Expression Of HSPG And Heparanase In The Kidney Of Rats With Membranous Nephropathy, And The Effect Of Sulodexide On It

Objective: Membranous nephropathy is a common cause of adultprimary nephrotic syndrome. Pathological examination showed diffuseglomerular immune complex deposited in layer of epithelial cells, glomerularbasement membrane thickening with "spikes", The main clinicalmanifestations is massive proteinuria, hypoalbuminemia andhypercholesterolemia. Proteinuria is an independent risk factor of progressionof renal disease, effective control of proteinuria can delay the kidney diseasedevelopment. In recent years, foreign study found heparan sulfateproteoglycans (HSPG) express in renal filtration device, especially in theglomerular basement membrane, biochemistry research shows that HSPG isan organic integral component of GBM.In a lot of nephropathies, includingdiabetic nephropathy, minimal change and membranous nephropathy, theexpression of HSPG decrease.HSPG expression is associated with lowerurinary protein levels. The study found heparanase（HPA） expresses inendothelial cells and renal epithelial cells in passive Heyemann nephritis (antibrush border antibody is directly injected in rats, which will caused bynephritis similar to human membranous nephropathy).The formation ofurinary protein is related to the expression of HPA. HPA can selectivly act onthe side chain of negative charged HSPG in basement membrane.The lack ofnegative charged HSPG can change the glomerular basement membranepermeability, resulting in leakage of protein. Sulodexide can inhibit theexpression of heparanase, repair the negative charge of endothelial cells andreduce the leakage of negatively charged protein.Whether application ofsulodexide can increase the expression of HSPG through reducing theexpression of HPA in renal endothelial and epithelial cells of membranousnephropathy,then decreas the urine protein? There is a lack of research in this field at home and abroad.To detect the expression of HSPG, HPA in thekidneys of rats with membranous nephropathy and to intervene usingsulodexide, to study the relationship between HSPG and HPA, to investigatethe mechanism of sulodexide reducing urine protein in the membranousnephropathy.Then provide a new idea to delay the progression of membranousnephropathy.Method:40clean level healthy male SD rats, body weight160±20g,After1week adaptive feeding, urinary protein was negative,were randomlydivided into four groups. The normal control group, model group, high doseof sulodexide group, low dose of sulodexide group, with10rats in each group.The model group, high dose of sulodexide group and low dose of sulodexidegroup were injected cationic bovine serum albumin (C-BSA) to immune, afterone week, the tail vein were injected with C-BSA, the normal control groupwere injected with equal volume of saline, after continuous4weeks,24hoursurine of rats was left in metabolic cages， to detect24hours urinaryprotein.After modeling sucessfuly, high dose of sulodexide group receivedgastric perfusion of sulodexide20mg/kg, low dose of sulodexide groupreceived gastric perfusion of sulodexide10mg/kg. The model group andnormal control group was administered with equal volume of saline, rats freefeeding and drinking water. After continuous4weeks,24hours urine of therats was left again in metabolic cages, to detect24hours urine proteinagain.Then the rats were sacrificed, nephridial tissue was left. Observing theglomerular basement membrane lesions in the light microscope respectivelyby HE, Masson and PAS staining. Observing the glomerular basementmembrane and podocyte pathology changes by electron microscope. Dtectingthe expression of HSPG and HPA by immunohistochemical method.Theresults of image analysis system IPP (Image-Pro Plus) was used for semiquantitative analysis. All the data were expressed as mean±standarddeviation, single factor analysis of variance was used among the three groups,two independent samples t-test between two groups, and non-parametric testsfor the data which did not meet the normality and homogeneity of variance. Statistical analysis software SPSS13.0was used for data statistics, withP<0.05as a statistical significance.Results:1.24hour urinary protein quantitative：The tail vein wereinjected with C-BSA for4weeks.The model group, high dose of sulodexidegroup and low dose of sulodexide group had massive proteinuria. There wassignificant difference compared with the normal control group (P<0.05). After4weeks of continuous sulodexide gavage,24hour urinary protein in highdose of sulodexide group and low dose of sulodexide group wasdecreased.There was significant difference compared with model group(P<0.05).Compared to low dose of sulodexide group,24hour urinary proteinof high dose of sulodexide group was reduced more obviously (P<0.05).2.Pathomorphological observation of renal tissue：Using the light microscopeand electron microscope to observe, normal control group have no obviousabnormal glomerular structure. Glomerular basement membrane in modelgroup was thickening under light microscope. There was a lot of abundantelectron dense deposits in glomerular basement membrane under electronmicroscope. And podocyte foot process fused and diffused. Compared withthe model group,in low dose of sulodexide group and high dose of sulodexidegroup,glomerular basement membrane was thickening slightly under lightmicroscope.The deposition of electron dense material was reduced. Thepodocyte foot process fused loss.3.The immunohistochemical results of HSPG,HPA in renal tissue: The expression of HSPG in glomerular basementmembrane of normal control group is massive.There was a few of expressionof HSPG in low dose of sulodexide group and high dose of sulodexidegroup.Compared to the normal control group,the expression wasdecreased.There was statistically significant difference (P<0.05). Compared tolow dose of sulodexide group and high dose of sulodexide,the expression ofHSPG in model group was decreased, there was significant difference(P<0.05). Compared to high dose of sulodexide group, the expresstion ofHSPG in low dose of sulodexide group was decreased, there was significantdifference (P<0.05). The expression of HPA in glomerular endothelial and epithelial cells in the model group was massive. There was a few ofexpression of HPA in normal control group. Compared with model group,theexpresstion of HPA in low dose of sulodexide group and high dose ofsulodexide was decreased, there was significant difference (P<0.05).Compared to the normal control group,the expresstion was increased, therewas statistically significant difference (P<0.05). Compared with the normalcontrol group,the expresstion in model group was very obvious, there wassignificant difference (P<0.05).4.Correlation analysis: Do the pearsoncorrelation analysis for the expression of HSPG and HPA, results showed that:the expression level of HSPG was negatively correlated with HPA (r=﹣0.932P<0.05).Conclusion:1.Sulodexide can decrease urinary protein of the rats ofmembranous nephropathy.2. Sulodexide probably inhibit the expression ofHPA in renal tissue, and thus increase the HSPG of glomerular basementmembrane, improve the permeability of glomerular basement membrane,reduce renal damage,decrease proteinuria.3.Compared to low dose ofsulodexide, the treatment effect of high dose of sulodexide is obvious.