Resveratrol Rescues Kidney Mitochondrial Function Following Hemorrhagic Shock

Abstract

Objective: Hemorrhagic shock may contribute to acute kidney injury (AKI) by profoundly altering renal mitochondrial function. Resveratrol (RSV), a naturally occurring sirtuin 1 (SIRT1) activator, has been shown to promote mitochondrial function and reduce oxidative damage in a variety of aging-related disease states. We hypothesized that RSV treatment during resuscitation would ameliorate kidney mitochondrial dysfunction and decrease oxidative damage following hemorrhagic shock.

Methods: Using a decompensated hemorrhagic shock model, male Long-Evans rats (n = 6 per group) were killed prior to hemorrhage (sham), at severe shock, and following either lactated Ringer's (LR) resuscitation or LR + RSV resuscitation (RSV: 30 mg/kg). At each time point, blood samples were assayed for arterial blood gases, lactate, blood urea nitrogen, and serum creatinine. Mitochondria were also isolated from kidney samples in order to assess individual electron transport complexes (complexes I, II, and IV) using high-resolution respirometry. Total mitochondria reactive oxygen species were measured using fluorometry, and lipid peroxidation was assessed by measuring 4-hydroxynonenal by Western blot. Quantitative polymerase chain reaction was used quantify mRNA from peroxisome proliferator-activated receptor γ coactivator 1-α (PGC1-α) SIRT1, and proteins known to mitigate oxidative damage and promote mitochondrial biogenesis.

Results: Resveratrol supplementation during resuscitation restored mitochondrial respiratory capacity and decreased mitochondrial reactive oxygen species and lipid peroxidation. Compared with standard LR resuscitation, RSV treatment significantly increased SIRT1 and PGC1-α expression and significantly increased both superoxide dismutase 2 and catalase expression. Although RSV was associated with decreased lactate production, pH, blood urea nitrogen, and serum creatinine values did not differ between resuscitation strategies.

Conclusions: Resuscitation with RSV significantly restored renal mitochondrial function and decreased oxidative damage following hemorrhagic shock.

Figure 1. Potential mechanisms of beneficial effects of resveratrol in mitochondrial protection and anti-oxidative properties

SIRT1, sirtuin 1; PGC1-α, peroxisome proliferator-activated receptor gamma coactivator 1-α; NRF, nuclear respiratory factor; TFAM, mitochondrial transcription factor A; SOD2, superoxide dismutase 2.

Figure 2. Resveratrol treatment during resuscitation restored renal mitochondrial function following hemorrhagic shock and resuscitation

Mitochondrial respiratory capacities in isolated intact mitochondria were assessed by high resolution respirometry. Hemorrhagic shock decreased the respiratory capacity of all complexes in this model of decompensated hemorrhagic shock. RSV supplementation significantly restored Complexes II and IV-dependent respiratory capacity. LR = Lactated Ringer’s solution; RSV = Resveratrol. Values are mean ± SEM. n = 6,*p<0.05 versus Sham; ^†p<0.05 versus Severe Shock; ^‡p<0.05 versus LR Resuscitation.

Figure 3. Resveratrol treatment during resuscitation ameliorated renal mitochondrial oxidative stress following hemorrhagic shock and resuscitation

A, Mitochondrial-derived reactive oxygen species (ROS) production was detected by measuring the fluorescent signal from dichlorofluorescein. RSV supplementation significantly reduced the production of ROS following hemorrhagic shock and resuscitation. B, LR+RSV resuscitation significantly increased the mRNA expression of dismutase 2 (SOD2) and catalase (CAT) in kidney tissue when compared to LR resuscitation. C, 4-hydroxynonenal (4-HNE) was measured by western blotas a marker of mitochondrial lipid peroxidation. 4-HNE levels robustly increased following Severe Shock and were significantly reduced with LR+RSV resuscitation. D, Expression levels of 3-nitrotyrosine (3-NT) in mitochondria were determined by western blot. 3-NT levels increased with LR resuscitation, but did not increase with LR+RSV resuscitation. LR = Lactated Ringer’s solution; RSV = Resveratrol; COX = cyclooxygenase. Values are mean ± SEM. n = 6, *p<0.05 versus Sham; ^†p<0.05 versus Severe Shock; ^‡p<0.05 versus LR Resuscitation.

Figure 4. Resveratrol supplementation enhanced the mRNA expression of Sirtuin 1(SIRT1) and increased the nicotinamide adenine dinucleotide (NAD+) -nicotinamide adenine dinucleotide dehydrogenase (NADH) ratio in kidney

A, The decline in the mRNA expression level of SIRT1 after severe shock and LR resuscitation was reversed with RSV administration during resuscitation. B, NAD+, NADH concentration and NAD+-NADH ratio in kidney tissue were obtainedby detecting the fluorescent signal of resazurin. LR+RSV resuscitation significantly decreased the NADH concentration and nearly doubled the NAD+-to-NADH ratio when compared to LR resuscitation. LR = Lactated Ringer’s solution; RSV = Resveratrol. Values are mean ± SEM. n = 6, *p<0.05 versus Sham; ^†p<0.05 versus Severe Shock; ^‡p<0.05 versus LR Resuscitation.

Figure 5. mRNA expression of mitochondrial biogenesis factors and mitochondria abundance in kidney

A, mRNA expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) was significantly elevated in LR+RSV resuscitation group compared to the LR resuscitation only group. However, the mRNA expressions of mitochondrial transcription factor A (TFAM), nuclear respiratory factor (NRF) 1 and 2 were not influenced by RSV supplementation. B, Citrate synthase activity was determined spectrophotometrically based on coenzyme A coupled to 5, 5′-dithiobis-2-nitrobenzoic acid. Citrate synthase activity did not significantly change with Severe Shock or with resuscitative strategy. LR = Lactated Ringer’s solution; RSV = Resveratrol. Values are mean ± SEM. n = 6, *p<0.05 versus Sham; ^†p<0.05 versus Severe Shock; ^‡p<0.05 versus LR Resuscitation.