Dynamics of Aquaporin 2 Phosphorylation at Serine 256 and Serine 261 upon Co-Expression of Constitutively Active Variants of the Calcium- Sensing Receptor (CaSR) in Renal Cells

Background: We have recently shown that in MCD4 renal cells, cell surface AQP2 expression in cells exposed to CaSR agonists was higher than in control cells and did not increase significantly in response to short term exposure to forskolin. Those findings were in line with data obtained in hypercalciuric subjects displaying at baseline significantly higher AQP2 excretion and no significant increase in AQP2 excretion and urinary osmolarity after acute DDAVP administration compared to normocalciurics (Procino et al Plos One 2012). This indicates that CaSR-AQP2 interplay represents an internal renal defense to mitigate the effects of rising of calcium during antidiuresis on the risk of calcium precipitation. Methods: Human wild-type CaSR (hCaSR-wt) and its constitutively active variants (hCaSR-R990G; hCaSR-N124K) were functionally expressed in renal HEK cells stably expressing hAQP2. The N124K mutation is one of eight naturally occurring activating mutations in subjects with autosomal dominant hypocalcemia, whereas R990G is a gain-of- function of the CASR gene polymorphism. Western blotting analysis of a crude membrane fraction was performed using phospho-specific antibodies. Results: Compared to mock cells, pS256-AQP2 abundance was significantly increased in cells expressing either the wt-CaSR or its activating variants. Of note, we also found a significant increase in pS261-AQP2 in hCaSR-wt expressing cells compared to mock. Interestingly, the expression of pS261-AQP2 was significantly higher in cells expressing the constitutively active CaSR variants with respect to wt-CaSR expressing cells. No change in the pS269 was observed. Conclusions: Since previous data demonstrated that the amount of pS261 significantly decreases in response to short-term vasopressin exposure, it can be speculated that the increase in pS261 observed in cells expressing constitutively active CaSR variants might counteract the vasopressin response.