Experimental strategies in the treatment of acute renal failure in sepsis
Johannes, T.
Publication date 2011
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Citation for published version (APA):
Johannes, T. (2011). Experimental strategies in the treatment of acute renal failure in sepsis.
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C
HAPTER
ACTIVATED PROTEIN C RESTORES KIDNEY FUNCTION IN A
DOSE-DEPENDENT MANNER IN ENDOTOXIN-INDUCED ACUTE RENAL
FAILURE IN THE RAT
Tanja Johannes, Emre Almac, Egbert G Mik, Matthieu Legrand, Klaus E Unertl, and
Can Ince
A
bstractIntroduction Activated protein C (APC) has been shown to have beneficial effects on the inflammatory process and coagulation during sepsis. Inflammation and coagulopathy impair the microvasculature and therefore disturb oxygen transport to the tissue. The hypothesis of our study was that APC-treatment improves renal microvascular oxygenation and kidney function in endotoxin-induced acute renal failure in the rat.
Methods In 21 anesthetized and ventilated (FiO2 0.5) male Wistar rats arterial blood pressure and
renal blood flow were recorded. The renal microvascular PO2 was continuously measured by
phosphorescence lifetime technique. All animals received a LPS-bolus (10mg/kg) to induce endotoxemic shock. All rats received fluid resuscitation (HES 130kD) 1h after LPS-application. In one group of animals APC (Drotrecogin Alpha, Xigris®, Eli-Lilly) was continuously infused in a
concentration of 10µg/kg/h. Another group received a continuous infusion of 100µg/kg/h APC. Results After LPS-bolus MAP and RBF progressively dropped to 40% and 60% of baseline at 1h respectively. Treatment with APC 100 in addition to fluid resuscitation prevented a further decline of both parameters. APC, independently of concentration, had no significant effects on average µPO2,
but prevented the occurrence of cortical microcirculatory hypoxic areas. All animals had a significant decrease in CLcrea 1h after LPS. Only in animals receiving APC 100, CLcrea was significantly restored
at the end of the experiment (3h).
Conclusion APC 100 significantly restored kidney function compared to standard fluid resuscitation during endotoxemia. This was accompanied by protection against the occurrence of cortical microcirculatory hypoxic areas. Furthermore, this application best improved MAP.
I
ntroductionThe prevalence of acute renal failure (ARF) in sepsis is high (approximately 40%) and the mortality reaches up to 75% in ICU patients with septic shock (1, 2). The pathogenesis of septic renal failure remains unclear, however, and current strategies to overcome renal dysfunction are mainly supportive rather then curative (3).
There is growing evidence that microcirculatory dysfunction accompanied by tissue dysoxia might play a key role in the development of septic ARF (4, 5). An inappropriate release of pro-inflammatory mediators is as well involved in the pathogenesis of sepsis as disturbances in the coagulation system, both leading to microcirculatory dysfunction with consecutive organ failure (6, 7).
It has been shown that the reduction in plasma levels of protein C is associated with an increased risk of death in patients with sepsis (8, 9). Activated protein C (APC) is an important endogenous protein that modulates coagulation and inflammation by promoting fibrinolysis and inhibiting thrombosis and inflammation (10, 11). Different experimental and clinical studies could demonstrate that APC improved outcome of severe sepsis (8, 12-15). Recently two studies showed beneficial effects of APC in acute kidney injury in a rat model of LPS-induced (16), and CLP-related renal failure (17). Furthermore activated protein C reduced ischemia/reperfusion-induced renal injury in rats (18).
Based on the physiological actions of activated protein C and the recently demonstrated beneficial effects of APC on renal endothelial dysfunction we hypothesized that recombinant human activated protein C might have a potential role in a renal protective strategy in sepsis. We therefore investigated in a rat model of endotoxin-induced acute renal failure 1) the influence of APC treatment on systemic and regional hemodynamics 2) the effects of APC on the renal microvascular oxygenation and 3) the influence of APC on renal function. Furthermore our study should unveil a possible relation between renal dysfunction and microvascular hypoxia.
