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UvA-DARE is a service provided by the library of the University of Amsterdam (http://dare.uva.nl)
UvA-DARE (Digital Academic Repository)
Renal perfusion and oxygenation during acute kidney injury
Aksu, U.
Link to publication
Citation for published version (APA):Aksu, U. (2015). Renal perfusion and oxygenation during acute kidney injury
General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s),other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons).
Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, statingyour reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Askthe Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam,The Netherlands. You will be contacted as soon as possible.
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
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
References
• Abuelo JG. Normotensive ischemic acute renal failure. N Engl J Med 2007;357:7975.
• Adler S, Huang H. Oxidant stress in kidneys of spontaneously hypertensive rats
involves both oxidase overexpression and loss of extracellular superoxide dismutase.
Am J Physiol Renal Physiol 2004;287:F907–F913.
• Bagshaw SM. The long-term outcome after acute renal failure. Curr Opin Crit Care
2006;12:561–566.
• Boldt J, Ince C. The impact of fluid therapy on microcirculation and tissue
oxygenation in hypovolemic patients: a review. Intensive Care Med 2010;36:1299–
1308.
• Cittanova ML, Leblanc I, Legendre C, Mouquet C, Riou B, Coriat P. Effect of
hydroxyethylstarch in brain-dead kidney donors on renal function in kidney-transplant
recipients. Lancet. 1996;348(9042):1620-2.
• Clanton TL. Hypoxia-induced reactive oxygen species formation in skeletal muscle. J
Appl Physiol 2007;102:2379–2388.
• Coca SG, Yalavarthy R, Concato J, Parikh CR: Biomarkers for the diagnosis and risk
stratification of acute kidney injury: a systematic review. Kidney Int 2008;73:1008–
1016.
• Cooper CE, Giulivi C. Nitric oxide regulation of mitochondrial oxygen consumption
II: molecular mechanism and tissue physiology. Am J Physiol Cell Physiol
2007;292:C1993–C2003.
• Deng A, Miracle CM, Suarez JM, Lortie M, Satriano J, Thomson SC, Munger KA,
Blantz RC. Oxygen consumption in the kidney: effects of nitric oxide synthase
isoforms and angiotensin II. Kidney Int 2005;68:723–730.
• Dubin A, Pozo MO, Casabella CA, Murias G, Pálizas F, Moseinco M, Pálizas F,
Kanoore Edul VS, Ince C. Hydroxyethyl starch 130/0.4 is superior to saline solution
for resuscitation of the microcirculation. J Crit Care, 2010;25(4):659.e1-8.
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
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.
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.
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.
56
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
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
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.
85
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
86
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].
87
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
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).
107
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
108
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.
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.
111
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.
References
• Adler S, Huang H (2002) Impaired regulation of renal oxygen consumption in
spontaneously hypertensive rats. J Am Soc Nephrol 13(7):1788–1794
• Aksu U, Demirci C, Ince C (2011) The pathogenesis of acute kidney injury and the
toxic triangle of oxygen, reactive oxygen species and nitric oxide. Contrib Nephrol
174:119–128
• Bagshaw SM, Mortis G, Doig CJ, Godinez-Luna T, Fick GH, Laupland KB (2006)
One-year mortality in critically ill patients by severity of kidney dysfunction: a
population-based assessment. Am J Kidney Dis 48(3):402–409
113
• Bell M, Martling CR (2007) Long-term outcome after intensive care: can we protect
the kidney? Crit Care 11(4):147
• Bezemer R, Faber DJ, Almac E et al (2010) Evaluation of multi-exponential curve
fitting analysis of oxygen-quenched phosphorescence decay traces for recovering
microvascular oxygen tension histograms. Med Biol Eng Comput 48(12):1233–1242.
• Chatterjee PK, Cuzzocrea S, Brown PA et al (2000) Tempol, a membrane-permeable
radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the
rat. Kidney Int 58(2):658–673
• Chatterjee PK, Patel NS, Kvale EO et al (2002) Inhibition of inducible nitric oxide
synthase reduces renal ischemia/ reperfusion injury. Kidney Int 61(3):862–871
• Chatterjee PK (2007) Novel pharmacological approaches to the treatment of renal
ischemia-reperfusion injury: a comprehensive review. Naunyn Schmiedebergs Arch
Pharmacol 376(1–2):1–43
• Fujii T, Takaoka M, Ohkita M, Matsumura Y (2005) Tempol protects against ischemic
acute renal failure by inhibiting renal noradrenaline overflow and endothelin-1
overproduction. Biol Pharm Bull 28(4):641–645
• Gomes EC, Silva AN, de Oliveira MR (2012) Oxidants, antioxidants, and the
beneficial roles of exercise-induced production of reactive species. Oxid Med Cell
Longev 2012:756132.
• Guz G, Demirogullari B, Ulusu NN et al (2007) Stobadine protects rat kidney against
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
119
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
120
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.
121
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,
122
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
124
microvasculaire oxygenatie verbeterde. Samengevat, de effecten van TEMPOL leiden tot een
bescheiden (maar niet statistisch significante) verbetering van de nierfunctie na I/R.
125
References list
• Abuelo JG. Normotensive ischemic acute renal failure. N Engl J Med 2007;357:7975.
