温肾健脾方对糖尿病肾病大鼠肾小球足细胞nephrin和podocin表达的影响

Effect of warming kidney and spleen recipe on the expressions of nephrin and podocin in glomerular podocyte of diabetic nephropathy rats

目的观察温肾健脾方对糖尿病肾病大鼠肾小球足细胞nephrin和podocin表达的影响。方法将链脲佐菌素诱导的30只糖尿病肾病模型大鼠分为五组,每组各6只,即模型对照组(等渗NaCl溶液10 mL·kg^(-1)·d^(-1))、阳性对照组(缬沙坦25 mg·kg^(-1)·d^(-1))及低剂量组(温肾健脾方7.5 g·kg^(-1)·d^(-1))、中剂量组(温肾健脾方15 g·kg^(-1)·d^(-1))和高剂量组(温肾健脾方30 g·kg^(-1)·d^(-1)),并以6只正常大鼠作正常对照组(等渗NaCl溶液10 mL·kg^(-1)·d^(-1))。各组大鼠连续灌胃8周后,分别采用免疫组织化学法和荧光定量PCR检测各组大鼠肾小球足细胞nephrin和podocin蛋白及mRNA的表达。结果免疫组织化学结果显示,六组大鼠肾脏组织足细胞nephrin和podocin蛋白表达比较,差异均有统计学意义(F=6.656、8.667,P=0.016、0.007)。进一步两两比较发现,模型对照组足细胞nephrin和podocin蛋白表达量均较正常对照组降低(P均<0.05);与模型对照组比较,中、高剂量组nephrin和podocin蛋白表达量均升高(P均<0.05),而低剂量组仅podocin蛋白表达量较模型对照组有所升高(P<0.05);与阳性对照组相比,低、中、高剂量组仅podocin蛋白表达量均较阳性对照组升高(P均<0.05)。荧光定量PCR结果显示,六组大鼠肾脏组织足细胞nephrin和podocin mRNA相对表达量比较,差异均有统计学意义(F=5.412、3.308,P=0.034、0.015);进一步两两比较发现,模型对照组足细胞nephrin和podocin mRNA相对表达量较正常对照组均显著降低(P均<0.05);与模型对照组比较,中、高剂量组及阳性对照组nephrin和podocin mRNA的相对表达量均显著升高(P均<0.05),而低剂量组仅podocin mRNA相对表达量有所升高(P<0.05);与阳性对照组相比,仅低剂量组nephrin mRNA相对表达量有所降低(P<0.05)。结论温肾健脾方对肾小球足细胞nephrin和podocin有不同程度的保护作用,从而维持肾小球的正常滤过功能,减轻尿蛋白排泄率,以达到防治糖尿病肾病的目的。

Objective To observe the effect of warming kidney and spleen recipe on the expressions of nephrin and podocin in glomerular podocyte of diabetic nephropathy rats Methods Thirty diabetic nephropathy rats induced by streptozotocin were randomly divided into 5 groups, which were the model control group （10 mL·kg-m·d-l isotonic sodium chloride solution）, positive control group （25 mL＂ kgl＇d-l valsartan）, low dose group （7.5 g·kg-m·d-l warming kidneyand spleen recipe）, medium dose group （15 g·kg-m·d-l warming kidney and spleen recipe） and high dose group （30 g·kg-m·d-lwarming kidney and spleen recipe） with 6 rats in each group, and 6 normal rats were served as the normal control group （10 mL·kg-m·d-l isotonic sodium chloride solution）. Then the nephrin and podocin proteins and mRNA expressions in glomerular podocytes of rats in each group were detected by immunohistochemistry and fluorescence quantitative PCR respectively, after all rats were given intragastric administration for 8 weeks. Results Immunohistochemistry results showed that the expressions of nephrin and podocin proteins in podocytes of rats in six groups were statistically different （F= 6.656, 8.667; P=0.016, 0.007）. The expressions of nephrin and podocin proteins in podocytes of rats in the model control group were lower than those in the normal control group （all P 〈 0.05）. Compared with the model control group, the nephrin and podocin expressions in medium and high dose groups increased （all P 〈 0.05）, while the podocin protein expression in the low dose group was higher than that in the model control group （P 〈 0.05）. In addition, podocin proteins expressions in the low, medium and high dose groups were higher as compared with the positive control group （all P 〈 0.05）. The results of quantitative PCR showed that the relative expressions of nephrin and podocin mRNA in podocytes of rats in six groups were statistically different （F= 5.412, 3.305, P = 0.034, 0.015）. Compared with the normal control group, the relative expressions of nephrin and podocin mRNA in podocytes in the model control group were significantly lower （all P 〈 0.05）. Furthermore, the relative expressions of nephr/n and podocin mRNA in medium and high dose groups and the positive control group significantly increased than those in the model control group （all P 〈 0.05）, while the relative expression of podocin mRNA only increased in the low dose group （P 〈 0.05）. At the same time, the relative expression of nephrin mRNA only decreased in the low dose group as compared with the positive control group （P 〈 0.05）. Conclusion Warming kidney and spleen recipe could provide a protective effect for nephrin and podocin in glomerular podocyte, which suggested that it might maintain the normal glomerular filtration function and reduce the urinary protein excretion rate in order to prevent and treat diabetic nephropathy.