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Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell BASIC RESEARCH www.jasn.org Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis Ming Huang,1,2 Shuai Zhu,1,2 Huihui Huang,2 Jinzhao He,1,2 Kenji Tsuji,2 William W. Jin,2 Dongping Xie,3 Onju Ham,2 Diane E. Capen,2 Weining Lu,4 Teodor G. Paunescu, 2 Baoxue Yang,1 and Hua A. Jenny Lu2 1Department of Pharmacology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing, China; 2Center for Systems Biology, Program in Membrane Biology, Division of Nephrology, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; 3Department of Physiology, Tongji University School of Medicine, Shanghai, China; and 4Renal Section, Departments of Medicine, and Pathology & Laboratory Medicine, Boston University Medical Center, Boston, Massachusetts ABSTRACT 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. BASIC RESEARCH 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-b signaling cas- cade 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. JASN 30: 2073–2090, 2019. doi: https://doi.org/10.1681/ASN.2018111162 Received November 26, 2018. Accepted July 15, 2019. CKD affects approximately 10% of the world’s M. H. and S. Z. contributed equally to this work. population and places a significant burden on Published online ahead of print. Publication date available at economy and health care worldwide.1,2 Effective www.jasn.org. therapies to prevent or halt CKD progression are Correspondence: Dr. Hua A. Jenny Lu, Program in Membrane lacking, largely due to limited understanding of Biology, Department of Medicine, Massachusetts General Hos- thepathologicandmolecularbasisofthedisease. pital, 185 Cambridge Street, CPZN 8150, Boston, MA 02114, or Dr. Baoxue Yang, Department of Pharmacology, School of Basic It is increasingly recognized that AKI is a major Medical Sciences, Peking University Health Science Center, 38 contributor to the development of CKD.3 There- Xueyuan Road, Beijing 100191, China. E-mail: Lu.Hua@mgh. fore, it is critical to understand the injury and re- harvard.edu or [email protected] covery mechanisms of renal tubular cells and their Copyright © 2019 by the American Society of Nephrology JASN 30: 2073–2090, 2019 ISSN : 1046-6673/3011-2073 2073 BASIC RESEARCH www.jasn.org roles in mediating inflammatory response and fibrosis in the Significance Statement kidney. There are two main types of cell death that occur during Necroptosis has emerged as an important cell death pathway that kidney tubular injury: apoptosis and necrosis. Apoptotic cells contributes to inflammation and injury of many organs, including the are frequently detected in the renal proximal tubule, distal kidney. The mechanisms underlying necroptosis are not well un- derstood. The authors have identified a previously unrecognized 3 tubule, and loop of Henle in various AKI models. Tubular important role of integrin-linked kinase (ILK) in mediating nec- cell apoptosis was once considered to be a key form of cell roptosis in collecting duct epithelial cell using genetically en- death leading to CKD.4 However, this point of view was re- gineered mice to lack Ilk in the collecting duct principal cells of the cently challenged because inhibition of apoptosis, e.g., by cas- kidney. These Ilk-knockout mice develop acute tubular injury, in- fi fl pase inhibitors, failed to alleviate renal injury.5 Therefore, an terstitial brosis and in ammation in the kidneys. Treating both the ILK inhibited cultured cells and ILK-deficient mice with a necroptosis alternative pathology has to be sought. In recent years, break- inhibitor, necrostatin-1, reduced the harmful effects associated with through studies show that necrosis can occur in a highly reg- the loss of ILK. The study shows that ILK plays an important role in ulated manner through complex molecular interactions, and regulating necroptosis in kidney tubular cells. it plays a critical role in mediating tissue injury and interstitial fibrosis.6,7 significant necroptosis of CD PCs instead. ILK