• Adler S, Huang H. Impaired regulation of renal oxygen consumption in spontaneously hypertensive rats. J Am Soc Nephrol 2002;13:1788–94.
• Adler S, Huang H. Oxidant stress in kidneys of spontaneously hypertensive rats
involves both oxidase overexpression and loss of extracellular superoxide dismutase. Am J Physiol Renal Physiol 2004;287:F907– F913.
• Aksu U, Demirci-Tansel C, Ince C. Effects of balanced and unbalanced colloid and
crystalloid solutions on renal microvascular perfusion in endotoxemic rats. Crit Care 2010;168(14 Suppl 1):501.
• Aksu U, Demirci C, Ince C (2011) The pathogenesis of acute kidney injury and the
toxic triangle of oxygen, reactive oxygen species and nitric oxide. Contrib Nephrol 174:119–128.
• Altun D, Uysal H, Askin H, Ayar A. 2011. Determination of the effects of genistein
on the longevity of Drosophila melanogaster meigen (Diptera; Drosophilidae). Bull Environ Contam Toxicol 86:120–123.
• Aminoff D. Methods for the quantitative estimation of N-acetylneuraminic acid and
their application to hydrolysates of sialomucoids. Biochem J. 1961;81:384–392.
• Aydin S, Yanar K, Atukeren P, Dalo E, Sitar ME, Uslu E, Caf N, Cakatay U. Comparison of oxidative stress biomarkers in renal tissues of D-galactose induced, naturally aged and young rats. Biogerontology. 2012;13:251–260.
• Augustyniak A, Bartosz G, Cipak A, Duburs G, Horakova L, Luczaj W, Majekova M,
Odysseos AD, Rackova L, Skrzydlewska E, Stefek M, Strosov´a M, Tirzitis G, Venskutonis PR, Viskupicova J, Vraka PS, Zarkovi´c N. Natural and synthetic antioxidants: an updated overview. Free Radic Res. 2010;44:1216–1262.
• Bagshaw SM: The long- term outcome after acute renal failure. Curr Opin Crit Care.
2006;12:561–566.
• Bell M, Martling CR (2007) Long-term outcome after intensive care: can we protect the kidney? Crit Care 11(4):147.
126
• Bezemer R, Faber DJ, Almac E et al (2010) Evaluation of multi-exponential curve fitting analysis of oxygen-quenched phosphorescence decay traces for recovering microvascular oxygen tension histograms. Med Biol Eng Comput 48(12):1233–1242.
• Bezemer R, Legrand M, Klijn E, Heger M, Post IC, van Gulik TM, Payen D, Ince C.
Real-time assessment of renal cortical microvascular perfusion heterogeneities using near- infrared laser speckle imaging. Opt Express. 2010;18:15054–15061.
• Boldt J, Ince C. The impact of fluid therapy on microcirculation and tissue
oxygenation in hypovolemic patients: a review. Intensive Care Med. 2010;36:1299–1308.
• Boldt J, Muller M, Heesen M, Heyn O, Hempelmann G. Influence of different volume
therapies on platelet function in the critically ill. Intensive Care Med. 1996;22:1075–81.
• Breusing N, Grune T. 2010. Biomarkers of protein oxidation from a chemical,
biological and medical point of view. Exp Gerontol 45:733–737.
• Brill SA, Stewart TR, Brundage SI, Schreiber MA. Base deficit does not predict mortality when secondary to hyperchloremic acidosis. Shock. 2002;17:459–62.
• Brunkhorst FM, Oppert M. Nephrotoxicity of hydroxyethyl starch solution. Br J
• Bullivant EM, Wilcox CS, Welch WJ. Intrarenal vasoconstriction during
hyperchloremia: Role of thromboxane. Am J Physiol. 1989;256(1 Pt 2):F152–7.
• Cakatay U. Pro-oxidant actions of alpha-lipoic acid and dihydrolipoic acid. Med Hypotheses. 2006;66:110–117.
• Cakatay U. Protein redox-regulation mechanisms in aging. In: Bondy S, Maiese K,
editors. Aging and age related disorders. New York: Springer, 2011;p 3–25.
• Cakatay U, Aydin S, Atukeren P, Yanar K, Sitar ME, Dalo E, Uslu E. Increased protein oxidation and loss of protein-bound sialic acid in hepatic tissues of D-galactose induced aged rats. Curr Aging Sci. 2013;6:135–141.
127
• Chatterjee PK, Cuzzocrea S, Brown PA et al (2000) Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat. Kidney Int 58(2):658–673
• Chatterjee PK, Patel NS, Kvale EO et al (2002) Inhibition of inducible nitric oxide
synthase reduces renal ischemia/ reperfusion injury. Kidney Int 61(3):862–871
• Chatterjee PK (2007) Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review. Naunyn Schmiedebergs Arch Pharmacol 376(1–2):1–43
• Cai B, Chen F, Lin X, et al. Anti-inflammatory adjuvant in resuscitation fluids
improves survival in hemorrhage. Crit Care Med. 2009;37:860–8.
• Carlstrom M, Brown RD, Yang T, Hezel M, Larsson E, Scheffer PG, Teerlink T, Lundberg JO, Persson AE. L-arginine or tempol supplementation improves renal and cardiovascular function in rats with reduced renal mass and chronic high salt intake. Acta Physiol (Oxf) 2013;207:732–741.
