Ischemia/reperfusion (IR) induces a cascade of events leading to multiple organ failure.1,2 Multiple inflammatory mechanisms are activated during ischemia and can be amplified for hours after reperfusion. Reperfusion promotes the release of free radicals, adhesion molecules, and inflammatory cytokines,3 resulting in endothelial cell damage4 and tissue injury. These mechanisms induce microvascular alterations, neutrophil recruitment, and increased vascular permeability, which then influence microvascular blood flow.5
Acute kidney injury (AKI) due to renal IR is frequently observed during abdominal aortic aneurysm surgery with suprarenal clamping.6,7 Postischemic microvascular damage is associated with swelling of endothelial cells and altered microvascular perfusion.8,9 These endothelial lesions have been associated not only with postoperative AKI but also with the development of chronic renal dysfunction.10,11 The renal microcirculation, particularly the peritubular circulation, can be markedly affected in IR.12 In humans undergoing renal transplantation, perioperative alterations in microcirculatory perfusion detected by videomicroscopy are a risk factor for early renal graft dysfunction.13
Strategies to improve renal function and renal microvascular perfusion during IR have thus been an intense topic of research, but few therapies have successfully transitioned from bench to bedside.14,15 Manipulation of nitric oxide (NO) pathways is an attractive option. During IR, decreased NO synthase (NOS) activity induces an imbalance between NO and superoxide anion (O2−) levels16 with increased xanthine oxidase activity.17 The resulting high superoxide anion concentrations play a key role in the observed IR injuries.18 During this process, direct endothelial cell lesions cause marked microvascular alterations.19
IR also decreases tetrahydrobiopterin (BH4) levels,20 inactivating NOS21 and causing a shift from NO to ROS production. In a murine model of renal IR, BH4 pretreatment restored renal endothelium-dependent vasodilation.22 Similarly, administration of sepiapterin, the precursor of BH4, prevented the decrease in cortical and outer medullary microvascular Po2, reduced renal infiltration by inflammatory cells, and decreased urine neutrophil gelatinase-associated lipocalin levels.23 However, these trials did not directly evaluate the impact on the renal microcirculation. The aim of this study was, therefore, to assess the effects of BH4 pretreatment on the renal microcirculation, redox state, and function in a sheep model of IR induced by suprarenal cross-clamping. We hypothesized that BH4 administration would decrease the renal microcirculatory damage induced by IR.
The study protocol was approved by the institutional review board for animal care of the Free University of Brussels (Brussels, Belgium). Care and handling of the animals followed the National Institutes of Health guidelines, and the study meets the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for animal research.
Nineteen female Ovis Aries sheep (aged 9–12 months) were fasted for 24 hours before the experiment with free access to water. On the day of the experiment, the animals were premedicated with 0.25 mg/kg intramuscular midazolam (Dormicum; Roche Pharmaceuticals, Attikis, Greece) and 20 mg/kg ketamine hydrochloride (Imalgine; Merial, Lyon, France) and then placed in the supine position. After induction of anesthesia by intravenous administration of 30 μg/kg fentanyl citrate (Janssen, Beerse, Belgium) and endotracheal intubation, continuous anesthesia was provided by intravenous administration of midazolam (0.5 mg/kg/h), ketamine hydrochloride (5 mg/kg/h), and morphine (0.5 mg/kg/h). Neuromuscular blockade was achieved using an intravenous bolus of 1 mg/kg rocuronium (Esmeron; Organon, Oss, the Netherlands) followed by 0.2 mg/kg/h throughout the experiment to avoid movement artifacts. Boluses of fentanyl (0.5 mg) were administered if needed. Volume-controlled mechanical ventilation (Servo 900 C ventilator; Siemens-Elema, Solna, Sweden) was provided, with ventilator conditions adjusted to ensure normoxia (80 mm Hg ≤ Pao2 ≤ 120 mm Hg) and normocapnia (35 mm Hg ≤ Paco2 ≤ 45 mm Hg) on repeated blood gas analysis.
