Nanjing Key Laboratory of Pediatrics, Children's Hospital of Nanjing Medical University, Nanjing, China.
Department of Nephrology, Children's Hospital of Nanjing Medical University, Nanjing, China.
Clin Sci (Lond). 2020 Jun 26;134(12):1357-1376. doi: 10.1042/CS20200030.
Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-β1 (TGF-β1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-β1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.
非特异性 Rho 相关激酶（ROCK）抑制剂可减轻单侧输尿管梗阻（UUO）模型中的肾纤维化，而 ROCK1 的基因缺失对小鼠的肾脏病理没有影响。因此，ROCK2 是否在肾小管间质纤维化中发挥作用尚需阐明。在本研究中，使用 ROCK2 的选择性抑制剂或基因敲除方法来研究 ROCK2 在肾小管间质纤维化中的作用。在慢性肾脏病（CKD）患者的纤维化肾脏中，我们观察到 ROCK2 的表达增强，与间质纤维化呈正相关。在小鼠中，ROCK2 蛋白水平在 UUO 模型中呈时间依赖性增加。通过腹腔注射 KD025（剂量为 50mg/kg/天）治疗 CKD 动物，Masson 三色染色显示的肾纤维化明显减轻，纤维化基因的表达减少。在体外，通过 KD025 或 ROCK2 敲降/敲除抑制 ROCK2，可显著减弱转化生长因子-β1（TGF-β1）刺激的小鼠肾近端肾小管上皮细胞（mPTCs）中的促纤维化反应。此外，细胞代谢受损被报道为 CKD 的一个重要致病因素。通过代谢组学分析，我们发现 KD025 恢复了代谢紊乱，包括 TGF-β1 刺激的肾小管上皮细胞中谷胱甘肽代谢受损。一致地，KD025 增加了抗氧化应激酶和核红细胞 2 相关因子 2（Nrf2）在纤维化模型中的表达。此外，KD025 减少了纤维化肾脏中巨噬细胞的浸润和炎症反应，并在体外抑制了巨噬细胞的激活。总之，抑制 ROCK2 可能成为 CKD 肾小管间质纤维化的一种潜在新型治疗方法。
