UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl)
Experimental strategies in the treatment of acute renal failure in sepsis
Johannes, T.
Publication date 2011
Link to publication
Citation for published version (APA):
Johannes, T. (2011). Experimental strategies in the treatment of acute renal failure in sepsis.
General rights
It 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 regulations
If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://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.
C
HAPTER
NONRESUSCITATED ENDOTOXEMIA INDUCES MICROCIRCULATORY
HYPOXIC AREAS IN THE RENAL CORTEX IN THE RAT
Tanja Johannes, Egbert G Mik, and Can Ince
A
bstractThe pathophysiology of acute renal failure (ARF) in sepsis is only partly understood. In several animal models of septic ARF, no profound tissue hypoxia or decrease in microcirculatory PO2
(µPO2) can be seen. We hypothesized that heterogeneity of microcirculatory oxygen supply to
demand in the kidney is obscured when looking at the average µPO2 during endotoxemia. In 20
anesthetized and ventilated rats, MAP, renal blood flow (RBF), and creatinine clearance (CLcrea)
were recorded. Renal µPO2 was measured by phosphorescence quenching, allowing measurement
of µPO2 distributions. Five animals received a 1-h LPS infusion (10 mg kg-1 h-1). In 5 rats, RBF was
mechanically reduced to 2.1 ± 0.2 mL min-1. Five animals served as time control. LPS infusion
significantly reduced RBF to 2.1 ± 0.2 mL min-1 and induced anuria. Average cortical µPO2
decreased from 68 ± 4 to 52 ± 6 mmHg, with a significant left shift in the cortical oxygen histogram toward hypoxia. This shift could not be observed in animals receiving mechanical RBF reduction. In these animals, CLcrea was reduced to 50%. An additional group of rats (n = 5) received fluid
resuscitation. In these animals, RBF was restored to baseline, CLcrea increased approximately 50%,
and the cortical microcirculatory hypoxic areas disappeared after resuscitation. In conclusion, endotoxemia was associated with the occurrence of cortical microcirculatory hypoxic areas that are not detected in the average PO2 measurement, proving the hypothesis of our study. These
I
ntroductionAcute renal failure (ARF) is an often-seen complication in critically ill patients. In sepsis, ARF has a prevalence ranging from 25% in severe sepsis to 50% in septic shock (1) and a mortality up to 75% in patients with acute septic renal failure (2, 3). The pathogenesis of sepsis-induced renal failure is multifactorial. An inappropriate release of various inflammatory mediators and an imbalance between vasoactive substances causing direct cytotoxic effects and impairment of the microvasculature seem to be key factors (4). Septic ARF is characterized by intrarenal vasoconstriction leading to renal hypoperfusion with a redistribution of the cortical blood flow toward the medulla (5-7). However, the relationship between renal oxygen delivery, consumption, and tissue oxygenation, especially with regard to biological response and functional consequences, is still poorly understood. Therefore, the role of microcirculatory dysfunction and subsequent oxygen deficiency in septic renal failure remains controversial (8-10). In a recent study, we only detected a minor influence of endotoxemia on mean microvascular PO2 readings in both cortex and outer medulla (11). Because of the far-reaching
consequences, it is necessary to have a more detailed look on intrarenal oxygenation during endotoxemia before drawing the conclusion that endotoxemia-induced ARF is not associated with local hypoxia.
In this study, we hypothesized that heterogeneity of microcirculatory oxygen supply to demand, leading to local microvascular hypoxia, in the kidney is obscured when looking at the average microvascular PO2 during endotoxemia. To investigate this hypothesis, we performed rat
experiments in which endotoxemia was induced by intravenous infusion of LPS. Heterogeneity of intrarenal oxygenation was studied by a phosphorescence quenching technique, allowing recovery of microvascular PO2 histograms from cortex and outer medulla (12). The analysis of renal oxygen
distribution and the appearance of cortical microcirculatory hypoxic areas during endotoxemia led to a second series of experiments in which a mechanical reduction in renal blood flow (RBF) to values comparable to the flow reduction seen during endotoxemia was induced. This second series of experiments should answer the question if the cortical microcirculatory hypoxic areas were specific for endotoxemia and not simply a nonspecific phenomenon due to reduction of RBF. A third group of fluidresuscitated animals was included to study the effect of fluid resuscitation on the reversibility of microcirculatory hypoxic areas and the possible influence of (relative) hypovolemia.
