser speckle imaging (LSI) on the renal cortex.

Materials and methods

Animals

The protocol of the present study was approved by the Animal Research Committee of the

Academic Medical Center at the University of Amsterdam. Animal care and handling were

performed in accordance with the national guidelines for the care of laboratory animals. The

experiments were performed in 20 male Wistar rats with a mean +/- SEM body weight of 385

+/- 1 g.

Preparation

The rats were anesthetized with an intraperitoneal injection of a mixture of 90 mg/kg

ketamine, 0.5 mg/kg medetomidine, and 0.05 mg/kg atropine. Anesthesia was maintained

with ketamine 50 mg/kg per hour administered intravenously. A tracheotomy was performed,

and a polyvinylchloride tube was inserted into the trachea to enable mechanical ventilation

with oxygen of FiO2 = 0.4. A capnometer (Capstar-100; CWE, Inc, Ardmore, Pa) was used to

measure end-tidal carbon dioxide partial pressure (EtCO2), which was used to adjust

ventilator settings to maintain an arterial PCO2 between 35 and 40 mmHg. Body temperature

was measured with a thermocouple placed in the rectum and was maintained at 37.0 ± 0.5 oC

with a heating pad below and a warming lamp above the animal.

A catheter was placed into the right carotid artery and connected to a pressure transducer for

continuous monitoring of arterial blood pressure and heart rate. A polyethylene catheter (outer

diameter, 0.9 mm) was inserted into the right jugular vein for intravenous administration of

fluid. To compensate for fluid loss, saline was infused continuously at a rate of 15 mL/kg per

hour. Catheters were advanced into both the right femoral artery and vein for withdrawal of

blood for blood gas measurements and administration of drugs, respectively.

After a left-sided laparotomy of lower abdomen, the left kidney was exposed from adipose

tissues, and a catheter was placed in the ureter to collect urine during the experiment. A 0.5-

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mm perivascular flow probe (Transonic Systems Inc, Ithaca, NY) was placed around the renal

artery and connected to a flow meter (T206; Transonic Systems Inc).

Study design

After the surgical preparation, the animals were randomly assigned to one of four groups. In

three groups (n = 5/group), endotoxemic shock was induced by infusion of 10 mg/kg LPS

over 30 min (Escherichia coli 055:B5; Sigma, Paris, France). When mean arterial pressure

(MAP) was decreased to 40 mmHg (after approximately 4 h), fluid resuscitation was started

in two groups of rats (unbalanced colloid and balanced colloid resuscitation), and in one

group, no resuscitation fluids were given as an LPS control group. An additional group (n = 5)

of animals was included as a sham-operated control group receiving no LPS and no additional

fluids.

Unbalanced colloid resuscitation (LPS + HES-NaCl) was done with 15 mL/h 6% HES

130/0.4 prepared in saline solution (Na+ 154 mmol/L, Cl- 154 mmol/L; Voluven, Fresenius

Kabi, Bad Homburg, Germany). Balanced colloid resuscitation (LPS + HES-RA) was done

with 15 mL/h balanced 6% HES 130/0.4 dissolved in RA preparation (Na+ 130 mmol/L, Cl-

112 mmol/L, K+ 5.36 mmol/L, Ca+2 0.912 mmol/L, Mg+2 0.984 mmol/L, acetate- 27.2

mmol/L; Plasma Volume; Baxter, Melsungen, Germany). The infusion rates were determined

in previous studies by our group [Johannes et al., 2006; Legrand et al., 2010]. Time points

were (a) baseline; (b) during endotoxemic shock at a MAP of 40 mmHg; and (c) after 30 min

of resuscitation.

Systemic hemodynamics

Arterial pressure was measured in the carotid artery. Mean arterial pressure (in mmHg) was

calculated as MAP = 2/3 x diastolic pressure + 1/3 x systolic pressure. Heart rate (in beats/min;

HR) was determined by derivation of arterial pressure signal. Blood samples (0.2 mL) were

taken from the femoral artery at each time point and replaced with the same volume of saline.

The samples were used to determine blood gas values, plasma ion levels, and pH (ABL505;

Radiometer, Copenhagen, Denmark) as well as hematocrit, hemoglobin concentration, and

hemoglobin oxygen saturation (OSM 3; Radiometer).

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Renal macrovascular and microvascular perfusions

Blood flow in the renal artery (RBF [in mL/min]) was measured continuously and normalized

to body weight. Renal vascular resistance (RVR [dyn/s per cm-5]) was estimated as

(MAP / RBF) x 100.

Laser speckle imaging was used to visualize the spatiotemporal characteristic of renal cortical

perfusion changes during endotoxemia and fluid resuscitation as described previously

[Bezemer et al., 2010; Legrand et al., 2011]. Typical laser speckle images of the renal

microcirculation before and during endotoxemic shock are provided elsewhere [Legrand et

al., 2011]. For LSI measurements, a commercially available system was used (Moor

Instruments, Devon, UK) in which a 785-nm class 1 laser diode was used for illumination of

the tissue to a depth of approximately 1 mm. Laser speckle images were acquired using a 576

x 768-pixel grayscale CCD camera at a frame rate of 25 Hz and converted to pseudo-color

speckle contrast images where the perfusion was scaled from blue (low perfusion) to red (high

perfusion). For LSI of the rat kidney, the field of view was set to ~1.8 x 2.4 cm

(corresponding to ~30 µm/pixel). Using a 5 x 5-pixel window to calculate speckle contrast,

the maximal image resolution was ~150 µm/pixel area. The laser speckle images were

analyzed for mean renal microvascular perfusion (arbitrary unit [AU]) and perfusion

heterogeneity (AU), calculated as the range in perfusion values divided by the mean perfusion

value.

Renal function

Creatinine clearance (clearcrea [mL/min]) was assessed as an index of the glomerular filtration

rate. The concentrations of creatinine in urine and plasma were determined by colorimetric

methods. Calculation of the clearance was done using the standard formula: clearcrea = (Ucrea x

V)\ Pcrea, where Ucrea is the concentration of creatinine in urine, V is the urine volume per unit

time, and Pcrea is the concentration of creatinine in plasma. In addition, excretion fraction of

Na+ (EFNa+ [%]) was calculated to use as a marker of tubular function as: EFNa+ = (UNa+ x

Pcrea) / (PNa+ x Ucrea) x 100, where UNa+ is Na+ concentration in urine, and PNa+ is the Na+

concentration in plasma.

Statistical analysis

Data were analyzed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, Calif) and

presented as mean ± SEM. Results obtained in different groups were compared using two-

  73  

way analysis of variance and, when appropriate, post hoc analyses with Bonferroni tests.

Differences were considered statistically significant at p < 0.05.

Results

Although LPS infusion induced significant effects on systemic and renal hemodynamics and

renal function, all animals survived for the duration of the experiment.

Systemic hemodynamics

Systemic hemodynamic parameters and blood acidic state parameters are presented in Tables

1 and 2, respectively, and plasma ion levels are presented in Table 3. Lipopolysaccharide

infusion decreased MAP in all animals but did not affect the heart rate. Hydroxyethyl

starch-NaCl and HES-RA resuscitation improved the MAP (p < 0.05 vs. LPS) but could not

restore MAP back to baseline (p < 0.05 vs. control).

Table 1. MAP and HR. *p < 0.05 vs. control group. †p < 0.001 vs. LPS group.

Baseline Shock Resuscitation MAP (mmHg)                  Time-Control 99 + 2 99 + 2 93 + 1 LPS 96 + 4 45 + 4 39 + 2* LPS+HES-NaCl 93 + 1 48 + 2 56 + 4*

† LPS+HES-RA 96 + 2 49 + 2 59 + 5*

† HR (BPM)                  Time-Control 263 + 3 268 + 2 277 + 7 LPS 264 + 7 257 + 26 253 + 26 LPS+HES-NaCl 260 + 11 239 + 11 285 + 27 LPS+HES-RA 278 + 11 275 + 19 256 + 19

Lipopolysaccharide infusion induced metabolic acidosis and increased plasma potassium

levels (p < 0.05 vs. control), which could not be restored by HES-NaCl or HES-RA

resuscitation (p > 0.05 vs. LPS and control). HES-NaCl resuscitation, moreover, slightly

increased the plasma chloride level (p > 0.05 vs. LPS and control), which was slightly

reduced by HES-RA resuscitation (p > 0.05 vs. LPS and control). Plasma chloride levels were

significantly lower after HES-RA resuscitation compared with after HES-NaCl resuscitation.

  74  

Table 2. Arterial pH, base excess, and HCO3- levels. *p < 0.05 vs. control group.

Baseline Shock Resuscitation pH

               Time-control 7.37 + 0.02 7.49 + 0.05 7.50 + 0.03 LPS 7.41 + 0.01 7.19 + 0.07 7.11 + 0.06* LPS + HES-NaCl 7.40 + 0.02 7.16 + 0.05 7.06 + 0.03* LPS + HES-RA 7.39 + 0.02 7.15 + 0.04 7.07 + 0.05* Base excess (mmol/L)

               Time-control -1.4 + 0.5 1.6 + 1.1 2.5 + 0.6 LPS -0.9 + 0.9 -12.3 + 0.5 -11.4 + 1.0* LPS + HES-NaCl 0.5 + 0.7 -13.4 + 1.9 -13.4 + 1.0* LPS + HES-RA -2.6 + 0.2 -12.4 + 2.1 -13.3 + 1.6* HCO3

-(mmol/L)                Time-control 21.8 + 1.1 22.6 + 1.0 23.1 + 0.4

LPS 21.7 + 0.8 16.3 + 2.2 13.2 + 2.1* LPS + HES-NaCl 21.7 + 1.2 15.9 + 0.8 14.9 + 0.9* LPS + HES-RA 19.5 + 1.4 15.6 + 1.2 19.6 + 1.0*

Renal hemodynamics

Renal macrocirculatory and microcirculatory perfusions are shown in Figure 1. Endotoxemia

was associated with a decreased RBF (p < 0.05 vs. control) and increased RVR (p < 0.05 vs.

control). Whereas HES-NaCl resuscitation was ineffective in restoring these parameters

(p > 0.05 vs. LPS), HES-RA resuscitation could significantly decrease RVR and increase

RBF (p < 0.05 vs. LPS). The reduction in RBF consequent to LPS infusion was accompanied

by a decrease in cortical microvascular perfusion, which could be partially restored by HES-

NaCl (p > 0.05 vs. LPS) and HES-RA (p < 0.05 vs. LPS). In addition, the increased cortical

perfusion heterogeneity that arose during endotoxemia could be countered by HES-NaCl and

HES-RA resuscitation (p < 0.05 vs. LPS).

Table 3. Plasma ion levels after resuscitation. *p < 0.05 vs. control group, †p < 0.05 vs. HES-NaCl group.

    Na+ (mmol/L) K+ (mmol/L) Cl- (mmol/L) Time-control 141.3 + 1.1 4.0 + 0.3 109.3 + 1.3 LPS 145.5 + 1.3 7.2 + 0.4* 110.3 + 0.8 LPS + HES-NaCl 147.6 + 1.0* 6.1 + 0.4* 113.5 + 1.3 LPS + HES-RA 141.3 + 1.4 6.9 + 0.3* 107.7 + 1.4†

  75  

Table 4. Clearcrea and fractional Na+ excretion after resuscitation *p < 0.05 vs. control group.

    Clearcrea(ml/min) Fractional Na+ excretion

Time-control 0.34 + 0.09 0.6 + 0.1 LPS Anuric Anuric

LPS + HES-NaCl 0.33 + 0.02 3.9 + 0.2* LPS + HES-RA 0.31 + 0.09 3.5 + 0.5*

Renal function

Parameters of renal function are reported in Table 4. As LPS infusion resulted in anuria,

fractional sodium excretion and clearcrea rate could not be determined for the endotoxemic

time point. Both resuscitation regimens resulted in an increased fractional sodium excretion

with respect to the time control group (p < 0.05), and none of the regimens affected clearcrea

rate.

Fig.1. Renal blood flow (A), RVR (B), cortical microvascular perfusion (C), and cortical perfusion

heterogeneity (D) at baseline (BL) and during shock and resuscitation (RS). *p < 0.05 vs. control

group, †p < 0.001 vs. LPS group, #p < 0.01 vs. HES-NaCl group.

