A role of store-operated Ca2+ entry in AVP-regulated renal water handling

AVP-regulated water transport in the collecting duct (CD) is a critical determinant of water homeostasis in the body. AVP stimulates water reabsorption in the CD through translocation of AQP2 to the luminal membrane of principal cells. This process requires prolonged elevation of [Ca2+]i via release from the endoplasmic reticulum (ER). Molecular mechanisms maintaining this AVP-induced [Ca2+]i response remain obscure. The accrued evidence points to the involvement of store-operated Ca2+ entry (SOCE) in AVP-stimulated [Ca2+]i signaling. This project exploits spontaneously hypertensive stroke-prone rats (SHR-A3) as an animal model of SOCE disruption. SHR-A3 has a natural mutation of STIM1 gene resulting in truncation of the STIM1 C-terminal region. The latter is responsible for interaction with plasma membrane Ca2+-permeable channels, such as ORAI1 and TRPC3. My experiments in freshly isolated split-opened CDs demonstrated that truncation of STIM1 in SHR-A3 line abolishes SOCE, while it is functional in stroke-resistant spontaneously hypertensive (SHR-B2) rats lacking the STIM1 mutation. AVP-stimulation elicits a sustained [Ca2+]i elevation in CD cells from SHR-B2 rats, but induces only a transient increase of [Ca2+]i in CDs from SHR-A3. SHR-A3 rats also have lower creatinine levels in urine, indicating reduced urinary concentration. Thus, I hypothesize that STIM1-activated Ca2+ entry via ORAI1/TRPC3 provides Ca2+ necessary to replenish ER stores, depleted by the initial AVP stimulation. This enables sustained Ca2+ release from ER necessary for proper AQP2 translocation to the apical membrane. Disruption of SOCE results in poor response at the cellular level and compromised ability of the kidney to concentrate water. To test this hypothesis I address 2 specific aims: 1) delineate molecular basis of SOCE in the CD; 2) determine physiological relevance of SOCE in AVP-dependent renal water handling and assess the consequences of SOCE disruption in the kidney. I will employ unique experimental arsenal, including Fura-2 based Ca2+ imaging, immunofluorescent confocal microscopy, systemic assessment of renal function and a rat model with defective SOCE. This allows me to fully probe my ideas in native tissue: freshly isolated split-opened CDs, as well as at the level of whole organism. Overall, current project will identify the molecular determinants of SOCE and establish a functional role of SOCE for renal water handling. (AHA Program: Postdoctoral Fellowship)