NHE8 attenuates calcium influx into NRK cells and the proximal tubule epithelium.

Abstract

To garner insight into the renal regulation of calcium homeostasis, we performed an mRNA micro-array on kidneys from mice treated with the calcium sensing receptor (CaSR) agonist cinacalcet. This revealed decreased gene expression of the sodium/proton 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 calcium fluxes, we employed the normal rat kidney (NRK) cell line. Cell surface biotinylation and confocal immunofluorescence microscopy demonstrated NHE8 apical expression. Functional studies found 5-(N-Ethyl-N-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 calcium balance, we measured changes in intracellular calcium uptake by live-cell calcium imaging with the fluorophore FURA-2 AM. Inhibition of NHE8 with EIPA or by removing extracellular sodium enhanced calcium influx into NRK cells. Calcium influx was mediated by a voltage-dependent calcium channel rather than directly via NHE8. NRK cells express Cav1.3 and display verapamil sensitive calcium influx and NHE8 inhibition augmented calcium influx via a voltage-dependent calcium channel. Finally, proximal tubules perused ex vivo demonstrated increased calcium influx in the presence of luminal EIPA at a concentration that would inhibit NHE8. These studies are consistent with NHE8 regulating calcium uptake into the proximal tubule epithelium.