• Celotto AC, Capellini VK, Baldo CF, Dalio MB, Rodrigues AJ, Evora PR. Effects of
acid–base imbalance on vascular reactivity. Braz J Med Biol Res. 2008;41:439–45.
• Cittanova ML, Leblanc I, Legendre C, Mouquet C, Riou B, Coriat P. Effect of hydroxyethylstarch in brain-dead kidney donors on renal function in kidney-transplant recipients. Lancet. 1996;348(9042):1620-2.
• Clancy D, Birdsall J. Flies, worms and the free radical theory of ageing. Ageing Res
Rev. 2013;12:404–412.
• Clanton TL. Hypoxia- induced reactive oxygen species formation in skeletal muscle. J Appl Physiol. 2007;102:2379–2388.
• Coca SG, Yalavarthy R, Concato J, Parikh CR. Biomarkers for the diagnosis and risk
stratification of acute kidney injury: a systematic review. Kidney Int. 2008;73:1008–1016.
• Coimbra R, Porcides R, Loomis W, et al. HSPTX protects against hemorrhagic shock
resuscitation-induced tissue injury: an attractive alternative to Ringer’s lactate. J Trauma. 2006;60:41–51.
immobilization of leukocytes at the endothelial surface. Arterioscler Thromb Vasc Biol. 2003;23:1541–7.
128
• Cooper CE, Giulivi C. Nitric oxide regulation of mitochondrial oxygen consumption
II: molecular mechanism and tissue physiology. Am J Physiol Cell Physiol. 2007;292:C1993–2003.
• Crimi E, Zhang H, Han RN, Del Sorbo L, Ranieri VM, Slutsky AS. Ischemia and
reperfusion increases susceptibility to ventilator-induced lung injury in rats. Am J Respir Crit Care Med. 2006;174:178–86.
• Cui X, Wang L, Zuo P, Han Z, Fang Z, Li W, Liu J. D-galactose-caused life
shortening in Drosophila melanogaster and Musca domestica is associated with oxidative stress. Biogerontology 2004;5:317–325.
• De Backer D, Creteur J, Preiser JC, Dubois MJ, Vincent JL. Microvascular blood flow
is altered in patients with sepsis. Am J Respir Crit Care Med. 2002;166:98-104.
• Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R, Reinhart K, Angus DC, Brun-Buisson C, Beale R, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008. Crit Care Med. 2008;36:296-32.
• Demoncheaux EA, Higenbottam TW, Kiely DG, et al. Decreased whole body
endogenous nitric oxide production in patients with primary pulmonary hypertension. J Vasc Res. 2005;42:133–6.
• Deng A, Miracle CM, Suarez JM, et al. Oxygen consumption in the kidney: effects of
nitric oxide synthase isoforms and angiotensin II. Kidney Int. 2005;68:723–730.
• Dorje P, Adhikary G, Tempe DK. Avoiding latrogenic hyperchloremic acidosis – call for a new crystalloid fluid. Anesthesiology. 2000;92:625–6.
• Dröge W. Free radicals in the physiological control of cell function. Physiol Rev.
2002;82:47–95.
• Dubin A, Pozo MO, Casabella CA, Murias G, Pálizas F, Moseinco M, Pálizas F, Kanoore Edul VS, Ince C. Hydroxyethyl starch 130/0.4 is superior to saline solution for resuscitation of the microcirculation. J Crit Care, 2010 Dec;25(4):659.e1-8.
• Dunphy I, Vinogradov SA, Wilson DF. Oxyphor R2 and G2: phosphors for measuring
oxygen by oxygen-dependent quenching of phosphorescence. Anal Biochem 2002;310:1918.
129
• Evans RG, Gardiner BS, Smith DW, O’Connor PM. Intrarenal oxygenation: Unique challenges and the biophysical basis of homeostasis. Am J Physiol Renal Physiol. 2008;295:F1259–70.
lipocalin at ICU admission predicts for acute kidney injury in adult patients. Am J Respir Crit Care Med. 2011;183:907– 914.
• Gomes EC, Silva AN, de Oliveira MR (2012) Oxidants, antioxidants, and the
beneficial roles of exercise-induced production of reactive species. Oxid Med Cell Longev 2012:756132.
• Gredilla R, Barja G. Caloric restriction, aging and oxidative stress. Endocrinology.
2005;146:3713–3717.
• Goswami K, Koner BC. Level of sialic acid residues in platelet proteins in diabetes, aging, and Hodgkin’s lymphoma: a potential role of free radicals in desialylation. Biochem Biophys Res Commun. 2002;297:502–505.
• Gunnerson KJ, Saul M, He S, Kellum JA. Lactate versus non-lactate metabolic
acidosis: a retrospective outcome evaluation of critically ill patients. Crit Care. 2006;10:R22.
• Guz G, Demirogullari B, Ulusu NN et al (2007) Stobadine protects rat kidney against
• Habib RH, Zacharias A, Schwann TA, et al. Role of hemodilutional anemia and transfusion during cardiopulmonary bypass in renal injury after coronary
130
revascularization: implications on operative outcome. Crit Care Med. 2005;33:1749–1756.
• Haisch G, Boldt J, Krebs C, Kumle B, Suttner S, Schulz A. The influence of
intravascular volume therapy with a new hydroxyethyl starch preparation (6% HES 130/0.4) on coagulation in patients undergoing major abdominal surgery. Anesth Analg. 2001;92:565–71.
