| Background and objectives:Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM) and an important cause of death in these patients . The pathological hallmarks of diabetic nephropathy are mesangial cell proliferation and extracellular matrix (ECM) accumulation, which lead to increased thickness of the glomerular and tubular basement membrane (GBM) and mesangial expansion, and finally result in glomerulosclerosis and loss of the renal function. These mainly result from an imbalance between the synthesis and degradation of ECM. Studies have shown that matrix metalloproteinases (MMPs) especially gelatinases and their specific tissue inhibitors of matrix metalloproteinases (TIMPs) are involved in the pathogenesis of some renal diseases. But their effects on the development and progression of diabetic nephropathy have not been fully explored. The present study was designed to investigate the renal MMP-2 activities, MMP-2, TIMP-2 protein and mRNA expression, and their serum levels at different course in diabetic rats induced by streptozotocin (STZ), in order to determine their relationships with collagen IV(IV-C) degradation , renal function and morphology ,to probe the role of MMP-2/TIMP-2 in the pathogenesis of diabetic nephropathy. Methods:1. The diabetic rat model: Six to eight week male Wistar rat were divided into two groups randomly, including normal control group(NC) and diabetic model group(DM). Then the two groups are both divided into 1,4,8,12,20 week groups respectively. The diabetic model was induced in rats by intraperitonealinjection with STZ in the dose of 60mg/kg. Rats with blood glucose above 16. 7mmol/L are included in diabetic rats.2. Renal function evaluation: Determining 24 hour urinary protein, urinary creatinine and serum creatinine, BUN to evaluate renal function. Kidneys were resected and weighed; the renal size was evaluated by calculating the kidney weight/body weight.3. Histopathologic evaluation: Renal morphology was examined by light microscope and transmission electron microscope (TEM).4. The protein expression of renal IV-C, MMP-2and TIMP-2 was observed by immunohistochemical stain.5. Determination of renal MMP-2 activities by gelatin zymography.6. Determination of the expression of renal MMP-2mRNA and TIMP-2mRNA by RT-PCR method.7. The levels of serum MMP-2and TIMP-2 were detected quantitatively by ELISA method.Results:1. The changes of Body weight (BW) and blood glucose level: In diabetic groups, BW was lower than that in normal control group from the first week, and declined gradually with the longer time of diabetes mellitus; the blood glucose was continuously higher than that in normal control group.2. The changes of renal function: Compaired with the normal rats, in DM rats, BUN, urinary protein/24hrs were significantly higer from the first week, and increased with the longer time of diabetes mellitus; while Ccr increased from the first week, reached the peak in 12 week, and then decreased, but still remained higer than that in normal control group.3. Histological changes of the kidney: The morphology of renal tissue was abnormal in diabetic rats. Under light microscope, glomerular enlargement, proliferation of messangial cell and matrix, increased basement membrane thickness and dilation of glomerular capillary vessels, hyalinization of tubular epithelial cell were observed. Electron microscope reveals hypertrophy and partretraction of epithelial cell foot processes, increase in basement membrane thickness. Changes of renal morphology became more and more severe with the longer time of diabetes mellitus.4. The changes of renal IV-C protein expression: In diabetic groups, the renal IV-C protein expression was higher than that in normal control group from the first week, and increased with the longer time of diabetes mellitus. The IV-C protein expression correlated positively with urinary protein/24hrs level.5. The changes of renal MMP-2 activities: Compaired with the normal rats, MMP-2 activities in rats with DM was lower from the first week, and declined with the longer time of diabetes mellitus. MMP-2 activities correlated negatively with renal IV-C protein expression and 24 hour urinary protein level.6. The protein and mRNA expression of renal MMP-2, TIMP-2: In diabetic groups, the protein and mRNA expression of renal MMP-2 were downregulated than that in normal control groups from the first week, and decline with the longer time of diabetes mellitus. And they correlated positively with renal MMP-2 activities, negatively with IV-C protein expression . The protein and mRNA expression of renal TIMP-2 was upregulated than that in normal control groups from the first week, and decline with the longer time of diabetes mellitus. They correlated positively with IV-C protein expression, negatively with renal MMP-2 activities.7. The changes of serum MMP-2 and TIMP-2 levels: In diabetic groups, the levels of serum MMP-2 and TIMP-2 were upregulated compaired with the normal control groups from the first week, and increase with the longer time of diabetes mellitus. The serum MMP-2 level correlated negatively with the protein and mRNA level of renal MMP-2; the serum TIMP-2 level correlated positively with the protein and mRNA level of renal TIMP-2.Conclusions:1. In diabetic rats, there were changes of renal functions and morphology in early stage. And these became more and more severe with the longer time of diabetes mellitus. This reveals that DM can be complicated by DN in the early stage.2. In diabetic rats, the renal IV-C protein expression increased conjunction with renal MMP-2 activities decreased.3. In diabetic rats, changes of urinary protein/24hrs and morphology were closely in related with increasing of renal IV-C protein expression and decreasing of renal MMP-2 activities.4. Relative analysis demonstrated the serum MMP-2 level correlated negatively with the protein and mRNA level of renal MMP-2; the serum TIMP-2 level correlated positively with the protein and mRNA level of renal TIMP-2.5. In diabetic rats, there were changes of renal MMP-2 activities and MMP-2, TIMP-2 protein and mRNA expression in early stage, this reveals MMP-2, TIMP-2 were involved in the pathogenesis of diabetic nephropathy.Innovations and meanings:1. This study first took a dynamic observation on renal MMP-2 activities, MMP-2, TIMP-2 protein and mRNA expression, IV-C protein expression in diabetic rats, and found they were associated with the changes of urinary protein/24hrs and renal morphology. It indicated that MMP-2, TIMP-2 are involved in the pathogenesis of diabetic nephropathy.2. This study firstly observed both the serum level of MMP-2, TIMP-2 and renal MMP-2, TIMP-2 protein and mRNA expression, and discussed their correlation.
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