Indoxyl sulfate influences susceptibility of proximal tubule epithelial cells to gentamicin cytotoxicity
Authors: Alge Joseph , Nguyen Loc 
Published: May 2023

Background: Patients with chronic kidney disease (CKD) are at increased risk of acute kidney injury (AKI) following exposure to nephrotoxic medications such as gentamicin, which can accelerate the progression of CKD. Therefore, there is a critical need to understand the molecular pathogenesis of acute-on-chronic kidney disease. A hallmark of CKD is the systemic bioaccumulation of uremic solutes, such as the tryptophan metabolite indoxyl sulfate (IS). Accumulating evidence suggests that IS has direct effects on the kidneys that contribute to CKD progression, and the objective of this study was to the determine the effect of IS on proximal tubule epithelial cell (PTEC) and test the hypothesis that IS exposure increases susceptibility to gentamicin-induced cytotoxicity. Methods: Cultured immortalized PTECs (HK-2 cells) were treated with IS and gentamicin at varying concentrations, and cell viability was assessed using the Trypan blue and MTT assays. TMT proteomic profiling with IBAQ quantitation and gene set enrichment analysis was used to identify differentially abundant proteins and cellular processes and pathways that are dysregulated by IS exposure and could mediate the cytotoxic effects of IS and influence susceptibility to gentamicin cytotoxicity. Results: Treatment with 100 μM IS had a modest cytotoxic effect (15-20% cell death after 24 hours over multiple experiments). Proteomic profiling of HK-2 cells identified 10,030 proteins, and IS treatment induced changes in the abundance of 60 proteins, including increased expression of kidney injury molecule-1, an established biomarker of PTEC injury, and VCAM-1, a marker of maladaptive repair after AKI. In contrast, expression of mitochondrial complex V ATPase Fo subunits C1, C2, and C3 and mitochondrial ribosomal proteins L18 and L50 were down regulated, and GSEA found that there was an overall decrease in mitochondrial translation following IS exposure. These results suggest that IS is directly cytotoxic to PTECs and negatively impacts mitochondrial dynamics and cellular respiration. Pretreatment with IS increased susceptibility of HK-2 cells to 10 mM gentamicin cytotoxicity as assessed by Trypan blue staining (30.1% +/- 6.4% cells stained positive without IS pretreatment vs 46.1 +/- 3.1% with IS pretreatment, p < 0.001), and this effect was confirmed using the MTT assay. Conclusion: IS has direct cytotoxic effects on PTECs and increases their susceptibility to gentamicin cytotoxicity, and systemic bioaccumulation of IS in CKD could therefore predispose to subsequent AKI following nephrotoxic medication exposure. This phenomenon could be mediated IS-induced downregulation of mitochondrial gene translation associated with dysregulated mitochondrial dynamics and impaired cellular respiration. This study was supported by the George M. O'Brien Kidney Translational Core Center at the University of Michigan (subaward of 5P30-DK081943 14). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.