The right carotid artery and jugular vein were surgically exposed under anesthesia. A 6F arterial catheter (Vygon, Cirencester, United Kingdom) was introduced into the carotid artery and connected to a pressure transducer (Edwards Lifesciences, Irvine, CA) zeroed at the midchest level. An introducer was inserted through the jugular vein, and a 7F Swan-Ganz catheter (Edwards Lifesciences) was advanced into the pulmonary artery.
A left lumbotomy was performed with the animal in the ventral position. A clamp was positioned around the suprarenal aorta. A probe was placed around the left renal artery (Transonic Systems, Ithaca, NY) to measure renal blood flow (RBF). The left kidney was exposed, and 2 microdialysis catheters (CMA 60 catheter; CMA Microdialysis, Solna, Sweden) were inserted: 1 in the cortex and the other in the medulla.
Monitoring and Measurements
Arterial and venous blood gas analyses were obtained hourly and analyzed using a Cobas-b 123 point-of-care system (Roche Diagnostics, Mannheim, Germany). Measurements included hemoglobin concentration, arterial (Sao2) and mixed venous oxygen saturation, Pao2 and Paco2, and arterial pH, lactate, potassium, and glucose. Arterial creatinine concentration was measured at baseline, 30 minutes, and 6 hours after reperfusion in the central laboratory of the hospital. Peak airway pressure, respiratory rate, plateau airway pressure, Fio2, heart rate, and urine output were recorded hourly. Mean arterial pressure (MAP), pulmonary arterial pressure, right atrial pressure, and pulmonary artery occlusion pressure were measured at end-expiration (Sirecust 404; Siemens, Erlangen, Germany). Core temperature and cardiac output (CO; Vigilance; Edwards Lifesciences) were continuously monitored. Body surface area, cardiac index, oxygen (O2) delivery, O2 consumption, and O2 extraction were calculated using standard formulas. For RBF, we express the relative (r) value from baseline (rRBF = [(RBFTn − RBFT0)/RBFT0] × 100), with measurements made at baseline (before clamping), before unclamping, and at 1, 4, and 6 hours after reperfusion.
The microvascular renal cortex was visualized using a side-stream dark field (SDF) videomicroscopy system (Microscan; MicroVision Medical Inc, Amsterdam, the Netherlands), with a ×5 imaging objective giving ×326 magnification. The lens of the imaging device was applied without pressure to the renal cortex. Image recording was performed at baseline and at 1, 4, and 6 hours after reperfusion. Five good-quality videos of 20 seconds were recorded from several areas in the renal cortex using a computer and a video card system (MicroVideo; Pinnacle Systems, Mountain View, CA). The images were stored under a random number for analysis. An investigator blinded to group/time allocation later analyzed these sequences semiquantitatively. Because of the specific morphology of renal capillaries, we counted the number of vessels that crossed a diagonal line to calculate vessel density (see Supplemental Digital Content, Figure S1, http://links.lww.com/AA/B765). The type of flow was defined as continuous (perfused vessels), intermittent, or absent (nonperfused vessels). Vessel size was determined using a micrometer scale, and vessels were separated into large and small, using a diameter cutoff value of 20 μm. The small vessel density was calculated as the number of small vessels crossing this diagonal, divided by the length of the diagonal. Small vessel perfusion was defined as the proportion of small perfused vessels (PPV) and calculated as the number of capillaries continuously perfused during the 20-second observation period, divided by the total number of vessels of the same type. Perfused small vessel density (PVD) was calculated as the product of capillary density and perfused vessel density for vessels of the same type. The heterogeneity index of the PPV was calculated as the difference between the maximum PPV and the minimum PPV, divided by the mean PPV of the 5 videos. In each animal, the data from the investigated areas were averaged for each time point.24
Renal metabolism was evaluated using microdialysis catheters in the cortex and the medulla of the kidney. Each was connected to a microdialysis pump (CMA 107; CMA Microdialysis) perfusing a balanced fluid (Perfusion fluid T1; CMA Microdialysis AB, Na+ 147 mmol/L, K+ 4 mmol/L, Ca2+ 2.3 mmol/L, Cl− 156 mmol/L, pH 6.0) at a rate of 0.3 µL/min. Effluents were collected hourly and analyzed immediately by an automatic microdialysis analyzer (CMA 600 Analyzer; CMA Microdialysis) for concentrations of lactate (L), pyruvate (P), glucose (G), glycerol, and computations of L/P and L/G ratios.25,26
After surgery, we waited at least 1 hour for stabilization before ischemia induction. During this period, we maintained a MAP ≥75 mm Hg, a mixed venous oxygen saturation ≥65%, and an arterial lactate <2 mEq/L. If this was not the case, we administered a bolus of 10 mL/kg of Ringer’s lactate over 15 minutes. If CO increased by at least 10%, we repeated this fluid challenge until the targets were obtained.