M
aterial and methods AnimalsAll experiments in this study were reviewed and approved by the Animal Research Committee of the Academic Medical Center at the University of Amsterdam. Handling and care of the animals were performed in accordance with the guidelines for Institutional and Animal Care and Use Committees. For the experiments, 20 male Wistar rats (Charles River, Maastricht, The Netherlands) with a body weight of 286 ± 18 g were used (mean ± SD).
Surgical preparation
Rats were anesthetized by injection of ketamine (90 mg kg-1, i.p.; Nimatek; Eurovet, Bladel, The
Netherlands), medetomidine (0.5 mg kg-1, i.p.; Domitor; Pfizer, New York, NY), and atropine sulfate
(0.05 mg kg-1, i.p.; Centrafarm, Etten-Leur, The Netherlands). Mechanical ventilation (FIO2, 0.4) was
Melsungen, Germany; outer diameter, 0.9 mm) for drug and fluid administration. Catheterization of the right carotid artery allowed monitoring of arterial blood pressure and heart rate. The right jugular vein and the right femoral artery and vein were cannulated and used for withdrawal of blood and continuous infusion of Ringer lactate (15 mL kg-1 h-1; Baxter, Uden, The Netherlands). Body
temperature was kept at 37 ± 0.5°C, and arterial PCO2 was maintained between 35 and 40 mmHg
by adjustment of ventilator settings.
The left kidney was exposed via a flank incision, decapsulated, and immobilized in a Lucite kidney cup (K. Effenberger, Pfaffingen, Germany). Under preservation of the nerves, the renal vessels were carefully separated from each other. A small piece of aluminum foil was placed on the dorsal side of the renal vein to prevent contribution of underlying tissue to the phosphorescence signal (venous PO2 measurement). For continuous measurement of RBF, a perivascular ultrasonic
transient time flow probe (type 0.7 RB; Transonic Systems, Inc., Ithaca, NY) was placed around the left renal artery and connected to a flow meter (T206; Transonic Systems Inc.). The left ureter was isolated, ligated, and cannulated with a polyethylene catheter to allow urine collection. The temperature of the kidney surface was measured and kept at approximately 37°C. The experiment was ended by infusion of 1 mL of 3 M potassium chloride inducing sudden cardiac arrest.
Hemodynamics, blood gas measurements, and kidney function
MAP (in millimeters of mercury) was continuously measured in the carotid artery and calculated as diastolic pressure + (systolic pressure - diastolic pressure) / 3. The RBF (in milliliters per minute) was measured and recorded continuously. At two different time points, an arterial blood sample was taken from the femoral artery: first time point, 0 min = baseline (t0); second time point, 60 min =
endotoxemia or time equivalent (t1). Samples were analyzed for blood gas values (ABL505 blood
gas analyzer; Radiometer, Bronshoj, Denmark) for determination of hemoglobin, hematocrit, hemoglobin oxygen saturation, sodium, and potassium concentration (OSM 3; Radiometer). The clearance of creatinine (CLcrea) was assessed as an index for glomerular filtration rate. For analysis
of urine volume and creatinine concentration, urine samples were collected at 10-min intervals. Plasma samples were obtained at the midpoint of each 10-min urine collection period and analyzed for creatinine levels. The concentrations of creatinine in urine and plasma were determined by colorimetric methods.
Measurement of renal microvascular oxygenation and renal venous PO2
The applied method of oxygen-dependent quenching of phosphorescence for detection of changes in microvascular PO2 (µPO2) and measurement of renal venous PO2 (PrvO2) is described in detail
elsewhere (12, 13). In brief, after intravenous infusion, a water-soluble phosphorescent dye (Oxyphor G2; Oxygen Enterprises, Ltd., Philadelphia, Pa) binds to albumin, forming a stable complex (14-16). This complex is well confined to the circulation and if excited by a flash of light emits phosphorescence with a wavelength approximately 800 nm (17). The phosphorescence decay depends on the surrounding oxygen concentration. The relationship between the measured decay time and the PO2 is given by the Stern-Volmer relation:
+ = 0 1 1 ! ! kq[O2]
where τ is the measured decay time, τ0 is the decay time at an oxygen concentration of zero, and kq
is the quenching constant. For oxygenation measurements within the rat renal cortex and outer medulla, a dual-wavelength phosphorimeter was used. This allowed the continuous and
simultaneous measurement of two different depths. The heterogeneity in oxygen pressure was analyzed by fitting a sum of small rectangular distributions to the distributions of quencher concentration in the phosphorescence data, an approach published by Golub et al. (18).