M
aterial and methodsExperiments were approved by the Animal Research Committee of the Academic Medical Center at the University of Amsterdam and conducted in accordance with the guidelines for Institutional and Animal Care and Use Committees (IACUC) care and handling of the animals. The experiments were performed in 23 Wistar male rats (Harlan, Horst, The Netherlands) with a body weight of 318 ± 37 g. Animal preparation and monitoring.
Rats were anesthetized by intraperitonal injection of a mixture of 90mg/kg ketamine, 0.5mg/kg medetomidine and 0.05mg/kg atropine-sulphate. Animals were mechanically ventilated with a FiO2 of
0.5 via tracheostomy. The right carotid artery was cannulated and used for monitoring of arterial blood pressure and heart rate. A catheter in the right femoral artery was used for blood gas sampling. The right femoral vein was cannulated for continuous infusion of Ringer’s lactate (15mL/kg/h; Baxter, Utrecht, The Netherlands) and ketamine (50mg/kg/h; Nimatek®; Eurovet, Bladel,
The Netherlands). The animal’s temperature was maintained at 37°C. The left kidney was exposed via flank incision, decapsulated and immobilized in a Lucite kidney cup. A perivascular flow probe connected to a flow meter (T206; Transonic Systems Inc., Ithaca, NY, USA) was used to measure renal blood flow (RBF). The left ureter was isolated, ligated and cannulated for urine collection. At the end of the experiment the left kidney was removed and weighed.
Experimental protocol.
The experimental schema is demonstrated in Figure 1.
surgical procedure and Oxyphor G2 infusion (=2h) LPS bol us ( 10 mg/kg)
fluid resuscitation 5 ml/kg bolus ! 5ml/kg/h infusion ± APC infusion (10 !g/kg/h) or (100 !g/kg/h)
2h 1h
t0 t1 t2
FIGURE 1. Experimental schema.
At start of the experiment rats were randomized between control (n=5), fluid resuscitation (n=6), APC 10 (n=6) and APC 100 (n=6) groups. All animals receiving APC received standard fluid resuscitation with hydroxyethl starch (HES 130 kD). At the end of surgery (1h) an intravenous infusion of Oxyphor G2 (10 mg/kg in 15 min; Oxygen Enterprises Ltd., Philadelphia, PA, USA), necessary for the oxygen measurements, was started. Forty minutes later µPO2 and rvPO2 were continuously recorded via
two optical fibers placed both 1mm above the kidney surface and 1mm above the renal vein. Then, the baseline blood sample (0.4mL) was taken. In 18 rats a bolus of lipopolysaccharide (LPS, 10 mg/kg; serotype 0127:B8, Sigma-Aldrich, Zwijndrecht, The Netherlands) was given to induce septic shock. One hour after the LPS-bolus three groups of animals received fluid resuscitation (5 mL/kg followed by 5 mL/kg/h; Voluven®, 6 % HES 130/0.4; Fresenius Kabi, Schelle, Belgium) for two hours.
In addition to the fluid resuscitation, in one group of animals APC (10 µg/kg/h; recombinant human activated protein C; Drotrecogin Alpha, Xigris®, Eli-Lilly Indianapolis, IN, USA) was continuously
infused. Another group received APC in a concentration of 100 µg/kg/h. All animals received the same fluid volume. The experiment was ended 10 min after stop of treatment or a corresponding time point for the control groups.
Measurement of renal microvascular oxygenation and renal venous PO2.
