From Fish Physiology to Human Disease: The Discovery of the NCC, NKCC2, and the Cation-Coupled Chloride Cotransporters.

The renal Na-K-2Cl and Na-Cl cotransporters are the major salt reabsorption pathways in the thick ascending limb of Henle loop and the distal convoluted tubule, respectively. These transporters are the target of the loop and thiazide type diuretics extensively used in the world for the treatment of edematous states and arterial hypertension. The diuretics appeared in the market many years before the salt transport systems were discovered. The evolving of the knowledge and the cloning of the genes encoding the Na-K-2Cl and Na-Cl cotransporters were possible thanks to the study of marine species. This work presents the history of how we came to know the mechanisms for the loop and thiazide type diuretics actions, the use of marine species in the cloning process of these cotransporters and therefore in the whole solute carrier cotransproters 12 (SLC12) family of electroneutral cation chloride cotransporters, and the disease associated with each member of the family.

Influence of administration time and sex on natriuretic, diuretic, and kaliuretic effects of diuretics.

Sex differences in renal function and blood pressure have been widely described across many species. Blood pressure dips during sleep and peaks in the early morning. Similarly, glomerular filtration rate, filtered electrolyte loads, urine volume, and urinary excretion all exhibit notable diurnal rhythms, which reflect, in part, the regulation of renal transporter proteins by circadian clock genes. That regulation is sexually dimorphic; as such, sex and time of day are not two independent regulators of kidney function and blood pressure. The objective of the present study was to assess the effect of sex and administration time on the natriuretic and diuretic effects of loop, thiazide, and K+-sparing diuretics, which are common treatments for hypertension. Loop diuretics inhibit Na+-K+-2Cl- cotransporters on the apical membrane of the thick ascending limb, thiazide diuretics inhibit Na+-Cl- cotransporters on the distal convoluted tubule, and K+-sparing diuretics inhibit epithelial Na+ channels on the connecting tubule and collecting duct. We simulated Na+ transporter inhibition using sex- and time-of-day-specific computational models of mouse kidney function. The simulation results highlighted significant sex and time-of-day differences in the drug response. Loop diuretics induced larger natriuretic and diuretic effects during the active phase. The natriuretic and diuretic effects of thiazide diuretics exhibited sex and time-of-day differences, whereas these effects of K+-sparing diuretics exhibited a significant time-of-day difference in females only. The kaliuretic effect depended on the type of diuretics and time of administration. The present computational models can be a useful tool in chronotherapy, to tailor drug administration time to match the body's diurnal rhythms to optimize the drug effect.NEW & NOTEWORTHY Sex influences cardiovascular disease, and the timing of onset of acute cardiovascular events exhibits circadian rhythms. Kidney function also exhibits sex differences and circadian rhythms. How do the natriuretic and diuretic effects of diuretics, a common treatment for hypertension that targets the kidneys, differ between the sexes? And how do these effects vary during the day? To answer these questions, we conducted computer simulations to assess the effects of loop, thiazide, and K+-sparing diuretics.

Elevated FGF23 Levels in Mice Lacking the Thiazide-Sensitive NaCl cotransporter (NCC).

Fibroblast growth factor 23 (FGF23) participates in the orchestration of mineral metabolism by inducing phosphaturia and decreasing the production of 1,25(OH)2D3. It is known that FGF23 release is stimulated by aldosterone and extracellular volume depletion. To characterize this effect further in a model of mild hypovolemia, we studied mice lacking the thiazide sensitive NaCl cotransporter (NCC). Our data indicate that NCC knockout mice (KO) have significantly higher FGF23, PTH and aldosterone concentrations than corresponding wild type (WT) mice. However, 1,25(OH)2D3, fractional phosphate excretion and renal brush border expression of the sodium/phosphate co-transporter 2a were not different between the two genotypes. In addition, renal expression of FGF23 receptor FGFR1 and the co-receptor Klotho were unaltered in NCC KO mice. FGF23 transcript was increased in the bone of NCC KO mice compared to WT mice, but treatment of primary murine osteoblasts with the NCC inhibitor hydrochlorothiazide did not elicit an increase of FGF23 transcription. In contrast, the mineralocorticoid receptor blocker eplerenone reversed excess FGF23 levels in KO mice but not in WT mice, indicating that FGF23 upregulation in NCC KO mice is primarily aldosterone-mediated. Together, our data reveal that lack of renal NCC causes an aldosterone-mediated upregulation of circulating FGF23.

Exploring the intricate regulatory network controlling the thiazide-sensitive NaCl cotransporter (NCC).

