Department of Molecular and Cellular Physiology, Albany Medical College , Albany, New York.
Department of Cellular and Molecular Biology, Northwestern University , Chicago, Illinois.
Am J Physiol Renal Physiol. 2018 Oct 1;315(4):F769-F780. doi: 10.1152/ajprenal.00340.2017. Epub 2018 Apr 4.
Most renal transplants ultimately fail secondary to chronic allograft nephropathy (CAN). Vimentin (vim) is a member of the intermediate filament family of proteins and has been shown to be important in the development of CAN. One of the pathways leading to chronic renal fibrosis after transplant is thought to be epithelial to mesenchymal transition (EMT). Even though vim expression is one of the main steps of EMT, it is unknown whether vim expression is required for EMT leading to renal fibrosis and allograft loss. To this end, the role of vim in renal fibrosis was determined via unilateral ureteral obstruction (UUO) in vim knockout mice (129 svs6 vim -/-). Following UUO, kidneys were recovered and analyzed via Western blotting, immunofluorescence, and transcriptomics. Cultured human proximal renal tubular (HK-2) cells were subjected to lentiviral-driven inhibition of vim expression and then treated with transforming growth factor (TGF)-β to undergo EMT. Immunoblotting as well as wound healing assays were used to determine development of EMT. Western blotting analyses of mice undergoing UUO reveal increased levels of vim soon after UUO. As expected, interstitial collagen deposition increased in control mice following UUO but decreased in vim -/- kidneys. Immunofluorescence analyses also revealed altered localization of β-catenin in vim -/- mice undergoing UUO without significant changes in mRNA levels. However, RNA sequencing revealed a decrease in β-catenin-dependent genes in vim -/- kidneys. Finally, vim-silenced HK-2 cell lines undergoing EMT were shown to have decreased cellular migration during wound healing. We conclude that vim inhibition decreases fibrosis following UUO by possibly altering β-catenin localization and downstream signaling.
大多数肾移植最终因慢性同种异体移植物肾病 (CAN) 而失败。波形蛋白 (vim) 是中间丝家族蛋白的成员，已被证明在 CAN 的发展中很重要。移植后导致慢性肾纤维化的途径之一被认为是上皮细胞到间充质转化 (EMT)。尽管 vim 表达是 EMT 的主要步骤之一，但尚不清楚 vim 表达是否是导致肾纤维化和移植物丢失的 EMT 所必需的。为此，通过 129 svs6 vim -/- 基因敲除小鼠的单侧输尿管梗阻 (UUO) 来确定 vim 在肾纤维化中的作用。UUO 后，通过 Western blot、免疫荧光和转录组学回收和分析肾脏。培养的人近端肾小管 (HK-2) 细胞通过慢病毒驱动的 vim 表达抑制，然后用转化生长因子 (TGF)-β 处理以进行 EMT。免疫印迹和划痕愈合试验用于确定 EMT 的发展。UUO 小鼠的 Western blot 分析显示 UUO 后不久 vim 水平升高。正如预期的那样，UUO 后对照小鼠的间质胶原沉积增加，但 vim -/- 肾脏减少。免疫荧光分析还揭示了 UUO 中 vim -/- 小鼠 β-连环蛋白的定位改变，而 mRNA 水平没有明显变化。然而，RNA 测序显示 vim -/- 肾脏中 β-连环蛋白依赖性基因减少。最后，显示 EMT 中的 vim 沉默 HK-2 细胞系在划痕愈合过程中细胞迁移减少。我们得出结论，vim 抑制通过可能改变 β-连环蛋白定位和下游信号来减少 UUO 后的纤维化。
