Fisiopatología Renal, Centre d'Investigacions en Bioquímica i Biologia Molecular, Institut de Recerca Vall d'Hebron, Hospital Universitari Vall d'Hebron, Barcelona, Spain.
PLoS One. 2010 Nov 10;5(11):e13930. doi: 10.1371/journal.pone.0013930.
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.
亲环素（Cyps）是环孢素 A（CsA）的细胞内受体，负责肽基脯氨酰顺反异构和几种膜蛋白的伴侣。CsA 结合后会抑制这些功能。尽管 CsA 作为免疫抑制剂具有巨大的益处，但由于其不良的肾毒性作用，包括钠潴留、高血压、高钾血症、间质纤维化和移植受者进行性肾功能衰竭，其使用受到限制。在本报告中，我们专注于鉴定新型 CypB 相互作用蛋白，以了解 CypB 在肾功能中的作用，并进一步深入了解 CsA 诱导毒性的分子机制。通过用人肾脏 cDNA 进行酵母双杂交筛选，我们发现 CypB 与膜 Na/K-ATP 酶β1 亚基蛋白（Na/K-β1）之间存在新的相互作用，该相互作用在近端肾小管衍生的 HK-2 细胞中通过下拉、共免疫沉淀和共聚焦显微镜得到了证实。Na/K-ATP 酶泵是一种关键的质膜转运体，负责维持膜两侧 Na+和 K+的电化学梯度。我们表明 CypB 沉默在 HK-2 细胞中对 Na/K-ATP 酶活性的影响与 CsA 处理相似。还观察到α和β亚基在 ER 中的富集，这表明泵可能在这个隔室中成熟和向质膜转运过程中出现了故障。这些数据表明，CypB 通过与 Na/K-β1 的相互作用可能调节泵的成熟和通过分泌途径的运输，为亲环素与 CsA 的肾毒性作用之间的关系提供了新的见解。
