The calcium sensing receptor modulates H⁺-ATPase activity in intercalated cells

Previous studies found that calcium sensing receptor (CaSR) it's expressed in intercalated cells of the collecting duct and that its activation by calcium in the luminal membrane promotes acidification of urine. Therefore, the aim of the study was to analyze the effects of CaSR stimulus on the biochemical activity of the vacuolar H⁺-ATPase in a cellular model of intercalated cells, MDCK-C11 cells. Biochemical activity of H⁺-ATPase was performed using cell homogenates and the inorganic phosphate released was determined by a colorimetric method. Changes in cytosolic ionized calcium ([Ca²⁺]_i) were also determined using Fluo-4. A significant increase of vacuolar H⁺-ATPase activity was observed when the CaSR was stimulated with agonists such as Gd³⁺ (300 μM), neomycin (200 μM) and by the calcimimetic R-568 (1 μM). This activity was also stimulated in a dose-dependent fashion by changes in extracellular Ca²⁺ concentration ([Ca²⁺]_o) between 10^-2 and 2 mM. The calciolytic NPS 2143 (150 nM) significantly reduced the vacuolar H⁺-ATPase activity observed with 2 mM [Ca²⁺]_o. Inhibition of phospholipase C (PLC) activity with U73122 (5 x 10^-7 M) reversed the increase in pump activity observed in the presence of Gd³⁺. Activation of CaSR by the specific CaSR agonist R-568 produced a sustained rise of [Ca²⁺]_i, an effect that disappears when extracellular calcium was removed in the presence of thapsigargin. In summary, CaSR stimulation induces an increase in the vacuolar H⁺-ATPase activity of MDCK-C11 cells, an effect that involves an increase in [Ca²⁺]_i and require PLC activity. The consequent decrease in intratubular pH could lead to increase ionization of luminal calcium, potentially reducing the formation of calcium phosphate stones.