| Objective:1.To evaluate the effect of pioglitazone hydrochloride(PIO)on the expression of renal nephrin mRNA and protein,and explore its possible reno-protective mechanism in streptozotocin(STZ)-induced diabetic rats.2.To observe the effect of pioglitazone on the urinary nephrin excretion in type 2diabetes and understand the protection role and discuss the mechanism of pioglitazone on diabetic nephropathy patients.Methods:1.Animal experiment part: 50 male Sprague-Dawley rats were randomly divided into normal control group and experimental group.8 rats were normal control group,42 diabetic models were fasted overnight and induced by a single i.p.injection of streptozotocin(65 mg/kg;Sigma Chemical,St.Louis,MO).Peripheral blood was harvested from vena caudalis 72 hr post-injection to assess BG level,rats with BG more than 16.7 mmol/L indicated the successful induction of diabetes.One week after the STZ injection,32 diabetic rats were randomly divided into the following four groups:vehicle(0.9% sodium chloride)treatment group 、 10 mg.kg–1.d–1 PIO treatment group(group DR1)、20mg.kg–1.d–1 PIO treatment group(group DR2)and 30 mg.kg–1.d–1PIO treatment group(group DR3)(n=8,respectively)for 8 weeks.Week 0 was defined as the start day of drug administration.10 ml morning urine sample was collected at the week 0th 、4th and 8th to test urinary albumin(UAlb),creatinine(Ucr)and sediment nephrin.Peripheral BG was tested weekly.After 8 weeks,all animals were anaesthetized(after 14 h of starvation)by using i.p.injection of chloral hydrate 300mg/kg,then blood sample was collected for the measurements of HbA1 c,lipid profile,blood urea nitrogen(BUN),serum creatinine(Scr).After the animals were sacrificed,the left kidney was immediately enucleated and weighed separately and then kidney hypertrophy index(KI)was calculated.Moreover,the mean glomerular cross-sectional area(MGA),mean glomerular volume(MGV),glomerular basement membrane thickness(GBMT)and foot process fusion ratio(FPFR)were calculated.Then the kidneys were surgically removed and weighed separately,then divided into two parts.One part was stored in 10% neutral buffered formalin for immunohistochemical and pathological analysis,and the other was quickly frozen in liquid nitrogen and stored at-80℃ for mRNA extraction.2.Clinical part: According to urine albumin excretion rate,114 diabetic patients were divided into normal group(n=25)、 microalbuminuria group(n=61)and macroalbuminuria group(n=28).We used enzyme-linked immunosorbent assay(ELISA)to detect urinary nephrin.61 diabetic patients with microalbuminuria were further randomly divided into control group(n=29,SU durg was added to the therapeutic regimen when FBG more than 7.0mmol/L)and pioglitazone group(n=32,pioglitazone was added to the therapeutic regimen when FBG more than 7.0mmol/L).Then we examined various clinical indexes(include FBG、HbA1C、urinary albumin、urinary nephrin and urinary creatinine)in control group and pioglitazone group.Results1.Animal experiment part:(1)Diabetic rats from group DM to PIO-treated groups had marked hyperglycaemia at any time point(P<0.01,versus group NC).HbA1 c levels elevated markedly at the 8th week in group DM(P < 0.01,versus group NC)with no significant difference between group DM and PIO –treated groups.Serum TG level was decreased in PIO treatedgroups(P < 0.05,versus group DM).The level of HDL-C in groups DR2 and DR3 increased significantly(P < 0.05,versus group DM).(2)At the 0th week,there was no difference in UACR among all five groups.But at the 8th week,the level of UACR increased significantly in group DM(P < 0.05,versusgroup NC),UACR in PIO-treatment groups decreased significantly(P < 0.05,versus DM group),which in group DR2 and DR3 were lower than that in group DR1(P <0.05),whereas no significant differences were found between group DR2 and DR3.(3)The structure and width of GBM and epithelial foot processes were mostly normal in group NC at the 8th week.In group DM,the ultrastructure of GBM became ambiguous,the FPFR increased significantly.In addition,GBMT and FPFR decreased markedly in PIO-treated groups compared with group DM(P < 0.05),but still increased compared with group NC(P < 0.05).Furthermore,the changes mentioned above in group DR2 and DR3 were superior to those in group DR1(P < 0.05).(4)At the 8th week,the KI、MGV and MGA in the four diabetes groups were increased signifcantly compared to the NC group(P<0.01).Besides the KI and MGA decreased markedly in PIO-treated groups compared with group DM(P < 0.05),and the KI and MGA in group DR2 and DR3 were significantly lower to those in group DR1(P <0.05).In addition,the MGV in group DR1 slightly lower than DM group(P>0.05),but the MGV in group DR2 and DR3 were inferior to DM group(P<0.01).(5)At the 8th week of the study,the UNERs in PIO-treated groups were decreased significantly as compared to group DM,in addition,UNERs in group DR2 and DR3 were lower than that in group DR1(P < 0.05),yet no statistical difference was detected between group DR2 and DR3.(6)At the 8th week,there was a significant decrease in nephrin protein content in group DM as compared to group NC,however there was a marked increase in PIO treated-groups as compared to group DM,whereas there were no significant differences among different doses of PIO-treated groups,The nephrin m RNA expression showed an significant increase in group DM as compared to group NC(P < 0.05),moreover the nephrin mRNA expressions were reduced in group DR1,DR2 and DR3 as compared to group DM,yet no differences among different dosages of PIO-treatment groups.(7)Pearson correlation analysis showed that UNER was related positively to UACR(r= 0.881,P < 0.01).2.Clinical part:(1)Nephrin was found in the urine of diabetes group,but didn’t be detected in normal control group.Meanwhile in macro-albuminuria group the UNER was significantly higher than that in the micro-albuminuria group..(2)There were no statistical difference about age、BMI、WHR、FBG、SBP、DBP and HbAlc between group A and B before treatment.However the FBG、HbAlc were significantly lower after treatment in the two group compared to before treatment(P<0.01),furthermore no statistical difference was found between the two groups after treatment(P>0.05).(3)The UNER and UACR were found no obvious change in the group A before and after treatment(p>0.05).But in group B,compared with before treatment,the UNER and UACR were found significantly decreased after treatment(P<0.05).(4)Pearson correlation analysis showed that UNER was related positively to UACR(r=0.624,P<0.01).Conclusions:(1)The enhanced nephrin is expressed in renal tissure duringthe condition of diabetes,meanwhile the excretion of urinary nephrin is increased.(2)Pioglitazone can inhibit the expression of nephrin of renal tissure as well as restraining the excretion of urinary nephrin in a dose-dependent manner and independent of its hypoglycemic effect.(3)Pioglitazone has protective effects on podocytes of diabetic rats,which may be related partly with its effects in excretion of urinary nephrin and regulating renal nephrin mRNA and protein expression.(4)Urinary nephrin excretion is increased in type 2 diabetic patients,which is associated with increased urinary albumin excretion.Moreover,pioglitazone can protect the kidney by reducing the urinary nephrin excretion in type 2 diabetic patients. |
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