Calculation of renal oxygen delivery and oxygen consumption per unit time
The renal oxygen delivery was calculated as DO2ren (in milliliters per minute) = RBF x arterial oxygen
content (1.31 x Hb x SaO2) + (0.003 x PaO2). The renal oxygen consumption per unit time was
calculated as VO2ren (in milliliters per minute per gram) = RBF x arterial - renal venous oxygen
content difference. The renal venous oxygen content was calculated as (1.31 x Hb x SrvO2) + (0.003
x PrvO2) (13).
Experimental protocol
After 60 min of surgery, two optical fibers for oxygenation measurements were placed both 1 mm above the decapsulated kidney surface and 1 mm above the renal vein. Oxyphor G2 (1.2 mg kg-1 in
15 min; Oxygen Enterprises) was infused intravenously. The measurement of µPO2 and PrvO2 was
started 40 min later. The baseline blood sample was then taken. At this time point, the rats were randomized between the LPS (LPS; n = 5) and time control groups (TC; n = 5).
In total, 5 animals received a 1-h infusion of LPS (10 mg kg-1; serotype 0127:B8; Sigma,
Zwijndrecht, The Netherlands) to induce endotoxemia. Five rats served as time controls. A second blood gas was taken 60 min after start of LPS infusion and analyzed as described before. In a second series of experiments, a third group of rats was prepared for a mechanical reduction in RBF (FR; n = 5). In detail, in these animals, a thin silastic catheter was placed loosely around the renal artery at its junction with the abdominal aorta. The movement of a small plastic ring surrounding the catheter loop allowed for controlled partial occlusion of the vessel and an RBF reduction to 2 mL min -1. The experiments were ended 60 min after start of LPS infusion or a corresponding time point by
intravenous bolus injection of 3 M KCl.
An additional group of animals (n = 5) received fluid resuscitation 1 h after LPS infusion (10 mg kg-1). For resuscitation, HES (130 kd; Voluven; 6% HES 130/0.4; Fresenius Kabi, Utrecht, The
Netherlands) was infused at a rate of 20 mL h-1 until a 5-min steady plateau in RBF was reached. In
this group, three measurement points were defined: first time point, 0 min = baseline (t0); second
time point, 60 min = endotoxemia (t1); and 90 min = after 30 min of fluid resuscitation (t2).
Statistical analysis
Values are reported as mean ± SD unless otherwise indicated. Analysis software for the monoexponential fit procedures of the phosphorescence curves was written in Labview 6.1 software (National Instruments, Austin, Tex). Statistics and the PO2 histogram recovery were performed using
GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, Calif). For testing differences within groups and intergroup differences, two-way ANOVA for repeated measurements with Bonferroni post test was performed. The oxygen histograms were analyzed using two-way ANOVA as previously described. Every bin of the histogram was separately tested in time and versus control. P values less than 0.01 were considered significant.
R
esultsSystemic hemodynamics
No differences in systemic hemodynamics existed between the three groups at baseline (Table 1). Endotoxemia induced a significant decrease in MAP from 116 ± 7 mmHg at baseline (t0) to 97 ± 17
mmHg at t1. Furthermore, RBF dropped significantly from 5.8 ± 0.8 to 2.1 ± 0.2 mL min-1 at t1 (1 h
after start of LPS infusion; P < 0.01 vs. baseline). A sample experiment showing the response of MAP and RBF during endotoxemia over time is shown in Figure 1A.
In the mechanical blood flow reduction group, RBF was reduced from 6.1 ± 0.7 to 2.1 ± 0.2 mL min-1 without affecting MAP (example shown in Fig. 3A). In the control group, MAP and RBF were
stable over time.
