Molecular pathogenesis of calcineurin inhibitor-induced hypertension

描述(由申请者提供)：本项目将研究钙调神经磷酸酶抑制剂，如他克莫司和环孢素，如何导致钾离子滞留的高血压。在已发表的初步研究中，我们发现他克莫司激活了肾脏中对噻嗪敏感的钠-氯协转运体(NCC)。这一效应被发现是导致高血压和钾滞留的关键因素。在这里，我们将研究涉及的机制。钙调神经磷酸酶是一种蛋白质磷酸酶，我们将测试它是否直接从NCC中去除磷酸盐，以抑制其作用，或者该途径是否涉及钙调神经磷酸酶对中间蛋白的作用。我们将确定其他蛋白磷酸酶，包括蛋白磷酸酶1A、2A和4是否从NCC中去除磷酸盐。蛋白磷酸酶抑制因子1(PPP1r1a)是钙调神经磷酸酶的典型靶点，在远端肾单位高表达。在我们的合作者的初步实验中，发现这种抑制物的基因缺失会减少体内磷酸化的NCC的丰度。因此，我们将测试钙调神经磷酸酶是否通过PPP1r1a调节NCC的活性。已知PPP1r1a可抑制蛋白磷酸酶1A的作用，而蛋白磷酸酶可作用于蛋白磷酸酶Spak。SPAK是典型的NCC激活蛋白。因此，我们将验证钙调神经磷酸酶抑制剂激活远端肾单位中的PPP1r1a，导致抑制PP1a，Spak激活，并增强NCC活性的总体假设。这一模型将在体外进行测试，使用我们最近开发的一种新的细胞系统，并在体内通过敲除信号通路的关键成分进行测试。无论是今天的患者治疗，还是明天的药物开发，这项提议都具有重大的临床意义。

DESCRIPTION (provided by applicant): This project will examine how calcineurin inhibitors, such as tacrolimus and cyclosporine, cause hypertension with potassium retention. In preliminary published studies, we showed that tacrolimus activates the thiazide-sensitive Na- Cl cotransporter (NCC) in the kidney. This effect was found to be essential for the resulting hypertension and potassium retention. Here we will examine the mechanisms involved. Calcineurin is a protein phosphatase and we will test whether it removes phosphates from NCC directly, to inhibit its actions, or whether the pathway involves calcineurin acting on intermediar proteins. We will determine whether other protein phosphatases, including protein phosphatases 1A, 2A, and 4 remove phosphates from NCC. Protein phosphatase inhibitor 1 (PPP1r1a) is a canonical target of calcineurin and is highly expressed along the distal nephron. In preliminary experiments by our collaborator, genetic deletion of this inhibitor was found to reduce the abundance of phosphorylated NCC in vivo. Therefore, we will test whether calcineurin regulates NCC activity via PPP1r1a. PPP1r1a is known to inhibit the actions of protein phosphatase 1A, which can act on the kinase SPAK. SPAK is the canonical NCC activating protein. Thus, we will test the over arching hypothesis that calcineurin inhibitors activate PPP1r1a in the distal nephron, leading to inhibition of PP1A, SPAK activation, and enhanced NCC activity. This model will be tested both in vitro, using a novel cell system that we developed recently, and in vivo, by knocking out key components of the signaling pathway. The proposal has substantial clinical implications, both in terms of patient treatment today, and in terms of drug development for tomorrow.