Experimental Study On The Protection Against Podocyte Injury And The Relation With Anti-inlfammatory And Anti-fibrosis Action By Pioglitazone In Diabetic Rats

Objective: To investigate the protection against podocyte injury and its relation withantiinflammatory action and anti-fibrosis by different dosages of hydrochloridepioglitazone in diabetic nephropathy(DN) rats.Methods: After an overnight fasting,8rats randomly allocated to normal control groupreceived citrate buffer intraperitoneal injection. Type1diabetic model was induced by asingle intraperitoneal injection of streptozotocin at a large dose of65mg/kg of bodyweight to42rats.72hours later, peripheral blood was harvested from vena caudalis toevaluate the blood glucose level. Animals were considered to be type1diabetic rats ifthey had peripheral blood glucose concentrations of16.7mmol/L or greater in additionto polyuria and other diabetic features. After7days of diabetic model establishment,experiment diabetes rats were then randomly divided into following four groups:diabetic rats without treatment (DM, diabetic model control group, n=8),10,20and30mg/kg/d pioglitazone (PIO) treated diabetic rats (group DR1, DR2and DR3, n=8respectively). The day the drug administration started was defined as week0. Theperipheral blood glucose, urinary albumin, nephrin, MCP-1, TGF-β1and urinarycreatinine were determined at the basal，the4th week and8th week among the fivegroups during the observation. To eliminate the impact of urine volume, urinaryalbumin, nephrin，MCP-1，TGF-β1were expressed as urinary UAlb/UCr (UACR),Unep/UCr (UNER), Umcp-1/UCr (UMCR), UTGF-β1/UCr (UTGR) respectively.Following8-week observation, all the animals were anaesthetized by intraperitoneal injection of chloral hydrate (300mg/kg body weight). The blood samples were collectedfor measuring HbA1c, lipid profile, serum creatinine and BUN. After the animals weresacrificed, the left kidney was immediately enucleated and weighed separately and thenkidney hypertrophy index(KI) was calculated. By virtue of electron microscope, thepathological changes of the kidney tissue were observed. Moreover, the meanglomerular cross-sectional area（MGA）, mean glomerular volume（MGV）, glomerularbasement membrane thickness (GBMT) and foot process fusion ratio (FPFR) werecalculated. Renal tissue nephrin mRNA，nephrin protein，MCP-1protein and TGF-β1protein expression were determined by RT-PCR and immunohistochemistryrespectively.Results：(1) The blood glucose levels throughout the study period and HbA1c at the8th week infour diabetic groups(group DM，DR1，DR2and DR3) were significantly higher thanthose in group NC (P<0.01), whereas no significant differences were found betweenPIO-treatment groups and group DM (P>0.05).(2) At the8th week, the parameters including SCr, BUN, TG, LDL-C were significantlyhigher, whereas serum HDL-C in four diabetic groups were lower compared to those ofNC groups (P<0.05or P<0.01). Pioglitazone therapy markedly reduced serum TGconcentration. Notably, serum HDL-C in group DR2and DR3were higher than that ofgroup DM (P<0.05).(3) At the baseline, UACR showed no significant difference among the five groups.Atthe4th week, UACR in group DR2and DR3were significantly lower than that of groupDM, whereas UACR in group DR1was slightly lower. At the8th week, the levels ofUACR in PIO-treatment groups were significantly lower compared to that of group DM(P<0.01), while UACR in group DR2and DR3were significantly lower than that ingroup DR1(P<0.05). (4) As shown in electronmicrographs, the glomerular basement membrane thickness(GBMT), ultrastructure of the podocyte and mesangial region in group NC were normaland foot process fusion rate (FPFR) was nearly0.03%. However, both the foot processeffacement and GBM incrassation were also observed in group DM. Meanwhile, somefoot processes were completely ruined, even vanished and the architecture of GBMbecame ambiguous. However, through PIO treatment, both FPFR and GBMT werereduced compared to those of group DM (P<0.01). More importantly, the decliningamplitudes of the parameters mentioned above were significantly greater in group DR2and DR3compared with those of group DR1(P<0.01).(5) At the baseline,UNER can be detected in five groups without no significantdifferences；At the4th week,UNER were significantly reduced in PIO-treated groups（P<0.05,versus DM group）,whereas no differences among the PIO-treated groups；At the8th week,UNER，UMCR and UTGR were significantly reduced in PIO-treatmentgroups compared to group DM（P<0.05）.UNER and UMCR both in DR2and DR3group were significantly lower than these of group DR1.(P<0.05),yet no differencebetween DR2and DR3group. UTGR among group DR1, DR2and DR3were not founddramatic difference.(6)The comparison of renal protein expression among the five groups was employed byintegrated optical density (IOD) detection：①Nephrin protein expressions：NC group＞PIO-treatment groups＞DM group（P＜0.05），whereas no differences among thePIO-treatment groups.②M CP-1protein expressions：DM group＞PIO-treatment groups＞NC group（P＜0.05），whereas no differences among the PIO-treatment groups.③T GF-β1protein expressions：DM group＞PIO-treatment groups＞NC group（P＜0.05），whereas no differences among the PIO-treatment groups.(7) RT-PCR analysis demonstrated that expression of renal nephrin mRNA in groupDM was significantly higher than that of group NC (P<0.05).8-week PIO treatmentreduce glomerular nephrin mRNA expression (P<0.05). No dramatic difference were found among group DR1, DR2and DR3.(8) Pearson correlation analysis shows：①UNER was positively correlated with UACRand KI(r=0.881，P<0.01；r=0.833，P<0.01).②UNER was positively correlatedwithUMCR (r=0.729，P<0.01).③UNER was positively correlated with UTGR (r=0.787，P<0.01).⑤The expression of renal nephein protein was negatively correlated with renalMCP-1protein.（r=-0.696，P＜0.01).⑥The expression of renal nephrin protein wasnegatively correlated with renal TGF-β1protein（r=-0.669，P＜0.01).Conclusions：(1) PIO has a protection aganist podocyte injury in STZ-induced diabetic rate with adose-dependent mode, which is dependent of its hypoglycemic effect.(2) Pioglitazone has protective effects on podocytes of diabetic rats, which may berelated partly with its effects in regulating renal nephrin mRNA and protein, this effectmay be again related with its effect in inhibiting renal local MCP-1and TGF-β1expression.