MAP (mmHg) RBF (ml/min) c!PO2 (mmHg) m!PO2 (mmHg) DO2ren (ml/min) VO2ren (ml/min/g) CLcrea (!l/min/g) TC (n = 5) t0 114 ± 3 6.5 ± 0.7 67 ± 3 52 ± 3 1.5 ± 0.2 0.12 ± 0.03 984 ± 199 t1 121 ± 3 5.8 ± 0.7 61 ± 9 48 ± 3 1.3 ± 0.2 0.17 ± 0.05 1169 ± 346 LPS (n = 5) t0 116 ± 7 5.8 ± 0.8 68 ± 4 55 ± 3 1.4 ± 0.2 0.10 ± 0.02 891 ± 119 t1 97 ± 17† 2.1 ± 0.2*† 52 ± 6* " 45 ± 2* 0.5 ± 0.1*† 0.07 ± 0.05† anuric FR (n = 5) t0 111 ± 5 6.1 ± 0.7 67 ± 7 49 ± 7 1.4 ± 0.2 0.20 ± 0.08 755 ± 134 t1 106 ± 17 2.1 ± 0.2*† 63 ± 4 46 ± 3 0.4 ± 0.1*† 0.07 ± 0.04* 427 ± 206†
Values represent mean ± SD. * P < 0.01 vs baseline; † P < 0.01 vs control; " P < 0.01 vs flow reduction.
TC = control group; LPS = group receiving LPS; FR = flow reduction group. t0 = baseline; t1= 1h after LPS-infusion or equivalent time.
MAP = mean arterial pressure; RBF = renal blood flow; c!PO2 = cortical microvascular PO2; m!PO2 = medullary microvascular PO2;
DO2ren = renal O2-delivery; VO2ren = renal O2-consumption; CLcrea = creatinine clearance.
TABLE 1. Systemic and regional variables
Renal oxygenation parameters
Data of the oxygenation parameters of the kidney are shown in Table 1. The baseline values in the experimental groups and the control group were not different. Renal oxygen delivery, VO2ren, and
µPO2 did not change in the control group. At the end of the 60-min infusion of LPS, there was a mild,
but significant, reduction in cortical and medullary µPO2 of 16 and 10 mmHg, respectively. An
example of the behavior of µPO2 during endotoxemia is shown in Figure 1B. Despite the only mild
reduction of 16 mmHg in the mean cortical microvascular PO2, 1 h after LPS infusion, the cortical
oxygen histogram was significantly (P < 0.01) shifted to the left compared with baseline and control (Fig. 2). This indicates the presence of microvascular hypoxic areas in which the detected PO2
values were less than 10 mmHg. The occurrence of microvascular hypoxic areas is accompanied by a significant reduction in microvascular regions with high PO2, denoted by bins 7 and 8 of the oxygen
histogram (97.5 and 112.5 mmHg, respectively). In contrast, the medullary oxygen histogram did not significantly change during endotoxemia. The distributions in the control group were stable over time.
Renal oxygen delivery decreased from 1.4 ± 0.2 at baseline to 0.5 ± 0.1 at t1 (P < 0.01 vs. baseline).
VO2ren did not change compared with baseline but significantly decreased compared with the control
group.
In the mechanical flow reduction group, the renal cortical and medullary µPO2 were not affected
and remained high with 63 ± 4 and 46 ± 3 mmHg, respectively (example shown in Fig. 3B). The oxygen distributions before and after RBF reduction are demonstrated in Figure 4. After flow reduction to 2 mL min-1, there was no significant change in the cortical or medullary µPO2 or in the
oxygen distribution of the two investigated kidney regions. Renal oxygen delivery significantly decreased from 1.4 ± 0.2 at t0 to 0.4 ± 0.1 upon flow reduction (P < 0.01 vs. baseline). Furthermore,
there was a significant drop in VO2ren from 0.20 ± 0.08 at t0 to 0.07 ± 0.04 mL min-1 g-1 at t1
compared with baseline.
FIGURE 1. Sample experiment for endotoxemia. MAP and RBF progressively dropped 10 min after start of LPS infusion. Whereas MAP slowly recovered after 10 min, RBF remained low approximately 2 mL min-1. The renal cortical
(cµPO2) and outer medullary (mµPO2) microvascular PO2 only slightly dropped during LPS infusion. t0 = baseline; t1 = 1
FIGURE 2. Histograms showing oxygen distributions in the cortex (A) and the outer medulla (B) of the rat kidney
for time control and for endotoxemia. t0 = baseline; t1 = 1 h after start of LPS infusion. *P < 0.01 versus baseline; †P <
0.01 vs. control; ‡P < 0.01 vs flow reduction.
