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Resveratrol inhibits TNF-α-induced inflammation to protect against renal ischemia/reperfusion injury in diabetic rats 1

Abstract Purpose To examine effects of resveratrol on renal ischemia/ reperfusion injury (I/R) in a streptozotocin (STZ)-induced diabetic rat model. Methods Twenty-four male Sprague Dawley rats were treated with STZ injection for the development of diabetes, and divided into the following groups: Sham group, I/R group and Resveratrol group (n=8). Resveratrol (RSV) was administered at a dose of 10 mg.kg-1.d-1 fourteen days prior to suffering from I/R. Renal function, histology, SOD, MDA, TUNEL assay and expression of TNF-α, IL-1β, NF-κB-P65, COX-2 and Caspase3, Bcl2 and Bax were analyzed. Results Administration of RSV significantly reduced the serum levels of renal dysfunction and injury markers, including creatinine, blood urea nitrogen and MDA; in the other hand, it significantly increased the serum levels of SOD. The protective effect of RSV was also reflected on histologic evaluation. RSV reduced the number of apoptotic cells as determined by TUNEL assay. RSV significantly reduced the protein expression of TNF-α, IL-1β, NF-κB-P65, COX-2 and Caspase3, and Bax. Meanwhile, RSV significantly increased the protein expression of Bcl2. Conclusion RSV attenuated I/R-induced renal injury in diabetic rats through the modulation of oxidative stress and TNF-α-stimulated inflammation.


■ Introduction
Acute kidney injury (AKI) is encountered in many clinical situations and leads to increase of health costs. As we know, renal ischemia/reperfusion (I/R) is one of the major causes of AKI. Diabetes is a common and increasing chronic metabolic disease. Diabetic nephropathy might gradually and unavoidably develop to end-stage renal failure 1 . Diabetes has been identified as an independent risk factor for AKI 2 ; meanwhile, it might not only cause renal damage and dysfunction, but also exacerbated oxidative stress, inflammation and apoptosis of I/R [3][4][5] .
The development mechanism of renal I/R is very complicated. Firstly, it is characterized by energy deficit, and then, it is followed by a series of physiological and pathological changes, including oxidative injury, inflammation, and metabolic dysfunction during reperfusion 6 . The underlying mechanism of I/R includes reactive oxygen species (ROS), abnormal lipid metabolism, inflammatory cascade, calcium overload and nitrosoredox imbalance from ischemic tissues. The tumor necrosis factor-α (TNF-α) was a major pro-inflammatory cytokine in kidney pathogenesis. Some studies have shown that TNF-α plays a key role in inflammation after renal I/R by up-regulating inflammatory genes such as cyclooxygenase 2 (COX-2) 7 . COX-2 plays an important role in the development of a serious of renal inflammatory diseases, especially in renal I/R. Inhibiting TNF-α activity has proved to have antioxidant and anti-inflammatory effect and protects kidneys from ischemic injury 8 .
Resveratrol was a polyphenol, which has been reported to possess a kind of pharmacological effects, such as anti-inflammatory properties, protection against coronary heart disease, modulation of lipid metabolism and prevention of cancer [9][10][11] . Resveratrol has significant protective effects against I/R injury in various organs [12][13][14][15][16] . Specifically, resveratrol has shown to protect against several types of renal injury by inhibiting oxidative stress and ameliorating inflammatory response, including diabetic nephropathy, drug-induced injury, and I/R injury [17][18] .
Nowadays, there is no research that reports the effects of resveratrol on renal ischemia/reperfusion injury in diabetic rats. In the present study, we aimed to investigate the effects of administration of resveratrol before I/R on renal structural and functional changes, oxidative stress, inflammatory response and apoptosis in diabetic rats. into 5µm thick sections according to the standard procedure. The sections were deparaffinized and hydrated gradually, and stained with hematoxylin and eosin (H&E). Morphologic assessments were performed blindly by an experienced renal pathologist.

Apoptosis assay
Renal apoptosis was examined by TUNEL assay using the in situ Apoptosis Detection kit from Roche Applied Science. TUNEL-positive cells were identified through the nucleus, which was stained either tan or brown. Five fields were randomly selected and the apoptosis index was calculated as the ratio of apoptotic-to-total cells.

Measurement of oxidative stress
The malondialdehyde (MDA) levels and superoxide dismutase (SOD) were detected using commercially available kits (Jiancheng Biotech, Nanjing, China), according to the manufacturer's instructions.

Immunohistochemistry
The expressions of COX-2 and Caspase3 were investigated. Tissue sections were stained by immunohistochemistry (IHC) using specific antibodies for COX-2 (1:1000 dilution; Cell Signaling Technology, Boston, MA) and Caspase3 (1:1000 dilution; Cell Signaling Technology, Boston, MA). Serial sections (thickness 5µm) were cut from the tissue blocks, deparaffinized in xylene, and hydrated in a graded series of alcohol. Staining was then performed using the DAB chromogenic agent (Dako Corp, Carpinteria, CA). Negative control experiments were routinely performed. For quantitation, the relative mean integrated optical density (IOD) of each group was divided by the average IOD of the control. All slides were evaluated by an experienced renal pathologist who was unaware of the origin of the slides.

Statistical analysis
All data are presented as mean±SEM. The means of the different groups are compared using one-way analysis of variance (ANOVA) and the Student-Newman-Keuls test. The Kruskal-Wallis ANOVA on ranks is used for nonnormally distributed data. The level of statistically significance is set at P<0.05.

Renal function
Cr and BUN levels were measured at 24 hours following I/R in diabetic rats. In this model, renal I/R caused a marked increase in serum Cr as well as BUN after I/R in diabetic rats. Treatment with resveratrol significantly reduced the serum Cr and BUN levels ( Fig. 1).