  76  

Discussion

The aim of this study was to investigate the acute effects of balanced versus unbalanced

colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in a rat model

of LPS-induced endotoxemia. To test the hypothesis that balanced colloid resuscitation would

be better for the kidney than unbalanced colloid resuscitation, we resuscitated with HES-NaCl

as an unbalanced colloid solution and HES-RA as a balanced colloid solution. The main

findings were that (a) LPS-induced endotoxemia was associated with deteriorated systemic

and renal hemodynamics, acid-base balance, mean cortical microvascular perfusion, and

perfusion heterogeneity and caused anuria; (b) both HES-NaCl and HES-RA resuscitation

improved systemic blood pressure, but only HES-RA resuscitation improved renal

macrovascular and microvascular perfusions; (c) neither HES-NaCl nor HES-RA

resuscitation could restore the metabolic acidosis or fractional sodium excretion; and (d)

plasma chloride levels were significantly lower after HES-RA resuscitation compared with

after HES-NaCl resuscitation.

As renal dysfunction is a common complication in intensive care patients, there is a

continuing research on the efficacy of fluid resuscitation strategies to protect the kidney

[Kellum, 2002; Thijs and Thijs, 1998]. As such, many recent studies underscore the

importance of early fluid resuscitation in severe sepsis and sepsis-induced tissue

hypoperfusion [Kortgen et al., 2006; Sebat et al., 2005; Dellinger et al., 2008]. However, the

type of fluid that should be used for resuscitation in sepsis to yield the best renal outcome

remains controversial today [Natanson et al., 1986; Mythen et al., 1993; Lacy and Wright,

1992; Khajavi et al., 2008]. This controversy includes both the use of crystalloid versus

colloid solutions and balanced versus unbalanced colloid solutions [Brunkhorst and Oppert,

2008; Mills, 2007; Wiedermann, 2004]. In pigs with severe sepsis, colloid solutions were

shown to have superior resuscitation capacity compared with saline solutions [Marx et al.,

2002]. Moreover, HES solutions have been shown to have antiinflammatory properties, and a

new HES solution (mean molecular weight, 130 kd; degree of substitution, 0.4; HES 130/0.4)

has been shown to improve microvascular perfusion and reduce macromolecular leakage

[Nohe et al., 2005; Hoffman et al., 2002]. However, contrastingly, various investigations have

concluded that HES solutions have potential adverse effects on renal function [Legendre et

al., 1993].

  77  

The present study was designed to assess the effects of a balanced volume replacement

regimen including a new balanced HES preparation (HES-RA) on renal macrocirculatory and

microcirculatory perfusions in comparison to an unbalanced fluid regimen. To this end, we

measured renal macrocirculatory blood flow using a transit-time ultrasound flow probe

around the renal artery, and we monitored cortical microvascular perfusion and perfusion

heterogeneity using LSI. Laser speckle imaging is a noninvasive technique for macroscopic

mapping of tissue perfusion, but sensitive to microcirculatory flow alterations, and allows

quantitative assessment of mean microcirculatory perfusion and microcirculatory perfusion

heterogeneity. In the present study, all rats suffered from LPS-induced endotoxemic shock as

indicated by reduced MAP and increased RVR and decreased RBF. The increased RVR was

caused by severe intrarenal vasoconstriction during septic acute renal failure as has been

shown previously [Kellum et al., 2004]. Using LSI, we showed acute beneficial effects of the

two HES solutions on cortical microvascular perfusion, which was most pronounced in the

HES-RA group.

The observed differences between HES-NaCl and HES-RA resuscitation with respect to their

effects on RVR, RBF, and cortical microvascular perfusion could be attributed to the

differential effects on the plasma chloride levels. Where HES-NaCl resuscitation slightly

increased the plasma chloride level, this was slightly decreased by HES-RA resuscitation,

which led to a significantly lower plasma chloride level in the HES-RA group. The role of

chloride in modulating vasoconstrictor responses to vasoactive agents has previously been

investigated in isolated rat kidneys [Quilley et al., 1993]. Hyperchloremic fluids induced

intrarenal vasoconstriction as indicated by increased RVR and decreased glomerular filtration

rate [Wilcox, 1983]. Furthermore, the observed acidemia affects a variety of vasoregulatory

mechanisms such as by increasing endogenous catecholamine release, which induces the

release of both proinflammatory and antiinflammatory cytokines [Le Tulzo et al., 1997] and

nitric oxide [Celotto et al., 2008], and by direct effects on the renal microvasculature [Quilley

et al., 1993]. Jensen and coworkers [Jensen et al., 1990] have shown that as a result of acid

loading, macrophages increase their tumor necrosis factor secretion, which affects

microvascular perfusion by the direct vasoactive properties and by direct tissue injury.

However, in the present study, we did not measure plasma tumor necrosis factor levels, and

the present study therefore does not permit testing of contribution of these molecules to the

observation of decreased renal microcirculatory perfusion.

  78  

The clearcrea rate, as an index of kidney function, could be restored to baseline levels

regardless of the type using HES-NaCl and HES-RA resuscitation, but fractional sodium

excretion, in contrast, remained elevated after HES resuscitation, which could indicate tubular

dysfunction. However, changes in creatinin clearance rate and fractional sodium excretion

should be regarded only as gross indicators of renal function in sepsis, although both are

accepted indicators of renal function as described by the AKIN and RIFLE criteria. As more

sensitive markers would be desirable, novel biomarkers are currently under investigation (e.g.,

neutrophil gelatinase-associated lipocalin and fatty acid binding protein), but the significance

of their detection in the context of endotoxemia is a matter of concern.

We are aware that our study suffers from several limitations inherent to the use of an animal

model of endotoxemia. First, LPS-induced endotoxemia may not reflect all the situations

encountered in human sepsis and may lack relevance in grampositive sepsis. Second, the

present study allows only assessment of the LPS-related effects in this short-term rat model of

acute endotoxemia. Third, LSI allows only assessment of cortical microvascular perfusion and

does not take changes in the medullar microcirculation into account. Furthermore, imaging

renal microcirculatory perfusion using LSI has been performed only in rats [Legrand et al.,

2011; Holstein-Rathlou et al., 2011], and clinical application of this technique would be

limited to surgical scenarios with exposed kidney (e.g., during renal transplantation). Fourth,

HES-based resuscitation strategies are controversial. However, only the old generation of

high-molecular-weight HES molecules has been reported to be associated with acute renal

failure in a dosedependent fashion. There is no evidence for such an association with the low-

molecular-weight (130/0.4) HES we used in this study, which actually has been shown to

have protective effects on the microcirculation. In line with this, we showed that fluid

resuscitation with HES-based solutions led to an improvement of renal macrocirculatory and

microcirculatory perfusion. Fifth, as vehicle control experiments (NaCl only, RA only) have

not been performed, it is difficult to determine what proportion of the improvement in renal

microvascular perfusion is due to the inherent properties of RA. However, the specific aim of

the present study was to investigate the potential beneficial effect of resuscitation with a

balanced colloid solution (HES-RA) compared with resuscitation with an unbalanced colloid

solution (HES-NaCl) on renal microvascular perfusion in endotoxemic rats. We have clearly

demonstrated this.

  79  

Conclusions

In endotoxemic rats, balanced colloid (HES) resuscitation was shown to be superior to

unbalanced colloid resuscitation in terms of improvement of renal macrovascular and

microvascular perfusions. However, whether this results in improved renal function in the

long-term warrants further study.

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Chapter 4

EFFECT OF TEMPOL ON REDOX HOMEOSTASIS AND STRESS TOLERANCE IN

MIMETICALLY AGED DROSOPHILA

Aksu U1, Yanar K2, Terzioglu D2, Erkol T3, Ece E3, Aydin S2, Uslu E2, Cakatay U2

1Department of Biology, Science Faculty, Zoology Division, Istanbul University, 2Department of Medical Biochemistry, Cerrahpasa Medical Faculty, Istanbul University, 3Department of Biology, Science Faculty, General Biology Division, Istanbul University,

Istanbul, Turkey

Published in: Arch Insect Biochem Physiol. 2014 Sep;87(1):13-25

  84  

Chapter 4

Effect Of tempol on redox homeostasis and stress tolerance in mimetically aged

Drosophila

Running title: Archives of Insect Biochemistry and Physiology.

We aimed to test our hypothesis that scavenging reactive oxygen species (ROS) with tempol,

a membrane permeable antioxidant, affects the type and magnitude of oxidative damage and

stress tolerance through mimetic aging process in Drosophila. Drosophila colonies were

randomly divided into three groups: (1) no D-galactose, no tempol; (2) D-galactose without

tempol; (3) D-galactose, but with tempol. Mimetic aging was induced by D-galactose

administration. The tempol-administered flies received tempol at the concentration of 0.2% in

addition to D-galactose. Thiobarbituric acid reacting substance (TBARS) concentrations,

advanced oxidation protein products (AOPPs), Cu,Zn-superoxide dismutase (Cu,Zn-SOD),

sialic acid (SA) were determined. Additionally, stress tolerances were tested. Mimetically

aged group without tempol led to a significant decrease in tolerance to heat, cold, and

starvation (p < 0.05), but tempol restored these parameters to control levels. The Cu,Zn-SOD

activity and SA concentrations were lower in both mimetically aged and tempol-administered

Drosophila groups compared to control (p < 0.05), whereas there were no significantly

difference between mimetically aged and tempol-administered groups. Mimetically aged

group without tempol led to a significant increase in tissue TBARS and AOPPs

concentrations (p < 0.05). Coadministration of tempol could prevent these alterations.

Scavenging ROS using tempol also restored redox homeostasis in mimetically aged group.

Tempol partly restored age-related oxidative injury and increased stress tolerance.

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Introduction

Free radical theory of aging is one of the widely accepted theories set forth in relation to

cellular effects of both natural and mimetic aging [Yanar et al., 2011; Aydin et al., 2012].

According to this theory, reactive oxygen species (ROS) is the cause of oxidative injury that a

living organism undergoes throughout its lifetime. Increased oxidative stress may cause

functional decline and various age-related disorders in humans and experimental animals

[Cakatay, 2011].

Aerobic organisms continuously produce ROS through their lifespan. Free radicals are

molecules with unpaired electron in the outermost molecular orbitals and these molecules

cause oxidative damage to cellular macromolecules such as DNA, proteins, and lipids

[Cakatay, 2011]. To protect itself against the harmful toxic effects of ROS and modulate the

physiological effects of ROS, the cell has developed endogenous antioxidant systems. Under

normal circumstances, ROS are metabolically formed but are removed efficiently by

antioxidant systems virtually instantly, so that no macromolecular damage occurs in the cell.

However, this homeostatic process becomes less efficient in aging favoring ROS formation

[Cakatay, 2011]. Impaired redox homeostasis originates both by the inefficiency of

antioxidant systems and by increased ROS formation due to the aging process. The ability of

amphipathic antioxidants to penetrate into cellular lipid bilayers is crucial to the protection

against macromolecular oxidation [Cakatay, 2006; Zhou et al., 2010].

Several routes of superoxide dismutase administration have been described, however Cu,Zn-

superoxide dismutase (Cu,Zn-SOD) cannot easily penetrate biological membranes to

attenuate the effects of intracellular production of superoxide radical anion [Fridovich, 1995].

Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl), a low molecular weight

piperidine nitroxide, can effectively penetrate biological membranes and scavenge superoxide

radicals. Mitochondrial ROS are known to be the main sources of all oxygen-related free

radicals, and antioxidant derivatives of tempol are accumulated in the mitochondria. The

possible proposed biochemical mechanism whereby tempol controls mitochondrial oxidative

stress is attributed to hydroxylamine reduction of tempol as well as nitroxide formation

[Wang et al., 2003; Wilcox, 2010]. Moreover, tempol has been reported to improve chronic

high salt intake induced kidney injury [Carlstrom et al., 2013], and to be effective in

preventing several of the adverse consequences of oxidative stress [Wilcox, 2010], and type 1

diabetes induced organ injury [Zheng et al., 2013] in animal models. Here, we demonstrated

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the beneficial effects on alleviation of oxidative protein damage by tempol and stress

tolerance in a mimetic aging model of Drosophila.

Biomarkers of oxidative protein damage are often measured to assess the status for study of

oxidative stress. Several oxidative protein modifications such as advanced oxidation protein

products (AOPPs) formation may result from ROS oxidative stress and lead to the formation

of the high molecular weight insoluble aggregates that are common in aging and age-related

disorders [Cakatay, 2011]. AOPPs contain a variety of protein oxidation products such as

protein carbonyl groups, dityrosine, and advanced glycation end products [AGEs; Selmeci,

2011]. Besides protein oxidation marker, other oxidative damage markers of lipid

peroxidation include malondialdehyde, lipid hydroperoxides, isoprostanes, and thiobarbituric

acid reacting substances [TBARS; Buege and Aust, 1978; Hanasand et al., 2012]. TBARS are

a group of reactive aldehydes resulting from ROS-induced degradation of polyunsaturated

membrane lipids [Buege and Aust, 1978; Hanasand et al., 2012].

