1. Resveratrol Ameliorated Diabetic Nephropathy By Activating Nrf2 Pathway 2. Increased Dietary Sodium Induces COX2 Expression By Activating NFκB In Renal Medullary Interstitial Cells

Part ⅠResveratrol ameliorated STZ induced diabetic nephropathyObjective To investigate the effect of resveratrol in STZ induced diabetes rat.Methods Normal male Sprague Dawley rats (n=60) were uninephrectomized. After one week, these rats were randomly divided into three groups:the control group (n=10, PBS 50mg/kg ip., physiological saline was administered by gavage at a dose of 5ml/kg/d), DN group (n=25, STZ 50mg/kg ip., physiological saline was administered by gavage at a dose of 5ml/kg/d) and DN plus resveratrol group (n=25, STZ 50mg/kg ip., resveratrol was administered by gavage at a dose of 20mg/kg/d). Eight weeks later, these rats were housed in metabolic cages and collected 24-hour urine. Then the rats were sacrificed under anesthesia and the kidneys were harvested for immunohistochemistry and immunoblotting, etc.Results 1.Resveratrol had no effect on the body weight and blood glucose level of STZ induced rats.2. Resveratrol decreased the urine albumin excretion rate and creatinine clearance rate of STZ induced diabetes rats.3. The expression of SIRT1 in STZ diabetes rats was lesser than that of normal control.Conclusion Treatment of resveratrol had no effect on the level of body weight and blood glucose of STZ induced diabetes rats. The urine albumin excretion rate of diabetes rats were decreased with the therapy of resveratrol. Resveratrol could also ameliorate the renal diseases of diabetes rats.Part IIResveratrol attenuated oxidative stress by activating Nrf2Objective To explore the mechanism of resveratrol ameliorate diabetic nephropathy. To observe if resveratrol could attenuate oxidative stress by activating Nrf2 in diabetic nephropathy.Methods 1. Animal model:Ten normal male SD rats were sacrificed under anesthesia and the kidneys were harvested for immunohistochemistry and immunoblotting2.in vitro study:In the cultured glomerular endothelia cells,A) Glucose stimulated model:Endothelia cells were treated with normal glucose, high glucose, and high glucose plus resveratrol for 24 hours, respectively. Then the expression of Nrf2 and SIRT1 were explored by immunoblotting, realtime PCR.B) Oxidative stress stimulated model:Endothelia cells were pre-treated with Nrf2 activator sulforaphane at different dose for 24 hours. Hydrogen peroxide was used to produce oxidative stress for 1 hour. Then the cells were harvested to test the expression level of Nrf2 and HO-1.C) Resveratrol stimulated model:Different dose of resveratrol was given to the endothelia cells for 24 hours. Immunoblotting and realtime PCR were used to detect the expression level of Nrf2 and HO-1.Results 1. SIRT1 and Nrf2 expressed rich in the normal renal cortex and medulla. Renal medulla expression of Nrf2 was higher than that of renal cortex (P<0.05). The expression of Nrf2 was similar with SIRT1 in normal kidney.2. High glucose decreased the expression of Nrf2 and SIRT1 in endothelia cells.3. Nrf2 activator could protect the endothelia cells by anti-oxidative stress. Nrf2 activator sulforaphane had no effect of the expression of Nrf2 but increased the level of HO-1 significantly.4. Resveratrol could activate Nrf2 by increasing the mRNA expression of Nrf2 and HO-1.Resveratrol amiloerated the decreasing of Nrf2 induced by high glucose in endothelial cells.Resveratrol drecreased the produce of ROS induced by high glucose in endothelial cells.5. There were some relationships between SIRT1 and Nrf2. These two moleculars could immunoprecipitate together.Conclusion Resveratrol attenuated oxidative stress by activating Nrf2, and then ameliorated diabetic nephropathy.Part ⅢCOX2 expression induced by high salt diet is localized to renal medullary interstitial cellsObjective To investigate the effect of high salt diet on the expression of COX2 and its cellular location.Methods Male C57B1/6j mice were fed either high salt diet (8% NaCl) or normal salt diet (0.3% NaCl) for 1 to 7 days. At the end of experiments, mice were sacrificed under anesthesia and the kidneys were harvested for immunohistochemistry, in situ hybridization, and immunoblotting.Results 1. Renal medullary COX2 expression was increased after