After baseline measurements, the animals were randomized (computer-generated randomization list) into 3 groups. Sham animals (n = 5) underwent surgical preparation, but aortic clamping was not performed. The IR group (n = 7) underwent suprarenal aortic clamping for 1 hour. In the BH4 group (n = 7), the same procedure was followed, but they received 20 mg/kg of BH4 (Schircks Laboratories, Geneva, Switzerland) immediately before aortic clamping. This dose was based on previous experiments.22 Ringer’s lactate was infused at least at 5 mL/kg/h throughout the experiment. If the MAP decreased to <75 mm Hg, we administered a bolus of 10 mL/kg of Ringer’s lactate over 15 minutes. If CO increased by at least 10%, we repeated a fluid challenge if the MAP remained <75 mm Hg. To prevent early hypotension, an additional bolus of 10 mL/kg of Ringer’s lactate was infused before unclamping. Animals undergoing IR were observed until the sixth hour after reperfusion, when they were euthanized by injecting a high dose of potassium chloride under deep anesthesia.
The primary outcome was to assess the effects of BH4 pretreatment on the renal microcirculation as evaluated by changes in PPV. Secondary outcomes were redox state and renal function. All analyses were predefined. All data are presented as mean (95% confidence intervals) unless otherwise stated. Mixed-effects polynomial regression models with restricted maximum likelihood estimation and first-order autoregressive covariance structure were used to examine the differences in all analyzed variables among the 3 groups (sham, IR, and BH4) at 3 time points: 1, 4, and 6 hours. When the trajectory of an analyzed variable was unlikely to follow a straight line, we considered up to the second-degree polynomial models of time (hour) so that the effects of hour and hour2 on that variable were tested as fixed effects. Interaction effects between groups and hour and hour2 were also tested. Baseline values were included in the model as covariates so that group comparisons at each particular point in time are adjusted for baseline values. Comparisons between baseline and the 3 time points within groups were also made using mixed-effects polynomial regression models. No correction was performed for the multiple comparison tests made across the groups at each particular point in time (1, 4, and 6 hours) or between time points within groups. Model checking was performed by inspection of residual and normal plots. When the normality of the residuals was rejected, the analyzed variable was log-transformed to fit the normality requirement of the mixed model. In this exploratory trial, we did not perform an a priori calculation of the sample size, but selected the number of animals based on our previous experience with this animal model. Assuming that a 10% reduction in PPV is a clinically important difference, group sample sizes of 5 (sham), 7 (IR), and 7 (IR + BH4) achieve 88% power to detect a difference of 9.5 in the IR group from a PPV of 95 in a design with 4 repeated measurements having a first-order autoregressive covariance structure when the standard deviation is 6, the correlation between observations on the same subject is 0.7, and the α level is .05.
All tests were 2 sided, and P < .05 was considered statistically significant. All analyses were performed using IBM SPSS 24 for Windows (IBM Corporation, Armonk, NY).
Baseline variables were similar in the 3 groups (Table 1). All animals survived to the end of the experiment, and all measurements were available for each animal at each time point. In the sham group, heart rate and MAP were lower at 1 and 6 hours than at baseline, but all the other variables remained stable over time. Compared to the sham group, the IR group developed a hyperdynamic response, characterized by a higher mean heart rate and MAP from 1 hour. BH4 attenuated this response, with no significant differences in heart rate or MAP compared to sham animals (Table 1). The mean O2 delivery, O2 consumption, or O2 extraction did not differ among groups (data not shown). The mean relative renal blood flow (Supplemental Digital Content, Figure S2, http://links.lww.com/AA/B765), urine output, or fluid balance (Table 1) also did not differ among groups. Mean creatinine levels had increased similarly in the IR and BH4 groups at 30 minutes compared to the sham group but were significantly lower in the BH4 group than in the IR group at 6 hours (P = .026; Figure 1).
