| N-Methyl-D-Aspartate Receptors (NMDARs) are Ca2+-permeable, ligand gated, non-selective cation channels that function as neuronal synaptic receptors but which are also expressed in multiple peripheral tissues. Here we show that NMDAR subunits NR3a and NR3b are highly expressed in the neonatal mouse kidney and that there is continued expression of NR3a in the renal medulla and papilla of the adult mouse. NR3a was also expressed in an inner medullary collecting duct cell line (mIMCD-3) where it was found that hypoxia and hypertonicity up-regulated NR3a expression. Using shRNA-based knockdown, a stable IMCD cell line was established that had ∼80% decrease in NR3a. Knockdown cells exhibited an increased basal intracellular free calcium concentration, reduced cell proliferation, and increased cell death. In addition NR3a knockdown cells exhibited reduced water transport in response to the addition of vasopressin, suggesting an alteration in aquaporin-2 (AQP2) expression/function. Consistent with this notion, we demonstrate decreased surface expression of glycosylated AQP2 in IMCD cells transfected with NR3a shRNA. To determine if this also occurred in vivo, we compared AQP2 levels in wild type versus in NR3a -/- mice. Total AQP2 protein levels in the outer and inner medulla were significantly reduced in knockout mice compared to control mice. Finally, NR3a-/- mice showed a significant delay in their ability to increase urine osmolality during water restriction. Thus, NR3a may play a reno-protective role in collecting duct cells. Under conditions that are associated with high vasopressin levels, NR3a, by maintaining low intracellular calcium levels, protects the function of the principal cells to reabsorb water and thereby increase medullary osmolality, which is essential for urinary concentration and dilution. |
