The Effects And Mechanisms Of Compound RapA On Tubulointerstitial Fibrosis Of Diabetic Nephropathy

Purpose:To analyze and discuss the effects of compound RapA on renal tubule interstitialfibrosis of diabetic nephropathy and its mechanisms by in vivo and in vitro experiments.Methods:1. In vivo experiment: Establish a diabetic nephropathy (DN) rat model andrandomly divide Wistar rats into the normal control group, the model group and the RapAtreatment group. Combine high fat forage feeding with low dose (35mg/kg) STZ injection toinduce Wistar rat model of Type2diabetes. Inject the normal control group isometriccitrate buffer (0.1mol/L, PH4.5). The measured postprandial blood glucose after72h is(PBG)≥11.1mmol/L so that it is identified as a diabetic model. After the modeling, theRapA treatment group are respectively given intragastric perfusion of RapA (2.5mg/kg/d,and5.0mg/kg/d). Observe the rats’ general condition and measure their weight, etc. Detectfasting glucose, fasting insulin, lipids and24h urine protein etc. by ELISA and chemicalassay. Examine the effects of long-term oral RapA on glucose tolerance function of Type2diabetic rats by oral glucose tolerance (OGTT) test. Sacrifice rats after the RapAintervention has been implemented for12weeks and keep rats’ kidney tissue for Massonstaining. Use Western blot to detect TGF-Smad signal pathway, MAPK, NADPH oxidaseand protein expression level of PKC.2. In vitro experiment: Target human proximal tubular epithelial cell line (HK-2) to determine the IC50of RapA on HK-2cells by using WST-1. Group cells into: A. normalglucose control group (NG, D-glucose5.5mM) B. vehicle group (DMSO), C. high glucose(HG, D-glucose30mM), C. high glucose+RapA group (HG+RapA200nM). Detectprotein expression levels of E-cadherin, α-SMA, FN and LN by Western blot and ROSlevel by using flow cytometry.Results:1. A general biochemical indicator showed that the model rats’ fasting insulin, fastingblood glucose, blood lipids,24-hour urinary protein excretion levels were far higher thanthat the normal control group, while its body weight decreased significantly (P <0.05).Compared to the model group, fasting insulin, fasting glucose, total cholesterol, FFA,24-hour urine protein levels in RapA treatment group decreased significantly (P <0.05),while its body weight had no significant difference (P>0.05).2. According to oral glucose tolerance test, it was showed that blood glucose levels ofthe model group at0,30,60,90and120minutes significantly increased compared to thenormal group (P <0.05). While the whole blood glucose levels of the RapA treatment groupat0,30,60,90and120minutes partially decreased compared with the model group but arestill higher than that of the normal group (P <0.05).3. Masson staining results showed that the normal control group had no obviousabnormalities, renal interstitial fibrosis of the model group was obvious, renal interstitialcollagen deposition of RapA treatment group reduced compared with the model group,especially for5.0mg/kg/d (P <0.01).4. In accordance with results of renal tissue by Western blot, it was found that, in theRapA treatment group, TGF-β1, phosphorylation of Smad2, phosphorylation of Smad3andSmad4, NADPH oxidation enzyme (p67phox, p47phox, p40phox, p22phox), MAPK andphosphorylation of PKC-alpha/beta, protein expression levels significantly decreasedcompared with the model group, while SOD-1protein expression increased (P <0.05).5. The correlation between drug concentration and cell number was calculated by using RapA with different concentrations to intervene HK-2cells. Besides, according to thecalculation, IC50of RapA in HK-2cells was361.7~381.7nM.6. Based on cells Western blot results and compared to normal glucose control group,high glucose could result in the increase of the HK-2cells expression of α-SMA, FN andLN and the decrease of E-cadherin protein levels (P <0.05). After the intervention of RapA200nM, α-SMA, FN and LN protein expression decreased, E-cadherin protein expressionincreased (P <0.05), there was no significant difference between normal glucose controlgroup and vehicle group (P>0.05).7. RapA could significantly reduce the production level of reactive oxygen species forHK-2cells under the stimulation of high glucose (P <0.05).Conclusions:1. RapA can reduce related metabolic risk factors, such as fasting insulin, fastingglucose, total cholesterol and free fatty acids. In addition, it can improve glucose toleranceabnormalities and significantly reduce the level of24h urinary protein excretion.2. RapA can decrease TGF-Smad pathway of the renal tissue, MAPK, NADPHoxidase, of PKC-related protein, reducing diabetic renal interstitial fibrosis.3. High glucose can increase α-SMA, FN and LN protein expression of HK-2cells,but reduce the E-cadherin protein expression, suggesting that high glucose can stimulateHK-2cells to generate EMT. The RapA can inhibit HK-2cell transdifferentiation tomyofibroblasts, reduce the expression of extracellular matrix and the generation level ofreactive oxygen species, suggesting that this may be one of the mechanisms of RapA toprevent and treat diabetic renal interstitial fibrosis.