Redox DAPK1 destabilizes Pellino1 to govern inflammation-coupling tubular damage during septic AKI

Abstract

Tubular damage initiated by inflammatory response and ischemic/hypoxic stress is a hallmark of septic acute kidney injury (AKI), albeit the molecular mechanism coupling the two events remains unclear. We investigated the intrinsic nature of tubular damage with respect to inflammatory/hypoxic stress during septic AKI. Methods: The apoptotic response of tubular cells to LPS stimuli was analyzed before and after hypoxia exposure. Cellular ubiquitination, co-immunoprecipitation, GST-pulldown, in vitro protein kinase assay, immunofluorescence and CRISPR technology were adopted to determine the molecular mechanism underlying this process. In vivo characterization was performed in wild-type and DAPK1 -/- mice models of cecal ligation and puncture (CLP). Results: We found that the MyD88-dependent inflammatory response couples to tubular damage during LPS stimuli under hypoxia in a Fn14/SCF Fbxw7α -dispensable manner via recruitment of caspase-8 with TRIF-RIP1 signalosome mediated by DAPK1, which directly binds to and phosphorylates Pellino1 at Ser39, leading to Pellino1 poly-ubiquitination and turnover. Either pharmacological deactivation or genetic ablation of DAPK1 makes tubular cells refractory to the LPS-induced damage in the context of hypoxia, while kinase activity of DAPK1 is essential for ruin execution. Targeting DAPK1 effectively protects mice against septic AKI and potentiates the efficacy of a MyD88 homodimerization inhibitor, ST2825. Conclusion: Our findings provide a rationale for the mechanism whereby inflammation intersects with hypoxic tubular damage during septic AKI through a previously unappreciated role of DAPK1-inducible Ser39 phosphorylation in Pellino1 turnover and underscore that combined targeting DAPK1 and MyD88 might be a feasible strategy for septic AKI management.

Interruption of neutrophil extracellular traps formation dictates host defense and tubular HOXA5 stability to augment efficacy of anti-Fn14 therapy against septic AKI

“…All animals were bred in the specific-pathogen-free animal facility of the Animal Experiment Center of Zhejiang University. Septic AKI models of cecal ligation and puncture (CLP) and the LPS-induced endotoxemia (LIE) were performed as described previously 17 - 20 . For CLP, a small midline incision was made in abdomen of mice after being anesthetized with ketamine (80 mg/kg).…”

“…Kidney tissues were excised from mice, fixed in 10% formalin, decalcified in 10% EDTA and embedded in paraffin for hematoxylin&eosin (H&E) and immunohistochemical staining as described in previous publications 17 , 18 , 20 . For immunohistochemical staining, renal sections were rehydrated and subjected to the heat-mediated antigen retrieval using citrate buffer (pH 6.0).…”

“…Immunocytofluorescence was performed as previously described 20 , 21 . In brief, cells in six-well plates were fixed with 4% formaldehyde in PBS for 15 min at 37 °C.…”

“…Human kidney proximal tubular epithelial (HK-2) cells were grown in RPMI-1640 (GIBCO, Carlsbad, CA) with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 g/mL streptomycin in a humidified incubator with 5% CO 2 at 37 °C as previously described 17 , 18 , 20 . Renal proximal tubular cells (RPTCs) were isolated from the indicated mice using a previously described procedure with minor modifications 19 .…”

“…Expression constructs and siRNAs were transfected into tubular cells using Lipofectamine 3000 transfection reagents (Invitrogen, Carlsbad, CA) by following the manufacturer's protocol as previously described 20 , 21 . Lentivirus carrying pLKO.1 plasmid was produced by co-transfecting HEK293T cells with pRSV-Rev, pMD2.G, pMDLg/pRRE, and pLKO.1-puro shRNA.…”