Morphologic features and immunohistochemistry
Morphologic features were evaluated using H&E (Fig. 2). The Sham group did not show any morphological changes. By contrast, the kidneys of untreated ischemic diabetic rats showed tubular cell swelling, cellular vacuolization, pyknotic nuclei, medullary congestion, and moderate to severe necrosis. Treatment with resveratrol preserved the normal morphology of the kidney and showed slight edema of the tubular cells and mild necrosis.

Caspase3
Resveratrol inhibits TNF-α-induced inflammation to protect against renal ischemia/reperfusion injury in diabetic rats Wang M et al.

Measurement of MDA and SOD
Renal I/R significantly increased the enzymatic activity of MDA, but it significantly decreased the enzymatic activity of SOD. However, these changes were significantly reversed by treatment with resveratrol (Fig. 3).

Resveratrol reduced cell apoptosis after renal ischemia/reperfusion
Apoptosis was evaluated by TUNEL assay. At 24 hours after I/R, a small quantity of TUNEL-positive cell was present in kidney obtained in Sham group. TUNEL assay showed an increase in the number of apoptotic cells after I/R and a decrease from RSV group (Fig. 5).
Renal I/R injury significantly up-regulated the levels of active caspase-3 and Bax expression in kidneys of diabetic rats. Resveratrol treatment inhibited the expression of active caspase-3 and Bax in diabetic rats. Meanwhile, Renal I/R injury significantly reduced cytosolic Bcl-2 levels in kidneys of diabetic rats. Resveratrol treatment restored the levels of Bcl-2 in diabetic rats (Fig. 5).

■ Discussion
Although resveratrol had demonstrated to possess significant protective effects against I/R injury of various organs, including kidney [12][13][14][15][16][17][18] , we demonstrated for the first time the protective effects of resveratrol in diabetic rats after renal I/R injury. Our study showed that administration of resveratrol proved to be an effective treatment against renal I/R in diabetic rats.
We demonstrated that resveratrol significantly reduced serum markers of renal dysfunction and injury, such as Cr and BUN. This was further supported by the preservation of renal histologic architecture in the treatment groups when compared to the Sham group and I/R group. Meanwhile, treatment with resveratrol was associated with a significant reduction in pro-inflammatory cytokines TNF-α, IL-1β and COX-2. Furthermore, administration of resveratrol was associated with a significant reduction in apoptosis induced by I/R injury in diabetic rats.
Oxidative stress played a key role in renal I/R injury 19,20 . As we know, free radicals played an important role in the process of lipid peroxidation reactions; it is very effective to protect renal function by inhibiting the oxidative stress-induced peroxidation of membrane lipids and other target macromolecules. During the reperfusion phase of ischemia, several potential sources of toxic oxygen species were involved in the injury tissue. ROS was one of these toxic oxygen species. It could not only deactivate some antiproteases, but also activate some proteases, which could lead to tissue damage 21,22 . Hyperglycemia was a notable characteristic of diabetes and could elevate the basal level of ROS in tissues and promote chronic oxidative stress. In the present study, MDA, an index of lipid peroxidation, was increased, but SOD was decreased in the kidney tissues, indicating the presence of I/R-induced oxidative damage in diabetic rats. In accordance with the previous observations, the findings of the present study showed that resveratrol was effective to reduce the I/R-induced renal damage and dysfunction in diabetic rats by reducing lipid peroxidation and scavenging oxygen free radicals.
Inflammatory response also played an important role in renal I/R injury. In the process of I/R injury, massive damage associated molecules were released. Then, inflammatory cells were recruited from the circulation into the injury renal tissue 23 . This was usually accompanied by the release of inflammatory cytokines, such as TNF-α, IL-1β and IL-6. Studies 4,24 showed that the inflammatory response induced by I/R injury was further exacerbated in diabetic rats, in which TNF-a and IL-1β levels in serum or renal tissues increased significantly. In this study, we have demonstrated that administration of resveratrol proved to be effective to inhibit the expression of the pro-inflammatory cytokines TNF-α and IL-1β elevated in the kidney of diabetic rats after I/R injury. Furthermore, COX-2 played an important role in renal inflammation, which was regulated by various stimuli, including TNF-α. Down-regulation of COX-2 proved to alleviate renal inflammation and reduce renal damage 25 . In the present study, resveratrol treatment proved to inhibit the expression of TNF-α, IL-1β, NF-κB-P65 and COX-2 after renal I/R in diabetic rats. So, we speculate that resveratrol might attenuate inflammatory response after renal I/R injury in diabetic rats via inhibiting TNF-αinduced NF-κB signaling and COX-2 activation.
Renal apoptosis was an important prognosticator in the development of ARF induced by I/R injury 26 . As oxidase load increased in the mitochondria, the outer membrane of mitochondria tended to be more permeable, resulting in the translocation of Bax from cytosol to the mitochondri. The Bcl-2 family proteins took part in the process of pro-apoptotic proteins translocation 27 . Our results showed that resveratrol significantly inhibited apoptosis caused by renal I/R injury in diabetic rats. In order to further clarify the reason of this change, we investigated the expressions of key apoptotic-related molecules, including active caspase-3, Bax and Bcl2. Our study showed that resveratrol increased the expression of anti-apoptotic Bcl-2 protein and inhibited the levels of Bax and active caspase-3.

■ Conclusions
We demonstrated that resveratrol protected diabetic rats against I/R-mediated renal injury. We further demonstrated that resveratrol possessed anti-oxidant, anti-inflammatory and anti-apoptotic properties after renal I/R injury in diabetic rats, which might be mediated via the modulation of oxidative stress, inhibition of TNFα-stimulated inflammation and inactivation of COX-2. These findings suggest the potential role of resveratrol against renal injury in diabetic patients.