人激肽释放酶基因对5／6肾切除大鼠肾间质纤维化的干预效应

Interference of human tissue kallikrein on renal interstitial fibrosis in rats with 5/6 nephrectomy

目的探讨人组织型激肽释放酶（HK）基因对5／6肾切除大鼠肾间质纤维化的干预效应及有关机制。方法将HK基因克隆入重组腺相关病毒载体（rAAV）中，经三质粒共转染方法在293细胞（HEK293）中包装成rAAV-HK病毒。雄性Wistar大鼠（n=24）按随机数字表法分为假手术组和5／6肾切除组，1个月后5／6肾切除组再按随机数字表法分为单纯手术组（n=6，不给予病毒注射）、实验组[n=6，单次尾静脉注射1×10^11蚀斑形成单位（pm）的rAAV—HK病毒1和对照组（n=6，单次尾静脉注射1×10^11pfu的rAAV—GFP病毒）。于术前、术后1个月（基因转染前）及转染后1～3个月测大鼠尾动脉血压。转染3个月后处死动物，取其心、肾等脏器，提取总RNA及蛋白质，经RT—PCR、Western印迹及ELISA方法检测HK基因在大鼠体内表达。肾组织切片行Masson染色观察大鼠肾间质病理变化，并行免疫组化染色检测缓激肽B2受体（BKB2R）和血管紧张素Ⅱ1型受体（AT1R）在肾脏的分布。Western印迹检测肾组织BKB2R、AT1R及磷酸化丝裂原活化蛋白激酶（p-MAPK）蛋白的表达水平。结果HK基因转染3个月后，与对照组相比，实验组大鼠血压显著下降[（163±13）mmHg比（217±16）mmHg，P〈0．01）1（1mmHg=0．133kPa）。与假手术组相比，单纯手术组和对照组肾间质有较多胶原沉积，呈现明显纤维化，而实验组胶原沉积较少，纤维化程度较轻，肾小管间质损伤指数显著小于对照组（1．33±0．73比3．01±O．62，P〈0．01）。免疫组化检测发现，BKB2R和ATIR主要分布于肾小管上皮细胞。基因转染后，肾组织BKB2R蛋白表达水平显著上调（P〈0．05），而AT1R蛋白表达水平显著下调（P〈0．05）；信号传导分子p-MAPK蛋白表达水平显著下调（P〈0．05）。结论HK基因导入明显减轻5／6肾切除大鼠肾间质纤维化程度，其机制可能与其调节BKB2R和AT1R蛋白在肾组织中的表达水平有关。上述作用可能与MAPK信号传导途径有关。

Objective To human tissue kallikrein （HK） gene Methods Human kallikrein eDNA based plasmid vector. The rAAV-HK investigate the interference and associated mechanism of on renal interstitial fibrosis in rats with 5/6 nephrectomy. was packed in a recombinant adeno-associated virus（rAAV）- was produced by transfection in 293 cells. Twenty-four male Wistar rats were divided into sham operation and operation groups. The rats with 5/6 nephrectomy were randomly divided into simple operation, control and experiment groups. The rats in experiment group received single dose rAAV-HK via the tail vein with 1 ×10^11 pfu. Before nephrectomy and every month after surgery until the rats were sacrificed, the caudal arterial pressure was measured using tail cuff blood pressure determinator. Three months after HK gene delivery, the rats were sacrificed. The expression of HK in rats was assessed by RT-PCR,Western blot and enzyme-linked immunosorbent assay （ELISA）. The pathological changes of renal interstitium were evaluated by Masson stainning, and the distribution of bradykinin B2 receptor （BKB2R） and angiotensin Ⅱ typel receptor （ATIR） was examined by immunohistochemistry. The expressions of BKB2R, ATIR, p-MAPK protein in renal tissue were detected by Western blot. Results Three months after HK gene delivery, the systolic blood pressure of experiment group was significantly decreased compared with the control group [（163±13） mm Hg vs （217±16） mm Hg, P〈0.01]（1 mm Hg=0.133 kPa）. Compared with sham rats, the rats in simple operation group and control group had much more renal interstitial collagen deposition and more serious fibrosis performance, but renal interstitial collagen deposition and fibrosis were significantly ameliorated in the rats of experiment group. In addition, the tubulointerstitial injury index of HK transgenic rats was significantly lower than that of the rats in control group （1.33±0.73 vs 3.01±0.62, P〈0.01）. Up-regulating expression of bradykinn B2 receptor protein and down-regulating expression of AT1 receptor and p-MAPK protein were found in renal tissues of experimental group after three months （P〈0.05）. Conclusion HK gene delivery significantly alleviates renal interstitial fibrosis in rats with 5/6 nephrectomy through regulating the expression of bradykinin B2 receptor, AT1 receptor and p-MAPK in renal tissue.