| Background and objective Diabetic nephropathy is one of serious complication of diabetes. It basic pathobiology change is that glomerular basement membrane thickening, mesenterium matrix hyperplasy, diffuse and nodular glomerulosclerosis. atherosclerosis and vascular calcification are the major complication of diabetes.The research of vascular lesion is focus on large vascular such as coronary and renal artery in diabetes. Renal arteries of the autopsy have been atheromatous plaque and calcification.But renal pathologist and nephrologist rarely describe or study on the relation between earlier period lesion of vascular clacification and renal small artery, and its effect in pathogenesis of diabetes and diabetic nephropathy. The paper observe continuously the expression of core-bind factor alpha-1 (Cbfa1),bone morphogenetic protein-2 (BMP-2 ),matrix Gla Protein ( MGP) and monocyte chemoattractant protein-1 (MCP-1) at the renal small artery in diabetic rat model through streptozotocin(STZ) to induction at different time points. The purpose is to research the relation between the change of these factor and renal small artery calcification in earlier diabetic nephropathy, to investigate the pathogenesy of vascular calcification in diabetic nephropathy.It may is a new idea and treatment target to development of diabetic nephropathy vascular lesion.Methods Adult Sprague-Dawley male rats were divided into diabetic group(DN) and felodipine treated group(DN-F), which were injected with a single intraperitoneal injection of streptozotocin, and control group(N). 24h Urinary protein, serum sugar, serum fat, serum urea, serum creatinine, serum phosphorus and calcium were measured respectively at 4, 8, 12, 16 and 24 weeks. The change of Cbfa1, BMP-2, MGP and MCP-1 were detected by immunohistochemistry, in-situ hybridization and Real-Time PCR in renal small artery at the same time points.Paraffin sections of renal tissue of rats at the same time points were stained by routine methods and alizarin Bordeaux calcium stain.the ratio of intima/tunica media thickness of small artery was measured by immunofluorescence-histochemistry, and quantified by computer image analysis.Results (1) Cbfal expressed obviously in renal small artery of DN and DN-F group by immunohistochemistry stain from 4 to 24 week compared with N group at the same time points, being peak at 24 week. (express slightly in glomeruli but not expressed in tubules). The result of in-situ hybridization show that it only express in renal small artery, and express weakly at 4 week, begin to enhance in endo-elastic layer and out-elastic laye at 8 week, muscular layer begin to enhance at 16,24 week. The expression of DN and DN-F group significantly increased compared with N group at other time point. But it was unsignificantly compared with other time point in N group. (2) The expression level of MGP was evidently increased at 4 week, slightly decreased at 8 week, lowest at 24 week in DN and DN-F group by Real-Time PCR. (3) The result of immunohistochemistry stain shows that BMP-2 was slight expressed at renal small artery in DN and DN-F group. The expression of BMP-2 began to increase at 4 week by in-situ hybridization. It was more evident with time lapse in renal small artery, and enhanced in endo- elastic layer and weaked in muscular layeat at 4, 8 week, began to enhance in muscular layeat at 12 week, to peak at 24 week. there is staining in N group. The result of Real-Time PCR showed that the expression level of BMP-2 began to increase from 4 to 24 week, 24 week being peak in DN and DN-F group, but not change in N group at other time points. (4) The result immunohistochemistry stain show that MCP-1 was expressed at renal small artery, glomeruli and tubules in DN and DN-F group, The expression of MCP-1 was evidently increased in renal small artery at 16, 24 week compared with 4, 8, 12 week. there was expressed at renal small vascular and glomeruli in N group, only slightly expressed at tubules. The result in-situ hybridization showed that the expression of MCP-1 was increased in renal small artery at 4 week, obviously increased at 16, 24 week in DN and DN-F group, The expression of MCP-1 had not difference between DN and DN-F group at other time points.there was expressed of MCP-1 in N group. (5) The lizarin Bordeaux calcium stainof renal tissue showed that the renal small artery had not calcium salt at 4 and 8 week, but have punctiform and lamellar calcium salts at 16 to 24 week in DN and DN-F group. (6) The result of immunofluorescence-histochemistry indicated that the intima of renal small artery showed red fluorescence, green fluorescence in tunica media. The ratio of intima and tunica media had not difference in DN and DN-F group compared with N group at 4 week, had difference at 12 week, obviously at 24 week. (7) A strong positive correlation between Cbfa1 and BMP-2 levels was founded, and it were obviously positive correlation with CHOL, TG and P, serum creatinine and Urinary protein, respectively in DN group. Cbfal and BMP-2 were significantly negative correlation with MGP. MGP was significantly negative correlation with CHOL, TG and P, serum creatinine and Urinary protein. Cbfa1 were significantly positive correlation with serum urea, serum calcium and MCP-1.But BMP-2 and MGP have not significantly correlation with it. The ratio of intima and tunica media was obviously positive correlation with Cbfa1, BMP-2, MCP-1, CHOL, TG and P.Conclusion 1. Cbfal and BMP-2 have already expressed in renal small artery in earlier of DN.The expression of MGP is gradually deceased with time 2. In earlier of DN and DN-F group, There is not calcium salt in small artery of renal, but increased calcium mineralization in mid-anaphase. 3. there is changed The ratio of intima and tunica media in earlier period, has changed slightly in mid-period, obviously in later period of DN. 4. MGP and BMP-2 have changed before clarification in earlier renal small artery of DN. The expression of MGP increased in earlier period, decreased in later period of DN. The expression of BMP-2 is just opposite with MGP. It suggested that MGP/BMP-2 is a pair of each orther antistatic factor in formation of vascular calcification. 5. Vascular calcification has obvious correlation with MCP-1, CHOL, TG, P and serum creatinine.These suggest that chronic inflammation and metabolic disturbance may promote vascular calcification at DN renal small artery. 6. The effect of felodipine is not obvious to vascular calcification in earlier renal small artery of DN. |
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