#1996 Identification of the biological bases associated with the mTOR-I-related podocytopathy through high-resolution mass spectrometry

Abstract

Abstract Background and Aims mTOR inhibitors (mTOR-I) often cause podocytopathy with the development of proteinuria (sometimes in the nephrotic range) in kidney transplant patients, but the biological mechanisms underlying this effect have been only partially defined. Method Immortalized human podocytes were treated in vitro with different everolimus (EVE) doses (10, 100, 200, 500 nM) for 24 h. Total cell lysates and supernatants were subjected to high-resolution mass spectrometry (Proteomics analysis). Innovative tests were used for bioinformatics analysis, including the Support Vector machine (SVM) and partial least squares discriminant analysis (PLS-DA). The results were validated in vitro and in human urine samples from 30 patients treated with EVE-based immunosuppressive treatment, using various molecular biology techniques (western blot, ELISA). Results Proteomic evaluation identified more than 7000 differentially expressed proteins. Among these, after a conservative statistical analysis, 78 showed an expression level significantly and directly correlated with EVE dosage. Functional analysis of the selected proteins showed a biological interconnection that involved several pathways, including kinases, cell cycle regulation, epithelial–mesenchymal transition, and protein synthesis. Osteopontin (SPP1) and Polo Like Kinase 2 (PLK2) were two of the most significantly correlated proteins (p < 0.00001, FDR<0.5%). The data were then confirmed through biomolecular experiments in vitro and in vivo in urine samples from kidney transplant recipients who received immunosuppressive treatment with EVE. Conclusion This study, for the first time, analyzed the biological impact of mTOR-I on podocytes, helping to understand not only the molecular basis of its therapeutic effect but also the biological reasons underlying their adverse events (particularly proteinuria). Validation of our data on larger cohorts of patients treated with this category of drugs will help identify new therapeutic targets to minimize adverse effects.