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Thiazide-type and thiazide-like diuretics are structurally distinct first-line antihypertensive drugs that target the sodium-chloride cotransporter (NCC) in the kidney. Thiazide-like diuretics are reported to have better cardioprotective effects than thiazide-type diuretics, but whether this is due to differences in NCC-inhibition mechanisms, if there is any, remains unclear. To understand the molecular mechanisms of NCC inhibition by thiazide-like diuretics, we determine the structures of human NCC (hNCC) bound to two of the most widely used thiazide-like diuretics, chlorthalidone and indapamide, using cryogenic electron microscopy (cryo-EM). Structural analyses reveal shared features and distinctions between NCC-inhibition by thiazide-like and thiazide-type diuretics. Furthermore, structural comparisons allow us to identify polymorphisms in hNCC that have substantial differential effects on the potencies of specific thiazide-like and thiazide-type diuretics. Our work provides important insights into the molecular pharmacology of NCC and a blueprint for developing precision medicine to manage hypertension with thiazide-like and thiazide-type diuretics. Thiazide-like diuretics, which target the sodium-chloride cotransporter (NCC), are first-line antihypertensive medications. Here, the authors determine the structures of human NCC in complex with two widely used thiazide-like diuretics, chlorthalidone and indapamide, and identify NCC polymorphisms that differentially affect drug-specific sensitivity.

High sodium intake is associated with the development of chronic diseases such as obesity. Although its role in obesity remains controversial, there may be a correlation between salt sensitivity and the early onset of chronic diseases in obese children. In all, 2,163 Korean children (1,106 boys and 1,057 girls) aged 8-9 years were recruited from seven elementary schools in Seoul. To evaluate whether obesity risk was modulated by the salt sensitivity, 11 SNPs related to salt sensitive genes (SSG) became the target of sodium intakes in obese children. BP, HOMA-IR, LDLc, TG, and the girls' sodium intake significantly increased, but HDLc significantly decreased with increase in BMI. Regardless of sex, the obesity risk was 5.27-fold (CI; 1.320-27.560) higher in the Q2 to Q5 of sodium intake adjusted by energy (4044.9-5058.9 mg/day) than in the lowest Q1 level (2287.6 mg/day) in obese children. BP was sensitively dependent on insulin resistance and lipid accumulation in all subjects; however, sodium intake may be an independent risk factor of obesity without increasing BP in girls. GRK4 A486V mutant homozygote was highly distributed in the obese group, but other SNPs had no impact. The obesity risk increased 7.06, 16.8, and 46.09-fold more in boys with GRK4 A486V, ACE, and SLC12A3 mutants as sodium intake increased. Among girls, the obesity risk increased in GRK4 A486V heterozygote and CYP11β-2 mutant homozygote although sodium intake was relatively lower, implying that ACE, SLC12A, CYP11β-2, and GRK4 A486V polymorphisms showed gender-based differences with regard to interaction between sodium intake and obesity. A high sodium intake markedly increased the obesity risk in variants of GRK4 A486V regardless of sex. The obesity risk increased with GRK4 A486V, ACE, and SLC12A3 variants in boys, whereas it increased with GRK4 A486V and CYP11B2 variants in girls as sodium intake increased. Obese children with the specific gene variants are recommended to reduce their sodium intake.

Gitelman syndrome (GS) is an autosomal recessive renal tubulopathy characterized by hypokalemic metabolic alkalosis with hypocalciuria and hypomagnesemia. GS clinical symptoms range from mild weakness to muscular cramps, paralysis or even sudden death as a result of cardiac arrhythmia. GS is caused by loss-of-function mutations in the solute carrier family 12 member 3 (SLC12A3) gene, but molecular mechanisms underlying such a wide range of symptoms are poorly understood. Here we report cryptic exon activation in SLC12A3 intron 12 in a clinically asymptomatic GS, resulting from an intronic mutation c.1669+297 T>G that created a new acceptor splice site. The cryptic exon was sandwiched between the L3 transposon upstream and a mammalian interspersed repeat downstream, possibly contributing to inclusion of the cryptic exon in mature transcripts. The mutation was identified by targeted next-generation sequencing of candidate genes in GS patients with missing pathogenic SLC12A3 alleles. Taken together, this work illustrates the power of next-generation sequencing to identify causal mutations in intronic regions in asymptomatic individuals at risk of developing potentially fatal disease complications, improving clinical management of these cases.

Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium \"DASH-like\" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.

Fibroblast growth factor‐23 (FGF23) is a bone‐derived hormone regulating renal phosphate reabsorption and vitamin D synthesis in renal proximal tubules. Here, we show that FGF23 directly regulates the membrane abundance of the Na + :Cl − co‐transporter NCC in distal renal tubules by a signaling mechanism involving the FGF receptor/αKlotho complex, extracellular signal‐regulated kinase 1/2 (ERK1/2), serum/glucocorticoid‐regulated kinase 1 (SGK1), and with‐no lysine kinase‐4 (WNK4). Renal sodium (Na + ) reabsorption and distal tubular membrane expression of NCC are reduced in mouse models of Fgf23 and α Klotho deficiency. Conversely, gain of FGF23 function by injection of wild‐type mice with recombinant FGF23 or by elevated circulating levels of endogenous Fgf23 in Hyp mice increases distal tubular Na + uptake and membrane abundance of NCC, leading to volume expansion, hypertension, and heart hypertrophy in a αKlotho and dietary Na + ‐dependent fashion. The NCC inhibitor chlorothiazide abrogates FGF23‐induced volume expansion and heart hypertrophy. Our findings suggest that FGF23 is a key regulator of renal Na + reabsorption and plasma volume, and may explain the association of FGF23 with cardiovascular risk in chronic kidney disease patients. Synopsis FGF23 serum levels are elevated in chronic kidney disease patients. FGF23 is here shown to be a regulator of the sodium‐chloride channel NCC in distal renal tubules, thus affecting renal sodium retention, plasma expansion, hypertension, and heart hypertrophy. FGF23 regulates membrane abundance and activity of the renal sodium‐chloride channel NCC through the ERK1/2‐SGK1‐WNK4 signaling pathway. FGF23 is a sodium‐conserving hormone. Elevated circulating FGF23 leads to volume expansion, hypertension, and cardiac hypertrophy in a Klotho‐dependent fashion. The NCC inhibitor chlorothiazide blunts FGF23‐induced hypertension. A low sodium diet aggravates the hypertensive action of FGF23 through crosstalk with aldosterone signaling at the level of SGK1. Graphical Abstract FGF23 serum levels are elevated in chronic kidney disease patients. FGF23 is here shown to be a regulator of the sodium‐chloride channel NCC in distal renal tubules, thus affecting renal sodium retention, plasma expansion, hypertension, and heart hypertrophy.

The Na + -Cl − cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Here we determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension, but it remains unknown whether/how phosphorylation transforms the NCC structure to accelerate ion translocation. We show that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation. The Na + -Cl − cotransporter (NCC) drives salt reabsorption in the kidney. Here the authors determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they occlude the NCC ion translocation pathway.

Recent studies suggest a role for T lymphocytes in hypertension. However, whether T cells contribute to renal sodium retention and salt-sensitive hypertension is unknown. Here we demonstrate that T cells infiltrate into the kidney of salt-sensitive hypertensive animals. In particular, CD8 + T cells directly contact the distal convoluted tubule (DCT) in the kidneys of DOCA-salt mice and CD8 + T cell-injected mice, leading to up-regulation of the Na-Cl co-transporter NCC, p-NCC and the development of salt-sensitive hypertension. Co-culture with CD8 + T cells upregulates NCC in mouse DCT cells via ROS-induced activation of Src kinase, up-regulation of the K + channel Kir4.1, and stimulation of the Cl − channel ClC-K. The last event increases chloride efflux, leading to compensatory chloride influx via NCC activation at the cost of increasing sodium retention. Collectively, these findings provide a mechanism for adaptive immunity involvement in the kidney defect in sodium handling and the pathogenesis of salt-sensitive hypertension. T cells contribute to development of high blood pressure but their role in salt-sensitive hypertension is less clear. Liu et al . show that CD8 + T cells upregulate and activate Na-Cl co-transporter NCC in distal convoluted tubules via direct cell-cell contact and ROS-Src activation, leading to Na + retention and salt-sensitive hypertension.