• Hanasand M, Omdal R, Norheim KB, Goransson LG, Brede C, Jonsson G. Improved
detection of advanced oxidation protein products in plasma. Clin Chim Acta. 2012;413:901–906.
Hydroxyethyl starch (130 kD), but not crystalloid volume support, improves microcirculation during normotensive endotoxemia.Anesthesiology. 2002;97:460–70.
• Holstein-Rathlou NH, Sosnovtseva OV, Pavlov AN, Cupples WA, Sorensen CM,
Marsh DJ: Nephron blood flow dynamics measured by laser speckle contrast imaging. Am J Physiol Renal Physiol. 2011;300:F319-F329.
• Horstick G, Lauterbach M, Kempf T, et al. Early albumin infusion improves global
and local hemodynamics and reduces inflammatory response in hemorrhagic shock. Crit Care Med. 2002;30:851–5.
• Hoste EA, Kellum JA (2006) Acute kidney injury: epidemiology and diagnostic
criteria. Curr Opin Crit Care 12(6):531–537
• Huangfu J, Liu J, Sun Z, Wang M, Jiang Y, Chen ZY, Chen F. Anti-ageing effects of astaxanthinrich alga Haematococcus pluvialis on fruit flies under oxidative stress. J Agric Food Chem. 2013;61(32):7800–7804.
• Huttemann M, Lee I, Grossman LI et al (2012) Phosphorylation of mammalian
cytochrome c and cytochrome c oxidase in the regulation of cell destiny: respiration, apoptosis, and human disease. Adv Exp Med Biol 748:237–264
• Izmaylov DM, Obukhova LK. Geroprotector efficiency depends on viability of control
population: life span investigation in D. melanogaster. Mech Ageing Dev. 1996;91(3):155–164.
• Jeong GY, Chung KY, Lee WJ, Kim YS, Sung SH (2004) The effect of a nitric oxide
donor on endogenous endothelin-1 expression in renal ischemia/reperfusion injury. Transplant Proc 36(7):1943–1945
• Johannes T, Mik EG, Ince C. Dual-wavelength phosphorimetry for determination of
cortical and subcortical microvascular oxygenation in rat kidney. J Appl Physiol. 2006;100:1301–10.
• Johannes T, Mik EG, Ince C. Nonresuscitated endotoxemia induces microcirculatory
hypoxic areas in the renal cortex in the rat. Shock 2009;31:97-103.
• Johannes T, Mik EG, Klingel K, Dieterich HJ, Unertl KE, Ince C. Endotoxin- induced acute renal failure reversed by low-dose dexamethasone. Shock. 2009;31:521– 528.
• Johannes T, Ince C, Klingel K, Unertl KE, Mik EG. Iloprost restores renal
oxygenation and kidney function in endotoxemia- related acute renal failure in the rat. Crit Care Med. 2009;37:1423– 1432.
• Johannes T, Mik EG, Nohe B, Raat NJ, Unertl KE, Ince C. Influence of fluid
resuscitation on renal microvascular PO2 in a normotensive rat model of endotoxemia. Crit Care. 2006;10:R88.
• Johannes T, Mik EG, Nohe B, Unertl KE, Ince C. Acute decrease in renal
microvascular PO2 during acute normovolemic hemodilution. Am J Physiol Renal Physiol. 2007;292:F796–803.
• Juca CA, Rey LC, Martins CV. Comparison between normal saline and a
polyelectrolyte solution for fluid resuscitation in severely dehydrated infants with acute diarrhoea. Ann Trop Paediatr. 2005;25:253–60.
• Kellum JA, Bellomo R, Kramer DJ, Pinsky MR. Etiology of metabolic acidosis during
saline resuscitation in endotoxemia. Shock. 1998;9:364–8.
• Kellum JA. Metabolic acidosis in the critically ill: lessons from physical chemistry. Kidney Int Suppl. 1998;66:S81–6.
• Kellum JA. Fluid resuscitation and hyperchloremic acidosis in experimental sepsis:
improved short-term survival and acid–base balance with Hextend compared with saline. Crit Care Med. 2002;30:300–5.
132
• Kellum JA. Saline-induced hyperchloremic metabolic acidosis.Crit Care Med. 2002;30:259-61.
• Kellum JA, Song M, Li J. Science review: Extracellular acidosis and the immune
response: clinical and physiologic implications. Crit Care. 2004;8:331–336.
• Kellum JA, Song M, Venkataraman R. Effects of hyperchloremic acidosis on arterial pressure and circulating inflammatory molecules in experimental sepsis. Chest. 2004;125:243-248.
• Kellum JA, Song M, Almasri E. Hyperchloremic acidosis increases circulating
inflammatory molecules in experimental sepsis. Chest. 2006;130:962–7.
• Kemming GI, Meisner FG, Wojtczyk CJ, et al. Oxygent as a top load to colloid and hyperoxia is more effective in resuscitation from hemorrhagic shock than colloid and hyperoxia alone. Shock. 2005;24:245–54.
• Khajavi MR, Etezadi F, Moharari RS, et al. Effects of normal saline vs lactated
ringer’s during renal transplantation. Ren Fail. 2008;30:535–9.
