| Background and ObjectiveDiabetic nephropathy (DN) is one of the common microvascular complications of diabetes mellitus and it is also the major cause of end stage renal disease (ESRD). Microalbuminuria is an earlier clinical diagnosis index for DN which signified for irreversible pathological changes of DN patients'glomerular, and patients who appeared macroalbuminuria will progress to ESRD quickly. So it is improtant to identify the pathogenesis of proteinuria for DN prevention and cure. Permselectivity of serum protein is dertermined by the integrity of glomerular filtration barrier .Heparan sulfate (HS) in the glomerular basement membrane(GBM) is improtant in maintaining the charge-dependent permeability. The beta-D-endoglycosidase heparanase has been proposed to be important in the pathogenesis of proteinuria by degrading the negatively charged HS side chains within the GBM selectivity. Similarly, podocin is one of important component of podocyte slit diaphragm, recent studies have showed that it was involved in several diseases such as nephrotic syndrom, and all of these diseases have close correlation with proteinuria. However, there is few reports about the role of heparanase and podocin in the pathogenesis of proteinuria in DN. Therefore, in this study we intend to approach the molecule mechanism of proteinuria in DN by examining the expressions of heparanase and podocin in DN rats, and we will analyze their correlation in the pathogenesis of proteinuria furtherly.Methods 1. To construct diebetic nephropathy rats model.Twenty six male 180-200g Sprague-Dawley rats (n=26) were divided randomly into normal group(n=6) and DN group(n=20).DN rats were induced by intraperitoneal injection of 60mg/kg 0.1mmol/L streptozotocin(STZ).The levels of blood and urine glucose were measured after 72 hours,we considered that the model were successful if the rat blood glucose was greater than or equal to 16.65mmol/L and urine glucose was 3+ to 4+.Then,the DN group was divided rondomly into DN 6 weeks group(n=10) and DN 12 weeks group (n=10) again.The rats of normal group were intraperitoneal injection partes aequales citric acid buffer as control.2. To collect 24 hour urine using metal metabolism cage and to collect blood sample of all rats and to exermine the quantitation of proteinuria and biochemical indicator after 6 and 12 week respectively.To sacrifice all rats, the left kidneys were freezed in liquid nitrogen after being weighed and the rights were fixed by 10% formaldehyde for light microscope and immunohistochemistry.3. The methods of immunohistochemistry and reverse transcription PCR were used to detect the expression of HPA and podocin of kidney tissue of all rats .Results1 . Compared with normal group,the levels of relative renal weight,24 hour urine volume and proteinuria quantitation of DN groups increased markedly (P<0.05);2,In contrast with the normal group,the expression of HPA protein and mRNA increased significantly (P<0.001). Average optical density value of HPA protein positive area for normal group was 24.57±1.99, but DN6 week group was 78.77±2.73 and DN12 week group was 87.59±10.22, there was a significant difference between groups (F=11.17 P<0.001). The semiquantitative analysis for HPA mRNA showed: normal group was 0.24±0.03,DN6 week group was 0.89±0.14,DN12 week group was 1.09±0.13, there was a significant difference between groups(F=11.19 P<0.001).In addition, we had found significant correlation between HPA protein and mRNA and the quantification of urinary protein (r=0.783 P<0.001; r=0.793 P<0.001).3. Podocin protein expression in DN group rats were decreased markedly compared with nomal group (P<0.001). Average optical density value of podocin protein positive area for normal group was 78.69±4.33, whereas DN6 week group was 68.57±5.75 and DN12 week group was 55.27±7.46, there was a significant difference between groups (F=11.65 P<0.001) and negative correlation between podocin protein expression and the proteinuria quantification (r=-0.833 P<0.001).The semiquantitative analysis of podocin mRNA showed that the gene expression increased in DN6 week and declined in DN12 week, there was no significant difference between groups(F=0.98 P>0.05) .When compared with normal group, there was also no significant difference (P>0.05). No correlation was found between podocin gene expression and the proteinuria quantification (r=0.320 P>0.05).4, There was significant inverse correlation between HPA and podocin protein expressions (r=-0.653 P<0.05),but no correlation was found in their gene expression (r=0.265 P>0.05).Conclusions1. Up-regulated expression of HPA had correlation with the pathogenesis of proteinuria in DN rats.2. Down-regulated expression of podocin protein might lead to the proteinuria in DN. The discoincidence between podocin mRNA and protein expression suggested that it might be a compensatory reaction on the mRNA level to down-regulated expression of podocin protein at the earlier stage of diebetic nephropathy in podocyte.3. HPA and podocin had a common pathway,through which they took part in theonset of proteinuria in DN.
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