Renal Thiazide-sensitive Na-Cl Cotransporter Research Articles

Regulation of the Renal NaCl Cotransporter and Its Role in Potassium Homeostasis.

Daily dietary potassium (K+) intake may be as large as the extracellular K+ pool. To avoid acute hyperkalemia, rapid removal of K+ from the extracellular space is essential. This is achieved by translocating K+ into cells and increasing urinary K+ excretion. Emerging data now indicate that the renal thiazide-sensitive NaCl cotransporter (NCC) is critically involved in this homeostatic kaliuretic response. This suggests that the early distal convoluted tubule (DCT) is a K+ sensor that can modify sodium (Na+) delivery to downstream segments to promote or limit K+ secretion. K+ sensing is mediated by the basolateral K+ channels Kir4.1/5.1, a capacity that the DCT likely shares with other nephron segments. Thus, next to K+-induced aldosterone secretion, K+ sensing by renal epithelial cells represents a second feedback mechanism to control K+ balance. NCC's role in K+ homeostasis has both physiological and pathophysiological implications. During hypovolemia, NCC activation by the renin-angiotensin system stimulates Na+ reabsorption while preventing K+ secretion. Conversely, NCC inactivation by high dietary K+ intake maximizes kaliuresis and limits Na+ retention, despite high aldosterone levels. NCC activation by a low-K+ diet contributes to salt-sensitive hypertension. K+-induced natriuresis through NCC offers a novel explanation for the antihypertensive effects of a high-K+ diet. A possible role for K+ in chronic kidney disease is also emerging, as epidemiological data reveal associations between higher urinary K+ excretion and improved renal outcomes. This comprehensive review will embed these novel insights on NCC regulation into existing concepts of K+ homeostasis in health and disease.

Regulation of the renal NaCl cotransporter by the WNK/SPAK pathway: lessons learned from genetically altered animals.

The renal thiazide-sensitive NaCl cotransporter (NCC) is the major salt transport pathway in the distal convoluted tubule of the mammalian nephron. NCC activity is critical for modulation of arterial blood pressure and serum potassium levels. Reduced activity of NCC in genetic diseases results in arterial hypotension and hypokalemia, while increased activity results in genetic diseases featuring hypertension and hyperkalemia. Several hormones and physiological conditions modulate NCC activity through a final intracellular complex pathway involving kinases and ubiquitin ligases. A substantial amount of work has been conducted to understand this pathway in the last 15 yr, but advances over the last 3 yr have helped to begin to understand how these regulatory proteins interact with each other and modulate the activity of this important cotransporter. In this review, we present the current model of NCC regulation by the Cullin 3 protein/Kelch-like 3 protein/with no lysine kinase/STE20-serine-proline alanine-rich kinase (CUL3/KELCH3-WNK-SPAK) pathway. We present a review of all genetically altered mice that have been used to translate most of the proposals made from in vitro experiments into in vivo observations that have helped to elucidate the model at the physiological level. Many questions have been resolved, but some others will require further models to be constructed. In addition, unexpected observations in mice have raised new questions and identified regulatory pathways that were previously unknown.

Mini-review: regulation of the renal NaCl cotransporter by hormones.

The renal thiazide-sensitive NaCl cotransporter, NCC, is the major pathway for salt reabsorption in the distal convoluted tubule. The activity of this cotransporter is critical for regulation of several physiological variables such as blood pressure, serum potassium, acid base metabolism, and urinary calcium excretion. Therefore, it is not surprising that numerous hormone-signaling pathways regulate NCC activity to maintain homeostasis. In this review, we will provide an overview of the most recent evidence on NCC modulation by aldosterone, angiotensin II, vasopressin, glucocorticoids, insulin, norepinephrine, estradiol, progesterone, prolactin, and parathyroid hormone.

Circadian exosomal expression of renal thiazide-sensitive NaCl cotransporter (NCC) and prostasin in healthy individuals.

A circadian timing system is involved in the maintenance of fluid and electrolyte balance and blood pressure control. Aldosterone and vasopressin modulate ion transporters and channels crucial in sodium (Na) and water reabsorption such as the epithelium Na channel and the renal thiazide-sensitive NaCl cotransporter (NCC). We analyzed in urinary exosomes the intraday variations of NCC and prostasin expression and the association with electrolytes and water balance parameters. Blood and urine samples were collected at five time points during the day from five healthy subjects. Blood renin, aldosterone, cortisol, ACTH, and plasmatic and urinary Na, potassium, creatinine, adiuretin (ADH), NCC, and prostasin were evaluated. ACTH and cortisol showed a circadian pattern, similarly to aldosterone, while exosomal NCC and prostasin pattern were similar to urinary ADH, decreased in the morning and subsequently increased in the afternoon and evening. In urinary exosomes, NCC and prostasin had a diurnal pattern parallel to ADH and aquaporin 2, confirming that, in healthy subjects, both prostasin and NCC relate to water balance. These results provide suggestions for a possible chronotherapeutic approach in patients treated with thiazides, diuretic drugs acting as specific inhibitors of NCC-mediated Na reabsorption.

