NKCC1在慢性肾脏病大鼠主动脉纤维化中的作用研究

Function of NKCC1 in the Aortic Fibrosis of Rats with Chronic Kidney Disease

该文探讨了Na-K-2Cl协同转运蛋白1(Na-K-2Cl cotransporter 1,NKCC1)在慢性肾脏病(chronic kidney disease,CKD)大鼠主动脉纤维化中的作用。采用5/6肾切除方法建立CKD大鼠模型,尾套法测定大鼠血压,脲酶法测定血清尿素氮(blood urea nitrogen,BUN),肌氨酸氧化酶法测定血清肌酐(serum creatinine,Scr);利用苏木素–伊红(hematoxylin and eosin,HE)染色观察组织形态,Masson染色观察组织纤维化情况;利用Real-time PCR、Western blot和免疫组织化学染色法检测大鼠肾脏和主动脉组织中NKCC1的表达;体外培养大鼠主动脉内皮细胞(endothelial cell,EC),通过免疫荧光实验观察细胞中Ⅰ型胶原(Collagen-Ⅰ,Col-Ⅰ)、Ⅲ型胶原(Collagen-Ⅲ,Col-Ⅲ)的表达,利用Western blot检测α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)、血小板–内皮细胞黏附分子(CD31)、钙黏蛋白(E-cadherin)、波形蛋白(vimentin)的表达。结果显示,CKD大鼠的平均收缩压(systolic blood pressure,SBP)较术前和假手术组(sham)均明显升高(P<0.05),BUN和Scr显著高于sham组(P<0.05);CKD大鼠肾脏和主动脉组织损伤严重且纤维化明显,NKCC1的表达较sham组明显增加;醛固酮(aldosterone)使EC中NKCC1、Col-Ⅰ和Col-Ⅲ增多,间质细胞标志物α-SMA和vimentin上调,内皮细胞标志物CD31和E-cadherin下调,在此基础上用布美他尼(bumetanide)使NKCC1、Col-Ⅰ和Col-Ⅲ减少,α-SMA和vimentin表达下降,CD31和E-cadherin升高。该研究表明,NKCC1可能通过增强内皮–间质转化(endothelial-to-mesenchymal transition,EndoMT)促进CKD主动脉纤维化。

This work explored the role of NKCC1 (Na-K-2Cl cotransporter 1) in aortic fibrosis in CKD (chronic kidney disease) rats.The CKD rat model was established by 5/6 nephrectomy.Blood pressure was measured by tail cuff method.BUN (blood urea nitrogen) was measured by urease method.Scr (serum creatine) was measured by sarcosine oxidase method.Tissue morphology was observed by HE (hematoxylin and eosin) staining,and fibrosis was observed by Masson-trichrome staining.The expression of NKCC1 in rat kidney and aorta was detected by Real-time PCR,Western blot and immunohistochemical staining.Rat aortic EC (endothelial cell) was cultured in vitro.Ⅰmmunofluorescence experiments were performed to observe the expression of Col-Ⅰ (collagen-Ⅰ) and Col-Ⅲ (collagen-Ⅲ) in the cells.Western blot was used to detect the expression of α-SMA (α-smooth muscle actin),CD31 (platelet-endothelial cell adhesion molecule),E-cadherin and vimentin.The results showed that the mean SBP (systolic blood pressure) of CKD rats was significantly higher than that of the preoperative and sham groups (P<0.05).The expression levels of BUN and Scr were significantly higher than those of the sham group (P<0.05).Kidney and aorta of CKD rats were severely damaged and showed obvious fibrosis.The expression of NKCC1 was significantly increased compared with that in the sham group.When NKCC1 in EC were activeted by aldosterone,the expression leves of Col-Ⅰ,Col-Ⅲ and the interstitial cell markers α-SMA,vimentin were up-regulated,while the endothelial cell markers CD31 and E-cadherin were down-regulated.On this basis,when NKCC1 activity were inhibited by bumetanide,the expression of Col-Ⅰ,Col-Ⅲ,α-SMA and vimentin was down-regulated,while CD31 and E-cadherin were up-regulated.This study shows that NKCC1 may promote CKD aortic fibrosis by enhancing EndoMT (endothelial-to-mesenchymal transition).