| Objective: To investigate the dynamic changes of urinary PCX excretion and toobserve the reno-protective effect of different dosages of hydrochloride pioglitazoneand its effect on renal PCX expression in diabetic rats. Methods: After an overnightfasting,8rats randomly allocated to normal control group received citrate bufferintraperitoneal injection. Type1diabetic model was induced by a single intraperitonealinjection of streptozotocin at a large dose of65mg/kg of body weight to42rats.72hours later, peripheral blood was harvested from vena caudalis to evaluate the bloodglucose level. Animals were considered to be type1diabetic rats if they had peripheralblood glucose concentrations of16.7mmol/L or greater in addition to polyuria and otherdiabetic features. After7days of diabetic model establishment, experiment diabetesrats were then randomly divided into following four groups: diabetic rats withouttreatment (DM, diabetic model control group, n=8),10,20and30mg/kg/d pioglitazone(PIO) treated diabetic rats (group DR1, DR2and DR3, n=8respectively). The day thedrug administration started was defined as week0. The peripheral blood glucose,urinary albumin, urinary sediment PCX, urinary creatinine were determined at the basal,the2nd week, the4th week and8th week in the five groups during the observation. Toeliminate the impact of urine volume, urinary albumin and urinary sediment PCX wereexpressed as urinary UAlb/UCr (UACR), UPCX/UCr (UPCR) respectively. Following8-week observation, all the animals were anaesthetized by intraperitoneal injection ofchloral hydrate (300mg/kg body weight). The blood samples were collected for 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 was calculated. By virtue of hematoxylin-eosin staining andelectron microscope, the pathological changes of the kidney tissue were observed.Moreover, the mean glomerular cross-sectional area, mean glomerular volume,glomerular basement membrane thickness (GBMT) and foot process fusion ratio (FPFR)were calculated. Renal tissue PCX protein and mRNA expression were determined byimmunohistochemistry and RT-PCR respectively.Results：(1) The blood glucose levels throughout the study period along with FBG and HbA1c atthe8th week in four diabetic groups were significantly higher than those in group NC(P<0.01), whereas no significant differences were found between PIO-treatment groupsand 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.Since the2nd weeks, the levels of UPCR in four diabetic groups were significantlyhigher than that of group NC. At the2nd week, UACR showed no dramatic differenceamong the four diabetic groups. At the4th week, UACR in group DR2and DR3weresignificantly lower than that of group DM, whereas UACR in group DR1was slightlylower. At the8th week, the levels of UACR in PIO-treatment groups were significantlylower compared to that of group DM (P<0.01), while UACR in group DR2and DR3were significantly lower than that in group DR1(P<0.05). (4) The capillary loops of normal glomerulus in group NC were distinct throughhematoxylin-eosin staining. The amount of endothelial cells, mesangial cells and thearchitecture of the glomerulus were normal. Mean glomerular cross-sectional area weresignificantly increased, accompanied by bowman’s space enlargement and mesangialregion proliferation in diabetic rats. At the8th week, KI, MGA and MGV in fourdiabetic groups were significantly higher than those of group NC (P<0.01). Followed8-week PIO administration, KI and MGA were significantly lower compared to those ofgroup DM (P<0.05), while the parameters mentioned above in group DR2and DR3were significantly lower than those of group DR1(P<0.05). In addition, MGV in groupDR2and DR3dramatically decreased as compared to that in group DM (P<0.01).(5) 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).(6) At the baseline, UPCR showed no significant difference among the five groups.Since the2nd weeks, the levels of UPCR in four diabetic groups were significantlyhigher than that of group NC. At the2nd week and4th week, UPCR showed nodramatic difference among the four diabetic groups. At the8th week, UPCR in groupDR2and DR3were significantly lower compared to that of group DM, whereas UACRin group DR1was slightly lower. In addition, the level of UPCR in group DR2and DR3were significantly lower than that of group DR1. (7) Immunohistochemistry analysis showed that PCX protein was detected within thevisceral surface of the bowman’s capsule, in the podocyte, with a membranous patternof staining in normal control samples. The comparison of renal PCX protein expressionamong the five groups was employed by integrated optical density (IOD) detection.PCX protein expressions were decreased significantly in four diabetic groups, especiallyin group DM (P<0.01). Renal PCX expressions were dramatically increased after8-week PIO administration and this was more prominent in group DR2and DR3incomparison with group DR1.(8) RT-PCR analysis demonstrated that expression of renal PCX mRNA in group NCwas significantly higher than that of group DM (P<0.01).8-week PIO treatmentimproved glomerular PCX mRNA expression (P<0.01). No dramatic difference werefound among group DR1, DR2and DR3(P>0.05).(9) UPCR was positively correlated with UACR and KI respectively (r=0.86, r=0.83,P<0.01).Conclusions：(1) Podocyte injury may be involved in the development of diabetic nephropathy.Increased urinary PCX excretion appears to be an early biomarker of diabetic kidneylesion.(2) Pioglitazone could alleviate kidney injury of diabetic rats, which may related to insome extent its effects of restraining the loss of PCX in the urine and inhibiting thedown-regulation of the mRNA and protein expression of podocyte PCX with a dose-dependent manner. |
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