Li Xiao-Long, Liu Jing, Chen Xiao-Shan, Cheng Li-Min, Liu Wei-Ling, Chen Xing-Feng, Li Yue-Jiao, Guan Yong-Yuan, Zeng Xin, Du Yan-Hua
Department of Pharmacology, Cardiac & Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, Guangdong, China.
Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China.
Br J Pharmacol. 2022 Jun;179(12):3043-3060. doi: 10.1111/bph.15786. Epub 2022 Feb 24.
Renal fibrosis is the final common outcome in most forms of chronic kidney disease (CKD). However, the underlying causal mechanisms remain obscure. The present study examined whether transmembrane member 16A (TMEM16A), a Ca -activated chloride channel, contributes to the progression of renal fibrosis.
Masson staining, western blot and immunohistochemistry were used to measure renal fibrosis and related proteins expression. MQAE was used to evaluate the intracellular Cl concentration.
TMEM16A expression was significantly up-regulated in fibrotic kidneys of unilateral ureteral obstruction (UUO) and high-fat diet murine models and in renal samples of IgA nephropathy patients. In vivo knockdown of TMEM16A with adenovirus harbouring TMEM16A-shRNA or inhibition of TMEM16A channel activity with inhibitors CaCCinh-A01 or T16Ainh-A01 effectively prevented UUO-induced renal fibrosis and decreased protein expression of fibronectin, α-SMA and collagen in the obstructed kidneys. In cultured HK2 cells, knockdown or inhibition of TMEM16A suppressed TGF-β1-induced epithelial-mesenchymal transition, reduced snail1 expression and phosphorylation of Smad2/3 and ERK1/2, whereas overexpression of TMEM16A showed the opposite effects. TGF-β1 increased [Cl ]i in HK2 cells, which was inhibited by knockdown or inhibition of TMEM16A. Reducing [Cl ]i significantly blunted TGF-β1-induced Smad2/3 phosphorylation and profibrotic factors expression. The profibrotic effects of TGF-β1 were also reduced by inhibition of serum- and glucocorticoid-inducible protein kinase 1 (SGK1). SGK1 was also suppressed by reducing [Cl ]i.
Blockade of TMEM16A prevented the progression of kidney fibrosis, likely by suppressing [Cl ]i/SGK1/TGF-β1 signalling pathway. TMEM16A may be a potential new therapeutic target against renal fibrosis.
肾纤维化是大多数形式的慢性肾脏病（CKD）的最终共同结局。然而，其潜在的因果机制仍不清楚。本研究检测了钙激活氯离子通道跨膜蛋白16A（TMEM16A）是否促进肾纤维化进展。
采用Masson染色、蛋白质印迹法和免疫组织化学法检测肾纤维化及相关蛋白表达。用6-甲氧基-N-（3-磺酸丙基）喹啉碘化物（MQAE）评估细胞内氯离子浓度。
在单侧输尿管梗阻（UUO）和高脂饮食小鼠模型的纤维化肾脏以及IgA肾病患者的肾组织样本中，TMEM16A表达显著上调。用携带TMEM16A短发夹RNA的腺病毒在体内敲低TMEM16A，或用抑制剂CaCCinh-A01或T16Ainh-A01抑制TMEM16A通道活性，可有效预防UUO诱导的肾纤维化，并降低梗阻肾脏中纤连蛋白、α-平滑肌肌动蛋白（α-SMA）和胶原蛋白的蛋白表达。在培养的人近端肾小管上皮细胞（HK2）中，敲低或抑制TMEM16A可抑制转化生长因子-β1（TGF-β1）诱导的上皮-间质转化，降低蜗牛蛋白1（snail1）表达以及Smad2/3和细胞外信号调节激酶1/2（ERK1/2）的磷酸化，而TMEM16A过表达则产生相反的效果。TGF-β1增加HK2细胞内的氯离子浓度，敲低或抑制TMEM16A可抑制这一作用。降低细胞内氯离子浓度可显著减弱TGF-β1诱导的Smad2/3磷酸化和促纤维化因子表达。抑制血清和糖皮质激素诱导蛋白激酶1（SGK1）也可降低TGF-β1的促纤维化作用。降低细胞内氯离子浓度也可抑制SGK1。
阻断TMEM16A可能通过抑制细胞内氯离子浓度/SGK1/TGF-β1信号通路来阻止肾纤维化进展。TMEM16A可能是抗肾纤维化的一个潜在新治疗靶点。