The IR group had a lower mean PVD and higher mean heterogeneity index of the PPV than the sham group at 1, 4, and 6 hours after reperfusion (Figure 2). BH4 blunted these microvascular alterations so that mean small vessel density, mean PVD, and mean PPV were higher than in the IR group from 1 to 6 hours after reperfusion. There were no statistically significant differences in microcirculatory variables between the BH4 and sham groups.
Mean cortical and medullary glucose were lower in the IR group than in the sham group at 1 hour, and mean cortical and medullary lactate and cortical pyruvate were higher (Table 2). The mean cortical and medullary L/P ratio and L/G ratio and cortical glycerol values were higher in the IR group from 1 hour than in the sham group (Table 2 and Supplemental Digital Content, Figure S3, http://links.lww.com/AA/B765). These alterations were blunted by BH4 administration, which was associated with lower levels of mean medullary and cortical lactate, cortical glycerol, and lower L/P and L/G ratios from 1 hour than in the IR group (Table 2 and Supplemental Digital Content, Figure S3, http://links.lww.com/AA/B765). Mean medullary glycerol was higher in the BH4 group than in the sham group at 1 hour (P= .008).
In this sheep model of renal IR induced by aortic cross-clamping, we demonstrated that BH4 administration improved the renal microcirculation and metabolism as well as organ function.
The IR induced by aortic cross-clamping was associated with early, marked alterations in the renal microcirculation with a decrease in the density of perfused capillaries and increased heterogeneity of perfusion, which persisted throughout the period of observation. These alterations were accompanied by altered cortical and medullary metabolism, which shifted toward an anaerobic profile, as shown by increased L/P and L/G ratios. Similar alterations in renal metabolism have been reported in models of experimental renal transplantation.27,28 We also observed an increase in glycerol, an essential component of the cellular membrane, suggestive of cellular necrosis.25 This increase was of short duration and concurrent with the increase in L/P ratio, suggesting hypoxic cell death. Taken together, our data suggest that anaerobic metabolism was the main IR-induced abnormality blunted by BH4 administration. The resolution of signs of anaerobic metabolism and necrosis despite persistent alterations in perfusion suggest either that there was enough flow for the remaining surviving cells or that there was oxygen conformance, with regulated hypometabolism obtained by shutting down cell functions not essential to cell survival, and allowing cell recovery when oxygen transport was restored.29
Interestingly, we observed similar metabolic alterations in the cortex and in the medulla, although the medulla is more sensitive to injury because of its lower baseline Po2.30 Indeed, in a rat model of IR, microvascular oxygenation was affected most in the medulla.23 Using intravital videomicroscopy in a rat model of renal IR, Yamamoto et al12 found that the microvascular alterations were more pronounced in the peritubular than in glomerular capillaries. Of note, we cannot be certain that the cortical microdialysis measurements were not influenced by the medullary region because of the close proximity or because of diffusion of the measured metabolites. Additionally, we only evaluated cortical microvascular perfusion and cannot be sure that medullary perfusion was similarly altered.
A crucial finding of this study is that BH4 limited the changes in the renal microcirculation 1 hour after reperfusion. Administration of BH4 resulted in greater microvascular density than was present in the IR group. In addition, BH4 limited the development of anaerobic metabolism and probably cellular necrosis (glycerol level). Our data do not allow us to determine whether BH4 improved renal metabolism due to an improvement in flow or through a direct cellular effect. Nevertheless, BH4 blunted the changes in renal function, as shown by lower creatinine levels compared to the IR group.