• Kiil F. Renal energy metabolism and regulation of sodium reabsorption. Kidney Int. 1977;11:153–60.
• Klenzak J, Himmelfarb J. Sepsis and the kidney. Crit Care Clin. 2005;21:211-222.
• Kortgen A, Niederprum P, Bauer M. Implementation of an evidence-based Bstandard
operating procedure and outcome in septic shock. Crit Care Med 2006;34:943-949.
• Krieter H, Brückner UB, Kefalianakis F, Messmer K. Does colloid-induced plasma hyperviscosity in haemodilution jeopardize perfusion and oxygenation of vital organs? Acta Anaesthesiol Scand. 1995;39:236–44.
• Kucuk HF, Kaptanoglu L, Ozalp F et al (2006) Role of glyceryl trinitrate, a nitric
oxide donor, in the renal ischemia- reperfusion injury of rats. Eur Surg Res 38(5):431–437
• Kumar A, Prakash A, Dogra S. 2010. Naringin alleviates cognitive impairment,
mitochondrial dysfunction and oxidative stress induced by D-galactose in mice. Food Chem Toxicol 48:626–632.
133
• Lacy JH, Wright CB. Use of plasma volume expanders in myocardial revascularisation. Drugs. 1992;44:720-727.
• Lameire N, Van Biesen W, Vanholder R. Acute renal failure. Lancet. 2005;365:417–
430.
• Lameire N, Van Biesen W, Vanholder R. Acute renal failure. Lancet. 2005;365:417–430.
• Lang K, Boldt J, Suttner S, Haisch G. Colloids versus crystalloids and tissue oxygen
tension in patients undergoing major abdominal surgery. Anesth Analg. 2001;93:405–9.
• Lassen UV, Thaysen JH. Correlation between sodium transport and oxygen
consumption in isolated renal tissue. Biochim Biophys Acta. 1961;47:616–618.
• Lawler JM, Kwak HB, Kim JH, Suk MH. Exercise training inducibility of MnSOD protein expression and activity is retained while reducing prooxidant signaling in the heart of senescent rats. Am J Physiol Regul Integr Comp Physiol. 2009;296:R1496–R1502.
• Le Dorze M, Legrand M, Payen D, Ince C (2009) The role of the microcirculation in
acute kidney injury. Curr Opin Crit Care 15(6):503–508.
• Legendre C, Thervet E, Page B, Percheron A, Noel LH, Kreis H. Hydroxyethyl starch and osmotic-nephrosis-like lesions in kidney transplantation. Lancet. 1993;342:248-249.
• Legrand M, Almac E, Mik EG, Johannes T, Kandil A, Bezemer R, Payen D, Ince C.
L- NIL prevents renal microvascular hypoxia and increase of renal oxygen consumption after ischemia- reperfusion in rats. Am J Physiol Renal Physiol. 2009;296:F1109– F1117.
• Legrand M, Bezemer R, Kandil A, Demirci C, Payen D, Ince C. The role of renal
hypoperfusion in development of renal microcirculatory dysfunction in endotoxemic rats. Intensive Care Med. 2011;7:1534-542.
• Legrand M, Mik EG, Balestra GM, et al. Fluid resuscitation does not improve renal
oxygenation during hemorrhagic shock in rats. Anesthesiology. 2010;112:119–27.
• Legrand M, Mik EG, Johannes T, Payen D, Ince C. Renal hypoxia and dysoxia following reperfusion of the ischemic kidney. Mol Med 2008;14:502– 516.
134
• Le Tulzo Y, Shenkar R, Kaneko D, et al. Hemorrhage increases cytokine expression in lung mononuclear cells in mice: involvement of catecholamines in nuclear factor-kappaB regulation and cytokine expression. J Clin Invest. 1997;99:1516–24.
• Lindseth RE, Hamburger RJ, Szwed JJ, Kleit SA. Acute renal failure following trauma. J Bone Joint Surg Am. 1975;57:830–5.
• Liskaser FJ, Bellomo R, Hayhoe M, et al. Role of pump prime in the etiology and
pathogenesis of cardiopulmonary bypass-associated acidosis. Anesthesiology 2000;93:11703.
• Liu LM, Ward JA, Dubick MA. Effects of crystalloid and colloid resuscitation on
hemorrhage-induced vascular hyporesponsiveness to norepinephrine in the rat. J Trauma 2003;54:S159–68.
• Li Y, Chen X. Sialic acid metabolism and sialyltransferases: natural functions and
• Long J, Wang X, Gao H, Liu Z, Liu C, Miao M, Cui X, Packer L, Liu J. D-galactose toxicity in mice is associated with mitochondrial dysfunction: protecting effects ofmitochondrial nutrient R-alpha-lipoic acid. Biogerontology. 2007;8:373–381.
• Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin
phenol reagent. J Biol Chem. 1951;193:265–275.
• Lushchak OV, Gospodaryov DV, Rovenko BM, Yurkevych IS, Perkhulyn NV, Lushchak VI. Specific dietary carbohydrates differentially influence the life span and fecundity of Drosophila melanogaster. J Gerontol A Biol Sci Med Sci. 2013;69(1):3–12.
• Maiese K, Chong ZZ. Nicotinamide: necessary nutrient emerges as a novel
cytoprotectant for the brain. Trends Pharmacol Sci. 2003;24:228–232.