Reply to “Letter to the Editor: ‘How does potassium supplementation lower blood pressure?’”

to the editor: We appreciate Dr. McDonough's and Dr. Nguyen's valuable comments and interest (6a) in our study. They raised several interesting issues regarding our recent paper ([4][1]), which explored the molecular mechanisms responsible for the blood pressure-lowering effect of potassium

Regulation of the renal Na+-Cl−cotransporter by phosphorylation and ubiquitylation

The activity of the renal thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule plays a key role in defining arterial blood pressure levels. Increased or decreased activity of the NCC is associated with arterial hypertension or hypotension, respectively. Thus it is of major interest to understand the activity of NCC using in vivo models. Phosphorylation of certain residues of the amino-terminal domain of NCC has been shown to be associated with its activation. The development of phospho-specific antibodies against these sites provides a powerful tool that is helping to increase our understanding of the molecular physiology of NCC. Additionally, NCC expression in the plasma membrane is modulated by ubiquitylation, which represents another major mechanism for regulating protein activity. This work presents a review of our current knowledge of the regulation of NCC activity by phosphorylation and ubiquitylation.

Enhanced FGF23 Serum Concentrations and Phosphaturia in Gene Targeted Mice Expressing WNK-Resistant Spak

Background: The WNK-dependent STE20/SPS1-related proline/alanine-rich kinase (SPAK) regulates the renal thiazide sensitive NaCl cotransporter (NCC) and the renal furosemide sensitive Na⁺,K⁺,2Cl^- cotransporter (NKCC2) and thus participates in the regulation of renal salt excretion, extracellular fluid volume and blood pressure. Inhibition of NCC leads to anticalciuria. Moreover, NCC is also expressed in osteoblasts where it is implicated in the regulation of bone mineralization. Osteoblasts further influence mineral metabolism by releasing the phosphaturic hormone FGF23. The present study explored, whether SPAK participates in the regulation of calcium-phosphate homeostasis. Methods: FGF23 serum levels and phosphate homeostasis were analyzed in gene targeted mice expressing SPAK resistant to WNK-dependent activation (spak^tg/tg) and in mice expressing wild type SPAK (spak^wt/wt). Results: Serum FGF23 level was significantly higher, urinary phosphate excretion significantly larger and serum phosphate concentration significantly lower in spak^tg/tg mice than in spak^wt/wt mice. Urinary calcium excretion was significantly decreased in spaktg/tg mice. Serum levels of calcitriol and PTH were not significantly different between the genotypes. Bone density was significantly increased in spak^tg/tg mice compared to spak^wt/wt mice. Treatment of spak^wt/wt mice with HCT increased FGF23 serum levels, and led to phosphaturia and hypophosphatemia. Conclusions: SPAK is a strong regulator of FGF23 formation, bone mineralization and renal Ca²⁺ and phosphate excretion.

Sex and Age Result in Differential Regulation of the Renal Thiazide-Sensitive NaCl Cotransporter and the Epithelial Sodium Channel in Angiotensin II-Infused Mice

Background/Aims: We determined the effects of age and sex on the blood pressure (BP) response to angiotensin II (Ang II) infusion and evaluated the potential mechanistic role of the thiazide-sensitive NaCl cotransporter (NCC) and the epithelial sodium channel (ENaC). Methods: Male and female mice (∼3 or 21 months of age) were infused with Ang II or control for 7 days. Results: Males had a greater BP response to Ang II, somewhat enhanced by aging. Mean systolic BPs (at 7 days) were (mm Hg): 161, 143, 172, and 157 in young male, young female, old male, and old female mice, respectively. Immunoblotting changes in the whole kidney that supported this BP profile included a 51 and 52% increase in NCC band density in the old females and old males (as compared to sex-respective controls) with Ang II infusion, while the young males and young females showed an increase of 40 and 0%, respectively. Young males also had a greater reduction in major bands of β- and γ-ENaC, than did young female mice. The natriuretic response to hydrochlorothiazide supported an increase in activity of NCC with Ang II in aged mice only. Conclusions: Increased sensitivity to Ang II in aging and male mice may involve overactivity of NCC.

