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View Was Recently Challenged Given the Curring in Proximal Tubules, We Have Unexpectedly Detected Discovery of a Regulated Necrosis Pathway, Necroptosis

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View Was Recently Challenged Given the Curring in Proximal Tubules, We Have Unexpectedly Detected Discovery of a Regulated Necrosis Pathway, Necroptosis BASIC RESEARCH www.jasn.org Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct Huihui Huang,1,2 William W. Jin,1 Ming Huang,1 Heyu Ji,1 Diane E. Capen,1 Yin Xia,3 Junying Yuan,4 Teodor G. Paunescu,˘ 1,2 and Hua A. Jenny Lu1,2 1Center for Systems Biology, Program in Membrane Biology and Division of Nephrology, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 2Harvard Medical School, Boston, Massachusetts 3Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China 4Department of Cell Biology, Harvard Medical School, Boston, Massachusetts ABSTRACT Background Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. Methods Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. Results Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine- protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addi- tion, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflam- mation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, 2 2 deletion of RIPK3 in the Ripk3 / mice significantly attenuated gentamicin-induced AKI. Conclusions A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin- induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis. JASN 31: 2097–2115, 2020. doi: https://doi.org/10.1681/ASN.2019020204 Received February 26, 2019. Accepted May 11, 2020. Aminoglycosides, such as gentamicin, are one of Published online ahead of print. Publication date available at the most commonly used classes of antibiotics to www.jasn.org. treat gram-negative bacterial infection. However, its clinical application has been greatly limited by Correspondence: Dr. Hua A. Jenny Lu, Center for Systems Bi- ology, Program in Membrane Biology and Division of Nephrol- its associated nephrotoxicity. The incidence of ogy, Department of Medicine, Massachusetts General Hospital, gentamicin-induced AKI ranges from 2% to as 185 Cambridge Street, CPZN 8150, Boston, MA 02114. Email: high as 55%.1 Itisestimatedthatupto30%of [email protected] patients treated with gentamicin for more than Copyright © 2020 by the American Society of Nephrology JASN 31: 2097–2115, 2020 ISSN : 1046-6673/3109-2097 2097 BASIC RESEARCH www.jasn.org 7 days exhibit signs of renal impairment.2 Multiple efforts to Significance Statement prevent gentamicin-induced AKI have not been effective due to the lack of understanding of the specific mechanism un- Gentamicin-induced AKI is a commonly recognized clinical prob- derlying gentamicin-induced tubular injury. It was observed lem, but the mechanism is not well understood. A mouse model of a long time ago that gentamicin was primarily taken up by gentamicin-induced AKI revealed a previously unrecognized role of necroptosis in mediating collecting duct epithelial cell death, in- and accumulated in proximal tubular cells and caused the terstitial inflammation, and fibrosis. Importantly, either inhibiting a proximal tubular injury. For many years, it was believed necroptotic pathway activator RIPK1 kinase with its inhibitor Nec-1 that the main mechanism of gentamicin-induced AKI is tu- or deleting a key necroptotic gene, Ripk3,significantly attenuated bular cell apoptosis, which occurs predominantly in proxi- gentamicin-induced AKI in mice and in cultured porcine and murine fi mal tubules in experimental animals and patients treated kidney tubular cells. Identi cation of a novel programmed nec- roptosis pathway in gentamicin-induced renal tubule injury could 3,4 with gentamicin. However, treatment to inhibit apoptosis provide a new therapeutic target. using various methods only partially improves gentamicin- induced AKI in animals.5–7 Additional and/or alternative tu- bular injury mechanisms underlying gentamicin-induced becomes oligomerized and then translocates from the cytosol AKI are under active investigation.8 to the plasma membrane where it disrupts the latter.19 In Very interestingly, unlike other types of acute tubular injury addition, a very recent study has demonstrated the existence resulting in oliguric AKI, gentamicin-induced AKI is frequently of a repair mechanism mediated by the endosomal sorting associated with polyuria and reduced urine osmolality, which complex required for transport (ESCRT-III). Through shed- implicates a possible defect of the urinary concentration mech- ding of MLKL-damaged membrane, ESCRT-III is able to anism, a main function of the collecting duct (CD). Whether sustain cell survival despite MLKL activation.20 When cells gentamicin-induced AKI could involve other type(s) of tubular undergo necroptosis, they present with organelle and cell cell injury mechanisms and/or other tubular segments, such as swelling, permeabilization of the plasma membrane, and CDs, besides proximal tubules is not clear. Using an anti- spilling of intracellular contents.21,22 Emerging studies have gentamicin antibody to stain gentamicin-treated mouse kid- revealed that blocking necroptosis using a RIPK1 inhibitor or ney, it was found that, besides a predominant accumulation of RIPK3-deficient mice largely alleviated multiple kidney in- gentamicin in proximal tubular cells, there was a significant juries induced by ischemia reperfusion; chemical injury accumulation of gentamicin in the CDs. Those CD cells that from cisplatin, cyclosporine, and contrast dye; and by unilat- – accumulated gentamicin appeared “round” or swollen with eral ureteral obstruction.23 25 prominent nuclei.9 However, how the CD tubular cells took It has been well appreciated that gentamicin causes signif- up gentamicin and whether they contributed to gentamicin- icant AKI and some of the patients with gentamicin-induced induced AKI remains unknown. This study suggests that AKI progress to ESKD. Massive inflammation and tubular uptake of gentamicin by CD leads to CD necroptosis. CD damage were reported in animals and patients treated with necroptosis likely contributes critically to the development of gentamicin.26 Whether necroptosis, in addition to apoptosis, is massive inflammation and interstitial fibrosis in gentamicin- involved in gentamicin-induced tubular injury has not been elu- induced AKI. cidated. To understand the pathologic mechanism underlying Several types of cell death, including apoptosis and necro- gentamicin-induced AKI, we performed a study to re-examine sis, occur during kidney tubular injury. Apoptosis is a regu- the tubular cell injury in gentamicin-treated mice. We demon- lated cell death that has been well studied for decades in many strated the presence of widespread interstitial inflammation, fi- tissues, including the kidney.10,11 Tubular cell apoptosis was brosis, and significant renal dysfunction after 7 days of treatment once considered to be a key form of cell death leading to CKD. with gentamicin in mice. Besides a low grade of apoptosis oc- However, this point of view was recently challenged given the curring in proximal tubules, we have unexpectedly detected discovery of a regulated necrosis pathway, necroptosis. Nec- widespread necroptosis in principal cells (PCs) of the CDs in roptosis is the most characterized pathway of regulated ne- gentamicin-treated animals. We further show that gentamicin crosis in higher eukaryotic cells.12,13 It is implicated in a wide induced upregulation of necroptotic signals, and inhibiting nec- range of high-grade tissue injuries such as myocardial ische- roptosis by RIPK1 inhibitor necrostatin-1 (Nec-1) or RIPK3 mia and reperfusion injury, amyotrophic lateral sclerosis, deficiency significantly alleviated gentamicin-induced inflam- sepsis, and intestinal inflammation.14–16 The best-studied mation, interstitial fibrosis, and kidney dysfunction. necroptotic pathway is the TNF-induced necroptosis through activation of TNF receptor 1.17,18 Induction of necroptosis is METHODS mediated through activation of TNF receptor 1 and subse- quent activation and interaction of the receptor-interacting Animals protein kinase 1 (RIPK1) and RIPK3. RIPK1 and RIPK3 in- C57/BL6J mice were purchased from The Jackson Laboratory teraction leads to recruitment and phosphorylation of the (Bar Harbor, ME). The mice were given free access to food and 2 2 mixed lineage kinase domain-like
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