Regulation of Urea Transport by PKC-alpha

描述（由申请人提供）：加压素通过激活两个环AMP （cAMP）依赖的信号通路来调节尿素运输：蛋白激酶A和cAMP激活的交换蛋白。这导致UT-A1尿素转运蛋白磷酸化和根尖质膜积累的增加。髓质内部的高渗性可以独立于抗利尿激素，作为尿素运输的有力刺激。高渗性通过增加UT-A1磷酸化和质膜积累来刺激尿素运输。然而，发生这种情况的信号通路尚不清楚。我们有新的初步数据表明，高渗性通过蛋白激酶C （PKC）刺激尿素通透性。我们也有初步的数据表明PKC1是特定的PKC同工酶，因为：1)高渗性激活大鼠内髓集管（imcd）中的PKC1；2) PKC1敲除小鼠存在尿浓缩缺陷和UT-A1蛋白丰度降低。因此，在终末IMCD中，尿素转运和UT-A1活性的最大刺激需要cAMP和PKC1的刺激。我们的总体目标是研究PKC对UT-A1的调控。我们将验证PKC1通过改变UT-A1磷酸化来刺激UT-A1功能的假设。Aim 1将确定PKC调控UT-A1磷酸化的机制。Aim 2将确定UT-A1中的位点和PKC磷酸化的细胞位置。目的3将确定PKC和PKA在UT-A1膜积累、活性、磷酸化和磷酸酶介导的去磷酸化调控中的相互依赖性。目的4将确定PKC基因敲除小鼠尿浓缩缺陷的机制。我们提出的研究是非常重要的，因为它们可能产生关于水稳态失调机制的新信息。阐明增加尿素转运的非camp机制，进而增加尿液浓缩能力，可以为未来先天性肾源性尿囊症新治疗方法的转化研究奠定基础。

DESCRIPTION (provided by applicant): Vasopressin regulates urea transport by activating two cyclic AMP (cAMP) dependent signaling pathways: protein kinase A and exchange protein activated by cAMP. This results in increases of both the phosphorylation and apical plasma membrane accumulation of the UT-A1 urea transporter. The hypertonicity present in the inner medulla can act independently of vasopressin as a powerful stimulus of urea transport. Hypertonicity stimulates urea transport by increasing both UT-A1 phosphorylation and plasma membrane accumulation. However, the signaling pathway by which this occurs is unknown. We have new preliminary data showing that hypertonicity stimulates urea permeability via protein kinase C (PKC). We also have preliminary data suggesting that PKC1 is the specific PKC isozyme involved since: 1) hypertonicity activates PKC1 in rat inner medullary collecting ducts (IMCDs); and 2) PKC1 knock-out mice have a urine concentrating defect and a reduction in UT-A1 protein abundance. Thus, maximal stimulation of urea transport and UT-A1 activity in the terminal IMCD requires stimulation by both cAMP and PKC1. Our overall goal is to investigate the regulation of UT-A1 by PKC. We will test the hypothesis that PKC1 stimulates UT-A1 function through changes in UT-A1 phosphorylation. Aim 1 will determine the mechanism by which PKC regulates UT-A1 phosphorylation. Aim 2 will determine the site(s) in UT-A1 and the cellular location of phosphorylation by PKC. Aim 3 will determine the interdependence of PKC and PKA in the regulation of UT-A1 membrane accumulation, activity, phosphorylation, and phosphatase-mediated dephosphorylation. Aim 4 will determine the mechanism for the urine concentrating defect in PKC" knock-out mice. Our proposed studies are highly significant as they are likely to yield new information on mechanisms underlying dysregulation of water homeostasis. Elucidation of non-cAMP mechanisms for increasing urea transport, which in turn would increase urine concentrating ability, could form the basis for future translational studies of novel therapeutic approaches to congenital nephrogenic diabetes insipidus.