The thiazide-sensitive NaCl cotransporter (NCC) plays key roles in renal electrolyte transport and blood pressure maintenance. Regulation of this cotransporter has received increased attention recently, prompted by the discovery that mutations in the with-no-lysine (WNK) kinases are the molecular explanation for pseudohypoaldosteronism type II (PHAII). Studies suggest that WNK4 regulates NCC via two distinct pathways, depending on its state of activation. Furthermore, an intact STE20-related proline-alanine-rich kinase (SPAK)/oxidative stress response 1 kinase (OSR1) pathway was found to be necessary for a WNK4 PHAII mutation to increase NCC phosphorylation and blood pressure in mice. The mouse protein 25α is a novel regulator of the SPAK/OSR1 kinase family, which greatly increases their activity. The phosphorylation status of NCC and the WNK is regulated by the serum- and glucocorticoid-inducible kinase 1, suggesting novel mechanisms whereby aldosterone modulates NCC activity. Dephosphorylation of NCC by protein phosphatase 4 strongly influences the activity of the cotransporter, confirming an important role for NCC phosphorylation. Finally, γ-adducin increases NCC activity. This stimulatory effect is dependent on the phosphorylation status of the cotransporter. γ-Adducin only binds the dephosphorylated cotransporter, suggesting that phosphorylation of NCC causes the dissociation of γ-adducin. Since γ-adducin is not a kinase, it is tempting to speculate that the protein exerts its function by acting as a scaffold between the dephosphorylated cotransporter and the regulatory kinase. As more molecular regulators of NCC are identified, the system-controlling NCC activity is becoming increasingly complex. This intricacy confers an ability to integrate a variety of stimuli, thereby regulating NCC transport activity and ultimately blood pressure.

A single residue in transmembrane domain 11 defines the different affinity for thiazides between the mammalian and flounder NaCl transporters.

Little is known about the residues that control the binding and affinity of thiazide-type diuretics for their protein target, the renal Na(+)-Cl(-) cotransporter (NCC). Previous studies from our group have shown that affinity for thiazides is higher in rat (rNCC) than in flounder (flNCC) and that the transmembrane region (TM) 8-12 contains the residues that produce this difference. Here, an alignment analysis of TM 8-12 revealed that there are only six nonconservative variations between flNCC and mammalian NCC. Two are located in TM9, three in TM11, and one in TM12. We used site-directed mutagenesis to generate rNCC containing flNCC residues, and thiazide affinity was assessed using Xenopus laevis oocytes. Wild-type or mutant NCC activity was measured using (22)Na(+) uptake in the presence of increasing concentrations of metolazone. Mutations in TM11 conferred rNCC an flNCC-like affinity, which was caused mostly by the substitution of a single residue, S575C. Supporting this observation, the substitution C576S conferred to flNCC an rNCC-like affinity. Interestingly, the S575C mutation also rendered rNCC more active. Substitution of S575 in rNCC for other residues, such as alanine, aspartate, and lysine, did not alter metolazone affinity, suggesting that reduced affinity in flNCC is due specifically to the presence of a cysteine. We conclude that the difference in metolazone affinity between rat and flounder NCC is caused mainly by a single residue and that this position in the protein is important for determining its functional properties.

The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex.

The pathogenesis of essential hypertension remains unknown, but thiazide diuretics are frequently recommended as first-line treatment. Recently, familial hyperkalemic hypertension (FHHt) was shown to result from activation of the thiazide-sensitive Na-Cl cotransporter (NCC) by mutations in WNK4, although the mechanism for this effect remains unknown. WNK kinases are unique members of the human kinome, intimately involved in maintaining electrolyte balance across cell membranes and epithelia. Previous work showed that WNK1, WNK4, and a kidney-specific isoform of WNK1 interact to regulate NCC activity, suggesting that WNK kinases form a signaling complex. Here, we report that WNK3, another member of the WNK kinase family expressed by distal tubule cells, interacts with WNK4 and WNK1 to regulate NCC in both human kidney cells and Xenopus oocytes, further supporting the WNK signaling complex hypothesis. We demonstrate that physiological regulation of NCC in oocytes results from antagonism between WNK3 and WNK4 and that FHHt-causing WNK4 mutations exert a dominant-negative effect on wild-type (WT) WNK4 to mimic a state of WNK3 excess. The results provide a mechanistic explanation for the divergent effects of WT and FHHt-mutant WNK4 on NCC activity, and for the dominant nature of FHHt in humans and genetically modified mice.