Abstract：
In the aftermath of acute kidney injury (AKI), surviving proximal tubule epithelia repopulate injured tubules to promote repair. However, a portion of cells fail to repair [termed failed-repair proximal tubule cells (FR-PTCs)] and exert ongoing proinflammatory and profibrotic effects. To better understand the molecular drivers of the FR-PTC state, we reanalyzed a mouse ischemia-reperfusion injury single-nucleus RNA-sequencing (snRNA-seq) atlas to identify Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in FR-PTCs but not in healthy or acutely injured proximal tubules after AKI (2 and 6 wk) in mice. We confirmed expression of Tnik protein in injured mouse and human tissues by immunofluorescence. Then, to determine the functional role of Tnik in FR-PTCs, we depleted TNIK with siRNA in two human renal proximal tubule epithelial cell lines (primary and immortalized hRPTECs) and analyzed each by bulk RNA-sequencing. Pathway analysis revealed significant upregulation of inflammatory signaling pathways, whereas pathways associated with differentiated proximal tubules such as organic acid transport were significantly downregulated. TNIK gene knockdown drove reduced cell viability and increased apoptosis, including differentially expressed poly(ADP-ribose) polymerase (PARP) family members, cleaved PARP-1 fragments, and increased annexin V binding to phosphatidylserine. Together, these results indicate that Tnik upregulation in FR-PTCs acts in a compensatory fashion to suppress inflammation and promote proximal tubule epithelial cell survival after injury. Modulating TNIK activity may represent a prorepair therapeutic strategy after AKI.NEW & NOTEWORTHY The molecular drivers of successful and failed repair in the proximal tubule after acute kidney injury (AKI) are incompletely understood. We identified Traf2 and Nck interacting kinase (Tnik) to be exclusively expressed in failed-repair proximal tubule cells after AKI. We tested the effect of siTNIK depletion in two proximal tubule cell lines followed by bulk RNA-sequencing analysis. Our results indicate that TNIK acts to suppress inflammatory signaling and apoptosis in injured renal proximal tubule epithelial cells to promote cell survival.
摘要：
在急性肾损伤（AKI）后，存活的近端小管上皮重新填充受损的小管以促进修复。然而,一部分细胞修复失败(称为修复失败的近端小管细胞，FR-PTC）并发挥持续的促炎和促纤维化作用。为了更好地理解FR-PTC状态的分子驱动因素，我们重新分析了小鼠缺血再灌注损伤单核RNA测序（snRNA-seq）图谱，以鉴定Traf2和Nck相互作用激酶，Tnik,仅在FR-PTC中表达，但在小鼠AKI后（2周和6周）健康或急性损伤的近端小管中没有。我们通过免疫荧光证实了Tnik蛋白在受损小鼠和人组织中的表达。然后，为了确定Tnik在FR-PTC中的功能作用，我们在两种人肾近曲小管上皮细胞系(原代和永生化hRPTEC)中使用siRNA去除Tnik,并通过批量RNA测序分析每种细胞.通路分析显示炎症信号通路显著上调,而与分化的近端小管相关的途径如有机酸转运显著下调。TNIK敲低导致细胞活力降低和细胞凋亡增加，包括差异表达的PARP家族成员，切割PARP-1片段并增加膜联蛋白V与磷脂酰丝氨酸的结合。一起,这些结果表明,Tnik在FR-PTC中的上调以代偿方式起作用,抑制炎症并促进损伤后的近端小管上皮细胞存活.调节Tnik活性可能代表AKI后的前修复治疗策略。