Increase in oxidative stress may be one of the reasons for the decrease in the stress tolerance,

which develops through natural and mimetic aging [Yanar et al., 2011; Aydin et al., 2012].

Research on D-galactose has shown that the optimum doses for establishing a mimetic aging

model of D-galactose can affect the redox homeostasis by increasing the formation of

hydrogen peroxide, galactitol, and AGEs [Yanar et al., 2011; Aydin et al., 2012]. Although

majority of the mimetic aging studies related to D-galactose administration were performed

by using rodents, D-galactose-induced aging model has also been applied to Drosophila [Cui

et al., 2004] where Cui and co-workers showed that D-galactose administration shortens the

lifespan of Drosophila. Although use of synthetic antioxidants has recently become

widespread, their effects on protecting and restoring cellular redox homeostasis is not entirely

known [Augustyniak et al., 2010].

Organisms such as Drosophila are mostly composed of postmitotic cells where studies from

this invertebrate support the free radical theory of aging much more so than results from

vertebrates. Additionally, postmitotic cells in vertebrate such as neurons and muscles are

more sensitive than other type of cells with regard to oxidative stress mediators [Cakatay,

2011].

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The aim of this study was to test the hypothesis whether tempol restores impaired redox

homeostasis and increases stress tolerance in a mimetic aging model of Drosophila. For this

reason, we investigated the extent of general oxidative stress and, specifically, oxidative

protein damage in mimetically aged flies following tempol administration. To this end

TBARS, AOPPs, Cu,Zn-SOD, and sialic acid (SA) were determined.

Materials and Methods

Chemicals and Apparatuses

Chemicals and solvents used in the experiments were of the highest purity and analytical

grade. All chemicals and reagents were purchased from Merck (Darmstadt, Germany) or

Sigma-Aldrich (St Louis, MO). Deionized water was used in the analytical procedures.

Reagents were stored at +4°C. The reagents were maintained in equilibrium at room

temperature for 0.5 h before use. All centrifugation procedures were performed with a Sigma

3–18 KS centrifuge (SIGMA Laborzentrifugen GmbH, Osterode am Harz, Germany).

Oxidative stress parameters were run in duplicate by using the Biotek SynergyTM H1 Hybrid

Multi-Mode Microplate Reader (BioTek US, Winooski, VT).

Animals

In this study Drosophila melanogaster (Diptera: Drosophilidae; fruit fly) of the Oregon-R

strain, was used. All the individuals making up the experimental groups were exposed to 60%

relative humidity and ambient temperature of 25°C during the experimental period. Animals

were kept in 25 × 100 mm glass bottles containing 2 ml standard nutrient.

Experimental Groups

_Control group: In standard feeding environment. Standard feedlot: contained 8.5 g corn

flour, 0.75 g agar, 0.75 g dry yeast, 6.5 g sucrose, 0.5 ml 100% propionic acid, and 90 ml

distilled water.

_ Mimetically aged Drosophilas: By adding same amount of D-galactose instead of sucrose to

the standard feeding environment.

_ Mimetically aged Drosophilas + Tempol administration: By adding tempol in the

concentration of 0.2% D-galactose [Izmalylov and Obukhova, 1996]. Two male and two

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female individuals were allowed to live for 4 weeks in organized environment described

above, and the experimental process was terminated. In this period, the flies were middle

aged. At the end of the fourth week, male flies were isolated at which time both stress

tolerance tests and biochemical analyzes were carried out. Female flies were discarded due to

possible antioxidant effect of estrogens [Altun et al., 2011].

Stress Tolerance Tests (Cold, Heat, and Starvation)

The stress response tests need to be run precisely and carefully so that no other physical

factors can contribute to the reason the flies are dying. Flies exposed to the different diets

were assayed simultaneously and percentage of survival ratio was calculated as Ns/Nt × 100,

where Ns is the number of survived flies and Nt is the number of total flies.

Cold Stress. Flies in put in an empty bottle, in groups of 10–15 individuals, and exposed to a

temperature of 0 °C for 2 h. At the end of the period, flies were taken into standard diet

medium. After 24 h, the flies that survived were counted and the percentage of ratio

calculated by taking the average of three repeated processes.

Heat Stress. Flies in groups of 10–15 individuals were exposed to 38.3°C and 60% relative

humidity for 2 h, and the survivors counted after 24 h and the percentage of ratio calculated

by taking the average of three repeated processes into account.

Starvation Stress. The flies in groups of 10–15 individuals were kept in bottles with no food.

Dehydration was prevented by putting water absorbed Whatman filter papers in the bottles.

After 24 h, the dead and surviving flies were counted and the percentage of ratio calculated by

taking the average of three repeated processes into account.

Biochemical Methods

Flies (25 in each group) were sacrificed by exposure to −80 °C for 15 min for three times. For

the frozen 25 flies, homogenization was performed in ice-cold phosphate buffer solution by a

glass homogenizer (Potter-Elvehjem). Afterwards, the resulting homogenates were

centrifuged at the rate of 7,000 rcf for 10 min and the resulting supernatants stored at −80 °C

until they were analyzed. The by-product of the centrifugation, supernatants of homogenates

was used for the biochemical assays.

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Measurement of Advanced Oxidative Protein Products (AOPPs). Modified method of

Hanasand et al. [2012] was performed for spectrophotometric determination of AOPPs

concentrations. According to the procedure, homogenates were diluted with citric acid, 10 µl

of 1.16 M KI was added to the diluted solution, 2 min later followed by 20 µl acetic acid. The

absorbance of the reaction mixture was immediately read at 340 nm against the blank

solution. AOPPs concentrations were expressed as micromoles per liter of chloramine-T

equivalents.

Measurement of Protein-Bound Sialic Acid. SA concentrations were determined by the

thiobarbituric acid (TBA) method of Tram et al. [1997], who have made some modifications

to previous methods [Aminoff, 1961] resulting in improved sensitivity and higher

reproducibility. Homogenate proteins (30 µl) were precipitated with 500 µl trichloroacetic

acid with the volume of 20% (w/v). The upper layer was removed and discarded. The

precipitated proteins were dissolved in 280 µl H2SO4 and then incubated in 80°C for 1 h for

hydrolysis. N-acetylneuraminic acid was used as a standard. The samples, standards, and

blank were treated with 70 µl of periodate reagent (25 mM periodic acid in 0.125N sulfuric

acid) and incubated at 37°C for 30 min. The reaction was terminated by adding 70 µl of

sodium arsenite (2% sodium arsenite in 0.22 M hydrochloric acid). Once the yellow color of

liberated iodine had disappeared, 140 µl of TBA (0.1 M, pH 9.0) was added and the solution

heated in temperature-controlled water bath for 7.5 min, and then cooled in icy water.

Dimethyl sulfoxide (560 µl) was added and corresponding absorbances were measured at 549

nm.

Assay of Thiobarbituric Acid Reacting Substances. The rate of lipid peroxidation was

determined by the procedure of Buege and Aust [1978]. One of the major secondary products

of lipid peroxidation is reactive aldehydes. TBARS, along with other by-products, react with

TBA to generate a colored product that absorbs maximally at 535 nm wavelength,

representing the color produced by all the TBARS. The coefficients of intra- and

interassayvariations for TBARS assay were 3.4 and 5.4%, respectively.

Assay of Superoxide Dismutase Activity (Cu,Zn-SOD). Determination of Cu,Zn-SOD (EC

1.15.1.1) activity was assayed in supernatant fractions based on the method developed by Sun

  90  

et al. [1988]. This assay involves the inhibition of nitroblue tetrazolium reduction, with

xanthine oxidase used as a superoxide radical generator. One unit of Cu,Zn-SOD is defined

as the amount of enzyme needed to exhibit a 50% dismutation of superoxide radical. The

coefficients of intra- and interassay variations were 2.4 and 2.7%, respectively.

Total Protein Assay. Supernatants were stored at −70°C for protein measurement. Total

protein was determined by the Folin phenol procedure [Lowry et al., 1951].

Statistical Analyses

Data sets are shown as mean ± SE. While the results were statistically evaluated, one-way

ANOVA and post hoc Bonferroni tests were performed. The significance level of p < 0.05

was considered as significant for the statistical evaluations.

Results

Stress Tolerance Test Results

Test results are shown in Figures 1–3. After the exposure to heat and cold, more flies in the

mimetic aging group died compared to the respective control group (p < 0.05). Although the

ratio of those that died of starvation was not much, it is statistically significant l (p < 0.05 vs.

control). Resluts showed that tempol administration caused the percentage of survival in all

three tests to increase to a level close to that of the control group (p > 0.05 vs. control).

Fig.1. Survival percentage of groups after exposure to heat. (The bars represent mean of 25 animals ±

SE. *p < 0.05 vs. control; #p < 0.05 vs. mimetically aged group.)

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Fig.2. Survival percentage of groups after exposure to cold. (The bars represent mean of 25 animals ±

SE. *p < 0.05 vs. control; #p < 0.05 vs. mimetically aged group.)

Fig.3. Survival percentage after the starvation stress. (The bars represent mean of 25 animals ± SE. *p < 0.05 vs. control group.)

Biochemical Results

Cu,Zn-SOD activities and TBARS concentrations for the experimental groups are shown,

respectively, in Figures 4 and 5. In comparison to the control group, the Cu,Zn-SOD activity

decreased (p < 0.05 vs. control) and the TBARS concentration significantly increased (p <

0.05 vs. control) by D-galactose administration. On the other hand, no significant variation

was observed in Cu,Zn-SOD activity by tempol administration compared to D-galactose

administration (p > 0.05), whereas TBARS concentration dropped to control group

concentration level (p < 0.05 in comparison to control group, p < 0.05 in comparison to

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mimetically aged group). SA and AOPPs concentrations for the experiment groups are

presented, respectively, in Figures 6 and 7. The SA concentrations showed no significant

difference between the mimetically aged and -administered groups (p >0.05), whereas SA

concentrations were found to be lower in both mimetically aged and tempol-administered

groups compared to the control group (p < 0.05). AOPPs concentrations in the mimetically

aged group male flies were significantly higher than those in the male control group (p <

0.05), and additionally tempol administration decreased AOPPs concentrations (p < 0.05 vs.

mimetically aged group.)

Fig.4. Superoxide dismutase (Cu,Zn-SOD) activity values of the groups. (The bars represent mean of

25 animals ± SE. *p < 0.05 vs. control group.)

Fig.5. Thiobarbituric acid reacting substances (TBARS) concentrations of the groups. (The bars

represent mean of 25 animals ± SE. *p < 0.05 vs. control; #p < 0.05 vs. mimetically aged group.)

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Fig.6. Protein-bound sialic acid (SA) values of the groups. (The bars represent mean of 25 animals ±

SE. *p < 0.05 vs. control group.)

Fig.7. Advanced oxidative protein products (AOPPs) concentrations of the groups. (The bars represent

mean of 25 animals ± SE. *p < 0.05 vs. control; #p < 0.05 vs. Mimetically aged group.)

Discussion

The idea that oxidative damage underlies mimetic aging is mainly supported by studies in

rodents [Yanar et al., 2011; Aydin et al., 2012; Cakatay et al., 2013]. Increased oxidative

protein damage and free radical mediated desialylation of cellular proteins is another

important mechanism thought to underlie cellular aging in rodents [Cakatay et al., 2013]. On

the other hand, Drosophila is used widely to examine the relationship between oxidative

stress and aging [Cui et al., 2004; Lushchak et al., 2013; Yamamato et al., 2013] because

Drosophila genetic systems are well known in postmitotic tissues [Clancy and Birdsall,

2013].

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When D-galactose is present at high levels, it can be converted to aldose hydroperoxides with

the catalysis of galactose oxidase, and generate superoxide radical anion and other ROS

[Zhong et al., 2009]. Previous studies showed that mitochondrial dysfunction maybe be a key

componenet in the mechanism of accelerated aging caused by D-galactose [Long et al., 2007;

Kumar et al., 2010]. Additionally, it has been demonstrated that D-galactose causes damage

on the integrity of the mitochondria and disturbs the efficiency of ATP production, which in

turn contributes to more ROS generation in mitochondria [Long et al., 2007].

