缬沙坦对糖尿病肾病大鼠足细胞相关蛋白Nephrin和TRPC6表达的研究

Effect of valsartan on the expression of Nephrin and TRPC6 in podocytes from diabetic nephropathy rats

目的观察足细胞相关蛋白Nephrin、TRPC6在糖尿病肾病(DN)大鼠肾小球中的表达,探讨缬沙坦对其干预的可能机制,为DN的防治提供理论依据。方法雄性SD大鼠40只按体重随机分为正常对照组(NC组,n=7)、DN组(n=11)、缬沙坦干预组H0组、缬沙坦干预组H1组。干预8周后检测大鼠血糖、体重、24 h尿蛋白、血肌酐、尿肌酐,计算肌酐清除率(Ccr);免疫组化法观察肾组织Nephrin、TRPC6的蛋白表达;反转录聚合酶链式反应(RT-PCR)法检测Nephrin、TRPC6的m RNA表达,并进行统计分析。结果 (1)与NC组相比,DN组大鼠Ccr明显减低、24 h尿蛋白定量增加(P<0.05);与DN组相比,H0组及H1组均出现Ccr增加、24 h尿蛋白定量减少(P<0.05),与H0组相比,H1组出现Ccr增加、24 h尿蛋白定量减少(P<0.05)。(2)与NC组比较,DN组大鼠肾脏Nephrin m RNA及蛋白表达均下调,TRPC6 m RNA及蛋白表达均上调(均P<0.05);与DN组比较,H0组及H1组Nephrin m RNA及蛋白表达均上调,TRPC6 m RNA及蛋白表达均下调(均P<0.05);H1组与H0组比较,Nephrin m RNA及蛋白表达均上调,TRPC6m RNA及蛋白表达均下调(均P<0.05)。(3)相关分析:采用直线相关分析后发现,24 h尿蛋白与肾脏Nephrin蛋白及基因表达呈负相关,与TRPC6蛋白及基因表达呈正相关;Ccr与肾脏Nephrin蛋白及基因表达呈正相关,与TRPC6蛋白及基因表达呈负相关。结论缬沙坦可通过上调Nephrin的表达及下调TRPC6表达维持足细胞裂孔膜的完整性,减轻足细胞裂孔膜的损伤,减少蛋白尿产生;在一定剂量范围内,缬沙坦的肾脏保护作用呈剂量依赖性。

Objective To observe the expression of podocyte associated proteins Nephrin and TRPC6 in the glomerular of the rats with diabetic nephropathy(DN) and explore the possible mechanism of the intervention of valsartan which provide a theoretical basis for the prevention and treatment of DN. Methods Male SD rats(40) were randomly divided into control group(group NC, n=7), diabetic nephropathy group(DN, n=11), valsartan intervention H0 group and valsartan intervention H1 group. After 8 weeks, to test the index, such as blood glucose, weight, urinary protein within 24 hours, serum creatinine, and urine creatinine; to observe the protein expression of Nephrin, TRPC6 with immunohistochemical methods; the m RNA expression of Nephrin, TRPC6 were detected by RT-PCR, analyzing all data. Results(1) Compared with subjects in group NC, those in group DN expressed obvious drop of creatinine clearance rate, increase in urinary protein within 24 hours(P<0.05). Compared with subjects in group DN, those in group H0 and group H1 expressed higher creatinine clearance rate, lower urinary protein(P<0.05) as well. Compared group H0 with group H1, subjects in group H1 expressed even higher creatinine clearance rate, lower urinary protein(P<0.05).(2) Compared with the subjects in group NC, those in group DN expressed drop in m RNA and protein but climb in that of TRPC6(P<0.05); Compared with the subjects in group DN, those in group H0 and group H1 expressed increase in m RNA and protein of Nephrin but decrease in that of TRPC6(P<0.05); Compared with subjects in group H0, those in group H1 expressed increase in m RNA and protein of Nephrin, but decrease in that of TRPC6(P<0.05).(3) Comprehensive analysis: through linear analysis, urinary protein with in 24 hours had the negative correlation with protein and gene expression of Nephrin, while had a positive correlation with those of TRPC6. On the other hand, Ccr expressed a positive correlation with protein and gene expression of Nephrin, while it had a negative correlation with those of TRPC6. Conclusions Valsartan can maintain the integrity of podocyte hiatus film, alleviate the damage to it and reduce the production of urinary protein by increasing the expression of Nephrin as well as decreasing that of TRPC6. Given the acceptable dosage, the valsartan’s protection to kidney tends to be dose-reliant.