| Objective: Diabetic nephropathy (DN) encompasses a complex of structure alterations, including renal hypertrophy in early stage and diffuse renal sclerosis in late stage. In early stage, the hypertrophy of the kidneys is caused by both of hyperplasia (an increase in cell number) and hypertrophy (an increase in cell size) of the renal cells. Early hypertrophy of the kidneys is closely related to late renal sclerosis. However, the exact pathogenesis of these pathological changes is not clear. Many investigations have indicated that DN involves such cooperative factors as high glucose, hymodynamic, growth factors, vascoactive substances and so on. Cell cycle proteins play important part in the regulation of cell cycle and cell growth. However, the role of cell cycle proteins on the pathogenesis of DN, especially on the hyperplasia and hypertrophy of renal cells at early stage ,has not been studied in detail. In the present study, we investigate the expression of cell cycle proteins, including cyclinD1, cyclin-dependent kinase 4 (CDK4), cyclin-kinase inhibitor p16 (p16) ,in the renal tissues at early stage of experimental diabetes, further to clarify their effects on hyperplasia and hypertrophy of renal cells in diabetes. Methods: Experimental diabetes was induced by streptozotocin (STZ) in male Sprague Dawley (SD) rats. The animals were divided into two groups: group A(control) and group B(diabetic). The rats of group B received a single intraperitoneal injection of STZ dissolved in 0.1mol/L sodium citrate (pH 4.5) at a dose of 65mg/kg body weight. The rats of group A only received an injection of the same volume of 0.1mol/L sodium citrate. The model of diabetes was considered to be successful when the blood glucose≥16.7mol/L and the glucose in urine was +++～++++ after 24 hours of the injection. The rats of both groups in the same number(n=6) were respectively sacrificed at day 5, day 15, and day 30 after injection. The kidneys were removed and prepared for light microscopy, immunohistochemistry, flow cytometry and in situ hybridyzation. The protein expression of proliferating cell nuclear antigen(PCNA), CDK4, cyclinD1 and p16 was evaluated by immunohistochemistry or flow cytometry. The mRNA expression of cyclinD1 was detected by in situ hybridyzation. The kidney/body weight ratio and the diameter of glomeruli and tubules were also evaluated. All the data were expressed as the form of . T test was used to analyse the difference between the two groups and within one group, which was finished with SPSS software. Results: 1. Compared with that of control rats, the kidney/body weight ratio increased in diabetic rats at day 5,which remained at a higher level at day 15 and day 30. 2. There was no apparent pathological changes observed in diabetic renal tissues at day 5 and day 15, but the glomeruli and tubules enlarged in diabetic rats at day 30. Morphometric analysis showed that the diameter of glomeruli and tubules increased at day 15 and day 30 compared with that of control. 3. PCNA protein was expressed in nucleus examined by immunohistochemistry. In control group, there was no positive cell observed in glomeruli and very few positive cells in tubules at day 5, day 15, day 30. Compared with that of control, positive cells increased both in glomerular and tubular cells in diabetic rats at day 5. At day 15, in the diabetic rats, positive cells in tubules decreased compared with that at day 5. But it was still higher than that of control. In contrast, there was no positive cells observed in glumeruli of diabetic rats at day 15. At day 30, in diabetic group, there was very few positive cells which can be seen only in tubules. 4. CDK4 was also expressed in nucleus examined by immunohistochemistry. No expression of CDK4 protein was found in glomerular cells both in control and diabetic rats at day 5, day 15, day 30. In contrast, The protein expression of CDK4 increased significantly in diabetic tubles at day 5 compared with that of control. However, at day 15, it decreased compared with that o...
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