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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. [ABSTRACT FROM AUTHOR]

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 μM 14,15- EET for 30 min reduced phosphorylation of NKCC2 at the AVPsensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis. [ABSTRACT FROM AUTHOR]

To garner insights into the renal regulation of Ca 2+ homeostasis, we performed an mRNA microarray on kidneys from mice treated with the Ca 2+ -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 Ca 2+ 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 Ca 2+ balance, we measured changes in intracellular Ca 2+ uptake by live cell Ca 2+ imaging with the fluorophore Fura-2 AM. Inhibition of NHE8 with EIPA or by removing extracellular Na + -enhanced Ca 2+ influx into NRK cells. Ca 2+ influx was mediated by a voltage-dependent Ca 2+ channel rather than directly via NHE8. NRK cells express Cav1.3 and display verapamil-sensitive Ca 2+ influx and NHE8 inhibition-augmented Ca 2+ influx via a voltage-dependent Ca 2+ channel. Finally, proximal tubules perused ex vivo demonstrated increased Ca 2+ 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 Ca 2+ uptake into the proximal tubule epithelium.