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

Am. J. Physiol. Renal Physiol.2019 Aug 01;317(2):F240-F253. doi:10.1152/ajprenal.00329.2018. Epub 2019 May 01
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摘要


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-(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 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.

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