high salt diet, while COX1 expression was not changed. Renal medullary COX2 expression was substantially increased after 2 days of high salt diet, and remained elevated throughout the 7 days study. On the contrary, COX1 immunoreactive protein level was constitutively high, and not altered by high salt diet.2. COX2 is localized exclusively in renal medullary interstitial cells after high salt diet. The results of in situ hybridization showed COX2 mRNA expression was primarily localized in the renal medulla, specifically in renal medullary interstitum between renal tubules. COX1 mRNA was also expressed in the renal medulla, but was detected primarily in the collecting ducts. The immunofluorescence staining of COX2 revealed perinuclear localization in subpopulation of renal medullary cells that arranged in rows, resembling the rungs of a ladder. COX2 immunofluorescence did not co-localize with AQP2, AQP1, CLC-K, CD31.Conclusion The renal medullary interstitial cells are the major sites of COX2 induction following high salt diet.Part ⅣIncreased Dietary Sodium Induces COX2 Expression by activating NFκB in Renal Medullary Interstitial CellsObjective To evaluate the role of NFκB in high salt diet induced renal medullary COX2 expression.Methods 1.The effect of high salt diet on renal medullary NFκB activity was examined in transgenic mice carrying a luciferase reporter driven by an NF□B response promoter, HLL mice. The cellular location of NFκB activation was examined using transgenic mice that carry an enhanced green fluorescent protein (EGFP) fusion protein under the control of an NFκB response promoter LTR. HLL mice were fed with either normal salt diet or high salt diet for 3 days, after which renal medullary luciferase activity was determined using a commercial luciferase assay kit. The expression of EGFP was determined by immunofluorescence staining using an anti-EGFP antibody.2.To test the hypothesis that NFκB is responsible for mediating high salt diet induced COX2 expression in the renal medulla, mice on normal salt diet were pretreated with an IκB kinase selective inhibitor, IMD-0354 or vehicle for 2 days, followed by high salt diet for 3 days in the presence of IMD-0354 or vehicle. These mice were housed in metabolic cages and collected 24-hour urine at the last day of the experiment. Urinary PGE2 concentration was measured using ELISA kit. Mice were sacrificed under anesthesia and the kidneys were harvested for immunoblotting.3. Thirty c57BL/6J mice on normal salt diet were pretreated with a proteasome inhibitor, Bortezomib or vehicle 0.5mg/kg iv biw. Then the mice were fed with either normal salt diet or high salt diet for 3 days. These mice were housed in metabolic cages and collected 24-hour urine at the last day of the experiment and then sacrificed under anesthesia and the kidneys were harvested for immunoblotting.Results 1. High salt diet increases NFκB activity in renal medullary interstitial cells. High salt diet significantly increased luciferase reporter activity in the renal medulla by 7 fold compared to the normal salt diet fold (3626±1045 vs 513±248 unit/mg protein, n=10, P<0.05), suggesting that NF□B was activated in renal medulla following high salt diet. NFκB EGFP reporter mice showed strong EGFP immunofluorescence in renal medulla of the mice fed with high salt diet, but not in mice on normal salt diet. The EGFP expression was mainly observed in the renal medullary cells that were arranged in a laddering pattern between renal tubules, consistent with renal medullary interstitial cells.2. NFκB blockade attenuates high salt diet induced renal medullary COX2 expression. Treatment with the NFκB inhibitor substantially attenuated COX2 induction following high salt diet, suggesting that NFκB activation is essential to renal medulla COX2 expression induced by high salt diet. Consistently, treatment with IMB-0354 also significantly reduced urinary PGE2 excretion following high salt diet (809±267 vs 1798±578 mg/24h, n=3, P<0.05)3. The proteasome inhibitor, bortezomib also substantially attenuated COX2 induction following high salt diet. Treatment with bortezomib also significantly decreased the 24 hours urinary sodium excretion following high salt diet. Conclusion The NFκB pathway plays a critical role in high salt diet induced COX2 expression.