Prooxidant–antioxidant homeostasis was determined to investigate prooxidation due to D-

galactose-induced mimetic aging, although there are some differences in results obtained from

various mitotic and postmitotic tissues [Yanar et al., 2011; Aydin et al., 2012; Cakatay et al.,

2013]. In this study, the effect of tempol on an aging model in terms of stress tolerance,

general oxidative stress, and specifically oxidative protein damage has been to our knowledge

investigated for the first time. Our results support the idea that tempol has a positive impact

on the increase of stress tolerance as well as the detrimental effects of oxidative damage in

mimetically aged flies.

The present study has shown that that stress tolerance levels of older individuals increases

with tempol application. Increase in this resistance l ensures survival and therefore extend

lifespan. In the current study, the lifespan of the fly colony was not been determined.

However, increases in the amount of flies resistant to cold, heat, and hunger could also be

explained by an increase in lifespan. Tempol administration improved stress tolerance

response against heat and cold. Although, the effect of tempol on starvation response was not

statistically significant, there was an increased trend on mean values. In fact, starvation is

considered favorable for organisms due to the lesser mitochondrial electron leakage during

decreased glucose uptake and utilization [Gredilla and Barja, 2005].

At high concentrations, free radicals and radical-derived, nonradical reactive species are

hazardous for living organisms and damage all major cellular constituents. At moderate

concentrations, however, nitric oxide (NO), superoxide anion, and related ROS play an

important role as regulatory mediators in nuclear signaling processes. Many of the ROS-

mediated responses actually protect the cells against oxidative stress and reestablish “redox

homeostasis.” Higher organisms, however, have evolved the use of NO and ROS also as

signaling molecules for other physiological functions [Dröge, 2002].

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There are many irreversible degenerative molecular processes in proteome of aging cells

[Cakatay, 2011]. The most important process is the formation of ROS and the fact that they

cause damage to lipid, protein, and DNA in cells [Huangfu et al., 2013; Na et al., 2013]. In

addition, more reactive secondary derived metabolites from such macromolecules form in

time due to disruption in redox homeostasis in aging cells. In our study, this case has been

observed by means of the increase in TBARS concentration, which is the indicator of cellular

membrane damage. Moreover, it has been originally shown in our study that mimetic aging

significantly accelerates free radical mediated deterioration of redox homeostasis in various

stress conditions.

Cu,Zn-SOD is an essential antioxidant enzyme in the front line defense system that converts

dismutates superoxide radical anion to hydrogen peroxide and molecular oxygen within the

mitochondrial matrix [Maiese and Chong, 2004]. It is well known that during the aging

process, there is a reduced Cu,Zn-SOD activity [Lawler et al., 2009] and antioxidant enzyme

expression [Fleenor et al., 2012; Ramesh et al., 2012]. In our study, tempol administration in

the mimetically aged flies had no significant effect on the activity of Cu,Zn-SOD, which was

already at lower levels compared to the untreated galactose administered rodents. Since it was

shown that free radicals have an impact on the aging process, the notion that it increases the

lifespan by the inhibition of these molecules is proposed. Therefore, the studies with synthetic

antioxidants are being undertaken. Although the use of antioxidants seems to be beneficial,

care has to be taken that the endogenic defense system becomes of secondary importance.

This was observed in our study as well since no significant change was found in Cu,Zn- SOD

activity in the tempol application. This could have been caused by a decreased Cu,Zn-SOD

activity and/or expression level through aging process [Uzun et al., 2013].

The spectral characteristics of AOPPs correspond to several chromophores, including

dityrosine, carbonyls, and pentosidine, although nitrotyrosine is not in this group [Breusing

and Grune, 2010]. Oxidative modifications of cellular proteins, as in AOPPs formation,

usually results in a loss of protein function. When mimetically aged flies are compared to the

tempol-administered group, the impaired redox homeostasis was reversed by tempol by

means of decreased AOPPs concentrations. Impaired protein redox homeostasis, which

appears to occur in mimetically aged group, may be an enhancing factor in the propagation of

protein oxidation, as indicated by the AOPPs concentrations.

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Protein-bound SA residues play a significant role in various biological functions [Li and

Chen, 2012]. Desialylation shows its effect not only by altering the structure of glycoforms

and also function of glycoproteins, but also by increasing the concentration of SA, which

leads to the emergence of pathologies in tissues [Goswami and Koner, 2002]. SA residues

occupying terminal positions in N-linked oligosaccharides of glycoproteins have been shown

to play an important role in a variety of biological functions [Aminoff, 1961]. In our study,

there was no significant change in protein-bound SA concentrations in mimetically aged flies

compared to tempol-administered groups. To maintain the critical functions of SA groups

mentioned above, this finding might be explained by the importance of the strict maintenance

of the redox homeostasis of glycoproteins in proteomes of flies. In other words, the current

results of our study suggest that in D-galactose induced mimetic aging, there is an association

between desialylation of protein-bound SA and increased protein oxidation that leads to a

clustering of age-related disorder results obtained in these aged flies.

Conclusion

In conclusion, our study has demonstrated that scavenging ROS using tempol not only partly

reduced organism oxidative damage during aging, but also directly scavenged the mediators

related to oxidative stress rather than improving the reduced endogenous defense system,

thereby causing an improved endurance against environmental stress. Taken together, these

effects led to a modest improvement of aging-related frailty.

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Chapter5

SCAVENGING ROS IN THE ACUTE PHASE OF RENAL I/R INJURY ALSO

PROTECTS KIDNEY OXYGENATION AND NO LEVELS

Aksu U1,2, Ergin B1,2, Bezemer R1, Kandil A2, Milstein D.M. J.1, Demirci-Tansel C2, Ince C1

1Department of Translational Physiology, Academic Medical Center, University of Amsterdam, The Netherlands

2Department of Biology, Faculty of Science, Istanbul University, Istanbul, Turkey

Published in: Intensive Care Medicine Experimental 2015;3:21:1:10

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Chapter 5

Scavenging ROS in the acute phase of renal I/R injury also protects kidney oxygenation

and NO levels

Running title: ROS, O2, and NO in I/R-induced AKI

Background: We aimed to test our hypothesis that scavenging ROS with tempol would also

protect renal oxygenation and nitric oxide (NO) levels in the acute phase of renal I/R.

Methods: Rats were randomly divided into four groups of six: 1) no I/R, no tempol; 2) no I/R,

but with tempol; 3) I/R without tempol; and 4) I/R with tempol. I/R was induced by 30-min

clamping of the renal artery. The tempol-treated animals received 200 µmol/kg/h tempol

intravenously 15 min prior to I/R. Results: I/R without tempol led to a significant decrease in

renal DO2 and microvascular oxygenation, but tempol was able to these parameters. At R90,

the creatinine clearance rate was lower in the I/R-subjected group that did not receive tempol

compared to that in the other groups. I/R injury without tempol led to a significant increase in

tissue malondialdehyde levels (marker of oxidative stress) and a significant decrease in tissue

NO levels. Tempol administration before I/R could prevent these alterations. Conclusions:

Scavenging ROS using tempol also protects renal oxygenation and NO levels in the acute

phase of renal I/R. This demonstrates that renal oxygen, ROS, and NO levels are strongly

related in conditions of I/R.

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Introduction

Acute kidney injury (AKI) and is a complex clinical complication and is associated with a

high incidence of morbidity and mortality [Bagshaw, 2006; Bell and Martling, 2007]. One of

the most common causes of AKI is renal ischemia/reperfusion (I/R) injury as can occur in

numerous scenarios such as during surgery and also as a result of shock and resuscitation

[Lameire et al., 2005; Hoste and Kellum, 2006]. Despite the identification of several

mechanisms underlying the development of AKI, the pathophysiology of AKI is still

incompletely understood. It is clear, however, that instead of a single mechanism being

responsible for its etiology, AKI is associated with an entire orchestra of failing cellular

mechanisms [Welch et al., 2001; Adler and Huang, 2002; Welch et al., 2003; Aksu et al.,

2011].

It is well known that reactive oxygen species (ROS) are fundamentally implicated as primary

culprits in the pathophysiology of renal I/R injury and consequent AKI. The excess generation

of ROS and decreases in antioxidant defenses are known to contribute to I/R injury.

Superoxide dismutase (SOD), a ubiquitous intrinsic biological antioxidant, catalyzes the

dismutation of superoxide anions into oxygen and hydrogen peroxide. Tempol (4-hydroxy-

2,2,6,6-tetramethyl piperidinoxyl) is a membrane-permeable, metal-independent SOD

mimetic specific for superoxide anions (O2-). Several studies have demonstrated that tempol

may reduce renal I/R injury through its free radical scavenging activity [Chatterjee et al.,

2000; Fujii et al., 2005].

In a series of recent reviews, we have described that our hypothesis that a disturbed balance

between oxygen, nitric oxide (NO), and ROS might form an important component of the

pathogenesis of I/R-induced AKI [Legrand et al., 2008; Le Dorze et al., 2009; Aksu et al.,

2011]. In the present study we aimed to test whether the proven protective effects of tempol

are indeed associated with improved renal oxygenation and NO levels in a short-term rat

model of renal I/R.

Materials and methods

Animals

All experiments in this study were approved by the institutional Animal Experimentation

Committee of the Academic Medical Center of the University of Amsterdam. Care and

handling of the animals were in accordance with the guidelines for Institutional and Animal

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Care and Use Committees. The study has been carried out in accordance with the Declaration

of Helsinki. Experiments were performed on 24 Sprague-Dawley rats (Harlan Netherlands

BV, Horst, The Netherlands) with a mean ± SD body weight of 348 ± 21 g.

Surgical preparation

All animals were anesthetized with an intraperitoneal injection of a mixture of 75 mg/kg

ketamine (Nimatek®, Eurovet, Bladel, The Netherlands), 0.5 mg/kg dexmedetomidine

(Dexdomitor, Pfizer Animal Health BV, Capelle aan den IJssel, The Netherlands), and 0.05

mg/kg atropine-sulfate (Centrafarm Pharmaceuticals BV, Etten-Leur, The Netherlands). After

preparing a tracheotomy the animals were mechanically ventilated with a FiO2 of 0.4. Body

temperature was maintained at 37±0.5 °C during the entire experiment by an external thermal

heating pad. Ventilator settings were adjusted to maintain end-tidal pCO2 between 30 and 35

mmHg and arterial pCO2 between 35 and 40 mmHg.

For drug and fluid administration and hemodynamic monitoring, vessels were cannulated with

polyethylene catheters with an outer diameter of 0.9 mm (Braun, Melsungen, Germany). A

catheter in the right carotid artery was connected to a pressure transducer to monitor mean

arterial blood pressure (MAP) and heart rate. The right jugular vein was cannulated for

continuous infusion of Ringer’s Lactate (Baxter, Utrecht, The Netherlands) at a rate of 15

mL/kg/hour and maintenance of anesthesia. The right femoral artery was cannulated for

drawing blood samples and the right femoral vein for fluid resuscitation.

The left kidney was exposed, decapsulated, and immobilized in a Lucite kidney cup (K.

Effenberger, Pfaffingen, Germany) via ~4 cm incision in the left flank in each animal. Renal

vessels were carefully separated under preservation of nerves and the adrenal gland. A

perivascular ultrasonic transient time flow probe was placed around the left renal artery (type

0.7 RB Transonic Systems Inc., Ithaca, NY, USA) and connected to a flow meter (T206,

Transonic Systems Inc., Ithaca, NY, USA) to continuously measure renal blood flow (RBF).

An estimation of the renal vascular resistance (RVR) was made as: RVR (dynes.sec.cm-5) =

(MAP/RBF) × 80. The left ureter was isolated, ligated, and cannulated with a polyethylene

catheter for urine collection.

After the surgical preparation one optical fiber was placed 1 mm above the decapsulated

kidney and another optical fiber was placed 1 mm above the renal vein to measure renal

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microvascular and venous oxygenation using phosphorimetry (explained in more detail

below). A small piece of aluminum foil was placed on the dorsal side of the renal vein to

prevent contribution of the underlying tissues to the phosphorescence signal in the venous pO2

measurements. Oxyphor G2, a two-layer glutamate dendrimer of tetra-(4-carboxy-phenyl)

benzoporphyrin (Oxygen Enterprises Ltd., Philadelphia, PA, USA) was subsequently infused

(i.e. 6 mg/kg IV over 5 min), followed by 30 min of stabilization time. The surgical field was

covered with a humidified gauze compress throughout the entire experiment to prevent drying

of the exposed tissues.

