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Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1in four APAs and one APN (p.L51_A57del, n= 3; p.L49_L55del, n= 2). SLC30A1encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57delvariant in a doxycycline-inducible adrenal cell system revealed pathological Na+influx. An aberrant Na+current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+activity, which stimulated CYP11B2mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.

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.

The tight junction proteins claudin-10 and -16 are crucial for the paracellular reabsorption of cations along the thick ascending limb of Henle’s loop in the kidney. In patients, mutations in CLDN16cause familial hypomagnesemia with hypercalciuria and nephrocalcinosis, while mutations in CLDN10impair kidney function. Mice lacking claudin-16 display magnesium and calcium wasting, whereas absence of claudin-10 results in hypermagnesemia and interstitial nephrocalcinosis. In order to study the functional interdependence of claudin-10 and -16 we generated double-deficient mice. These mice had normal serum magnesium and urinary excretion of magnesium and calcium and showed polyuria and sodium retention at the expense of increased renal potassium excretion, but no nephrocalcinosis. Isolated thick ascending limb tubules of double mutants displayed a complete loss of paracellular cation selectivity and functionality. Mice lacking both claudin-10 and -16 in the thick ascending limb recruited downstream compensatory mechanisms and showed hypertrophic distal convoluted tubules with changes in gene expression and phosphorylation of ion transporters in this segment, presumably triggered by the mild decrease in serum potassium. Thus, severe individual phenotypes in claudin-10 and claudin-16 knockout mice are corrected by the additional deletion of the other claudin.

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 AVP-sensitive 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.

Tenapanor inhibits sodium/hydrogen exchanger isoform 3 to reduce intestinal phosphate absorption via reduction of passive paracellular phosphate flux.