| Background: Acute renal ischemia reperfusion(IR) injury is a common complication following some surgeries, including kidney transplantation. Once happened, the outcome of patients will be seriously affected. However, there is still a lack of effective drugs at present. And in clinical practice, patients mainly depend on renal replacement therapies, including hematodialysis and peritoneal dialysis, to passively wait for the damaged renal function to recover by itself. Therefore, if specific drugs can be found in treating renal IR injury, it will have a great prospect in clinical application.Polydatin(PD), a natural antioxidant, which has multiple pharmacological activities, has beneficial effects in some ischemic organs. Recently, it has been demonstrated that PD also exerts nephroprotective effects in renal IR injury Rats. But there is still a lack of systematic pharmacodynamic research of PD in renal IR injury mice and the underlying molecular mechanisms remains to be further explored.Objective: In this study, to evaluate the nephropretective effects of PD in renal IR injury and to explore the mechanisms underlying, renal IR injury models were established both in mice and in cultured cells. By detecting the levels of serum creatinine(Scr) and blood urea nitrogen(BUN) and performing the evaluation of histological score of kidney injuries(HSK) and terminal deoxynucleotidyl transferase d UTP nick end labeling(TUNEL) apoptosis staining in kidney specimen, the effects of polydatin in improving renal function and dreasing the damage degree of renal tissues were assessed. To evaluate the effects of PD in anti-oxidative stress and anti-inflammation in renal IR injury, some indicators of oxidative stress and molecules assocated with inflammation were detected. And by using the specific inhibitor of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt), wortmannin(Wort), to block the PI3K/Akt signaling pathway, or adding human recombination sonic hedgehog(Shh) protein to treat cells in vitro to activate the Shh signaling pathway, the underlying molecular mechanisms of the effects of PD in acute renal IR injury were further explored.Methods: Experiment One, male BALB/c mice weighting 20~25 g were randomly divided into five groups as follows:(1) Sham group;(2) Vehicle group;(3) PD low dose(10 mg/kg) group;(4) PD medium dose(20 mg/kg) group;(5) PD high dose(40 mg/kg) group. By clamping bilateral renal pedicles for ischemia 30 minute time followed by releasing clamps to allow blood reperfusion to establish renal IR injury mouse models. For mice in sham group, pedicles were also dissected but not clamped. Animals were ethically sacrificed at 3 d after renal IR injury, and whole blood and kidneys were harvested for further analysis. By detecting the levels of Scr and BUN and performing the evaluation of HSK and the percentage of positive cells of TUNEL apoptosis staining in kidney specimen, the renal function and damage degree of renal tissues were assessed. Malondialdehyde(MDA) and nitric oxide(NO) levels and superoxide dismutase(SOD) activity were measured by using colorimetric analysis. The expression of cyclooxygenase-2(COX-2) and inducible nitric oxide(i NOS) and the phosphorylation level of Akt were detected by using Western blot analysis. By using ELISA assay, the contents of tumor necrosis factor-?(TNF-?), interleukin-1?(IL-1?),(prostaglandin E-2) PGE-2 in renal tissues were measured.Experiment two, male BALB/c mice weighting 20~25 g were randomly divided into six groups as follows:(1) Sham group;(2) Sham group with Wort treatment;(3) IR group;(4) IR group with Wort treatment;(5) PD(40 mg/kg) group;(6) PD(40 mg/kg) group with Wort treatment. In this part of experiments, 1 mg/kg of wortmannin, the specific inhibitor of PI3K/Akt signaling, was given to mice intraperitoneally to block the activation of Akt in vivo. Animals were ethically sacrificed at 3 d after renal IR injury, and whole blood and kidneys were harvested for further analysis. By detecting the levels of Scr and BUN and performing the evaluation of HSK and the percentage of positive cells of TUNEL apoptosis staining in kidney specimen, the renal function and the damage degree of renal tissues were assessed. The levels of MDA and NO and the activity of SOD were measured by using colorimetric analysis. The expression of COX-2 and i NOS and the phosphorylation level of Akt were detected by using Western blot assay. By using ELISA analysis, the contents of TNF-?, IL-1?, PGE-2 in renal tissues were measured.Experiment three, human kidney tubular epithelial cells(HK-2 cells) were cultured under normal or oxygen-glucose deprivation/re-oxygenation(OGD/R) condition with different drug(including PD, Wort and Shh) administration methods. MTT assay was used to detect the survival ability of cells in different groups. The contents of TNF-? and IL-1? in supernatant fluid of cultured cells were detected by using ELISA assay. And Western blotting was used to detect the protein levels of total Akt(t-Akt), phospho-Akt(p-Akt), and Shh in cells of different groups.Results: Experiment one, compared with Vehicle group, the high dose of PD significantly decreased the Scr, BUN, HSK and the percentage of positive cells in TUNEL staining, obviously inhibited IR induced increase of MDA, and markedly improved the activity of SOD(P < 0.05). The high dose of PD significantly up-regulated the phosphorylation level of Akt, markedly inhibited the protein expression of COX-2 and i NOS, and significantly decreased the levels of their downstream factors PGE-2 and NO in kidney tissues(P < 0.05). The results of ELISA analysis showed that compared with Sham group, the expression of TNF-? and IL-1? in kidneys after IR was significantly higher(P < 0.05); compared with Vehicle group, both 20 and 40 mg/kg of PD obviously decreased the expression of TNF-? and IL-1? in kidneys after IR injury(P < 0.05).Experiment two, the results of Western blot showed that intraperitoneal injection of Wort(1 mg/kg) markedly inhibited the phosphorylation of Akt in kidneys of model mice. Compared with the PD group without Wort treatment, the administration of PD with Wort together significantly increased the BUN, Scr, HSK and the percentage of positive cells in TUNEL staining, abolished the antioxidative effects of PD including decreasing the content of MDA and increasing the activity of SOD, and counteracted the anti-inflammatory effects of PD including inhibiting the expression of TNF-?, IL-1?, COX-2 and i NOS and decreasing the contents of PGE-2 and NO(P < 0.05).Experiment three, PD significantly improved the viability of cells with OGD/R treatment, and apparently inhibited the secretion of TNF-? and IL-1? induced by OGD/R(P < 0.05). The inhibition of PI3K/Akt signaling pathway counteracted the anti-inflammation and pro-survival effects of PD, and blocked the protein expression of Shh which was up-regulated by PD in HK-2 cells(P < 0.05). The exogenous treatment of human recombination Shh protein not only improved the survival of cells with OGD/R treatment, but also inhibited the inflammation induced by OGD/R in HK-2 cells(P < 0.05).Conclusion: PD improved the renal function, decreased the renal tissue damage, and exerted beneficial effects of anti-oxidative stress and anti-inflammation in renal IR injury mice, which might be regulated by PI3K/Akt pathway. And we further identified that PD up-regulated the expression of Shh protein in HK-2 cells to exert pro-survival and anti-inflammatory effects, which were, at least partly, dependent on the activation of PI3K/Akt pathway. |
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