特异性敲除Apelin基因对单侧输尿管梗阻小鼠肾间质纤维化的影响

目的观察多肽Apelin基因缺失对单侧输尿管梗阻(UUO)小鼠肾间质纤维化的影响,探讨在梗阻性肾病肾纤维化形成中Apelin的作用和可能机制。方法利用CRISPR/Cas9技术构建Apelin基因敲除小鼠(KO)共20只,同时选取野生型小鼠(WT)20只,均为8周龄雄性C57BL/6J种系,采用随机数字表法分为:KO+Sham组、KO+UUO组、WT+Sham组、WT+UUO组,每组各10只。分别对小鼠行单侧输尿管结扎(UUO)或假手术(Sham)。术后2周将小鼠处死,取血浆和手术侧肾脏进行检测。采用Masson染色观察肾脏纤维化程度,免疫组织化学染色检测肾脏基质成分纤维连接蛋白(FN)、Ⅰ型胶原(Col-I)的分布和表达情况。采用Western blotting法检测肾小管上皮标志物E-钙黏素(E-cadherin)、间充质标志物α-平滑肌肌动蛋白(α-SMA),以及致纤维化细胞因子--转化生长因子-β1(TGF-β1)及其受体TGFβ-R1的蛋白表达量。采用酶联免疫吸附法(ELISA)检测小鼠血浆肌酐(Cr)、尿素氮(BUN)水平。结果KO+UUO组小鼠肾脏可见明显的肾小管萎缩和肾间质纤维化,基质成分FN和Col-I表达量显著升高。WT+UUO组小鼠肾脏的纤维化程度和基质的表达量均低于KO+UUO组,差异具有统计学意义(P<0.05)。KO+Sham组和WT+Sham组肾脏无明显的纤维化改变和基质的沉积。KO+UUO组小鼠,可见显著的肾小管上皮-间充质转分化(EMT)表现,E-cadherin减少,α-SMA增多,TGF-β1和TGFβ-R1受体的表达量也明显升高。与KO+UUO组相比,WT+UUO组肾小管上皮E-cadherin的表达较多,α-SMA、TGF-β1和TGFβ-R1受体的表达均较低,差异具有统计学意义(P<0.05)。而WT+Sham组和KO+Sham组小鼠上述标志物的表达趋于正常水平。KO+UUO组小鼠血浆Cr、BUN的水平最高,WT+UUO组小鼠上述标志物的水平均低于KO+UUO组,差异具有统计学意义(P<0.05)。KO+Sham组小鼠血浆Cr、BUN与WT+Sham组相比,差异无统计学意义(P>0.05)。结论Apelin在正常状态下对肾功能无明显影响,但在输尿管梗阻状态下其基因缺失可能是引起肾间质纤维化加重的因素。

Effect of specific Apelin gene knockout on renal interstitial fibrosis in mice model of unilateral ureteral obstruction

Objective To observe the effect of polypeptide Apelin deficiency on the progression of renal interstitial fibrosis in mice model of unilateral ureteral obstruction( UUO),and to investigate the role and possible mechanism of Apelin in the development of renal fibrosis in obstructive nephropathy. Methods Twenty Apelin knockout mice( KO) were constructed by CRISPR/Cas9 technology. At the same time,twenty wild-type mice( WT) were selected,all of them were 8-week-old male C57 BL/6 J germline. They were divided into KO + Sham group,KO+ UUO group,WT + sham group and WT + UUO group randomly,with 10 mice in each group. Unilateral ureteral ligation or sham operation were performed on them. The mice were sacrificed 2 weeks after the operation,and the plasma and operative kidneys were taken for various analyses.Masson staining was performed to observe the degree of renal fibrosis,and immunohistochemical staining was performed to detect the distribution and expression of fibronectin( FN) and collagen type I( Col-I) in the extracellular matrix of kidney. Western blotting was performed to detect the expression of renal tubular epithelial protein E-cadherin,mesenchymal markers α-SMA,and fibrogenic cytokines TGF-β1 and its receptor TGFβ-R1. Enzyme linked immunosorbent assay( ELISA) was performed to determine the concentration of Cr and BUN in blood plasma. Results In KO + UUO group,atrophy of kidney tubules and interstitial fibrosis were evidently observed,while the level of FN and Col-I in renal interstitium were also increased. The degree of renal fibrosis and the expression of extracellular matrix in WT + UUO group were lower than those in KO+ UUO group,the difference was statistically significant( P < 0. 05). In KO + Sham and WT + Sham group,tubulointerstitium was normal,and interstitial matrix expression was only slightly. In KO + UUO group,there were significant changes of tubular epithelial-mesenchymal transition( EMT),and the dramatic suppression of E-cadherin and upregulation of α-SMA could be observed. There was also an obvious increase of TGF-β1 and TGFβ-R1. Compared with KO + UUO group,the expression of E-cadherin was higher,while the expression of α-SMA,TGF-β1 and TGFβ-R1 was lower in WT + UUO group,the difference was statistically significant( P < 0. 05). The expression of these molecules in WT+ Sham group and KO + Sham group tended to be normal. The plasma levels of Cr and BUN were the highest in KO + UUO group,and were lower in WT + UUO group,the difference was statistically significant( P < 0. 05). There was no significant difference in plasma Cr and BUN between KO + Sham group and WT + Sham group( P > 0. 05). Conclusion Apelin has no significant effect on renal function in normal condition,but the gene deletion in ureteral obstruction condition may be a factor contributing to deterioration of renal interstitial fibrosis.