Expression of protein p27 in glomerular cells of diabetic rats in relation to cell proliferation and apoptosis

Xiaofeng HE; Xiaocheng LIU; Jufang SHAO

ABSTRACT

BACKGROUND:

The p27, one of the cyclin-dependent kinase inhibitors in renal disease, regulates cell growth through affecting cellular events. Abundant evidence has suggested that p27 has great role in regulating re nal cell proliferation, hypertrophy, differentiation and apoptosis, etc.

OBJECTIVE:

To investigate the relationship of protein 27 expressions in renal tissue with cell proliferation and apoptosis.

DESIGN:

Completely randomized grouping design, control animal experi ment.

SETTING:

Nephrology Laboratory, Tongji Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology. MATERIALS This experiment was carried out at Nephrology Laboratory of Internal Medicine, Tongji Hospital and Micro-Pathology Center in Tongji Medical College from September 2004 to January 2005. A total of 48 adult male SD rats were enrolled, all belonged to cleaning grade.

METHODS:

①Establishing diabetic models diabetic rat models were es tablished with streptozotocin, 60 mg/Kg, given through intraperitoneal in. Jection in the experimental group. Blood glucose and urine glucose from caudal vein were measured after 48 hours. Animals in experimental group whose random serum glucose at least 16.7 mmol/L and urine glucose ()- ()-() were regarded as successful models. ②Six rats in experimental group and the control group, respectively were sacrificed in the experimental pe riod of the 3rd, 7th, 14th and 28th days. Before sacrificed, creatinine clearance (Ccr) was measured. Concentration of Retinal binding protein (Rbp) in urine was measured. Proteinuric elements were analyzed by sodium dodecyl sulfate-polyacrlamide gelelectrophoresis (SDS-PAGE). ③Renal tissues were observed pathologic changes through light microscopy and electronic mi croscopy. ④Expression of p27 and proliferation cellnuclear antigen (PC NA) were detected by immunohistochemical staining method, and apoptosis was determined by Terminal deoxynucleotidy 1 transferase-mediated dUTP nick end labeling (TUNEL). We use positive glomerular cells / total glomerular cells to present positive rate and apoptosis rate. ⑤The data were analyzed for statistically significant group difference using unpaired t test and Spearman correlative-analysis. MAIN OUTCOME

MEASURES:

Renal function, urine protein, p27 ex pression in glomerular cells, positive rate and apoptosis rate of proliferating cell nuclear antigen (PCNA) expression and compared with the control group.

RESULTS:

Totally 48 rats were involved in the result analysis. ①Changes of pathology At the 7th day after establishing models, glomerular hypertro phy was found through light microscopy and focal fusion of epithelial foot processes through electron microscopy. At the 14th day, mesangial region, which kept expanding through electronic microscopy, was seen under electron microscope. At the 28th day, slight segmental hypercellularity be gan to show in light microscopy. ②Ccr and Rbp levels of diabetic rats were higher significantly than those in the control group at the 7th, 14th and 28th days (t=2.143-3.004,P ＜ 0.05 ). The results of SDS-PAGE showed that middle and low molecular protein could be observed in every group from the 3rd day. ③The level of p27 expression in glomerular cells were lower obviously at the 3rd day(t=2.536,P ＜ 0.05), but higher at the 7th, 14th and 28th days in diabetic rats, which was higher remarkably than that in the control group (t=2.704-2.823,P ＜ 0.05). The number of PCNA positive cell in diabetic glomerulus became the largest at the 3rd day, then decreased continuously, it was still higher than that in the control group at the 7th dan 14th days (t=2.681,2.525,P ＜ 0.05), but became normal at day 28. The number of TUNEL positive cells in glomerulus also increased significantly at the 3rd day (t=2.764,P ＜ 0.05), but it was smaller than that in the control group at the 7th, 14th and 28th days(t=2.096-2.627,P ＜ 0.05). ④The level of p27 expression and the number of PCNA positive cells in glomerulus were negative correlated (r=-0.446, P ＜ 0.05). The level of p27 expression and the number of TUNEL positive cells in glomerulus were also negative correlated (r=-0.517, P ＜ 0.05).

CONCLUSION:

Functional impairment of reabsorption induced by proximal convoluted renal tubule and high filtering of glomerulus in early diabetics occurs synchronously. Protein p27 may be participated in the occurring and developing of diabetic nephropathy by checking of proliferation and apoptosis of renal cell in the early stage of diabetes.