Calcineurin-dependent regulation of renal Na-(K-)Cl-cotransporters (II)

盐和水的动态平衡和血压的调节关键依赖于肾脏的肾小管上皮功能。远端肾单位(粗大的升支[TAL]，远端的曲管[DCT])通过盐的重吸收有效地决定容量调节。肾脏特异的Na-(K-)Cl-共转运体(阳离子-氯离子共转运体，CCC)包括TAL中的NKCC2和DCT中的NCC。两者都被一系列磷酸化蛋白激酶(WNK/Spak/OSR1)激活。级联中的缺陷会导致动态平衡的改变。在具有管状盐滞留的单基因伪醛固酮增多症(PHA-II)中，我们表征了WNK1和WNK4的功能，建立了与它们的cullin/kelch样介导的降解有关的联系，并描述了一个新的过度激活的WNK1突变体。钙依赖的丝氨酸理论磷酸酶钙调神经磷酸酶控制着这些激活步骤。钙调神经磷酸酶抑制剂(CNI、他克莫司、环孢素A)被成功地用于移植后的免疫抑制，但副作用常见，如容量滞留和高血压。我们已经证明，CNI可能部分地具有管状盐滞留的表型PHA-II，这可以通过用噻嗪类利尿剂抑制NCC来减少。NKCC2也参与其中。为了分析相关机制，我们定位了钙调神经磷酸酶异构体和CNI结合的免疫亲和素，并对免疫亲和素缺陷的小鼠模型进行了评估。通过分析钙调神经磷酸酶、蛋白激酶和CCC之间的结合特性，我们发现了一些重要的、非冗余的中介蛋白，如参与NKCC2转运和脂筏结合的膜联蛋白A2，以及在NKCC2上传递钙调神经磷酸酶作用的钙调神经磷酸酶同源蛋白1(CHP1)和SorLA。在更新期间，我们将遵循钙调神经磷酸酶在TAL和DCT中不同地发挥重要调节功能的假设。Ccc、钙调神经磷酸酶和激酶之间的相互作用将从机制上得到验证。钙调神经磷酸酶的活性将在分子水平上进行测定和指定。支架蛋白的作用以及膜联蛋白A2、CHP1和SorLA的影响将在CCC磷酸调节的背景下得到验证。考虑到钙调神经磷酸酶和WNK-1功能的参与，将在DCT根据速尿治疗和饮食诱导的运输刺激进行适应性重组的过程中，研究蛋白酶体的降解、侵袭体的形成和自噬。将结合WNK-Spak/OSR1-CN-CCC级联的调节适应对高盐、低钾饮食(“西方饮食”)造成的容量保留进行分析。将研究持续的CNI效应，以登记已识别产品的状态和相互作用，并将考虑内分泌效应。针对免疫抑制治疗副作用的治疗选择应基于本项目的结果而实现。

Regulation of salt and water homeostasis and blood pressure critically depends on tubular epithelial functions in the kidney. The distal Nephron (thick ascending limb [TAL], distal convoluted tubule [DCT]) determines volume regulation effectively via salt reabsorption. The kidney-specific Na-(K-)Cl-cotransporters (cation-chloride cotransporters, CCC) comprise NKCC2 in TAL and NCC in DCT. Both are activated by a cascade of phosphokinases (WNK/SPAK/OSR1). Defects in the cascade cause alterations in homeostasis. In monogenetic pseudohypoaldosteronism (PHA-II) with tubular salt retention we have characterized WNK1 and WNK4 functions, established a link to their cullin/kelch-like-mediated degradation, and described a new, overactivating WNK1 mutant. The calcium-dependent serine-theorine phosphatase calcineurin controls these activating steps. Calcineurin Inhibitors (CNI; tacrolimus, cyclosporin A) are successfully administered for immunosuppression after transplantation, but side effects such as volume retention and hypertension are common. We have shown that CNI may in part phenocopy PHA-II with tubular salt retention, which could be reduced by inhibiting NCC with thiazide diuretics. An involvement of NKCC2 was demonstrated as well. To analyze the mechanisms involved, calcineurin isoforms and CNI-binding immunophilins were localized, and an immunophilin-deficient mouse model evaluated. Analysis of the binding properties between calcineurin, the kinases, and the CCC led us to the identification of essential, non-redundant mediator proteins such as annexin A2 for the trafficking and lipid-raft association of NKCC2, as well as calcineurin homologous protein 1 (CHP1) and SORLA to transmit calcineurin effects on NKCC2. During the renewal period, we will follow the hypothesis that calcineurin differntially exerts important regulatory functions in TAL as well as in DCT. The interactions between CCC, calcineurin, and kinases will be validated mechanistically. The phosphatase activity of calcineurin will be determined and specified at the molecular level. The role of scaffold proteins as well as the impact of annexin A2, CHP1 and SORLA will be verified in the context of CCC phosphoregulation. Proteasomal degradation, aggresome formation and autophagy will be studied during adaptive restructuring of DCT upon furosemide therapy and diet-induced transport stimulation, considering an involvement of calcineurin and WNK-1 functions. Volume retention caused by a high-salt, low-potassium diet ("Western diet") will be analyzed with respect to regulatory adaptations of the WNK-SPAK/OSR1-CN-CCC cascade. Sustained CNI effects will be studied to register status and interactions of the identified products, and endocrine effects will be considered. Therapeutic options addressing the side effects of immunosuppressive therapy shall be achieved based on the results of this project.