• Maiese K, Chong ZZ. Insights into oxidative stress and potential novel therapeutic targets for Alzheimer’s disease. Restor Neurol Neurosci. 2004;22:87–104.
• Mark LA, Robinson AV, Schulak JA (2005) Inhibition of nitric oxide synthase
reduces renal ischemia/reperfusion injury. J Surg Res 129(2):236–241.
• Marx G, Cobas Meyer M, Schuerholz T, Vangerow B, Gratz KF, Hecker H, Sumpelmann R, Rueckoldt H, Leuwer M. Hydroxyethyl starch and modified fluid gelatin maintain plasma volume in a porcine model of septic shock with capillary leakage. Intensive Care Med. 2002;28:629 635.
135
• Marx G. Fluid therapy in sepsis with capillary leakage. Eur J Anaesthesiol.
2003;20:429-442.
• McFarlane C, Lee A. A comparison of Plasma Lyte 148 and 0.9% saline for intraoperative fluid replacement. Anaesthesia. 1994;49:779–81.
• Michel F, Bonnefont-Rousselot D, Mas E, Drai J, Therond P (2008) Biomarkers of
lipid peroxidation: analytical aspects. Ann Biol Clin (Paris) 66(6):605–620
• Mik EG, Johannes T, Ince C. Monitoring of renal venous PO2 and kidney oxygen consumption in rats by a near-infrared phosphorescence lifetime technique. Am J Physiol Renal Physiol. 2008;294:F676–81.
• Mik EG, van Leeuwen TG, Raat NJ, Ince C. Quantitative determination of localized
tissue oxygen concentration in vivo by two-photon excitation phosphorescence lifetime measurements. J Appl Physiol. 2004;97:1962–9.
• Mills GH. Hydroxyethyl starch: does our choice of colloid prevent or add to renal
impairment? Br J Anaesth. 2007;98:157-159.
• Morariu AM, Maathuis MH, Asgeirsdottir SA, et al. Acute isovolemic hemodilution triggers proinflammatory and procoagulatory endothelial activation in vital organs: role of erythrocyte aggregation. Microcirculation. 2006;13:397–409.
• Morgan TJ. The meaning of acid-base abnormalities in the intensive care unit: part
IIIVeffects of fluid administration. Crit Care. 2005;9:204-211.
• Morris JA, Mucha P, Ross SE, et al. Acute posttraumatic renal failure: a multicenter perspective. J Trauma. 1991;31:1584–90.
• Mythen MG, Salmon JB, Webb AR. The rational administration of colloids. Blood
Rev. 1993;7:223-228.
• Na HJ, Park JS, Pyo JH, Lee SH, Jeon HJ, Kim YS, Yoo MA. Mechanism of metformin: inhibition of DNA damage and proliferative, activity in Drosophila midgut stem cell. Mech Ageing Dev. 2013;134(9):381–390.
negative bacteremia produces both severe systolic and diastolic cardiac dysfunction in a canine model that simulates human septic shock. J Clin Invest. 1986;78:259-270.
136
• Nath KA, Norby SM (2000) Reactive oxygen species and acute renal failure. Am J Med 109(8):665–678
• Naylor JM, Forsyth GW. The alkalinizing effects of metabolizable bases in the healthy
calf. Can J Vet Res. 1986;50:509–51.
• Nieuwdorp M, Mooij HL, Kroon J, et al. Endothelial glycocalyx damage coincides with microalbuminuria in type 1 diabetes. Diabetes. 2006;55:1127–32.
• Nohe B, Johannes T, Reutershan J, et al. Synthetic colloids attenuate leukocyte–
endothelial interactions by inhibition of integrin function. Anesthesiology. 2005;103:759–767.
• Noiri E, Nakao A, Uchida K et al (2001) Oxidative and nitrosative stress in acute renal
ischemia. Am J Physiol Renal Physiol 281(5):F948–F957
• Patel NS, Chatterjee PK, Chatterjee BE et al (2002) TEMPONE reduces renal dysfunction and injury mediated by oxidative stress of the rat kidney. Free Radic Biol Med 33(11):1575–1589
• Payen D, de Pont AC, Sakr Y, Spies C, Reinhart K, Vincent JL. A positive fluid
balance is associated with a worse outcome in patients with acute renal failure. Critical Care. 2008;12:R74.
• Pedoto A, Caruso JE, Nandi J, et al. Acidosis stimulates nitric oxide production and lung damage in rats. Am J Respir Crit Care Med. 1999;159:397–402.
• Ramesh T, Kim SW, Sung JH, Hwang SY, Sohn SH, Yoo SK, Kim SK. Effect of fermented Panax ginseng extract (GINST) on oxidative stress and antioxidant activities in major organs of aged rats. Exp Gerontol. 2012;47:77–84.
metabolism in normal human subjects. Am J Kidney Dis. 1982;2:47–57.
• Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, Peterson E, Tomlanovich M: Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001;345:1368-1377.
• Rubio-Gayosso I, Platts SH, Duling BR. Reactive oxygen species mediate
modification of glycocalyx during ischemia–reperfusion injury. Am J Physiol Heart Circ Physiol. 2006;290:H2247–56.