Experimental protocol

After a stabilization period of 30 min, the animals were randomly divided into four groups of

six: 1) no I/R, no tempol (CTRL); 2) no I/R, but with tempol (TMPL); 3) I/R without tempol

(I/R); and 4) I/R with tempol (I/R+TMPL). Ischemia/reperfusion was induced by 30-min non-

destructive clamping of the renal artery. The tempol-treated animals received 200 µmol/kg/h

of 4-hydroxy-TEMPO (tempol) intravenously 15 min prior to initiation of I/R. Measurements

were performed up to 90 min post-ischemia and after the experiments, the kidneys were

isolated and renal tissue malondialdehyde and nitric oxide levels were measured.

Blood variables

Arterial blood samples (0.5 ml) were taken from the femoral artery at baseline (BSLN) and

after 15 and 90 min of reperfusion (R15 and R90, respectively). The blood samples were

replaced by the same volume of Ringer’s Lactate. Samples were analyzed for blood gas

values (ABL505 blood gas analyzer; Radiometer, Copenhagen, Denmark), hemoglobin

concentration, and hemoglobin oxygen saturation (OSM3; Radiometer, Copenhagen,

Denmark). Additionally, plasma creatinine and sodium concentrations were determined in all

samples.

Renal microvascular and venous oxygenation

Microvascular oxygen tension in the renal cortex (CµPO2), outer medulla (MµPO2), and renal

venous oxygen tension (PrvO2) were measured by oxygen-dependent quenching of

phosphorescence lifetimes of the systemically infused albumin-targeted (and therefore

circulation-confined) phosphorescent dye Oxyphor G2 [Johannes et al., 2006]. Oxyphor G2

has two excitation peaks (λexcitation1 =440 nm, λexcitation2 =632 nm) and one emission peak

(λemission =800 nm). These optical properties allow (near) simultaneous lifetime measurements

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in microcirculation of the kidney cortex and the outer medulla due to different optical

penetration depths of the excitation light [Johannes et al., 2006]. For the measurement of renal

venous PO2 (PrvO2), a mono-wavelength phosphorimeter was used [Mik et al., 2008]. Oxygen

measurements based on phosphorescence lifetime techniques rely on the principle that

phosphorescence can be quenched by energy transfer to oxygen resulting in shortening of the

phosphorescence lifetime. A linear relationship between reciprocal phosphorescence lifetime

and oxygen tension (i.e., the Stern-Volmer relation) allows quantitative measurement of PO2

[Bezemer et al., 2010].

Renal oxygen delivery and consumption

Arterial oxygen content (AOC) was calculated by (1.31×hemoglobin×SaO2)+(0.003×PaO2),

where SaO2 is arterial oxygen saturation and PaO2 is arterial partial pressure of oxygen. Renal

venous oxygen content (RVOC) was calculated as (1.31×hemoglobin×SrvO2)+(0.003×PrvO2),

where SrvO2 is venous oxygen saturation and PrvO2 is renal vein partial pressure of oxygen

(measured using phosphorimetry). Renal oxygen delivery was calculated as DO2

(mL/min)=RBF×AOC. Renal oxygen consumption was calculated as VO2 (mL/min)

=RBF×(AOC–RVOC).

Renal function

For analysis of urine volume, creatinine concentration, and sodium (Na+) concentration at the

the end of the protocol, urine samples from the left ureter were collected for 10 min.

Creatinine clearance rate (CCR) per gram of renal tissue was calculated with standard

formula: CCR [mL/min] = (UC×V)/PC, where UC is the urine creatinine concentration, V is

the urine volume per unit time, and PC is the plasma creatinine concentration. Renal sodium

reabsorption (TNa+, [mmol/min]) was calculated as TNa+ = (PNa+×CCR)-(UNa+×V) , where UNa+

is the urine sodium concentration and PNa+ is the plasma sodium concentration.

Renal tissue oxidative stress

Renal tissue malondialdehyde (MDA) levels were determined to assess lipid peroxidation as a

measure of renal oxidative stress. All kidneys were homogenized in cold 5 mM sodium

phosphate buffer. The homogenates were centrifuged at 12,000 g for 15 min at 4 ºC and

supernatants were used for MDA determination. The level of lipid peroxides was expressed as

micromoles of MDA per milligram of protein (Bradford assay).

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Renal tissue NO levels

NO undergoes a series of reactions in biological tissues leading to the accumulation of the

final products nitrite and nitrate. Thus, the index of the total NO accumulation is the sum of

both nitrite and nitrate levels in the tissue samples. To reduce the nitrate and nitrate pressnet

in the tissue samples to NO, the samples were put in the reducing agent vanadium (III)

chloride (VCl3) in 1 mol/L HCl at 90 oC. The VCl3 reagent converts nitrite, nitrate, and S-

nitroso compounds to NO gas which is guided towards an NO chemiluminescence signal

analyzer (Sievers 280i analyzer, GE Analytical Instruments) allowing the direct detection of

NO [Yang et al., 1997]. Within the reaction vessel, NO reacted with ozone to generate oxygen

and excited-state NO species, of which the decay is associated with the emission of weak

near-infrared chemiluminescence. This signal is detected by a sensitive photodetector and

converted to millivolts (mV). The area under the curve of the detected chemiluminescence

(mV·s) represents the amount of NO-ozone reactions in time and thus the amount of

bioavailable NO in the tested samples. The ratio of tissue NO to tissue protein content was

used to for standardization of the NO measurements.

Data analysis

Data analysis and presentation were performed using GraphPad Prism (GraphPad Software,

San Diego, CA, USA). Values are reported as the mean ± SD. Two-way ANOVA for repeated

measurements with a Bonferroni post hoc test were used for comparative analysis between

groups. A p-value of <0.05 was considered statistically significant.

Results

Systemic and renal hemodynamics and oxygenation

All systemic and renal hemodynamic and oxygenation variables are presented in Tables 1 and

2. MAP and renal VO2 remained stable throughout the entire protocol in all groups. Tempol

administration in the sham-operated animals (i.e., without I/R) did not affect any of the

systemic and renal hemodynamic and oxygenation variables. I/R without tempol

administration led to a significant decrease in RBF (2.5 ± 0.6 mL/min at R15 and 2.4 ± 0.3

mL/min at R90) and DO2 (1.05 ± 0.28 mL O2/min at R15 and 0.90 ± 0.22 mL O2/min at R90)

and a significant increase in RVR (3298 ± 955 dyn·s·cm-5 at R15 and 3352 ± 426 dyn·s·cm-5

at R90). Tempol administration prior to I/R was able to preserve RBF (4.0 ± 0.9 mL/min at

R15 and 4.1 ± 1.6 mL/min at R90), DO2 (1.61 ± 0.46 mL O2/min at R15 and 1.75 ± 0.70 mL

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O2/min at R90), and RVR (1999 ± 471 dyn·s·cm-5 at R15 and 2200 ± 1046 dyn·s·cm-5 at

R90).

Table 1: Mean arterial pressure (MAP), renal blood flow (RBF), renal vascular resistance (RVR), renal oxygen delivery (DO2), and renal oxygen consumption (VO2) at baseline (Bsln) and after 15 and 90 min of reperfusion (R15 and R90, respectively). Cp<0.05 vs CTRL, Tp<0.05 vs TMPL, Ip<0.05 vs I/R.

Bsln R15 R90 MAP [mmHg] CTRL 103 ± 7 103 ± 5 96 ± 6 TMPL 103 ± 8 96 ± 8 93 ± 4 I/R 101 ± 10 96 ± 6 98 ± 6 I/R+TMPL 105 ± 11 95 ± 16 96 ± 16 RBF [mL/min] CTRL 4.3 ± 1.3 4.1 ± 1.4 3.8 ± 0.5 TMPL 4.2 ± 0.7 3.8 ± 1 3.7 ± 1.3 I/R 4.0 ± 0.6 2.5 ± 0.6 CT 2.4 ± 0.3 CT I/R+TMPL 4.4 ± 1 4 ± 0.9 I 4.1 ± 1.6 I RVR [dyn.s.cm-5] CTRL 2060 ± 583 2143 ± 542 2070 ± 240 TMPL 1989 ± 379 2189 ± 712 2223 ± 733 I/R 2064 ± 414 3298 ± 955 CT 3352 ± 426 CT I/R+TMPL 1968 ± 454 1999 ± 471 I 2200 ± 1046 I DO2 [mL O2/min] CTRL 1.77 ± 0.53 1.65 ± 0.52 1.52 ± 0.22 TMPL 1.75 ± 0.20 1.54 ± 0.18 1.45 ± 0.21 I/R 1.62 ± 0.33 1.05 ± 0.28 CT 0.9 ± 0.22 CT I/R+TMPL 1.88 ± 0.42 1.61 ± 0.46 I 1.75 ± 0.70 I VO2 [mL O2/min/g] CTRL 0.12 ± 0.04 0.11 ± 0.02 0.12 ± 0.02 TMPL 0.13 ± 0.07 0.13 ± 0.03 0.11 ± 0.03 I/R 0.13 ± 0.04 0.1 ± 0.03 0.1 ± 0.03 I/R+TMPL 0.14 ± 0.04 0.13 ± 0.05 0.13 ± 0.04

Renal microvascular oxygenation in the cortex and medulla were decreased quickly during

ischemia but normalized immediately upon reperfusion. However, at R90, microvascular

oxygenation was significantly decreased in the I/R-subjected group that did not receive

tempol (44 ± 11 mmHg in the cortex and 41 ± 5 mmHg in the medulla) while this was

maintained in the I/R-subjected group that did receive tempol (57 ± 4 mmHg in the cortex and

51±2 mmHg in the medulla).

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Table 2: Microvascular oxygen tension in renal cortex (CµpO2) and medulla (MµpO2) at baseline

(Bsln), at the end of 30 min of ischemia (Isch), and after 15 and 90 min of reperfusion (R15 and R90,

respectively). Cp<0.05 vs CTRL, Tp<0.05 vs TMPL, Ip<0.05 vs I/R.

Bsln Isch R15 R90 CµpO2 [mmHg] CTRL 76 ± 2 70 ± 4 70 ± 4 62 ± 7 TMPL 76 ± 3 71 ± 8 73 ± 6 58 ± 6 I/R 79 ± 4 11 ± 4 CT 59 ± 4 44 ± 11 CT I/R+TMPL 77 ± 6 10 ± 4 CT 66 ± 9 57 ± 4 I MµpO2 [mmHg] CTRL 61 ± 5 57 ± 6 54 ± 4 51 ± 4 TMPL 57 ± 9 56 ± 10 55 ± 7 50 ± 7 I/R 59 ± 6 7 ± 1 CT 50 ± 3 C 41 ± 5 CT I/R+TMPL 59 ± 5 7 ± 1 CT 59 ± 7 I 51 ± 2 I

Renal function

The renal function variables are presented in Table 3. Tempol administration in the sham-

operated animals (i.e., without I/R) did not affect renal function. I/R without tempol

administration led to a significant decrease in CCR (0.3 ± 0.1 mL/min at R15) and TNa+ (0.04

± 0.01 mmol/min at R15) and tempol administration prior to I/R could not prevent these

reductions in CCR (0.4 ± 0.2 mL/min at R15) and TNa+ (0.06 ± 0.03 mmol/min at R15). At

R90 these decreases were mostly normalized except for the CCR in the I/R-subjected group

that did not receive tempol.

Table 3: Creatinine clearance rate (CCR) and sodium reabsoption (TNa+) at baseline (Bsln) and after 15 and 90 min of reperfusion (R15 and R90, respectively). Cp<0.05 vs CTRL, Tp<0.05 vs TMPL, Ip<0.05 vs I/R. Bsln R15 R90 CCR [mL/min] CTRL 1.2 ± 0.7 1.3 ± 0.3 1.5 ± 0.7 TMPL 1.1 ± 0.3 1.1 ± 0.3 1.2 ± 0.4 I/R 1.2 ± 0.4 0.3 ± 0.1 CT 0.7 ± 0.4 C I/R+TMPL 1.4 ± 0.6 0.4 ± 0.2 CT 1.0 ± 0.3 TNa+ [mmol/min] CTRL 0.18 ± 0.09 0.18 ± 0.09 0.14 ± 0.07 TMPL 0.15 ± 0.04 0.14 ± 0.04 0.13 ± 0.03 I/R 0.16 ± 0.06 0.04 ± 0.01 CT 0.09 ± 0.04 I/R+TMPL 0.20 ± 0.09 0.06 ± 0.03 CT 0.14 ± 0.05

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Renal oxidative stress and NO levels

The renal microvascular oxygenation, oxidative stress, and NO levels at the end of the

protocol are presented in Figure 1. Tempol administration without I/R injury led to a

significant decrease in tissue MDA levels (1.6 ± 0.17) and I/R injury in the absence of tempol

led to a significant increase in tissue MDA levels (3.8 ± 0.9). Tempol administration before

I/R could partially prevent this increase in MDA levels (2.4 ± 0.7). Tissue NO levels were not

affected by tempol administration without I/R injury (240 ± 100), but were significantly

decreased after I/R in the absence of tempol (72 ± 21). Tempol administration before I/R

could completely normalize the tissue NO levels (265 ± 143). Hence, tempol administration

prior to I/R injury reduced renal oxidative stress and normalized renal oxygenation and tissue

NO levels.