FIGURE 3. Sample experiment for RBF reduction. MAP transiently increased after reducing the RBF to values approximately 2 mL min-1. The intervention had no influence on renal cortical (cµPO2) and outer medullary (mµPO2)
FIGURE 4. Histograms showing oxygen distributions in the cortex (A) and the outer medulla (B) of the rat kidney
during reduction in RBF (FR) by ligation of the renal artery. t0 indicates baseline; t1, 1 h after start of blood flow
reduction. *P < 0.01 vs. control.
Kidney function
Creatinine clearance was stable during the experimental period in the control group. In the endotoxemia group, CLcrea was 891 ± 119 µL min-1 g-1 at baseline and became zero due to the
occurrence of anuria at t1 (Table 1).
In the flow reduction group, CLcrea was 50% reduced compared with baseline (P < 0.01 vs.
baseline).
Fluid resuscitation
In an additional group of endotoxemic rats, fluid resuscitation was given to prove the reversibility of microcirculatory hypoxic areas and to exclude hypovolemia as a cause of this phenomenon (Fig. 5). Endotoxemia was accompanied by a slight reduction in MAP and a significant drop in RBF of 62% at t1 (P < 0.01 vs. baseline). Fluid resuscitation had no effect on MAP, but restored RBF to baseline,
and animals started to urinate after anuria. Creatinine clearance increased approximately 50%, which was significantly lower than at baseline. Cortical and medullary µPO2 dropped 12 and 9
FIGURE 5. Fluid resuscitation. A, Influence of fluid resuscitation on systemic and regional variables. t0 indicates
baseline; t1, 1 h after start of LPS infusion; t2, after 30 min of fluid resuscitation. Histograms showing oxygen distributions
in the cortex (B) and the outer medulla (C) of the rat kidney during fluid resuscitation. *P < 0.01 vs. baseline (ANOVA for repeated measurements with Newman-Keuls posttest).
The cortical oxygen histogram, however, was significantly (P < 0.01) shifted to the left compared with baseline (Fig. 5B) at t1. This left shift was reversed by fluid resuscitation. In contrast, the medullary
oxygen histogram did not significantly change during endotoxemia and resuscitation. Renal oxygen delivery significantly decreased approximately 62% at t1 and increased 50% upon fluid resuscitation
(P < 0.01 vs. baseline). VO2ren was unaffected by endotoxemia and resuscitation.
D
iscussionIn a rat model, we studied the effect of endotoxemia on regional microvascular oxygenation of the kidney. Local µPO2 is depending on both VO2 and DO2, and changes in µPO2 therefore denote
changes in their balance. Classically, VO2 becomes only dependent on available oxygen at very low
PO2 levels (several millimeters of mercury) (19, 20). Previous studies on renal oxygenation in sepsis
reported PO2 values of several tens of millimeters of mercury (6, 21), arguing against tissue hypoxia
to a level that affects cellular metabolism and induces oxidative stress. However, insight in the heterogeneity of PO2 is lacking, and the occurrence of profound local hypoxia cannot be excluded. A
rejecting hypoxia as a possible contributing factor in the pathogenesis of sepsis-induced ARF. We tested the hypothesis that the occurrence of local microvascular hypoxia due to heterogeneity in microcirculatory oxygen supply to demand in the kidney is obscured when looking at the average microvascular PO2.
In this study, endotoxemia induced a significant reduction in both the average cortical and outer medullary µPO2, obtained by monoexponential analysis of the phosphorescencesignal. In
accordance with our previous report (11), both average renal cortical and medullary PO2 during
endotoxemia are well more than 40 mmHg, not at all suggestive of hypoxia-induced metabolic impairment. A more sophisticated analysis of the phosphorescence signals, allowing the recovery of heterogeneity in µPO2 values, shows the occurrence of regions with profound hypoxia in the cortex
(approximately 10% of the signal originated from regions with µPO2 values less than 15 mmHg). In
contrast, the µPO2 distribution in the outer medulla did not show a significant left shift during
endotoxemia.