• Sakr Y, Dubois MJ, De Backer D, Creteur J, Vincent JL. Persistent microcirculatory
alterations are associated with organ failure and death in patients with septic shock. Crit Care Med. 2004;32:1825-1831.
survival with early fluid resuscitation following hemorrhagic shock. World J Surg. 2001;25:592–7.
• Scheingraber S, Rehm M, Sehmisch C, Finsterer U. Rapid saline infusion produces
hyperchloremic acidosis in patients undergoing gynecologic surgery. Anesthesiology. 1999;90:1265–70.
• Schindler R. Causes and therapy of microinflammation in renal failure. Nephrol Dial
Transplant. 2004;19(Suppl 5):V34-V40.
• Schortgen F, Lacherade JC, Bruneel F, et al. Effects of hydroxy- ethylstarch and gelatin on renal function in severe sepsis: a multi- centre randomised study. Lancet. 2001; 357: 911–6
138
• Sebat F, Johnson D, Musthafa AA, Watnik M, Moore S, Henry K, Saari M. A multidisciplinary community hospital program for early and rapid resuscitation of shock in nontrauma patients. Chest. 2005;127:1729-1743.
• Selmeci L. Advanced oxidation protein products (AOPP): novel uremic toxins, or
components of the non-enzymatic antioxidant system of the plasma proteome? Free Radic Res. 2011;45(10):1115– 1123.
• Senturk LM, Seli E, Gutierrez LS, Mor G, Zeyneloglu HB, Arici A. Monocyte
chemotactic protein-1 expression in human corpus luteum. Mol Hum Reprod. 1999;5:697–702.
• Smith I, Kumar P, Molloy S, et al. Base excess and lactate as prognostic indicators for
patients admitted to intensive care. Intensive Care Med. 2001;27:74–83.
• Spahn DR, Cerny V, Coats TJ, et al. Management of bleeding following major trauma: a European guideline. Crit Care. 2007;11:R17.
• Stern SA. Low-volume fluid resuscitation for presumed hemorrhagic shock: Helpful
or harmful? Curr Opin Crit Care. 2001;7:422–30.
• Stewart PA. Modern quantitative acid–base chemistry. Can J Physiol Pharmacol. 1983;61:1444–61.
• Sun Y, Oberley LW, Li Y. A simple method for clinical assay of superoxide
dismutase. Clin Chem. 1988;34:497–500.
• Svensen C, Hahn RG. Volume kinetics of Ringer solution, dextran 70, and hypertonic saline in male volunteers Anesthesiology. 1997;87:204–12.
• Taylor KR, Gallo RL. Glycosaminoglycans and their proteoglycans: hostassociated
molecular patterns for initiation and modulation of inflammation. FASEB J. 2006;20:9–22.
• Terada LS, Guidot DM, Leff JA, et al. Hypoxia injures endothelial cells by increasing
endogenous xanthine oxidase activity. Proc Natl Acad Sci USA. 1992;89:3362–6.
• Thijs A, Thijs LG. Pathogenesis of renal failure in sepsis. Kidney Int Suppl. 1998;66:S34-S37.
• Tram TH, Brand Miller JC, McNeil Y, McVeagh P. Sialic acid content of infant
saliva: comparison of breast fed with formula fed infants. Arch Dis Child. 1997;77:315–318.
139
• Tsai AG, Acero C, Nance R, et al. Elevated plasma viscosity in extreme hemodilution
increases perivascular nitric oxide concentration and microvascular perfusion. Am J Physiol Heart Circ Physiol. 2005;288:H1730–9.
• Tsai AG, Intaglietta M. High viscosity plasma expanders: volume restitution fluids for
lowering the transfusion trigger. Biorheology. 2001;38:229–37.
• Ulloa L, Tracey KJ. The “cytokine profile”: a code for sepsis. Trends Mol Med. 2005;11:56–63.
• Uzun D, Korkmaz GG, Sitar ME, Cebe T, Yanar K, Cakatay U, Aydın S. Oxidative
damage parameters in renal tissues of aged and young rats based on gender. Clin Interv Aging. 2013; 8:809–815.
• Van Bommel J, Siegemund M, Henny ChP, Ince C. Heart, kidney and intestine have
different tolerances for anemia. Transl Res. 2008;151:110–7.
• Vinas JL, Sola A, Genesca M, Alfaro V, Pi F, Hotter G (2006) NO and NOS isoforms in the development of apoptosis in renal ischemia/reperfusion. Free Radic Biol Med 40(6):992–1003
• Vincent JL, Gerlach H. Fluid resuscitation in severe sepsis and septic shock: an
evidence- based review. Crit Care Med. 2004;32(suppl 11):S451– S454.
• Vink H, Duling BR. Identification of distinct luminal domains for macromolecules,erythrocytes, and leukocytes within mammalian capillaries. Circ Res. 1996;79:581–9.
• Vinogradov SA, Fernandez-Seara MA, Dupan BW, Wilson DF. A method for
measuring oxygen distributions in tissue using frequency domain phosphorometry. Comp Biochem Physiol A Mol Integr Physiol. 2002;132:147–52.
• Wan L, Bagshaw SM, Langenberg C, Saotome T, May C, Bellomo R.
Pathophysiology of septic acute kidney injury: what do we really know? Crit Care Med. 2008; 36(suppl 4):S198-S203.