BH4 can exert a number of effects. Oxidative stress present during IR transforms BH4 into its inactive form (dihydrobiopterin [BH2]).31 As a result, endothelial NOS becomes uncoupled. Increased ROS generation by NOS in the absence of BH4 can induce microcirculatory damage32 and organ failure.1 It is likely that the exogenous load of BH4 in our animals counterbalanced the deleterious effects of the increased ROS or limited their production. Importantly, BH4 administration did not produce any major hemodynamic alterations and was well tolerated.
Our data are consistent with other trials which have also reported beneficial effects of BH4 in IR. In a murine model of renal ischemia, BH4 administration restored endothelium-dependent vasodilation mediated by NO release.20 In prolonged muscular ischemia in rats, BH4 improved microvascular perfusion and muscle viability.33 Finally, in a rat model of IR, sepiapterin, a precursor of BH4, improved cortical and outer medullary oxygenation and decreased renal oxygen extraction.23 However, those studies differ from ours in using rodent models with less hemodynamic monitoring. Because of the impact of aortic cross-clamping on hemodynamics, we investigated the effects of BH4 in a large animal model in which the impact of systemic hemodynamics could be explored. In other models,34 including in humans,35,36 BH4 increased microvascular perfusion. Our results are in line with these observations and further add that BH4 protects the kidney from IR via microvascular and cellular mechanisms independent of its systemic effects.
This study has several limitations. First, our SDF data analysis was semiquantitative. Nevertheless, this methodology has been previously validated, and the interobserver variability remains less than 5%.37 Second, the number of animals was limited, which may explain why some differences only achieved a trend to significance. However, all the evaluated variables (SDF, microdialysis, and biochemical variables) changed concordantly. Third, animals were healthy before the IR, which may limit direct extrapolation to patients with chronic vascular disease who would be most likely to require aneurysmal surgical intervention. Nevertheless, previous studies have shown positive effects of BH4 in patients with hypercholesterolemia38 and atherosclerosis.39 Fourth, we did not evaluate the effects of IR and BH4 in other organs. In a model of aortic cross-clamping, Siegemund et al40 explored the intestinal effects of abdominal aortic clamping. They found alterations in the intestinal microcirculation similar to those seen in the renal microcirculation in our study. Fifth, we did not explore the effects of BH4 in the sham group and cannot exclude that the effects of BH4 were independent of IR. Nevertheless, our results suggest that BH4 may be effective in preventing some of the deleterious effects of IR. Finally, our monitoring continued for 6 hours after reperfusion, and we cannot comment on the longer-term consequences of this intervention. Although we did not test whether BH4 could be effective if given after reperfusion, the usefulness of BH4 as a pretreatment already offers interesting clinical perspectives. Renal transplantation and abdominal aortic aneurysm surgery are clinical models of IR injury in which BH4 administration would be possible before aortic or renal clamping. Some experimental data show that BH4 may be beneficial in other common surgical procedures, such as cardiac surgery.41 These possibilities should be tested in clinical trials.
In conclusion, renal IR induced by aortic cross-clamping decreases renal microvascular density and increases perfusion heterogeneity. This process is accompanied by a shift toward anaerobic metabolism and renal dysfunction. These alterations can be prevented by administration of BH4 before IR.
The authors thank Hassane Njimi, MSc, PhD, Erasme Hospital, Brussels, Belgium, for his help with the statistical analyses.
Name: Lokmane Rahmania, MD.
Contribution: This author helped design the study, collect and analyze the data, and write the first draft of the manuscript.
Name: Diego Orbegozo, MD.
Contribution: This author helped design the study, collect and analyze the data, and write the first draft of the manuscript.
Name: Fuhong Su, MD, PhD.
Contribution: This author performed the animal experiments and revised the manuscript for critical content.
Name: Fabio Silvio Taccone, MD, PhD.
Contribution: This author helped design the study and revised the manuscript for critical content.
Name: Jean-Louis Vincent, MD, PhD.
Contribution: This author helped design the study and revised the manuscript for critical content.
Name: Daniel De Backer, MD, PhD.
Contribution: This author helped design the study and analyze the data, and revised the manuscript for critical content.
All authors read and approved the final text.
This manuscript was handled by: Avery Tung, MD, FCCM.
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