Indoxyl Sulfate Influences Susceptibility of Proximal Tubule Epithelial Cells to Gentamicin Cytotoxicity
- AKI: Mechanisms - I
November 02, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
- Alge, Joseph, Baylor College of Medicine, Houston, Texas, United States
Patients with chronic kidney disease (CKD) are at increased risk of acute kidney injury (AKI) following exposure to nephrotoxic medications, and there is a critical need to understand the molecular pathogenesis of acute-on-chronic kidney disease. CKD leads to systemic bioaccumulation of uremic solutes, such as the tryptophan metabolite indoxyl sulfate (IS), which have direct effects on the kidneys that promote CKD progression. 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.
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 pathways that are dysregulated by IS exposure and could mediate the cytotoxic effects of IS influence susceptibility to gentamicin cytotoxicity.
Treatment with 100 μM IS had a modest cytotoxic effect (15-20% cell death after 24 hours). Proteomic profiling identified 10,030 proteins, and IS treatment induced changes in the abundance of 60 proteins, including increased expression of kidney injury molecule-1 and VCAM-1 and down regulated mitochondrial complex V ATPase subunits and mitochondrial ribosomal proteins. GSEA found that there was a 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.
IS induces modest cytotoxicity to PTECs and increases their susceptibility to gentamicin cytotoxicity. These effects are associated with downregulation of mitochondrial gene translation that could reflect broad perturbations in mitochondrial dynamics and impaired cellular respiration.
- NIDDK Support