Novel Mechanism for Tubular Injury in Nephropathic Cystinosis

Abstract

Understanding the unique susceptibility of the human kidney to pH dysfunction and injury in cystinosis is paramount to develop new therapies to preserve renal function. Renal proximal tubular epithelial cells (RPTECs) and fibroblasts, isolated from patients with cystinosis were transcriptionally profiled. Lysosomal fractionation, immunoblotting, confocal microscopy, intracellular pH, TEM, mitochondrial stress test, and membrane integrity assays were performed for validation. CRISPR, CTNS-/- RPTECs were generated. A new compound, Astaxanthin (ATX), was evaluated for rescue of the CTNS-/- phenotype. Alterations in cell stress, pH, autophagic turnover, and mitochondrial energetics, highlighted key changes in the vacuolar (V)-ATPases in patient derived and CTNS-/- RPTECs. ATP6V0A1 was significantly downregulated in cystinosis and highly co-regulated with loss of CTNS. Overexpression of ATP6V0A1 rescued cell stress and mitochondrial function. Treatment of CTNS-/- RPTECs with ATX, induced ATP6V0A1 expression and the resulting rescue of the RPTE cell and mitochondrial injury.In conclusion, our exploratory transcriptional and in vitro cellular and functional studies confirm that loss of cystinosin in RPTEC, results in a reduction in ATP6V0A1 expression, with changes in intracellular pH, mitochondrial integrity, mitochondrial function, and autophagosome-lysosome clearance. ATX can rescue the cystinotic RPTEC injury, at least partially mediated by upregulating ATP6V0A1 expression. The availability of ATX as a well-tolerated oral supplement, offers its further clinical evaluation as a potential novel therapeutic to limit renal tubular injury in cystinosis, independent of cysteine depletion alone.