Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis

Significance Statement Necroptosis has emerged as an important cell death pathway that contributes to inflammation and injury of many organs, including the kidney. The mechanisms underlying necroptosis are not well understood. The authors have identified a previously unrecognized important role of integrin-linked kinase (ILK) in mediating necroptosis in collecting duct epithelial cell using genetically engineered mice to lack Ilk in the collecting duct principal cells of the kidney. These Ilk -knockout mice develop acute tubular injury, interstitial fibrosis and inflammation in the kidneys. Treating both the ILK inhibited cultured cells and ILK-deficient mice with a necroptosis inhibitor, necrostatin-1, reduced the harmful effects associated with the loss of ILK. The study shows that ILK plays an important role in regulating necroptosis in kidney tubular cells. Background Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. Methods We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. Results Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF- β signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals’ injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. Conclusions The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.