The renal microvascular PO2 (µPO2) within the kidney cortex and outer medulla were measured by
oxygen-dependent quenching of phosphorescence using a dual-wavelength-phosphorimeter (19). The renal venous PO2 (rvPO2) was detected by the same method (20). Briefly described, Oxyphor
G2 (Oxygen Enterprises Ltd., Philadelphia, PA, USA), intravenously infused, binds to albumin (21). If excited by a flash of light (wavelength ~ 440 or 630 nm) the Oxyphor G2-albumin complex emits phosphorescence (wavelength ~800nm). Dependent on the oxygen concentration the phosphorescence intensity decreases and the relationship between the measured decay-time and the PO2 can be estimated using the Stern-Volmer relation (22-24). Heterogeneity in oxygen pressure
was analyzed by fitting a sum of small rectangular distributions to the distributions of quencher concentration in the phosphorescence data (5, 25, 26).
Blood gas measurements.
An arterial blood sample (0.4 mL) was taken via the femoral artery at three different time points. First time point: 0 minutes = baseline (t0), second time point: 1h after LPS-bolus (t1) and third time point:
3h after LPS-bolus or equivalent time (t2). The same volume of hydroxyethyl starch replaced the
sampling volume of blood. Samples were analyzed for determination of blood gas values (ABL505 blood gas analyzer; Radiometer, Copenhagen, Denmark), hematocrit, Hb-concentration, and HbSO2
(OSM3; Radiometer, Copenhagen, Denmark). Furthermore in each sample the plasma creatinine concentration was measured.
Calculations of renal oxygenation.
Renal oxygen delivery (DO2ren) was calculated as RBF x arterial O2-content: RBF x (1.31 x Hb x
SaO2) + (0.003 x PaO2). Renal oxygen consumption (VO2ren) was calculated as RBF x (arterial –
renal venous O2-content difference). Renal venous O2-content is: (1.31 x Hb x SrvO2) + (0.003 x
rvPO2) (20). An estimation of the vascular resistance (RVR) of the renal artery flow region was
made: RVR = MAP /RBF.
Measurement of renal function.
Creatinine clearance (CLcrea) was assessed as an index of glomerular filtration rate (GFR).
Calculations of the clearance were done with standard formula: CLcrea (mL/min) = (U x V) /P, where
U is the urine creatinine concentration, V is the urine volume per unit time and P is the plasma creatinine concentration. The specific elimination capacity for creatinine of the left kidney was normalized to the organ weight. For analysis of urine volume and creatinine concentration urine samples from the left ureter were collected at 10-min intervals. At the midpoint of each 10-min-interval plasma creatinine concentration was analyzed. Analysis of samples was performed using Jaffé method.
Data presentation and statistics.
Values are presented as mean ± SEM, unless otherwise indicated. Analysis of the monoexponential fit procedures of the phosphorescence curves was performed using Labview 6.1 software (National Instruments, Austin, TX, USA). For PO2 histogram recovery and statistics GraphPad Prism version
4.0 for Windows (GraphPad Software, San Diego, CA, USA) was used. For data analysis within each group and intergroup differences two-way ANOVA for repeated measurements with Bonferroni post test was performed. P values < 0.05 were considered significant.
R
esultsSystemic and regional variables.
Table 1 is showing the data for the measured systemic and regional variables. There was no statistically significant difference in baseline readings between the different groups. One hour (t1)
after LPS-bolus mean arterial pressure (MAP) was in all group significantly reduced compared to baseline and control. There was no change in heart rate (HR) at t1. After LPS-bolus renal blood flow
(RBF) progressively decreased and was at t1 in all experimental groups, reduced by more than 60%.
At t1 renal vascular resistance (RVR) was significantly increased in all experimental groups
compared to baseline and/or control. Between t1 and t2 MAP was not significantly different in the
experimental groups although the animals receiving APC 100 showed a slight recovery in MAP at t2
groups. At t2 RBF was in all experimental groups significantly reduced compared to baseline and
control. RVR was normalized after receiving resuscitation (t2).
