Multi‐omics Analysis Reveals Regulatory Mechanisms in Chronic Cyclosporine A‐induced Nephrotoxicity Studied a Rat Model

Chronic calcineurin inhibitor (CNI) nephrotoxicity is a major drawback in current immunosuppressive regimens. In the chronic setting, arteriolar hyalinosis, decreased glomerular filtration rate, interstitial fibrosis and tubular dedifferentiation are the major adverse side effects. Since CsA is still widely used in transplant recipients, particularly in posttransplant diabetes, mechanistic insights into nephrotoxic mechanisms are required. To identify regulatory mechanisms in CsA nephrotoxicity we used quantitative transcriptomic, proteomic and phosphoproteomic methods. We tested the hypothesis that tubulointerstitial pathomechanisms play a predominant role in chronic CNI nephropathy. Whole transciptome RNA‐seq as well as global proteomic and phosphoproteomic methodologies were performed on kidney extracts from normal Wistar rats receiving CsA (25mg/kg b.w./day) or vehicle for 3 weeks. Differentially expressed genes and proteins as well as their phosphorylation status were obtained. Products of interest were tested using Western blot, qPCR, and immunohistochemistry. CsA treatment stimulated genome‐wide alterations in rat kidney according to RNA‐seq data. We identified 342 transcripts upregulated (padj<0.05) which included Ribosome (padj<0,0001) and Oxidative phosphorylation (padj<0,0001) pathways, whereas 331 transcripts were downregulated, with enrichment in genes critical for amino acid metabolism. Data were controlled by the established upregulation of renin and downregulation of calbindin (pdaj<0,05) in global proteomics. KEGG pathway and GO analysis from proteomics largely corresponded to the RNA‐seq results. Upregulated proteins were further related to ECM‐receptor interaction and focal adhesion pathways (padj<0,05). Phosphoproteomics demonstrated functional phosphorylation of components from unfolded protein response pathways, indicating an activation of the integrated stress response upon CsA. Products were verified at the tissue‐ and cell level. In sum, using integrated ‐omics analysis in CsA nephrotoxicity proved to be a powerful approach. Chronic CsA treatment was associated with enhanced energy metabolism and activation of the unfolded protein‐response pathways. A tubulointerstitial focus has been demonstrated. Potential biomarker candidates have been obtained.