Differential proteomic analysis of cyclosporine A-induced toxicity in renal proximal tubule cells.
ABSTRACT The use of cyclosporine A (CsA) as a potent immunosuppressant has been limited by its severe nephrotoxic effects. The mechanisms involved are haemodynamic but also related to direct toxic effects of CsA on proximal tubule epithelial cells. We focused on defining a proteomic profile in CsA-treated proximal tubule cells to distinguish the direct impact of CsA on these cells from overlapping haemodynamically mediated phenomena that occur in an in vivo system.
By means of high-throughput differential proteomic analyses and mass spectrometry techniques in CsA and vehicle-treated proximal tubule-derived cell lines of human and mouse origin, we determined proteins that change their expression in the presence of CsA.
CsA-induced toxicity analyses revealed that 10 mM CsA for 24 h was the threshold condition to induce significant changes in cell viability and proteomic profile. We identified 38 differentially expressed proteins on CsA-treated mouse PCT3 and human HK-2 cells, related to protein metabolism, response to damage, cell organization and cytoskeleton, energy metabolism, cell cycle and nucleobase/nucleoside/nucleotidic metabolism. 1D and 2D western blot assays in crude extracts from CsA-treated cells or kidneys with impaired function upon CsA treatment revealed a correlation with proteomic changes or differential isoform expression, in randomly selected proteins.
Proteins identified in this work might be useful markers to eventually distinguish CsA toxicity from chronic allograft nephropathy in protocol biopsies of transplanted patients, facilitating the adjustment of CsA doses to non-toxic ranges, as well as to study the impact of potential therapeutic interventions in an animal model.
Article: Cyclophilin B interacts with sodium-potassium ATPase and is required for pump activity in proximal tubule cells of the kidney.[show abstract] [hide abstract]
ABSTRACT: Cyclophilins (Cyps), the intracellular receptors for Cyclosporine A (CsA), are responsible for peptidyl-prolyl cis-trans isomerisation and for chaperoning several membrane proteins. Those functions are inhibited upon CsA binding. Albeit its great benefits as immunosuppressant, the use of CsA has been limited by undesirable nephrotoxic effects, including sodium retention, hypertension, hyperkalemia, interstial fibrosis and progressive renal failure in transplant recipients. In this report, we focused on the identification of novel CypB-interacting proteins to understand the role of CypB in kidney function and, in turn, to gain further insight into the molecular mechanisms of CsA-induced toxicity. By means of yeast two-hybrid screens with human kidney cDNA, we discovered a novel interaction between CypB and the membrane Na/K-ATPase β1 subunit protein (Na/K-β1) that was confirmed by pull-down, co-immunoprecipitation and confocal microscopy, in proximal tubule-derived HK-2 cells. The Na/K-ATPase pump, a key plasma membrane transporter, is responsible for maintenance of electrical Na+ and K+ gradients across the membrane. We showed that CypB silencing produced similar effects on Na/K-ATPase activity than CsA treatment in HK-2 cells. It was also observed an enrichment of both alpha and beta subunits in the ER, what suggested a possible failure on the maturation and routing of the pump from this compartment towards the plasma membrane. These data indicate that CypB through its interaction with Na/K-β1 might regulate maturation and trafficking of the pump through the secretory pathway, offering new insights into the relationship between cyclophilins and the nephrotoxic effects of CsA.PLoS ONE 01/2010; 5(11):e13930. · 4.09 Impact Factor