The role of capacitative calcium entry in AVP-dependent water transport by the kidney

AVP regulated water reabsorption in the collecting duct (CD) via translocation of AQP2 to the luminal membrane of principal cells is pivotal for bodily fluid homeostasis. Existing evidence points to the involvement of capacitative Ca2+ entry (CCE) in AVP-regulated signaling. Decreased sensitivity of CD cells to AVP results in a devastating disorder ' nephrogenic Diabetes insipidus (NDI). A number of NDI cases lack a recognized environmental or genetic mechanism of disease. The objective of this application is to establish the significance of CCE in AVP-dependent renal water handling and reveal a possible role of this mechanism in NDI. The proposal benefits from the animal model with disrupted CCE ' spontaneously hypertensive stroke-prone rats (SHR-A3). SHR-A3 have a nonsense mutation truncating C-terminal region of STIM1 protein ' endoplasmic reticulum Ca2+ sensor, responsible for interaction with plasma membrane Ca2+ channels. My pilot experiments in CDs freshly isolated from SHR-A3 show that truncation of STIM1 disrupts CCE. CD cells from SHR-A3 fail to sustain [Ca2+]i in response to AVP. SHR-A3 animals have lower urine osmolality and creatinine levels, paralleled by elevated circulating AVP levels, indicating reduced urinary concentration due to a nephorgenic defect. Pharmacological activation of STIM1 plasma membrane partners restores CCE and rejuvenates the sustained Ca2+ response. I hypothesize that CCE is a vital determinant of renal water handling, as it provides Ca2+ necessary to sustain AVP-induced signal in CD cells. CCE disruption impairs AVP signaling at the cellular level, compromises the ability of the kidney to concentrate water and results in NDI. Stimulation of CCE rescues AVP [Ca2+]i signal in CD cells with defective STIM1 and, at least partially, restores renal water transport at the systemic level. To test this hypothesis I address 3 specific aims: 1) establish the main molecular determinants of CCE and define the consequences of its disruption in CD cells; 2) delineate the physiological role of CCE in renal water handling and reveal the pathophysiological ramifications of dysfunctional CCE; 3) define how activation of CCE affects AVP-regulated signaling and water transport in the animals with dysfunctional STIM1. Current project will reveal the molecular basis of CCE, establish its functional role in renal water handling and target CCE to rescue NDI manifestations observed in experimental animals. (AHA Program: Scientist Development Grant)