MAP (mmHg) HR (bpm) RBF (ml/min) RVR (dyne/sec/cm5) rvPO2 (mmHg) TC (n = 5) t0 109 ± 1 254 ± 12 6.3 ± 0.1 17 ± 1 74 ± 4 t1 112 ± 7 261 ± 11 5.8 ± 0.3 19 ± 1 73 ± 2 t2 112 ± 5 267 ± 16 6.0 ± 0.3 19 ± 1 64 ± 5 LPS + FR (n = 6) t0 102 ± 5 247 ± 16 7.2 ± 0.2 14 ± 1 69 ± 6 t1 71 ± 10*† 275 ± 15 2.9 ± 0.8*† 35 ± 12* 52 ± 6*† t2 67 ± 11*† 296 ± 9* 3.9 ± 0.7*† 19 ± 4 42 ± 3*† APC 10 (n = 6) t0 101 ± 4 257 13 6.8 ± 0.7 16 ± 1 67 ± 4 t1 62 ± 8*† 272 ± 18 3.3 ± 0.8*† 27 ± 6* 58 ± 4 t2 58 ± 7*† 296 ± 15* 3.6 ± 0.5*† 19 ± 4 43 ± 3*† APC 100 (n = 6) t0 102 ± 1 262 ± 7 6.3 ± 0.3 16 ± 1 65 ± 7 t1 73 ± 7*† 291 ± 22 2.7 ± 0.6*† 35 ± 7*† 47 ± 7*† t2 80 ± 7*† 320 ± 17*† 3.9 ± 0.3*† 21 ± 1 41 ± 4*†
Values represent mean ± SEM. * P < 0.05 vs baseline; † P < 0.05 vs control.
TC = time control; LPS + FR = group receiving lipopolysaccharide + fluid resuscitation; APC 10 = treatment with 10 !g/kg/h activated protein C; APC 100 = treatment with 100 !g/kg/h activated protein C. Animals receiving APC also received LPS + FR. t0 = baseline; t1 = 1h after LPS-bolus; t2 = 3 h after LPS-bolus or equivalent time. MAP = mean arterial pressure; HR = heart rate; RBF = renal blood flow; RVR = renal vascular resistance; rvPO2 = renal venous PO2.
TABLE. 1 Systemic and regional variables
Renal oxygenation.
Table 1 and Figure 2 are showing the measured renal oxygenation parameters. There was no statistically significant difference in baseline readings between the different groups. Average cortical µPO2 did significantly drop at t1. In the fluid resuscitation and the APC 100 group the rvPO2 was
significantly reduced compared to baseline and control after LPS at t1 (Table 1).
At t2 cµPO2 and mµPO2 were in all groups significantly lower than at baseline and in the APC 10
group significant for the cµPO2 compared to control. The reduction in the average µPO2 after
LPS-bolus was accompanied by a significant left shift in the cortical oxygen histogram of the experimental groups (Fig. 3A) at t2. This left shift was not present in the outer medullary region (Fig. 3B). In
animals receiving APC 10 or 100 there was a significant reduction in the appearance of cortical microcirculatory hypoxic areas compared to the fluid resuscitation group.
The renal venous PO2 dropped in all experimental groups significantly compared to baseline and
control at t2. The renal oxygen delivery (DO2ren) was at t1 and t2 in all experimental groups
significantly reduced compared to baseline and control (Fig. 4A). In contrast, the renal oxygen consumption (VO2ren) did not significantly change during endotoxemia and resuscitation (Fig. 4B). At
the end of the experiment (t2) there was a significant increase in renal oxygen extraction (O2ERren) in
all groups compared to baseline and control (Fig. 4C).
FIGURE 2. Measured renal microvascular oxygenation. Values represent mean ± SEM. * P < 0.05 vs baseline. † P < 0.05 vs control. LPS + FR = group receiving lipopolysaccharide + fluid resuscitation; APC 10 = treatment with 10 µg/kg/h activated protein C, APC 100 = treatment with 100 µg/kg/h activated protein C. Animals receiving APC also received LPS + FR. t0 baseline; t1 1h after LPS-bolus; t2 3h after LPS-bolus or equivalent time.