LPS infusion significantly decreased RBF and DO2ren, reduced MAP by approximately 9 mmHg
from baseline, and was accompanied by anuria. In contrast, mechanical RBF reduction, with a reduction in DO2ren comparable to the effect of LPS, did not induce hypoxic areas in neither cortex
nor outer medulla and was not associated with anuria (CLcrea did decrease compared with baseline,
but not significantly). Mechanical flow reduction is known to significantly alter renal perfusion pressure (22, 23), and in view of the modest effect of 50% reduction of RBF, a small reduction in MAP is not likely to hamper kidney function. These findings argue against prerenal causes being the predominant factor in the appearance of microcirculatory hypoxic areas and the impairment in kidney function (anuria). However, because prerenal acute kidney injury is always associated with changes in intrarenal hemodynamics, the strict separation between prerenal and/or intrarenal causes from the hemodynamic point of view is difficult. Concerning intrarenal changes, a distorted relationship between oxygen supply and demand on a local level due to endotoxin, probably due to increased intrarenal vasoconstriction, can explain our findings. In this view, the lack of large PO2 changes in
the mechanical flow reduction group can be explained by concomitant falls in DO2 and VO2 (reduced
filtration and resorption) in the presence of a good functioning microcirculation.
Although macrohemodynamic alterations do not seem to be the primary cause of anuria and the occurrence of hypoxic areas during endotoxemia, hypovolemia might be a contributing factor. For this reason, and to investigate whether the hypoxic areas were reversible, we studied the effects of fluid resuscitation. In comparison, and in reference to our previous studies, RBF increased upon resuscitation to baseline value (11). Fluid resuscitation restored kidney function to 50% of baseline and was accompanied by reduction of hypoxic areas in the cortex. These findings suggest a correlation between the occurrence of hypoxic areas and functional impairment of the kidney. Although fluid resuscitation had no effect on MAP, arguing against severe hypovolemia, the partial recovery of renal function does suggest some prerenal, that is, hemodynamically mediated component.
Although reports on heterogeneity in microvascular/tissue PO2 in sepsis are lacking, more data
exist on heterogeneity in microvascular blood flow due to the availability of several measurement techniques (24, 25). In various pathologies, a maldistribution in microvascular blood flow was demonstrated in tissues other than kidney (26, 27). Several animal studies reported an intrarenal redistribution of blood flow during endotoxemia (28, 29). The distribution of blood flow from cortex toward the medulla is suggesting that endotoxemia causes an increase in arteriovenous shunting,
resulting in a fall in tissue perfusion. Those effects add to preglomerular arteriovenous diffusive oxygen shunting, described by Schurek et al. (30) under physiological conditions. Shunting in combination with locally reduced blood flow (21) (theoretically) makes the cortex prone to hypoxia during endotoxemia.
When interpreting our findings, the reader should keep in mind some limitations of our animal model. Short-term models of rodent endotoxemia, like in our study, are accompanied by hypodynamic state and marked reduction in RBF with extensive intrarenal vasoconstriction (31). In contrast, studies in large animal models show a hyperdynamic response to endotoxemia without reduction in RBF (32, 33). For this reason, the results of animal studies have to be interpreted carefully, and still, a direct clinical relevance remains equivocal. Furthermore, due to the lack of a steady state of creatinine balance, changes in CLcrea as an index of glomerular filtration should only
be regarded as a gross indication.
Overall, we demonstrate that endotoxemia is associated with the occurrence of microcirculatory hypoxic areas that are not detected using techniques that provide an average PO2 value, proving the
hypothesis of our study. Our data are suggesting a role for regional hypoxia in the development of endotoxemia-induced renal dysfunction. However, a causal relation between local hypoxia and loss of kidney function remains to be proven. Nevertheless, our findings are significant because previous studies (5, 6, 11) failed to demonstrate profound tissue hypoxia to levels that might impair mitochondrial respiration. Because the used phosphorescence technique allows detection of local hypoxia but is not able to identify the anatomical location, we can only speculate regarding which part of the cortex might be affected. The peritubular capillaries are identified as being prone to damage and dysfunction during endotoxemia (34-36) and seem to be a good candidate for further research. Furthermore, the question remains if 20% of the PO2 values less than 22.5 mmHg and
10% of the values less than 7.5 mmHg are sufficient to induce anuria. A possible explanation can be the recently described phenomenon of “oxygen conformance of cellular metabolism” (37-39). If already mild hypoxia reduces cellular oxygen consumption (as opposed to the classical view [19, 20]), then our PO2 histograms show indeed that a large portion of the renal cortex might be in an
oxygen-dependent state.