deficiency in Necroptosis is the most-characterized pathway of regulated PCs activates necroptotic signaling and promotes inflammation necrosis in higher eukaryotic cells.6 Necroptosis is activated via and interstitial fibrosis in the kidney. More importantly, blocking the formation of the necrosome, which includes receptor in- necroptosis using necrostatin-1 (Nec-1), a chemical inhibitor of teracting protein kinase 1 (RIPK1), RIPK3, and mixed lineage RIPK1, attenuates CD injury and fibrosis in Ilk KO mice. There- kinase domain-like pseudokinase (MLKL). The homotypic fore, our data support a previously unrecognized, important func- interaction of RIPK3 and RIPK1 drives RIPK3 phosphoryla- tion of ILK in mediating necroptosis in CD epithelial cells. tion, which in turn recruits and phosphorylates MLKL.8 Phos- phorylated MLKL oligomerizes and translocates to the plasma membrane, eventually resulting in membrane rupture.9 When METHODS cells undergo necroptosis, they present with organelle and cell swelling, permeabilization of the plasma membrane, and spill- See Supplemental Methods for a detailed description. ing of intracellular contents.10 Recent studies indicate necrop- tosis is an important player in some high-effect diseases, such Experimental Animals as myocardial ischemia and reperfusion injury (IRI), sepsis, All animal experiments were approved by the Massachusetts and intestinal inflammation.11–13 Furthermore, it has been General Hospital (MGH) Subcommittee on Research Animal shown to contribute to the pathogenesis of renal IRI and Care, in compliance with the National Institutes of Health AKIs induced by nephrotoxic agents such as cisplatin and io- (NIH) Guide for the Care and Use of Laboratory Animals.All dinated contrast.14–16 The involvement of many signaling mice were on the C57BL/6J background. B6N;129-Ilktm1Star/J pathways that are critically associated with cell adhesion and mice carrying loxP sites have been previously described and survival, such as integrin and its downstream integrin-linked are available from the Jackson laboratory (stock number kinase (ILK), has not yet been studied in necroptosis. 023310).29 Transgenic mice harboring aquaporin 2 (Aqp2) ILK is a critical scaffold protein located in focal adhesions. Cre recombinase (Aqp2-Cre+) were generated by Dr. Günther Through interacting with the cytoplasmic domain of Schütz’s group (Heidelberg, Germany)30 and obtained from b-integrins, ILK transduces integrin signaling to the interior Dr. Wenzheng Zhang (Albany, NY).31 Homozygous, floxed Ilk fl fl of the cell and mediates diverse cellular processes, including mice (Ilk / ) were crossed with Aqp2-Cre+ mice to generate fl fl cell survival, proliferation, adhesion, differentiation, and migra- PC Ilk KO mice (Ilk / ; Aqp2-Cre+). The littermates without fl fl tion.17 In vivo studies have suggested that ILK plays a critical role Aqp2-Cre (Ilk / ; Aqp2-Cre2) were used as control. in developing and maintaining the structure and function of For the Nec-1 (Sigma-Aldrich, St. Louis, MO) treatment fl fl many organs, including the kidney.18–20 For example, inactivation experiment, 2-week-old wild-type and Ilk / ; Aqp2-Cre+ mice of ILK in mouse kidney podocytes leads to podocyte damage, were given 1.65 mg/kg Nec-1 or PBS containing 1% DMSO progressive proteinuria, glomerulosclerosis, and severe tubuloin- through intraperitoneal injection every 2 days for 2 weeks. terstitial fibrosis.21–23 Aberrant expression of ILK is associated Mice were euthanized at the age of 4 weeks. with renal tubulointerstitial fibrosis, renal cell carcinoma, dia- betic glomerulopathy, and congenital nephrotic syndrome.24–28 Kidney Tissue Preparation To further investigate the potential function of ILK in collecting Kidney tissues were collected as previously described.32 Briefly, duct (CD) principal cells (PCs), we generated knockout (KO) mice were anesthetized using isoflurane (3% inhalant). The right mice with Ilk deletion in PCs. We found that deletion of
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