NHE8 attenuates Ca²⁺influx into NRK cells and the proximal tubule epithelium

Abstract

To garner insights into the renal regulation of Ca²⁺ homeostasis, we performed an mRNA microarray on kidneys from mice treated with the Ca²⁺-sensing receptor (CaSR) agonist cinacalcet. This revealed decreased gene expression of Na⁺/H⁺ exchanger isoform 8 (NHE8) in response to CaSR activation. These results were confirmed by quantitative real-time PCR. Moreover, administration of vitamin D also decreased NHE8 mRNA expression. In contrast, renal NHE8 protein expression from the same samples was increased. To examine the role of NHE8 in transmembrane Ca²⁺ fluxes, we used the normal rat kidney (NRK) cell line. Cell surface biotinylation and confocal immunofluorescence microscopy demonstrated NHE8 apical expression. Functional experiments found 5-(<i>N</i>-ethyl-<i>N</i>-isopropyl)amiloride (EIPA)-inhibitable NHE activity in NRK cells at concentrations minimally attenuating NHE1 activity in AP-1 cells. To determine how NHE8 might regulate Ca²⁺ balance, we measured changes in intracellular Ca²⁺ uptake by live cell Ca²⁺ imaging with the fluorophore Fura-2 AM. Inhibition of NHE8 with EIPA or by removing extracellular Na⁺-enhanced Ca²⁺ influx into NRK cells. Ca²⁺ influx was mediated by a voltage-dependent Ca²⁺ channel rather than directly via NHE8. NRK cells express Cav1.3 and display verapamil-sensitive Ca²⁺ influx and NHE8 inhibition-augmented Ca²⁺ influx via a voltage-dependent Ca²⁺ channel. Finally, proximal tubules perused ex vivo demonstrated increased Ca²⁺ influx in the presence of luminal EIPA at a concentration that would inhibit NHE8. The results of the present study are consistent with NHE8 regulating Ca²⁺ uptake into the proximal tubule epithelium.