Protective Effects Of Resveratro1on The Rats With Early-Stage Diabetic Nephropathy And The Underlying Mechanisms

Objective: To investigate the protective effects of resveratrol on the ratswith early-stage diabetic nephropathy and the underlying mechanisms. Method （1）Sprague-Dawley （SD） male rats were given a single intraperitoneal injection ofstreptozotocin（STZ）65mg kg^-1, dissolved in ice cold sodium citrate buffer0.01MpH7.4, after12h of food deprivation to set up animal models of diabetes. Twelveweeks after STZ injection, the serum creatinine （Scr）,24h urinary albuminexcretion（UAE） and renal cortex morphological studies of diabetic rats showed theearly features of DN and rat models of DN were established successfully. Rats withDN were randomized into DN control group （group DN, n=6） and DN group withRes intervention （group DR, n=6）. The non-diabetic animals were divided into twogroups, one group remained normal controls （group NC, n=6） while the other groupwas treated with Res （group NR, n=6）. Rats in DR and NR groups were administeredintragastricly with Res10mg kg^-1 d^-1while those in DM and NC groups with equalvolume of0.5%sodium carboxymethylcellulose regularly for2weeks. At the end of14weeks, the blood glucose （BG）, body weight （BW）, kidney weight （KW）, serumcreatinine（Scr）, blood urea nitrogen （BUN）,24h urinary albumin excretion（UAE） ofall rats were measured. Renal cortex from four groups were embedded in paraffin andsectioned for morphological studies to inviestigated the early features of DN. Theactivity of malondialdehyde （MDA）, superoxide dismutase （SOD） and catalase （CAT）in the renal cortex was assayed by chromatometry. The expression of NOX4andGRP78proteins in the renal cortex were examed by immunohistochemistry. Thelevels of NOX4and GRP78proteins in the renal cortex were determined by Westernblot.（2） The rat renal tubular epithelial cells （NRK-52E） were cultured in vitro. Whenreached near85%confluency, the cells were serum-depleted for24h to synchronize cell growth. The NRK-52E cells were divided into four groups: normal glucose （NG5.6mmol/L）, normal glucose plus resveratrol （NG+Res20μmol/L）, high glucose （HG30mmol/L）, high glucose plus resveratrol （HG+Res20μmol/L）. After treatment for24h, the rate of cell proliferation was examined by MTT method，the rate of cellapoptosis was determined by flow cytometry and the expression of NOX4，GRP78proteins in the NRK-52E cells were analysed by Western blot. Results：（1） Comparedwith those in group NC, the BW in DN group were decreased, while BG, KW、KW/BW, Scr, BUN and24h UAE increased significantly （all P value <0.01）. The24h UAE, Scr and BUN, KW/BW in group DR decreased significantly as comparedwith those in group DN （all P value <0.01）. But there was no significant differencesin BG and BW between DR and DN group. The glomerular basement membrane wasintact in NC group, enhanced glomerular mesangial matrix, cell proliferation andenlarged renal tubule were observed in group DN and which were attenuated in thegroup DR.（2） The MDA activity in DN group increased while the SOD and CATactivity decreased significantly as compared with those in group NC （all P value<0.01）. The SOD and CAT activity was significantly higher（P<0.05） while MDA （Pvalue <0.01） was significantly lower in group DR than those in DN group. Rats withDN treated with resveratrol showed a robust decrease in NOX4and GRP78expression in renal cortex compared with untreated rats with DN. The proteinexpression of NOX4and GRP78in renal cortex of group DN were up-regulated thanthose in group NC （P value <0.01）. After the administration of resveratrol, theexpression of NOX4（P value <0.05） and GRP78（P value <0.01） protein weredown-regulated in group DR than those in group DN.（3） Compared to normal groupcells，the rate of cell proliferation and apoptosis in HG group increased significantly.Simultaneous incubation with resveratrol abrogated high glucose-induced NRK-52Ecell proliferation and inhibited cell apoptosis （all P value <0.01）. As compared to the NG group，a significant increase of NOX4、GRP78protein levels in HG group but asignificant decrease in HG plus resveratrol group compared to the HG group （all Pvalue <0.05）. Conclusions These findings suggest the possibility that resveratrolexerts anti-proliferative and anti-apoptosis effects on rat renal tubular epithelial cellsby suppressing oxidative stress and endoplasmic reticulum stress, thus alleviates renalinjuries of the rats with early stage of diabetes nephropathy.