Fig.1. Renal oxygenation, oxidative stress, and nitric oxide (NO) levels at the end of the protocol. (A) Microvascular oxygen tensions (µpO2) in the renal cortex; (B) Microvascular oxygen tensions (µpO2) in the renal medulla; (C) renal tissue malondialdehyde (MDA) levels normalized to the tissue protein content; and (D) tissue NO levels normalized to the tissue protein content. *p<0.05 versus all other groups; Cp<0.05 versus the CTRL group; Tp<0.05 versus the TMPL group.

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Discussion

In the present study we aimed to test the hypothesis scavenging ROS using tempol would be

associated with improved renal oxygenation and NO levels in a short-term rat model of renal

I/R. We have found that I/R was associated with a significant increased in tissue MDA

(marker of oxidative stress) and a significant decrease in tissue NO. The decrease in tissue

NO was followed by an increase in RVR and consequent decrease in RBF, renal DO2, and

renal microvascular oxygenation. These disturbances were associated with reduced renal

function in terms of sodium reabsorption and creatinine clearance. Pre-ischemic

administration of tempol, a known superoxide scavenger, was able to decrease oxidative

stress and increase renal tissue NO and microvascular oxygenation and thereby improve renal

function. Furthermore, we have shown that administration of tempol in the absence of I/R

leads to a reduction in the renal MDA levels normally present in renal tissue, but did not

affect any of the other parameters.

I/R injury is a multi-pathway process in which decreased ROS scavenging and increased ROS

generation are particularly important mediators leading to tissue injury [Nath and Norby,

2000; Aksu et al., 2011]. ROS are created in mitochondria [Yoshikawa et al., 2012], and

excess ROS injure the mitochondria themselves, impair cellular function, and promote

apoptosis [Huttemann et al., 2012]. It has previously been shown that antioxidants can

decrease cellular and tissue damage by decreasing intracellular ROS levels and suppressing

oxidative stress [Patel et al., 2002; Chatterjee, 2007; Guz et al., 2007; Roth et al., 2011;

Gomes et al., 2012; Riccioni et al., 2012]. In this study, we showed that tempol reduced renal

lipid peroxidation in renal tissue after renal I/R as reflected by decreased tissue MDA levels

[Michel et al., 2008]. In line, Patel et al. have previously shown that administration of

tempone, an unmetabolized form of tempol, reduced I/R-induced injury to peritubular cells by

thereby reduced renal dysfunction [Patel et al., 2002]. They showed, moreover, that this was

without the adverse cardiovascular effects observed when using other nitroxyl radical

scavenging agents. Noiri et al. also demonstrated that both L-NIL (i.e., a selective iNOS

inhibitor) and lecithinized SOD administration improve renal function due to scavenging of

peroxynitrite and thereby preventing lipid peroxidation and oxidative damage to DNA [Noiri

et al., 2001].

In this study tempol effectively inhibited an I/R-induced decrease in tissue NO concentration.

Decreased NO production via eNOS during renal I/R contributes to renal hypoperfusion and

  112  

renal injury. This has been confirmed by studies showing that L-arginine (i.e., a precursor of

NO) and NO donors improve renal function after I/R [Chatterjee et al., 2007; Kucuk, et al.,

2006; Jeong et al., 2004]. On the other hand, also the administration of iNOS inhibitors has

been shown protect the kidney against I/R injury [Chatterjee et a., 2002; Mark et al., 2005;

Vinas et al., 2006; Noiri et al., 2001]. In the present study, however, the protocol was too

short for iNOS expression to occur. Nonetheless, the administration of tempol did scavenge

the excess ROS generated during I/R and thereby prevented the interaction of eNOS-derived

NO and ROS forming peroxynitrite and leaving the NO available for maintenance of

microvascular perfusion. Hence, scavenging ROS has a double beneficial effect.

Our study has of course a number of limitations. First, this study was performed in rats and

the effects of tempol could be different in humans. Second, the duration of renal ischemia was

30 min and measurements were performed up to 90 min post-ischemia and thus long-term

effects of I/R and tempol were not studied. Additionally, a longer duration of ischemia might

have caused more severe renal dysfunction. Third, we did not measure ROS directly but

instead measured MDA as a marker of lipid peroxidation as a result of oxidative stress.

Conclusions

In conclusion, our study clearly demonstrated that scavenging ROS using tempol not only

reduced renal oxidative stress following I/R, but also normalized renal tissue NO levels and

thereby reduced RVR and improves RBF, renal DO2, and renal microvascular oxygenation.

Taken together, these effects led to a modest (albeit not statistically significant) improvement

of renal function after I/R.

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  117  

Summary and conclusions

In Chapter 1, an acetate- and gluconate-balanced crystalloid solution was tested for its effects

on the plasma ion levels and acid–base balance, renal oxygenation, oxidative stress status,

glycocalyx integrity, and systemic cytokine levels in a rat model of hemorrhagic shock. The

main findings of our study were that: (1) both the balanced and unbalanced crystalloid

solutions successfully restored the blood pressure, but renal blood flow was only recovered by

the balanced solution although this did not lead to improved renal oxygenation; (2) less

balanced fluid was required to restore blood pressure; (3) while unbalanced crystalloid

resuscitation induced hyperchloremia and worsened metabolic acidosis in hemorrhaged rats,

balanced crystalloid resuscitation prevented hyperchloremia, restored the acid–base balance,

and preserved the anion gap and strong ion difference in these animals; (4) neither balanced

nor unbalanced crystalloid resuscitation could normalize systemic inflammation (TNF-a and

IL-6); (5) only balanced crystalloid resuscitation significantly reduced renal oxidative stress,

as reflected by reduced L-FABP reactivity, but none of the fluids could restore the increased

NGAL, MDA, and hyaluronan levels; and (6) balanced crystalloid resuscitation significantly

improved renal oxygen consumption (increased VO2 , decreased EFNa+), but none of the

fluids was able to restore creatinine clearance rate in this short-term protocol. In conclusion,

while unbalanced crystalloid resuscitation induces hyperchloremia and worsens metabolic

acidosis in hemorrhaged rats, balanced crystalloid resuscitation prevents hyperchloremia,

restores the acid–base balance, and preserves the anion gap and strong ion difference in these

animals. Balanced crystalloid resuscitation prevents renal hypoperfusion better than

unbalanced crystalloid resuscitation. However, although the balanced preparation improves

some parameters, it does not improve oxidative stress and systemic inflammation.

In Chapter 2, we examined the acute effects of acetate-balanced colloid and crystalloid

resuscitation on renal oxygenation in a rat model of hemorrhagic shock. We tested the

hypothesis that acetate-balanced solutions would be superior in correcting impaired renal

perfusion and oxygenation after severe hemorrhage compared to unbalanced solutions. Our

main findings were that: (1) hemorrhagic shock was associated with acute decreases in blood

pressure, renal perfusion and oxygenation, and urine production; (2) volume replacement

therapy with balanced and unbalanced crystalloid and colloid solutions partially corrected

these parameters; and (3) the acetate-balanced colloid solution HES-RA was the only

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resuscitation fluid that could restore renal blood flow back to ∼85% of baseline level which

was associated with the most prominently improved renal oxygenation. While resuscitation

with the NaCl and RA (crystalloid solutions) and the HES-NaCl (unbalanced colloid solution)

led to hyperchloremic acidosis, resuscitation with the HES-RA (acetate-balanced colloid

solution) did not. In conclusion, the acetate-balanced colloid solution, HES-RA, was

furthermore the only fluid restoring renal blood flow back to ∼85% of baseline level and most

prominently improved renal microvascular oxygenation.

The aim of Chapter 3 was to investigate the acute effects of balanced versus unbalanced

colloid resuscitation on renal macrocirculatory and microcirculatory perfusions in a rat model

of LPS-induced endotoxemia. To test the hypothesis that balanced colloid resuscitation would

be better for the kidney than unbalanced colloid resuscitation, we resuscitated with HES-NaCl

as an unbalanced colloid solution and HES-RA as a balanced colloid solution. The main

findings were that (1) LPS-induced endotoxemia was associated with deteriorated systemic

and renal hemodynamics, acid-base balance, mean cortical microvascular perfusion, and

perfusion heterogeneity and caused anuria; (2) both HES-NaCl and HES-RA resuscitation

improved systemic blood pressure, but only HES-RA resuscitation improved renal

macrovascular and microvascular perfusion; (3) neither HES-NaCl nor HES-RA resuscitation

could restore the metabolic acidosis or fractional sodium excretion; and (4) plasma chloride

levels were significantly lower after HES-RA resuscitation compared with after HES-NaCl

resuscitation. In conclusion, this confirmed our hypothesis that balanced colloid resuscitation

is superior to unbalanced colloid resuscitation in terms of improvement of renal

macrovascular and microvascular perfusions. However, whether this results in improved renal

function in the long-term warrants further study.

A role for oxidative damage in mimetic aging is mainly supported by studies in rodents.

Increased oxidative protein damage and free radical mediated desialylation of cellular proteins

is another important mechanism for cellular aging in rodents. On the other hand, the

Drosophila is used widely to examine the relationship between oxidative stress and aging,

because the Drosophila genetic systems are well-known and postmitotic tissues. When D-

galactose is present at high levels, it can be converted to aldose hydroperoxides with the

catalysis of galactose oxidase, resulting in the generation of a superoxide radical anion and

other ROS. Previous studies showed that mitochondrial dysfunction maybe a key issue in the

mechanism of accelerated aging caused by D-galactose. Additionally, it was demonstrated

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that D-galactose causes damage to the integrity of the mitochondria and disturbs the

efficiency of ATP production, which in turn contributes to more ROS generation in the

mitochondria. In Chapter 4, the aim of the study was to test the hypothesis whether tempol

restores impaired redox homeostasis and increases stress tolerance in a mimetic aging model

of Drosophila. For this reason, we investigated the extent of general oxidative stress and,

specifically, oxidative protein damage in D-galactose administered mimetically aged flies

following tempol administration. Our study demonstrated that scavenging ROS using tempol

not only (1) reduced oxidative damage during aging, but also (2) directly scavenged the

mediators related to oxidative stress, rather than only improving the reduced endogenous

defense systems. This led to (3) an improved endurance against environmental stress. In

conclusion, the restoration of redox homeostasis led to a modest improvement of aging-

related frailty upon tempol administration.

In Chapter 5, we aimed to test the hypothesis that scavenging ROS using tempol would be

associated with improved renal oxygenation and NO levels in a short-term rat model of renal

I/R. We have found that (1) I/R was associated with a significant increase in tissue MDA

(marker of oxidative stress) and (2) a significant decrease in tissue NO. The decrease in tissue

NO was followed by (3) an increase in RVR and consequent decrease in RBF, renal DO2, and

renal microvascular oxygenation. (4) These disturbances were associated with reduced renal

function in terms of sodium reabsorption and creatinine clearance. Pre-ischemic

administration of tempol, a known superoxide scavenger, was able to decrease oxidative

stress and increase renal tissue NO and microvascular oxygenation and thereby improve renal

function. Furthermore, we have shown that administration of tempol in the absence of I/R

leads to a reduction in the renal MDA levels normally present in healthy renal tissue, but did

not affect any of the other parameters. In conclusion, our study clearly demonstrated that

scavenging ROS using tempol not only reduced renal oxidative stress following I/R, but also

normalized renal tissue NO levels and thereby reduced RVR and improves RBF, renal DO2,

and renal microvascular oxygenation. Taken together, these effects led to a modest (albeit not

statistically significant) improvement of renal function after I/R.

In conclusion, this thesis presents the findings of various experimental therapatic approaches

on in the treatment of acute kidney injury in different experimental models. The findings

indicate that the resuscitation fluids commonly used with the idea of protecting the kidney

actually do not correct systemic inflammation or oxidative stress, and therefore do not prevent

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renal ischemia and hypoxia. Nonetheless, eventhough fluid resuscitation does not have any

effects on renal oxygenation, it is of course better than not resuscitating at all. Therefore,

optimization of fluid therapy, such as balancing fluids, and other therapeutic approaches

aimed to protect the kidney, is utmost important. Taking into account our studies on

antioxidants, a new generation of fluids could be developed, incorporating antioxidant

properties. However, the long-term effects of balanced and antioxidant-enriched resuscitation

on renal function warrants further study.