FIGURE 3. Renal microvascular oxygen histograms. Panel A cortical oxygen histogram. Values represent mean ± SEM. * P < 0.05 vs baseline. † P < 0.05 vs control. # P < 0.05 vs FR. LPS + FR = group receiving lipopolysaccharide + fluid resuscitation; APC 10 = treatment with 10 µg/kg/h activated protein C, APC 100 = treatment with 100 µg/kg/h activated protein C. Animals receiving APC also received LPS + FR. t0 baseline; t1 1h after LPS-bolus; t2 3h after LPS-bolus or equivalent time. µPO2 = microvascular PO2.
FIGURE 3. Renal microvascular oxygen histograms. Panel B outer medullary oxygen histogram.
FIGURE 4. Calculated renal oxygenation parameters. Values represent mean ± SEM. * P < 0.05 vs baseline. † P < 0.05 vs control. LPS + FR = group receiving lipopolysaccharide + fluid resuscitation; APC 10 = treatment with 10 µg/kg/h activated protein C, APC 100 = treatment with 100 µg/kg/h activated protein C. Animals receiving APC also received LPS + FR. t0 baseline; t1 1h after LPS-bolus; t2 3h after LPS-bolus or equivalent time. DO2ren = renal oxygen delivery; VO2ren = renal oxygen consumption; O2ERren = renal oxygen extraction.
Kidney function.
There was no significant change in creatinine clearance (CLcrea) in the time control. All animals of the
experimental groups had a significant reduction in CLcrea and decrease in urine output after
LPS-bolus (t1). All treated animals started to increase urine volume after start of resuscitation. Only in the
APC 100 group CLcrea was totally restored to baseline compared to animals receiving fluid
resuscitation alone (Fig. 5).
FIGURE 5. Kidney function. Values represent mean ± SEM. * P < 0.05 vs baseline. † P < 0.05 vs control. # P < 0.05 vs FR. LPS + FR = group receiving lipopolysaccharide + fluid resuscitation; APC 10 = treatment with 10 µg/kg/h activated protein C, APC 100 = treatment with 100 µg/kg/h activated protein C. Animals receiving APC also received LPS + FR. t0 baseline; t1 1h after LPS-bolus; t2 3h after LPS-bolus or equivalent time. CLcrea = creatinine clearance.
D
iscussionIn the present study we demonstrated that recombinant human activated protein C in a concentration of 100µg/kg/h restored kidney function in a rat model of endotoxemia. The application of APC 100 added to standard fluid resuscitation maintained mean arterial pressure and preserved cortical microvascular oxygenation by preventing the occurrence of hypoxic areas.
Renal hypoperfusion is considered to be one of the most important pathogenetic factors in the development of ARF in sepsis (27). Disturbances in the autoregulation of microvessels, endothelial damage, tubular necrosis or disseminated fibrin thrombi leading to blockade of the intrarenal microvasculature (28, 29). The serine protease protein C plays an important role in controlling thrombosis and inflammation. Furthermore it could be
demonstrate that protein C exhibits cyto-protective effects (11, 30). There is evidence that reduction in plasma levels of protein C is prognostic for outcome in sepsis (31). In a recent study of LPS-induced kidney injury the ability of activated protein C to restore impaired renal hemodynamics could be shown. It is postulated that this restoration was due to suppression of local NO and the angiotensin system, as well as inhibition of leukocyte-endothelial cell interactions (16). Another study of the same group could demonstrate a clear link between an acquired protein C deficiency and renal dysfunction in a rat model of polymicrobial sepsis (17).
