The Experimental Study Of Felodipine To The Expression Of Bone Matrix Protein In Renal Small Artery Of Streptozocin-induced Diabetic Nephropathy Rats

Objective Vascular disease such as atherosclerosis is the major complication of diabetes mellitus(DM), involving aorta and coronary artery,while microvascular dysfunctional is closely connected with the development of diabetic nephropathy(DN), the important complication of DM. There is a growing body of evidence that vascular calcification is a common feature in advanced atherosclerosis and also a predictor of future cardiovascular events in diabetes. Diabetic patients seem to have increased prevalence of coronary calcification(CAC) when compared with non-diabetic patients and CAC are common and severe in diabetic patients with chronic kidney disease(CKD).Currently the study of vascular caicification are focus on coronary artery and renal artery, but we know little about small artery in organ such as renal. So we construct the model of streptozocin(STZ)-induced DN rats to observe the expression of osteoprotegerin(0PG), osteocalcin(OC) and core-binding factorα₁(Cbfal) in small artery of renal. We supposed that the change of these bone marix protein is associated with the development of DN and we choose felodipinethe to observe if it can prevent vascular calcification of DN.Methods Adult Sprague-Dawley male rats were divided into diabetic group and felodipinethe threpay group, which were injected with a single intraperitoneal injection of streptozotocin, and control group. Felodipinethe group were received felodipinethe (10mg.kg^-1.d^-1 by daily gastric gavage,n=35) while other rats were received vehicle. Urinary protein, blood sugar,blood calcium and phosphonium, blood cholesterol and triglyceride, serum urea creatinine were monitored respectively at 4, 8, 12, 16, and 24 weeks.Paraffin sections of renal tissue of rats at the same time points were stained by routine methods and immunohistochemistry, hybridization in situ. The changes of Cbfal, OC and OPG were quantificationally analyzed. The expression of OC and OPG mRNA in renal at the same time points were detected by Real-Time PCR.Results (1) From 4-week to 24-week, the number of Cbfal staining in diabetic renal small artery was significantly increased as compared with control group, being maximal at 24-week. Cbfal mRNA in diabetic renal was significantly upregulated at 24-week, compared with diabetic group at other time points or control group at the same time points, while there is weakly staining in renal of control group. (2) From 4-week to 12-week,there is no OC's protein or mRNA expression in three group's renal,detected by immunohistochemistry and Real-Time PCR. OC's protein and mRNA in diabetic renal was only detected at 16 and 24 weeks. (3) OPG-expressing small artery in diabetic renal cortex was found by immunohistochemistry and hybridization in situ from 4 -week to 24-week with peak levels at 12 weeks, subsequently decreased after 12 week. The level of OPG mRNA was significantly upregulated at 8 weeks, with peak levels at 12 week, and then downregulated gradually after 12 weeks. There is weakly staining of OPG in control renal. (4) In diabetic group, there were a strong correlation among Cbfal, OPG, OC and blood P, TG, CHOL and BUN, Crea. Cbfal correlated with OPG and OC positively, OPG correlated with OC positively. (5) In comparison with the diabetic group, felodipinethe threpay didn't ameliorate blood glucose, blood-fat, blood Ca and P, urine protein, pathological change of renal tissue, deposition of calcium salts and expression of Cbfal, OC. Only at 16 and 24 weeks, felodipinethe upregulated the expression of OPG. (6) Alizarin red staining of renal tissue indicated that there was no vascular calcification in nomal rats, while at 16-24W, light to middle vascular calcifation appered in renal small artery in DN and DN-F groups.Conclusion (1) Bone matrix protein has already expressed in renal small artery before the formation of vascular calcification. (2) Before vascular calcification, Cbfal in diabetic renal small artery was significantly upregulated following OC's occurrence, which indicated that Cbfal was a key regulating factor of diabetic vascular calcification. (3) OPG are early upregulation, which may be the compensative mechanism under pathological conditions and late down-regulation, which may related to hyperphosphatemia and hyperlipohaemia. (4) Alteration of the expression of Cbfal, OC and OPG was related to urine protein, renal function and hyperphosphatemia, which indicated that them participated the development of DN. (5) We didn't find that felodipinethe ameliorated diabetic vascular calcification in renal small artery in this model.