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Samenvatting en conclusies

In hoofdstuk 1 werd een acetaat- en gluconaat-gebalanceerde kristalloïdoplossing getest op de

effecten op de plasma-ion niveaus, het zuur-base-evenwicht, the renale oxygenatie, oxidatieve

stress status, glycocalyx integriteit, en systemische cytokine niveaus in een rat model van

hemorragische shock. De belangrijkste bevindingen van ons onderzoek waren dat: (1) zowel

de gebalanceerde en ongebalanceerde kristalloïde oplossingen met succes de bloeddruk

herstelde, maar de doorbloeding van de nier werd alleen hersteld door de gebalanceerde

oplossing, hoewel dit niet leidde tot een betere renale oxygenatie; (2) minder gebalanceerde

vloeistof nodig was om de bloeddruk te herstellen vergeleken met ongebalanceerde vloeistof;

(3) terwijl ongebalanceerde kristalloïde therapie hyperchloremie veroorzaakte en metabole

acidose verslechterde in hemorrhagische ratten, gebalanceerde kristalloïde therapie voorkwam

hyperchloremie, herstelde het zuur-base-evenwicht, en bewaarde de anion gap en het sterke

ion verschil in deze dieren; (4) zowel niet-gebalanceerde als gebalanceerde kristalloïdtherapie

konden niet de systemische inflammatie (TNF-a en IL-6) normaliseren; (5) gebalanceerde

kristalloïde oplossing verminderde aanzienlijk de renale oxidatieve stress, zoals weerspiegeld

door de gereduceerde L-FABP reactiviteit, maar geen van de vloeistoffen kon de verhoogde

NGAL, MDA en hyaluronzuur niveaus herstellen; en (6) gebalanceerde kristalloïde therapie

verbeterde aanzienlijk het renale zuurstofverbruik (gestegen VO2, gedaalde EFNa+), maar

geen van de vloeistoffen in staat was om de creatinineklaring te herstellen in dit kortduurende

protocol. In conclusie, terwijl ongebalanceerde kristalloïde therapie hyperchloremie

induceerde en metabole acidose verslechterde in ratten met hemorrhagische shock,

gebalanceerde kristalloïd therapie voorkwam hyperchloremie, herstelde het zuur-base-

evenwicht en bewaarde het anion gap en sterke-ionen verschil in deze dieren. Gebalanceerde

kristalloïde therapie voorkwam renale hypoperfusie beter dan ongebalanceerde kristalloïde

therapie. Hoewel de gebalanceerde oplossingen een aantal parameters verbeterde, geen een

oplossing kon de systemische oxidatieve stress en inflammatie verbeteren.

In hoofdstuk 2 hebben we onderzocht wat de acute effecten van acetate-gebalanceerde

colloïde en kristalloïde therapie op de renale oxygenatie zijn, in een ratmodel van

hemorragische shock. We hebben de hypothese getest dat acetaat-gebalanceerde oplossingen

superieur zijn in het corrigeren van insufficiënte nierperfusie en -oxygenatie na ernstige

bloeding, in vergelijking met ongebalanceerde oplossingen. De belangrijkste bevindingen

waren dat: (1) hemorrhagische shock was geassocieerd met acute verlaging van de bloeddruk,

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renale perfusie en oxygenatie en urineproductie; (2) volume-therapie met gebalanceerde en

ongebalanceerde kristalloïde en colloïdale oplossingen gedeeltelijk deze parameters

cirrigeerde; en (3) acetaat-gebalanceerde colloïdale oplossing (HES-RA) de enige

therapievloeistof is die de nierperfusie tot ~85% van het baseline-niveau kon brengen, wat

geassocieerd was met de sterkst verbeterde renale oxygenatie. Terwijl therapie met de NaCl

en RA (kristalloïde oplossingen) en de HES-NaCl (ongebalanceerde colloïdale oplossing)

leidde tot hyperchloremische acidose, therapie met de HES-RA (acetate- gebalanceerde

colloïdale oplossing) deed dat niet. De acetaat-gebalanceerde colloïdale oplossing HES-RA

was bovendien de enige vloeistof die de renale perfusie terug naar ~85% van het baseline

niveau kon herstellen en het sterkst de renale microvasculaire oxygenatie verbeterde. Echter,

de lange-termijn effecten van HES-RA therapie op de nierfunctie dient verder bestudeerd te

worden.

Het doel van het hoofdstuk 3 was om de acute effecten van gebalanceerde versus

ongebalanceerde colloïde therapie op de renale macro- en microcirculatie te testen in een

ratmodel van LPS-geïnduceerde endotoxemie. Om de hypothese te testen dat gebalanceerde

colloïde therapie beter is voor de nieren dan ongebalanceerde colloïde therapie, hebben we de

dieren behandeld met HES-NaCl als een ongebalancerde colloïde oplossing en HES-RA als

gebalanceerde colloïde oplossing. De belangrijkste bevindingen waren dat (1) LPS-

geïnduceerde endotoxemie was geassocieerd met verslechterde systemische en renale

hemodynamiek, zuur-base-evenwicht, gemiddelde corticale microvasculaire perfusie en

perfusie-heterogeniteit, en urineproductie; (2) zowel HES-NaCl als HES-RA therapie de

systemische bloeddruk verbeterde, maar alleen HES-RA therapie de renale macro- en

microcirculatie verbeterde; (3) noch HES-NaCl noch HES-RA therapie de metabole acidose

of de fractionele excretie van natrium kon herstellen; en (4) plasma-chloride niveaus

significant lager waren na HES-RA therapie opzichte van na HES-NaCl therapie.

In hoofdstuk 4 was het doel van de studie om de hypothese te testen dat TEMPOL de

verstoorde redox homeostase kan herstellen en de stresstolerantie kan verhogen in een

mimetische-veroudering model van Drosophila. Daarom onderzochten we de omvang van de

algemene oxidatieve stress en met name oxidatieve schade aan eiwitten in deze vliegen na

TEMPOL toediening. Een rol voor oxidatieve schade in mimetische veroudering wordt

voornamelijk ondersteund door studies met knaagdieren. Verhoogde oxidatieve schade eiwit

en vrije radicalen gemedieerde desialylation van cellulaire eiwitten is een ander belangrijk

  123  

mechanisme voor cellulaire veroudering bij knaagdieren. Aan de andere kant worden

Drosophila veel gebruikt om de relatie tussen oxidatieve stress en veroudering te

ouderzoeken, omdat de genetische systemen van Drosophila bekend zijn. Wanneer D-

galactose aanwezig is in hoge concentratie kan het omgezet worden naar aldose

hydroperoxiden met katalyse galactoseoxidase, resulterend in de vorming van een superoxide

radicaal anion en andere ROS. Eerdere studies toonden aan dat mitochondriale dysfunctie een

essentieel onderdeel is van het mechanisme van versnelde huidveroudering door D-galactose

kan zijn. Bovendien werd aangetoond dat D-galactose schade veroorzaakt aan de integriteit

van de mitochondriën en de efficiëntie van ATP-productie verstoort, wat weer bijdraagt aan

ROS generatie in de mitochondria. Tot slot, onze studie toonde aan dat het wegvangen van

ROS met behulp van TEMPOL niet slechts gedeeltelijk oxidatieve schade vermindert tijdens

veroudering, maar ook direct de factoren gerelateerd aan oxidatieve stress verminderen in

plaats van alleen het verbeteren van het verminderde endogene afweersysteem, waardoor een

verbeterde bescherming tegen de omgeving bewerkstelligd wordt. Samengevat, deze effecten

leiden tot een bescheiden verbetering van de verouderings-gerelateerde kwetsbaarheid van

cellen.

In hoofdstuk 5 hebben we geprobeerd om de hypothese te testen dat het wegvangen van ROS

met behulp van TEMPOL geassocieerd zou zijn met een verbeterde nierfunctie,

zuurstoftransport, en NO productie in een kortdurend ratmodel van renale ischemie/reperfusie

(I/R) schade. We hebben gevonden dat I/R geassocieerd was met een significante toename in

weefsel MDA (marker van oxidatieve stress) en een significante afname in weefsel NO. De

afname in weefsel NO werd gevolgd door een toename in de RVR en daaruit voortvloeiende

verlaging van de RBF, renale DO2, en renale microvasculaire oxygenatie. Deze verstoringen

waren geassocieerd met een verminderde nierfunctie in termen van natriumreabsorptie en

creatinineklaring. Pre-ischemische toediening van TEMPOL, een bekende superoxide

scavenger, kon oxidatieve stress verlagen en nierweefsel NO en microvasculaire oxygenatie

beschermen en daardoor de nierfunctie verbeteren na I/R. Verder hebben we aangetoond dat

toediening van TEMPOL in afwezigheid van I/R leidt tot een verlaging van de renale MDA-

niveaus normaal aanwezig in nierweefsel, maar dit had geen effect op de andere parameters.

Onze studie heeft duidelijk aangetoond dat het wegvangen van ROS met behulp van

TEMPOL niet alleen renale oxidatieve stress verminderde na I/R, maar ook nierweefsel NO

niveaus genormaliseerde alsmede de RVR verminderde en de RBF, nier-DO2, en renale

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microvasculaire oxygenatie verbeterde. Samengevat, de effecten van TEMPOL leiden tot een

bescheiden (maar niet statistisch significante) verbetering van de nierfunctie na I/R.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Acknowledgement

I am grateful for this moment in my life while I am writing this page of my thesis and reminisce moments of this wonderful chapter of my life. I am thankful to all the incredible people who played a vital role in helping to bring this project to finalize.

First of all, I would like to express my sincere gratitude to my supervisor, değerli hocam, Prof. Dr. Can Ince, for all motivation and supports, for all discussions, suggestions and feedbacks during last 14 years, especially my PhD study. Also, thank you for the patience during the whole period, despite of problems that I believe normally is not part of a supervisor responsibility. Thank you for trusting and giving me the possibility to work in an international research center and allowed me to develop my scientific career in a high standard and pleasant environment and you introduced me to clinical world and you made my dream came true. For me, hocam, you are a true mentor.

My thanks are also for my co-promoter Dr. Rick Bezemer who guided me through the process. Thank you for suggestions and all the comments, you put in the drafts, really helpful in improving my skills. I wish you all the best in your career!

I would like to thank to all PhD committee members for their willingness to join the committee: Prof. dr. F. Toraman, Prof. dr. J.H. Ravesloot, Prof. dr. S. Florquin, Prof. dr. E.T. van Bavel, Dr. E.G. Mik, Dr. C.T.P. Krediet.

Thanks to Dr. Jesse Ashruf. You came at the most trouble time of mine. Thank you very much for the contribution of the thesis to be published.

My sincere gratitude is also for Prof. Dr. Fevzi Toraman. Thank you for all opportunities me to add the clinical insight and you have always been a role model. It is honor for me to be a member of your team.

I also owe a debt of gratitude to Dr. Mathieu Legrand and Dr. Emre Almac for teaching a very hard surgery technique. Open abdominal surgery was really hard to perform, especially in rats.

Special thanks to Floris De Vries, Late Bas Bartels, Sema Aydin, Roos Koopman, Koray Yuruk and Bulent Ergin for warm friendship and fun work environment. Without you, my job would have undoubtedly been more difficult. I wish you lots of success with your further careers and rest in peace Bas!

Dear Peter, it was a great pleasure to know a person like you. I will never forget you and the period when we shared the room in AMC. Thanks for the heritage of NO analyzer. Rest in peace!

Special thanks for Prof. Dr. Gulderen Sahin. I will always remember your belief and motivation to me, which gave hope and strength.

I would also like to thank my dear friend Umut Naci. You always became a good example for me. I hope our friendship will last forever.

Finally, I would like to thank my family that in any way supports my PhD journey during all these years. Especially thank to my dear father. All successes in my life have something from you. Surely, you were with me from the beginning. Rest in peace!

Thanks to my dear sister Çiğdem. Maybe I could not show you my gratitude but surely I will need your support and constant love forever. Thanks to my dear mother. Great woman with great patience at all times.

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And special thanks to my dear wife Berna. Living with you in Amsterdam was amazing. Your personal support and motivation, especially during my stay in Amsterdam for months is one of the spines of this thesis.

