DataSheet3_Polydatin attenuates tubulointerstitial fibrosis in diabetic kidney disease by inhibiting YAP expression and nuclear translocation.ZIP

The activation of Yes-associated protein (YAP) pathway is mutually causal with the increase of extracellular matrix (ECM) stiffness. Polydatin (PD) has been proved to have anti-fibrosis effect in diabetic kidney disease (DKD), but it is still a mystery whether PD participates in YAP-related mechano-transduction. Therefore, this study intends to solve the following two problems: 1) To construct an in vitro system of polyacrylamide hydrogels (PA gels) based on the true stiffness of kidneys in healthy and DKD rats, and observe the effect of PD on pathological matrix stiffness-induced YAP expression in renal fibroblasts; 2) Compared with verteporfin (VP), a pharmacological inhibitor of YAP, to explore whether the therapeutic effect of PD on DKD in vivo model is related to the regulation of YAP. In this study, the in vitro system of PA gels with 3 kPa, 12 kPa and 30 kPa stiffness was constructed and determined for the first time to simulate the kidney stiffness of healthy rats, rats with DKD for 8 weeks and 16 weeks, respectively. Compared with the PA gels with 3 kPa stiffness, the PA gels with 12 kPa and 30 kPa stiffness significantly increased the expression of YAP, α-smooth muscle actin (α-SMA) and collagen I, and the production of reactive oxygen species (ROS) in renal fibroblasts, and the PA gels with 30 kPa stiffness were the highest. PD significantly inhibited the above-mentioned changes of fibroblasts induced by pathological matrix stiffness, suggesting that the inhibition of PD on fibroblast-to-myofibroblast transformation and ECM production was at least partially associated with regulating YAP-related mechano-transduction pathway. Importantly, the inhibitory effect of PD on YAP expression and nuclear translocation in kidneys of DKD rats is similar to that of VP, but PD is superior to VP in reducing urinary protein, blood glucose, blood urea nitrogen and serum creatinine, as well as decreasing the expression of α-SMA and collagen I, ROS overproduction and renal fibrosis. Our results prove for the first time from the biomechanical point of view that PD is a potential therapeutic strategy for delaying the progression of renal fibrosis by inhibiting YAP expression and nuclear translocation.Yes相关蛋白（Yes-associated protein, YAP）通路的激活与细胞外基质（extracellular matrix, ECM）硬度升高互为因果关系。虎杖苷（Polydatin, PD）已被证实可在糖尿病肾病（diabetic kidney disease, DKD）中发挥抗纤维化作用，但其是否参与YAP相关的机械转导过程仍未明确。为此，本研究拟解决两大科学问题：其一，基于健康大鼠与DKD大鼠肾脏的真实硬度，构建聚丙烯酰胺水凝胶（polyacrylamide hydrogels, PA凝胶）体外模型，观察PD对病理性基质硬度诱导的肾成纤维细胞YAP表达的影响；其二，以YAP的药理学抑制剂维替泊芬（verteporfin, VP）作为对照，探究PD在体内DKD模型中的治疗效应是否与调控YAP通路相关。本研究首次构建并验证了硬度分别为3 kPa、12 kPa与30 kPa的PA凝胶体外系统，分别模拟健康大鼠、DKD造模8周及16周大鼠的肾脏刚度。相较于3 kPa组PA凝胶，12 kPa与30 kPa组PA凝胶可显著上调肾成纤维细胞中YAP、α-平滑肌肌动蛋白（α-smooth muscle actin, α-SMA）及I型胶原的表达，同时促进活性氧（reactive oxygen species, ROS）的生成，且30 kPa组的效应最为显著。PD可显著抑制病理性基质硬度诱导的上述成纤维细胞异常变化，提示PD对成纤维细胞向肌成纤维细胞转化及ECM生成的抑制作用，至少部分通过调控YAP相关的机械转导通路实现。值得注意的是，PD对DKD大鼠肾脏中YAP表达及核转位的抑制效果与VP相当，但在降低尿蛋白、血糖、血尿素氮及血清肌酐水平，下调α-SMA与I型胶原表达、改善ROS过度生成及肾纤维化方面，PD的疗效均优于VP。本研究首次从生物力学视角证实，PD通过抑制YAP表达与核转位，有望成为延缓肾纤维化进展的潜在治疗策略。