Interleukin 18 function in atherosclerosis is mediated by the interleukin 18 receptor and the Na-Cl co-transporter. Interleukin-18 (IL18) participates in atherogenesis through several putative mechanisms 1 , 2 . Interruption of IL18 action reduces atherosclerosis in mice 3 , 4 . Here, we show that absence of the IL18 receptor (IL18r) does not affect atherosclerosis in apolipoprotein E–deficient ( Apoe −/− ) mice, nor does it affect IL18 cell surface binding to or signaling in endothelial cells. As identified initially by co-immunoprecipitation with IL18, we found that IL18 interacts with the Na-Cl co-transporter (NCC; also known as SLC12A3), a 12-transmembrane-domain ion transporter protein preferentially expressed in the kidney 5 . NCC is expressed in atherosclerotic lesions, where it colocalizes with IL18r. In Apoe −/− mice, combined deficiency of IL18r and NCC, but not single deficiency of either protein, protects mice from atherosclerosis. Peritoneal macrophages from Apoe −/− mice or from Apoe −/− mice lacking IL18r or NCC show IL18 binding and induction of cell signaling and cytokine and chemokine expression, but macrophages from Apoe −/− mice with combined deficiency of IL18r and NCC have a blunted response. An interaction between NCC and IL18r on macrophages was detected by co-immunoprecipitation. IL18 binds to the cell surface of NCC-transfected COS-7 cells, which do not express IL18r, and induces cell signaling and cytokine expression. This study identifies NCC as an IL18-binding protein that collaborates with IL18r in cell signaling, inflammatory molecule expression, and experimental atherogenesis.

Chronic kidney disease (CKD) is a common clinical disease with an increasing incidence, affecting 10 to 15% of the world’s population. Hypertension is the most common and modifiable risk factor for preventing adverse cardiovascular outcomes in patients with CKD. A survey from developed countries shows that 47% of hypertensive patients over the age of 20 have uncontrolled blood pressure (BP), and the control rate is even lower in developing countries. CKD is both a common cause of uncontrolled hypertension and a risk factor for altered sequelae. In particular, studies have demonstrated that abnormal blood-pressure patterns in CKD patients, such as non-dipping-blood-pressure patterns, are associated with a significantly increased risk of cardiovascular (CV) disease. The distal convoluted tubule (DCT) is a region of the kidney, and although only 5–10% of the sodium (Na+) filtered by the glomerulus is reabsorbed by DCT, most studies agree that Na-Cl cotransporter (NCC) in human, rabbit, mouse, and rat kidneys is the most important route of sodium reabsorption across the DCT for maintaining the homeostasis of sodium. The regulation of NCC involves a large and complex network structure, including certain physiological factors, kinases, scaffold proteins, transporter phosphorylation, and other aspects. This regulation network includes various levels. Naturally, cross-talk between the components of this system must occur in order to relay the important signals to the transporter to play its role. Knowledge of the mechanisms regulating NCC activation is critical for understanding and treating hypertension and CKD. Previous studies from our laboratory have investigated the mechanisms through which NCC is activated in several different models. In the following sections, we review the literature on the mechanisms of NCC in relation to hypertension in CKD.

Background Gitelman syndrome (GS) is an inherited renal tubular disorder characterized by hypokalemic alkalosis and hypomagnesemia, due to biallelic pathogenic variants in the solute carrier family 12 member 3 ( SLC12A3 ) gene encoding a sodium-chloride (Na-Cl) cotransporter (NCC). This work aimed at identifying SLC12A3 variants in the GS pedigree and reveal the effect of the mutations on protein structure and function. Methods Whole-exome sequencing (WES) and Sanger sequencing were performed in the pedigree. Configuration prediction of two mutant NCC proteins were achieved using SWISS-MODEL. The SLC12A3 missense mutants were generated by site-specific mutagenesis, and the protein expression, location and Na + uptake activity were assessed by using the HEK293T cell line. Results Genetic analysis identified novel compound heterozygous SLC12A3 variants (c.718G > A/p.E240K and c.2675T > C/p.L892P) in the patient with typical GS phenotype. Both of her parents, elder brother and her son carried the heterozygous p.L892P variant, but only the elder brother exhibited mild hypokalemia. Bioinformatics tools predicted that both mutations were highly species conserved and pathogenic. The prediction of mutant protein indicated that p.E240K and p.L892P altered protein’s secondary and three-dimensional (3D) structure and stability. Functional experiments revealed decreased protein expression and Na + uptake activity caused by these two variants, especially the p.L892P variant. Conclusion Our study presents the genetic and functional evidence for the novel compound heterozygous loss-of-function variants in SLC12A3 that may synergistically cuase GS, and expands the mutation spectrum of SLC12A3 variants in patients with GS.