Last but not least, I would like to express my gratitude to the Dutch culture as well as the research systems. Since 2001, the continuous interaction between us has shaped me and transformed me into the person that I am today.

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Curriculum vitae Ugur Aksu was born on February 1, 1979 in Istanbul, Turkey. He attended high school at

Haydarpasa Lycee in Istanbul. In 1996, he started his bachelor studies at the University of

Istanbul. During this time he developed an interest in physiology and participated in several

projects. The first project such project was “The effects of TNF-alpha on leukocyte

endothelial cell interactions during sepsis” project in which he was responsible for building a

research system in his department besides doing the experiments. This project was partly

supported by Prof. Ince from Academic Medical Centre in the Netherlands. He developed

skills in experimentation, enhanced his theoretical knowledge in cardiovascular system and

microcirculation and was oriented to scientific research environment. In 2003, he received his

master degree wıth the thesis titled “The effects of different nitric oxide synthase inhibitors

upon hemodynamic of rats received lipopolysaccaride“. In the same year, he started his PhD

studies at the Department of Biology, University of Istanbul. Besides his academic activities,

between November 2000 and January 2010, he was employed at the University of Istanbul as

a research assistant. In this position, his primary responsibility was to participate in teaching

activities and also to supervise laboratory work. In June 2009, he receveid PhD degree in

Biology with the thesis titled “Effects of β-3-agonists on cardiovascular system and adhesion

molecules in hyperglycemic rats.“ His thesis was about the investigation of β3-ARs’ effects

on the cardiovascular system and immunologic state during hyperglycemia. From July 2009

to August 2010, he worked on the basis of a research grant in the Department of Translational

Physiology at the Academic Medical Center, University of Amsterdam, The Netherlands.

Until present his research has focused lies on kidney perfusion and oxygenation changes in

various rat models of acute kidney injury. His research was supported by Dutch Kidney

Foundation and published in numerous international journals. Currently he is employed as an

associated professor in Istanbul University.

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Portfolio

Name PhD student: Uğur AKSU

PhD period: September 2009-November 2015

Name PhD supervisor: Prof.Dr. Can Ince

1) PhD Training

General courses

- Project management

- Biotek Epoch

- Biotek Synergy H1-M

- Course on Laboratory Animal Science

- Course on Neurobiology

- Course on Biostatistics

- Course on Cognitive Electrophysiology: ERP in the Evaluation of Cognitive Disorders

- PowerLab Training Course

- III. Ege Biennial Int. Neuroscience Grd. Summer School

- Symposium of Evolution on Biology Education

- Flow Cytometry Training VII

- National Student Scientific Session with International Participation

Specific courses

- CELL AND TISSUE PATOLOGY

- INTRACELLULAR TRAFFIC OF PROTEINS

- INTRO. TO CANCER BIOLOGY

- HORMONES OF VERTEBRATES

- ADVANCED PHYSIOLOGY I

- MEMBRANE PHYSIOLOGY

- ADVANCED PHYSIOLOGY II

(in doctorate)

- THE USE OF ANIMALS FOR EXPERIMENTS

- TISSUE CULTURES AND APPLICATION FIELDS

- TRACE ELEMENTS

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- ANTIOXIDATIONS AND DETOXIFICATIONS IN BIOLOGICAL SYSTEMS

- IN VIVO TECHNIQUES

- CYTOLOGICAL TECHNIQUES

- NEUROPHYSIOLOGY

Presentations

- "Fluoxetine Reduces The Lung Injury Induced By Infrarenal Abdominal Aortic Ischemia-Reperfusion In Rats.", 37th International Union of Physiological Sciences (IUPS) Congress,England

- "The effects of balanced and unbalanced colloid and crystalloid solutions on renal microvascular perfusion in endotoxemic rats", 31st ISICEM (International Symposium on Intensive Care and Emergency Medicine) 168 pp., Belgium 2010

- "The effects of balanced and unbalanced colloid and crystalloid solutions on renal oxygenation in a rat model of hemorrhagic shock and resuscitation" 31st ISICEM (International Symposium on Intensive Care and Emergency Medicine) 168 pp., Belgium, 2010

- "Atorvastatin improves development of pentylentetrzol-induced kindling, learning and memory disorders in rats" The 36th Congress of the International Union of Physiological Sciences P3PM-6-6 pp., Kyoto-Japan, 2009

- "Evaluation of the effects of α-lipoic acid and pycnogenol supplementation on NO release with ONOO-, 3-NTyr and total nitrite/nitrate levels in experimental cerebral ischemia-reperfusion subjected to diabetic rats", HSSR/AIST-NIEHS/NIH Joint International Symposium "Biomarkers of Oxidative Stress in Health and Disease" P4-6-1 pp., Osaka-Japan, 2008

- "Effects of lipoic acid on oxidative and nitrosative in cerebral ischemia reperfusion exposed diabetic rats" 17th IFCC-FESCC European Congress of Clinical Chemistry and Laboratory Medicine-Euromedlab, T128 pp., Amsterdam-Holland 2007

- "Exploring the recovering effects of pycnogenol on cerebral ischemia reperfusion in experimental diabetes model", 17th IFCC-FESCC European Congress of Clinical Chemistry and Laboratory Medicine-Euromedlab T127 pp., Amsterdam-Holland 2007

- "Effects of glucocorticoids on alpha adrenergic response during sepsis", 11th Annual Meeting of the European-Council- for Cardiovascular Research 769 pp., Nice-France, 2006

- "Lipoic acid attenuates oxidative and nitrosative stress, simultaneously sialic acid content in liver tissues of diabetic rats" 31st Congress of the Federation-of-European-Biochemical-Societies (FEBS), 179 pp., İstanbul-Türkiye, 2006

- "Comparative effects of nitric oxide inhibition by aminoguanidine before and after dopamine infusion on intestinal perfusion during endotoxemia", 24th Conference of the European-Society for Microcirculation 50 pp., Amsterdam-Holland, 2006

- "Alpha-Lipoic acid prevents oxidative injury in diabetic rats subjected to cerebral ischemia-reperfusion" 16th European Congress of Clinical Biochemistry and Laboratory Medicine (EUROMEDLAB 2005, 174 pp., Glasgow, 2005

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- "Mesenteric blood flow can be affected by aminoguanidine during sepsis", XXXV. International Congress of Physiological Sciences 11 pp., San Diego-USA, 2005

- "Oxidative injury in cerebral ischemia reperfusion exposed to diabetic rats", 13th Balkan Biochemical Biophysical Days & Meeting on Metabolic Disorders, 158 pp., Kuşadası-Türkiye, 2003.

Invited Oral presentations:

- Microcirculation in health and disease, Cerrahpasa Med. School, Istanbul, Turkiye, 2015.

- Perfusion heterogeneity in sepsis, İzmir Inovation meeting, Izmir, Turkiye, 2014.

- A biological mask: Glycocalyx, Acibadem University, Turkiye, 2013.

- Oxidative stress in disease, Acibadem University, Turkiye, 2013.

2. Teaching

Lecturing

- Animal Physiology (2015- )

- Selected Topics in Nervous System (2014- )

- The Modelling in Experimental Animals (2014- )

- Professional English (2010) (2013- )

- Supervising Student laboratory (2000-2013)

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List of publications

• Aksu U., Ergin B., Bezemer R., Milstein D., Ince C. Scavenging Reactive Oxygen Species Using Tempol In The Acute Phase Of Renal Ischemia/Reperfusion And Its Effects On Kidney Oxygenation And Nitric Oxide Levels, Intensive Care Medicine Experimental, vol.3 , pp.1-10, 2015

• Ischemia Modified Albumin; Does it Change During Pneumoperitoneum in Robotic

Prostatectomies? Accepted in International Braz J Urol, 2015

• Arıtürk C, Ozgen ZS, Kilercik M, Ulugöl H, Ökten EM, Aksu U, Karabulut H, Toraman F. Comparative Effects of Hemodilutional Anemia and Transfusion during Cardiopulmonary Bypass on Acute Kidney Injury: A Prospective Randomized Study. Arıtürk C, Ozgen ZS, Kilercik M, Ulugöl H, Ökten EM, Aksu U, Karabulut H, Toraman F. Heart Surg Forum. 2015 Aug 30;18(4):E154-E160.

• Erman H., Guner I., Yaman M.O., Uzun D.D., Gelisgen R., Aksu U., Yelmen N.,

Sahin G., Uzun H. The Effects Of Fluoxetine On Circulating Oxidative Damage Parameters In Rats Exposed To Aortic Ischemia-Reperfusion, European Journal of Pharmacology, vol.749, pp.56-61, 2015

• Toraman F., Aksu U. Monitoring of tissue oxygenation and perfusion. Turkiye

Klinikleri J Anest Reani, vol.8, pp.8-14, 2015

• Almac E., Bezemer R., Kandil A., Aksu U., Milstein D.M.J., Bakker J., Demirci-Tansel C., Ince C. Bis Maltolato Oxovanadium (Bmov) And Ischemia/Reperfusion-Induced Acute Kidney Injury In Rats. Intensive Care Medicine Experimental, vol.2, pp.1-9, 2014

• Aksu U., Guner I., Yaman O.M., Erman H., Uzun D., Sengezer-Inceli M., Sahin A.,

Yelmen N., Gelisgen R., Uzun H., Sahin G. Fluoxetine ameliorates imbalance of redox homeostasis and inflammation in an acute kidney injury model. J Physiol Biochem. 2014 Dec;70(4):925-34.

• Aksu U., Yanar K., Terzioglu D., Erkol T., Ece E., Aydin S., Uslu E., Cakatay U.

Effect of tempol on redox homeostasis and stress tolerance in mimetically aged Drosophila. Arch Insect Biochem Physiol. 2014 Sep;87(1):13-25.

• Guner I., Yaman M.O., Aksu U., Uzun D., Erman H., Inceli M., Gelisgen R., Yelmen

N., Uzun H., Sahin G. The effect of fluoxetine on ischemia-reperfusion after aortic surgery in a rat model. J Surg Res. 2014 Jun 1;189(1):96-105.

• Aksu U., Bezemer R., Ince C. Reply to: crystalloid resuscitation in hemorrhagic

shock. Resuscitation. 2012 Aug;83(8):e173. Epub 2012 May 4. No abstract available.

• Almac E., Aksu U., Bezemer R., Jong W., Kandil A., Yuruk K., Demirci-Tansel C., Ince C. The acute effects of acetate-balanced colloid and crystalloid resuscitation on renal oxygenation in a rat model of hemorrhagic shock. Resuscitation. 2012 Sep;83(9):1166-72. Epub 2012 Feb 19.

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• Aksu U., Bezemer R., Yavuz B., Kandil A., Demirci C., Ince C. Balanced vs unbalanced crystalloid resuscitation in a near-fatal model of hemorrhagic shock and the effects on renal oxygenation, oxidative stress, and inflammation. Resuscitation. 2012 Jun;83(6):767-73.

• Aksu U., Bezemer R., Demirci C., Ince C. Acute effects of balanced versus

unbalanced colloid resuscitation on renal macrocirculatory and microcirculatory perfusion during endotoxemic shock. Shock. 2012 Feb;37(2):205-9.

• Aksu U., Demirci C., Ince C. The pathogenesis of acute kidney injury and the toxic

triangle of oxygen, reactive oxygen species and nitric oxide. Contrib Nephrol. 2011;174:119-28. Epub 2011 Sep 9. Review.

• Uzum G., Akgun-Dar K., Aksu U. The effects of atorvastatin on memory deficit and

seizure susceptibility in pentylentetrazole-kindled rats. Epilepsy Behav. 2010 Nov;19(3):284-9.

• Guner I., Sahin G., Yelmen N.K., Aksu U., Oruc T., Yildirim Z.

Intracerebroventricular serotonin reduces the degree of acute hypoxic ventilatory depression in peripherally chemodenervated rabbits. Chin J Physiol. 2008 Jun 30;51(3):136-45. Erratum in: Chin J Physiol. 2008 Aug 31;51(4):261.

• Diler A.S., Uzüm G., Akgün Dar K., Aksu U., Atukeren P., Ziylan Y.Z. Sex

differences in modulating blood brain barrier permeability by NO in pentylenetetrazol-induced epileptic seizures. Life Sci. 2007 Mar 13;80(14):1274-81. Epub 2007 Jan 25.

• Guner I., Sahin G., Karaturan-Yelmen N., Aksu U., Oruc T., Yildirim Z. The Role of

Central Serotonin on Respiratory Regulation in Anaesthetized Rabbits. Cerrahpasa J Med 2006; 37: 98 – 102.