• Wang W, Jittikanont S, Falk SA, Li P, Feng L, Gengaro PE, Poole BD, Bowler RP,
Day BJ, Crapo JD, Schrie RW. Interaction among nitric oxide, reactive oxygen species, and antioxidants during endotoxemia-related acute renal failure. Am J Physiol Renal Physiol. 2003;284:F532–F537.
• Waters JH, Gottlieb A, Schoenwald P, Popovich MJ, Sprung J, Nelson DR. Normal
saline versus lactated Ringer’s solution for intraoperative fluid management in patients undergoing abdominal aortic aneurysm repair: an outcome study. Anesth Analg. 2001;93:817-22.
• Waters JH, Miller LR, Clack S, Kim JV. Cause of metabolic acidosis in prolonged
surgery. Crit Care Med. 1999;27:2142–6.
• Welch WJ, Baumgartl H, Lubbers D, Wilcox CS. Nephron PO2 and renal oxygen usage in the hypertensive rat kidney. Kidney Int. 2001;59:230–7.
• Welch WJ, Mendonca M, Aslam S, Wilcox CS (2003) Roles of oxidative stress and
AT1 receptors in renal hemodynamics and oxygenation in the postclipped 2K,1C kidney. Hypertension 41(3 Pt 2):692–696
• Weinberg JR, Boyle P, Thomas K, Murphy K, Tooke JE, Guz A. Capillary blood cell
velocity is reduced in fever without hypotension. Int J Microcirc Clin Exp. 1991;10:13-19.
• Wettstein R, Erni D, Intaglietta M, Tsai AG. Rapid restoration of microcirculatory
blood flow with hyperviscous and hyperoncotic solutions lowers the transfusion trigger in resuscitation from hemorrhagic shock. Shock. 2006;25:641–6.
• Wiedermann CJ. Hydroxyethyl starch; can the safety problems be ignored? Wien Klin
Wochenschr. 2004;116:583-594.
• Wilcox CS. Regulation of renal blood flow by plasma chloride. J Clin Invest 1983;71:726-735.
• Wilcox CS, Peart WS. Release of renin and angiotensin II into plasma and lymph
during hyperchloremia. Am J Physiol. 1987;253(4 Pt 2):F734–41.
• Wilcox CS. Effects of tempol and redox-cycling nitroxides in models of oxidative stress. Pharmacol Ther. 2010;126:119–145.
• Williams EL, Hildebrand KL, Mc Cormick SA, Bedel MJ. The effect of intravenous
lactated ringers solution versus 0.9% sodium chloride on serum osmolarity in human volunteers. Anesth Analg. 1999;88:999–1003.
141
• Winkelmayer WC, Glynn RJ, Levin R, Avorn J. Hydroxyethyl starch and change in renal function in patients undergoing coronary artery bypass graft surgery. Kidney Int. 2003;64(3):1046-9.
• Xiao N, Wang XC, Diao YF, Liu R, Tian KL. Effect of initial fluid resuscitation on
subsequent treatment in uncontrolled hemorrhagic shock in rats. Shock. 2004;21:276 80.
• Yada-Langui MM, Anjos-Valotta EA, Sannomiya P, Rocha e Silva M, Coimbra R.
Fluid resuscitation affects microcirculatory polymorphonuclear leukocyte behavior after hemorrhagic shock: role of hypertonic saline and pentoxifylline. Exp Biol Med (Maywood). 2004;229:684–93.
• Yamamoto R, Bai H, Dolezal AG, Amdam G, Tatar M. Juvenile hormone regulation
of Drosophila aging. BMC Biol. 2013;11:85.
• Yanar K, Aydin S, Cakatay U, Mengi M, Buyukpinarbasili N, Atukeren P, Sitar ME, Sonmez A, Uslu E. Protein and DNA oxidation in different anatomic regions of rat brain in a mimetic model. Basic Clin Pharmacol Toxicol. 2011;109:423–433.
• Yang F, Troncy E, Francoeur M et al (1997) Effects of reducing reagents and temperature on conversion of nitrite and nitrate to nitric oxide and detection of NO by chemiluminescence. Clin Chem 43(4):657–662
• Yoshikawa S, Muramoto K, Shinzawa-Itoh K (2012) Reaction mechanism of mammalian mitochondrial cytochrome c oxidase. Adv Exp Med Biol 748:215–236
• Yunos NM, Kim IB, Bellomo R, et al. The biochemical effects of restricting chloride-
rich fluids in intensive care. Crit Care Med. 2011;39:2419–24.
• Zander R. Base excess and lactate concentration in infusion solutions and blood products. Anasthesiol Intensivmed Notfallmed Schmerzther. 2002;37:359–63.
• Zheng H, Liu X, Patel KP. 2013. Centrally mediated erectile dysfunction in rats with
type 1 diabetes: Role of angiotensin II and superoxide. J Sex Med. 10(9):2165–2176.
• Zhong SZ, Ge QH, Qu R, Li Q, Ma SP. Paeonol attenuates neurotoxicity and ameliorates cognitive impairment induced by D-galactose in ICR mice. J Neurol Sci. 2009;277:58–64.
142
• Zhou Z, Lenk RP, Dellinger A, Wilson SR, Sadler R, Kepley CL. Liposomal formulation of amphiphilic fullerene antioxidants. Bioconjug Chem. 2010;21:1656–1661.
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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.
150
• 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.