We conclude that hypoxia should not be discarded as a contributing cause in the pathogenesis of endotoxemia induced ARF, and further studies into the cause of hypoxia, its localization, and its role in the development of sepsis-induced ARF are indicated.
R
eferences1. Uchino S, Kellum JA, Bellomo R, Doig GS, Morimatsu H, Morgera S, Schetz M, Tan I, Bouman C, Macedo E, et al.: Acute renal failure in critically ill patients: a multinational, multicenter study. JAMA 294:813-818, 2005.
2. Neveu H, Kleinknecht D, Brivet F, Loirat P, Landais P: Prognostic factors in acute renal failure due to sepsis. Results of a prospective multicentre study. The French Study Group on Acute Renal Failure. Nephrol Dial Transplant 11:293-299, 1996.
3. Brivet FG, Kleinknecht DJ, Loirat P, Landais PJ: Acute renal failure in intensive care unitsVcauses, outcome, and prognostic factors of hospital mortality; a prospective, multicenter study. French Study Group on Acute Renal Failure. Crit Care Med 24:192-198, 1996.
4. Wan L, Bellomo R, Di Giantomasso D, Ronco C: The pathogenesis of septic acute renal failure. Curr Opin Crit Care 9:496-502, 2003.
5. Gullichsen E, Nelimarkka O, Halkola L, Niinikoski J: Renal oxygenation in endotoxin shock in dogs. Crit Care Med 17:547-550, 1989.
6. James PE, Bacic G, Grinberg OY, Goda F, Dunn JF, Jackson SK, Swartz HM: Endotoxin-induced changes in intrarenal PO2, measured by in vivo electron paramagnetic resonance oximetry and magnetic resonance imaging.
Free Radic Biol Med 21:25-34, 1996.
7. Linder MM, Hartel W, Alken P, Muschaweck R: Renal tissue oxygen tension during the early phase of canine endotoxin shock. Surg Gynecol Obstet 138:171-173, 1974.
8. Gullichsen E: Renal perfusion and metabolism in experimental endotoxin shock. Acta Chir Scand Suppl 560:7-31, 1991.
9. Heemskerk AE, Huisman E, van Lambalgen AA, van den Bos GC, Hennekes M, Thijs LG, Tangelder GJ: Renal function and oxygen consumption during bacteraemia and endotoxaemia in rats. Nephrol Dial Transplant 12:1586-1594, 1997.
10. Weber A, Schwieger IM, Poinsot O, Klohn M, Gaumann DM, Morel DR: Sequential changes in renal oxygen consumption and sodium transport during hyperdynamic sepsis in sheep. Am J Physiol 262:F965-F971, 1992. 11. 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 10:R88, 2006.
12. Johannes T, Mik EG, Ince C: Dual-wavelength phosphorimetry for determination of cortical and subcortical microvascular oxygenation in rat kidney. J Appl Physiol 100:1301-1310, 2006.
13. 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 294:F676-F681, 2008.
14. Rietveld IB, Kim E, Vinogradov SA: Dendrimers with tetrabenzoporphyrin cores: near infrared phosphors for in vivo oxygen imaging. Tetrahedron 59: 3821-3831, 2003.
15. Rozhkov V, Wilson D, Vinogradov S: Phosphorescent Pd porphyrin-dendrimers: tuning core accessibility by varying the hydrophobicity of the dendritic matrix. Macromolecules 35:1991-1993, 2002.
16. Vinogradov SA, Lo LW, Wilson DF: Dendritic polyglutamic porphyrins: probing porphyrin protection by oxygen-dependent quenching of phosphorescence. Chem Eur J 5:1338-1347, 1999.
17. Dunphy I, Vinogradov SA, Wilson DF: Oxyphor R2 and G2: phosphors for measuring oxygen by oxygen-dependent quenching of phosphorescence. Anal Biochem 310:191-198, 2002.
18. Golub AS, Popel AS, Zheng L, Pittman RN: Analysis of phosphorescence in heterogeneous systems using distributions of quencher concentration. Biophys J 73:452-465, 1997.
