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Renal Role of Gprc5c in Regulating Systemic pH Homeostasis

Recent studies have identified orphan G‐protein coupled receptors (GPRs) as sensors of physiological metabolites. In this study, our goal is to elucidate the functional role of an understudied renal GPR, Gprc5c, which we previously found to be highly expressed in the kidney (Rajkumar et al., 2014). By immunofluorescence (IF), we find that Gprc5c localizes to the apical renal proximal tubules (PTs) in wild‐type (WT) mice, where it colocalizes with two PT markers ‐ LTA & megalin. Staining is absent in Gprc5c knockout (KO) mice kidneys. Gprc5c also localizes to the apical PTs by immunofluorescence in both rats and humans, suggesting an evolutionarily conserved role. In vivo, Gprc5c KO were not different from WT with respect to blood pressure, kidney/body weight, blood levels of Na+, Cl− & glucose, and urine levels of creatinine & glucose. However, Gprc5c KO have higher urine pH (WT: 5.48±0.01, n=6; KO: 5.81±0.06, n=6; p<0.05). In addition, the blood pH of Gprc5c KO mice trends acidic compared to WT in unanaesthetized mice (WT: 7.44±0.04, n=4; KO: 7.34±0.02, n=4; p=0.07), and in mice under isofluorane (WT: 7.22±0.03, n=5; KO: 7.03±0.04, n=5; p<0.05). In an in vitro GPCR internalization assay, alkaline pH (but not other chemicals tested) triggered Gprc5c internalization (an index of activation), with maximal internalization seen above pH 7.4 (pH 8.0>pH 7.4>pH 6.5). Other GPCRs tested as controls showed no changes in internalization over the pH range tested. On the apical membrane of the renal PT, sodium hydrogen exchanger 3 (NHE3) plays a key role in bicarbonate reabsorption, and therefore in maintaining pH homeostasis. Thus, we examined whether Gprc5c alters sodium proton exchanger activity. Using BCECF in a ratiometric fluorescence assay in Hek293T cells, we find that Gprc5c increases sodium proton exchange by 1.6 fold compared to empty vector controls in alkaline conditions (pH 8.0) (p<0.05), but not at pH 7.4. The increase seen at pH 8 is Tenapanor‐sensitive, implying that it is mediated via NHE3. In preliminary data, we also find that Gprc5c KO mice exhibit blunted changes in urinary pH when challenged with an alkali load for 7 days. Together, these data imply that Gprc5c localizes to the renal PTs apically where it contributes to systemic pH homeostasis via modulation of NHE3 function.