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.
Highlights
Cyclosporine A (CsA) is a potent immunosuppressive cyclic undecapeptide mainly used to prevent graft rejection after transplant surgery [1]
Experimental models of CsA nephrotoxicity in rats, in which glomerular hemodynamics were analyzed by renal micropuncture techniques, revealed that CsA administration is associated with afferent and efferent arteriolar vasoconstriction, with predominating preglomerular vasoconstriction that results in a significant reduction of renal plasma flow
Cyclophilins, first discovered as the intracellular receptors of the immunossupressant CsA, facilitate protein folding via their peptidyl prolyl cistrans isomerases (PPIases) activity and contribute to the maturation of several proteins [13,14]
Summary
Cyclosporine A (CsA) is a potent immunosuppressive cyclic undecapeptide mainly used to prevent graft rejection after transplant surgery [1] It has changed the outcomes of transplantation and autoimmune diseases, with the drawback of significant nephrotoxicity [2]. CsA nephrotoxic effects include a reduction in glomerular filtration rate by increasing arteriolar vasoconstriction and renal vascular resistance, microvascular arteriolopathy, interstitial fibrosis, and vacuolization of proximal tubule cells [3,4]. We hypothesized that identification of Cyp-interacting proteins in kidney could be relevant to understand CsA nephrotoxicity at the molecular level and, eventually, these proteins might become therapeutical targets to avoid toxicity while maintaining the drug’s immunosupresive effects in CsAtreated patients. We describe that CypB interacts with the Na/K-ATPase pump in human kidney and that this cyclophilin is crucial for pump location and activity
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