19. Jones DP, Mason HS: Gradients of O2 concentration in hepatocytes. J Biol Chem 253:4874-4880, 1978.
20. Wilson DF, Erecinska M, Drown C, Silver IA: The oxygen dependence of cellular energy metabolism. Arch Biochem Biophys 195:485-493, 1979.
21. Nitescu N, Grimberg E, Guron G: Low-dose candesartan improves renal blood flow and kidney oxygen tension in rats with endotoxin-induced acute kidney dysfunction. Shock 30:166-172, 2008.
22. Moosavi SM, Johns EJ: Effect of renal perfusion pressure on renal function, renin release and renin and angiotensinogen gene expression in rats. J Physiol 520(Pt 1):261-269, 1999.
23. Blaine EH, Zimmerman MB: Renin secretion during dynamic changes in renal perfusion pressure. Am J Physiol 236:F546-F551, 1979.
24. Millar CG, Thiemermann C: Intrarenal haemodynamics and renal dysfunction in endotoxaemia: effects of nitric oxide synthase inhibition. Br J Pharmacol 121:1824-1830, 1997.
25. Di Giantomasso D, Morimatsu H, May CN, Bellomo R: Intrarenal blood flow distribution in hyperdynamic septic shock: effect of norepinephrine. Crit Care Med 31:2509-2513, 2003.
26. Lamm WJ, Bernard SL, Wagner WW Jr, Glenny RW: Intravital microscopic observations of 15-microm microspheres lodging in the pulmonary microcirculation. J Appl Physiol 98:2242-2248, 2005.
27. Ellis CG, Bateman RM, Sharpe MD, Sibbald WJ, Gill R: Effect of a maldistribution of microvascular blood flow on capillary O(2) extraction in sepsis. Am J Physiol Heart Circ Physiol 282:H156-H164, 2002.
28. Millar CG, Thiemermann C: Carboxy-PTIO, a scavenger of nitric oxide, selectively inhibits the increase in medullary perfusion and improves renal function in endotoxemia. Shock 18:64-68, 2002.
29. Cronenwett JL, Lindenauer SM: Distribution of intrarenal blood flow during bacterial sepsis. J Surg Res 24:132-141, 1978.
30. Schurek HJ, Jost U, Baumgartl H, Bertram H, Heckmann U: Evidence for a preglomerular oxygen diffusion shunt in rat renal cortex. Am J Physiol 259: F910-F915, 1990.
31. Heyman SN, Darmon D, Goldfarb M, Bitz H, Shina A, Rosen S, Brezis M: Endotoxin-induced renal failure. I. A role for altered renal microcirculation. Exp Nephrol 8:266-274, 2000.
32. Langenberg C, Wan L, Egi M, May CN, Bellomo R: Renal blood flow and function during recovery from experimental septic acute kidney injury. Intensive Care Med 33:1614-1618, 2007.
33. Offner PJ, Robertson FM, Pruitt BA Jr: Effects of nitric oxide synthase inhibition on regional blood flow in a porcine model of endotoxic shock. J Trauma 39:338-343, 1995.
34. Richman AV, Gerber LI, Balis JU: Peritubular capillaries. A major target siteof endotoxin-induced vascular injury in the primate kidney. Lab Invest 43:327-332, 1980.
35. Achparaki A, Kotzampassi K, Eleftheriadis E, Foroglou C: Ultrastructural changes of the renal cortex after septic shock in rats. Histol Histopathol 3: 133-146, 1988.
36. Wu L, Tiwari MM, Messer KJ, Holthoff JH, Gokden N, Brock RW, Mayeux PR: Peritubular capillary dysfunction and renal tubular epithelial cell stress following lipopolysaccharide administration in mice. Am J Physiol Renal Physiol 292:F261-F268, 2007.
37. Chandel NS, Budinger GR, Choe SH, Schumacker PT: Cellular respiration during hypoxia. Role of cytochrome oxidase as the oxygen sensor in hepatocytes. J Biol Chem 272:18808-18816, 1997.
38. Schumacker PT, Chandel N, Agusti AG: Oxygen conformance of cellular respiration in hepatocytes. Am J Physiol 265:L395-L402, 1993.
39. Budinger GR, Chandel N, Shao ZH, Li CQ, Melmed A, Becker LB, Schumacker PT: Cellular energy utilization and supply during hypoxia in embryonic cardiac myocytes. Am J Physiol 270:L44-L53, 1996.
