Effects And Mechanisms Of Sulodexide On Glomerulus Extracellular Matrix Accumulating In DM Rats

Diabetic nephropathy (DN) was not only serious complication of diabetes mellitus (DM), but also the common cause of end-stage renal disease(ESRD). But there's no effective therapy on it so far. Therefore, exploring the effective treatment measures of diabetic nephropathy has become an urgent problem to be resolved in the field of nephropathy. The previous researches confirmed sulodexide was able to decrease urinary albumin level of DN to protect kidney, but mechanisms of protection about it on kidney only existed in some hypothesizes, and were not clear. The principal of pathological changes of DN included glomerulus hypertrophy, glomerulus extracellular matrix accumulating, basement membrane thickening and glomerulosclerosis. Among them, glomerulus extracellular matrix accumulating holds a crucial position on glomerulosclerosis. Currently, there're not relevant reports about that sulodexide inhibit glomerulus extracellular matrix accumulating in DM. Therefore, we established rat model of DM, then we researched effects of sulodexide glomerulus extracellular matrix accumulating and explored molecular mechanisms of sulodexide , based on the rat model of DM.Objectives: To establish rat model of type 2 diabetes mellitus. To investigate sulodexide,s protective effect on renal damage in DM rats and the effect of glomerular extracellular matrix accumulating. To experimentally explore molecular mechanism that sulodexide inhibit glomerulus extracellular matrix accumulating in DM rats.Methods: First, removed 10 from the SD rats randomly, set to the normal control group (group C), those 50 rats feed forage of high sugar and high fat for one month, then, the diabetic mellitus model of the male Sprague-Dawley (SD) rats were established by intra-abdominal injection of STZ. Those 40 rats model of diabetes mellitus were randomly divided into four groups: diabetic model group (DM group), low-dose sulodexide treatment group (DMS1 group), high-dose sulodexide treatment group (DMS2 group), and telmisartan treatment group (DMT group). We intramuscularly injected with sulodexide of 5mg/kg and 10mg/kg poured telmisartan of 5mg/kg every day. After 8 weeks, we measured blood urinary albumin, nephron injury index, glucose, glycosylated hemoglobin, urea nitrogen and serum creatinine by immunoradioassay. The pathological changes of renal tissue were observed under light microscopy and electron microscopy. The expressions of TGF-β1, CTGF, MMP-9 and TIMP-1 in each group's nephridial tissue were examined by immunocytochemical staining and western blot.Results:1. At the end of experimentation(8th weeks), the laboratory's result reveals the level of 24h urinary albumin and renal weight/body weight (RW/KW) had increased remarkably in group DM and decreased remarkably in every therapy groups, among them, group DMS2 and group DMT had reduced obviously, but were higher than group C (P<0.05orP<0.01). However, there was no significant difference between group DMS2 and group DMT (P>0.05). The level of blood glucose and glycosylated hemoglobin had increased remarkably in group DM (P<0.01). However, threr was no significant difference in every therapy groups (P>0.05). The level of blood urea nitrogen were no diversity in every groups (P>0.05).The level of serum creatinine had increased remarkable in group DM (P<0.05orP<0.01). The level of serum creatinine of every therapy groups were lower than the model group, however, there was no significant difference among every therapy groups and group DM (P>0.05).2. Morphology changes were examined under light microscope. In the C group, glomerulus has complete structural, without augmentation and anomaly change. In the DM group, glomerulus has mesangial cell ground substance hyperplasia,mesangial region widen,capillary wall increased thickness, glomerulus augmentation,parts of glomerular capillaries circumferential to appear eosinophilic protein substance sedimentation. Every therapy groups reduced degree of renal pathological morphology. Group DMS2 and group DMT reduced obviously.The area of glomerulus and area of glomerulus extracellular matrix had increased remarkably in group DM and decreased remarkably in every therapy groups, among them, group DMS2 and group DMT had reduced obviously, but were higher than group C(P<0.05orP<0.01). However, there was no significant difference between group DMS2 and group DMT (P>0.05).Morphology changes were examined under electron microscope. In the C group, glomeruli have complete structural, without augmentation and anomaly change. In the DM group, glomerular basement membrane suffusion or limitations increased thickness, mesenteria hyperplasia,podocyte foot process fused and disappeared. Every therapy groups reduced degree of renal pathological morphology. Group DMS2 and group DMT reduced obviously.3. After the DM rats'therapy over, the immunohistochemiscal staining results had showed that the expression of TGF-β1, CTGF, MMP-9 and TIMP-1 increased remarkable in group DM and the expression of TGF-β1, CTGF and TIMP-1 decreased remarkable in every therapy groups. Among them, group DMS2 and group DMT had reduced obviously, but were higher than group C (P<0.01or P<0.05). However, there was no significant difference between group DMS2 and group DMT (P>0.05). The expression of MMP-9 were no diversity in every therapy groups (P>0.05). The ratio of MMP-9/TIMP-1 had decreased remarkably in group DM and increased remarkably in every therapy groups. Among them, group DMS2 and group DMT had increased obviously , but were lower than group C (P<0.01).4. When the DM rats therapy over, the western blot results had showed that the expression intensity of TGF-β1, CTGF, MMP-9 and TIMP-1 increased remarkable in group DM and the expression intensity of TGF-β1, CTGF and TIMP-1 decreased remarkable in every therapy groups. Among them, group DMS2 and group DMT had reduced obviously, but were higher than group C (P<0.01). However, there was no significant difference between group DMS2 and group DMT(P>0.05). The expression intensity of MMP-9 were no diversity in every therapy groups (P>0.05). The ratio of MMP-9/TIMP-1 had decreased remarkably in group DM and increased remarkably in every therapy groups. Among them, group DMS2 and group DMT had increased obviously, but were lower than group C (P<0.01 or P<0.05).Conclusions:1. Sulodexide can decrease level urinary albumin of DM rats to protect kidney, and present dose-effect relationship, The renoprotection has no relative with blood glucose level of DM rats.2. Sulodexide can decrease the ratio of KW/BW and improve mesenteric hyperplasia, glomerular basement membrane increased thickness and podocyte foot process fused that prompt sulodexide possibly can inhibit glomerulosclerosis through inhibiting glomerulus extracellular matrix accumulating, and protect kidney.3. Sulodexide can inhibit the expression of TGF-β1, CTGF, and then inhibit glomerulus extracellular matrix accumulating. Sulodexide can improve the ratio imbalance of MMP-9/TIMP-1 and increase activity of MMP-9, and then strengthen glomerulus extracellular matrix degradation. This prompt that sulodexide possibly can inhibit glomerulus extracellular matrix accumulating through the mechanisms, then protect kidney.