Based on the findings of these previous studies and the known acting mechanisms of protein C we wanted to investigate, if recombinant human activated protein C in addition to standard fluid resuscitation can be used as a therapeutic strategy in the treatment of endotoxin-related ARF. In the present study, rats received 1h after LPS-bolus standard fluid resuscitation supplemented by continuous infusion of 10 or 100µg/kg/h APC. After 3h the clearance of creatinine restored to baseline values in the APC 100 group. In animals receiving fluid resuscitation alone the creatinine clearance was only 50% of baseline. In our investigations neither APC 10 nor APC 100 could significantly restore mean arterial pressure or renal blood flow. Similar findings for mean arterial pressure were demonstrated for endotoxin-induced shock in rabbits; here APC was infused in a concentration of 160µg/kg/h (32). Recently, we showed the occurrence of cortical microcirculartory hypoxic areas in endotoxin-induced renal failure in the rat (5). In that study a shift in the cortical oxygen histogram toward anoxia could be demonstrated in non-resuscitated animals 5h after LPS-infusion. Such a shift in the cortical oxygen histogram was also present in the fluid resuscitation group of our current study and was attenuated in rats receiving APC 10 or 100.
There is not much published data about APC in sepsis-induced ARF. We did not investigate the direct acting mechanisms of APC in our present study. However, the results of our study and the previous mentioned study of Gupta et al. (16) are two fitting pieces in the puzzle of explaining the changes in the microvascular oxygen histograms and in understanding more about the pathophysiology of septic ARF. Septic ARF is characterized by severe intrarenal vasoconstriction in face of a profound vasodilatation in the systemic circulation (28). These mechanisms can be explained by activation of the renin-angiotensin-system (RAS) in counteraction to peripheral vasodilation mediated by NO. In the septic kidney RAS activation is leading to an aggravation of renal hypoperfusion via vasoconstriction by angiotensin II (ANG II) (33). Gupta et al. could demonstrate that APC treatment blocked the LPS-induced activation of the renal RAS and suppressed the induction of renal iNOS. Using two-photon intravital microscopy they showed that this was accompanied by improved intrarenal blood flow and reduced leukocyte adhesion. For our
observations this could mean that counteracting the vasoconstrictive action of ANG II with APC might explain the protection against the occurrence of microcirculatory hypoxic areas in the renal cortex. This is in agreement with our previously published speculation that the peritubular capillary region of the cortex can be the anatomical correlate to the observed hypoxic areas (5). In their work Gupta et al. could furthermore demonstrate that treatment with APC reduced peritubular dysfunction. Peritubular capillary dysfunction is an early event in endotoxemia that contributes to tubular stress and renal injury (34). The interpretation of the above-demonstrated results emphasizes once more the possible important role of hypoxia in the development of acute renal failure in sepsis.
When interpreting our findings, the reader should keep in mind some limitations of our animal model. Such models can never be directly translated to the human pathophysiology and the results of animal studies have to be interpreted carefully. Another limitation of our study is that it does not investigate long-term survival and outcome. However, the aim of our study was to investigate the acute effects of APC treatment in a model of endotoxemic shock in which treatment is started in the presence of severe hemodynamic alterations like seen in clinical practice. Early goal-directed therapy using fluid resuscitation to prevent hypoperfusion of vital organs is the standard therapy in sepsis (35). Therefore, fluid resuscitation was the primary therapeutic approach in our model after profound septic shock had developed. So treatment with activated protein C supplemented standard fluid resuscitation. In contrast to a recently published protocol of our group (36), we gave 10 mg/kg LPS as a bolus resulting in a much more severe model of endotoxemic shock. As the lethality of non-resuscitated animals reached more than 50% such group was not included in the present study. 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.
Our data demonstrate that the continuous infusion of the activated protein C in a concentration of 100µg/kg/h substantially restored kidney function in a rat model of endotoxemia. This observation was associated by preserved cortical microvascular oxygenation and a significant protection against the occurrence of microcirculatory hypoxic areas. Based on these results one could hypothesize that APC 100 infusion as adjuvant to standard fluid resuscitation might be useful as a renal protective strategy to preserve renal oxygenation and kidney function in the early stage of sepsis.
A
cknowledgementWe acknowledge the support by Eli Lilly Co. in form of an educational grant to Can Ince.
R
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