Introduction: Kidney injury molecule 1 (KIM-1) is a phosphatidylserine receptor upregulated on the apical membrane of injured proximal tubule epithelial cells (PTECs).KIM-1 can sense dead cells by binding to PS that is exposed on the outer membrane of apoptotic cells.We previously showed that KIM-1 expression is crucial for mitigating tissue damage and promoting repair during AKI.Though KIM-1 can transduce intracellular signalling in PTECs, the mechanisms by which KIM-1 protects against AKI remains incompletely understood.This study aimed to identify novel KIM-1-interacting proteins to address this knowledge gap.Methods: KIM-1 immunoprecipitates from human PTECs (HK-2) endogenously expressing KIM-1 were stimulated with apoptotic cells and analyzed using liquid chromatography tandem mass spectrometry.Proteins were identified using PEAKS DB proteomics software.Proteinprotein interactions were confirmed using Western blot.KIM-1 and its putative interacting protein were silenced using siRNA.Protein knockdown was confirmed using western blot, and cell viability was assayed using kinetic live cell imaging using a BioTek Cytation 5 multimode reader.Results: The co-immunoprecipitation of KIM-1 and phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4) was confirmed by western blot and mass spectrometry analysis.PI3KR4 interacted with KIM-1 in HK-2 cells independent of apoptotic cell stimulation.Silencing KIM-1 in HK-2 cells increased PIK3R4 expression, whereas silencing PIK3R4 decreased KIM-1 expression.Moreover, silencing PIK3R4 did not affect HK-2 cell proliferation and cell death, but silencing KIM-1 led to increased cell proliferation and death.However, the simultaneous silencing of KIM-1 and PIK3R4 rescued proliferation and cell death.Conclusion: Our study identified PIK3R4 as a novel and constitutive KIM-1-interacting protein in PTECs.A negative feedback loop in HK-2 cells regulates KIM-1 and PIK3R4 expression.PIK3R4 may be a potential activator of cell proliferation, which KIM-1 inhibits.Further study is required to understand the role of PI3KR4 in vivo during AKI.I have no potential conflict of interest to disclose.I did not use generative AI and AI-assisted technologies in the writing process.
Li et al. (Wed,) studied this question.