Cellular and subcellular localization of N‐terminally phosphorylated NCC in normal rat kidney

N‐terminal phosphorylation of the renal thiazide‐sensitive NaCl cotransporter NCC (or TSC) (Slc12a3) is proposed to play a major role in its transport activity and cellular localization, as seen for its Slc12‐family homologues NKCC1 and NKCC2. We generated novel phospho‐specific antibodies against two N‐terminally conserved phosphorylation sites (Thr53, Thr58 and Thr53/Thr58 together) of NCC. Dot blot analysis confirmed the phospho‐specificity of the antibodies. Immunohistochemistry showed distinct staining of the apical plasma membrane domain of distal convoluted tubule (DCT). Confocal microscopy with specific tubule markers confirmed phosphorylated NCC (pNCC) was localized predominantly to DCT. At the transition between DCT and connecting tubule, pNCC was also detected, but in non‐AQP2 containing cells. Using immunogold electron microscopy, pNCC (Thr53 and Thr58) was localized only to apical plasma membrane domains and was not observed within the cell. Immunoblot analysis of rat kidney homogenates showed a protein band of 160 kDa in cortex, the expected size for glycosylated NCC. Acute vasopressin exposure increased pNCC abundance approximately 300%. Studies in Brattleboro rats confirmed that vasopressin regulates NCC phosphorylation in the DCT.

Metabolic, renal, and cardiovascular effects of selective estrogen receptor agonists

The estrogen receptor alpha (ERα)-selective agonist PPT decreases body weight gain (BWG) in ovariectomized (OVX) rats. Since increased body fat is associated with insulin resistance (IR) and hypertension (HT), we tested the hypothesis that PPT improves glucose tolerance (GT) and decreases blood pressure (BP) in OVX rats. Sham operated (SO, n=3) or ovariectomized (OVX) rats were treated with vehicle (DMSO, n=2), estradiol (E2, 2 μg, n=3), or PPT (0.13 mg, n=2) every 4 days and were fed a low fat diet (10%, LF) for 3 weeks then switched to a high fat (60%, HF) diet for 4 weeks. Locomotor activity (LA) and BP were measured by radiotelemetry, and GT was measured in response to an ip injection. There were no effects of diet (D) or treatment (T) on LA. After LF, BWG was 15 ± 3 gm in SO rats, increased (p<0.05) to 70 ± 13 gm in OVX rats, and was reduced (p<0.05) in OVX rats treated with E2 (25 ± 4 gm) or PPT (27 ± 15 gm). There were no effects of T on BWG after HF. There was an effect of T (p<0.01), but not D, on the area under the curve (AUC) for the GT response. The AUC was increased (p<0.01) in OVX rats compared with SO rats, and was normalized by E2, but not PPT. There were effects of T (p<0.001) and D (p=0.03) on BP. After LF, BP was 111.3 ± 0.5 mm Hg in SO rats, and was decreased to 104.8 ± 1.1 mmHg in OVX rats. BP was not different from OVX in E2-treated rats (104.6 ± 0.5 mmHg), but PPT increased (p=0.002) BP to 115.6 ± 1.3 mmHg. After HF, BP was reduced (p=0.03). The abundance of the renal thiazide-sensitive Na-Cl cotransporter (NCC), a transporter that we have found is increased in obese, IR, and HT Zucker rats, was increased (p<0.01) by PPT. These results demonstrate that although the ERα agonist PPT attenuates BWG, PPT also fails to normalize GT and increases renal NCC abundance and BP. The role of ERβ is currently under study. Supported by AG022624.

Regulation of the renal thiazide-sensitive Na-Cl cotransporter, blood pressure, and natriuresis in obese Zucker rats treated with rosiglitazone

Previously, we showed an increase in protein abundance of the renal thiazide-sensitive Na-Cl cotransporter (NCC) in young, prediabetic, obese Zucker rats relative to lean age mates (Bickel CA, Verbalis JF, Knepper MA, and Ecelbarger CA. Am J Physiol Renal Physiol 281: F639-F648, 2001). To test whether this increase correlated with increased thiazide sensitivity (NCC activity) and blood pressure, and could be modified by insulin-sensitizing agents, we treated lean and obese Zucker rats (9 wk old) with either a control diet or this diet supplemented with 3 mg/kg body wt rosiglitazone (RGZ), a peroxisomal proliferator-activated receptor subtype gamma agonist and potent insulin-sensitizing agent, for 12 wk (n = 9/group). The rise in blood pressure, measured continuously by radiotelemetry, was significantly blunted in the RGZ-treated obese rats. Similarly, blood glucose and urinary albumin were markedly decreased in these rats. RGZ-treated rats whether lean or obese excreted a NaCl load faster but excreted less sodium in response to hydrochlorothiazide, applied as a novel in vivo measure of NCC activity. Obese rats had increased renal protein abundance and urinary excretion of NCC; however, this was not significantly reduced by RGZ (densitometry in cortex homogenate - %lean control): 100 +/- 9, 93 +/- 4, 124 +/- 9, and 141 +/- 14 for lean control, lean RGZ, obese control, and obese RGZ, respectively. Subcellular localization, as evaluated by confocal microscopy and immunoblotting following differential centrifugation, of NCC was not different between rat groups. Overall, RGZ reduced blood pressure and thiazide sensitivity; however, the mechanism(s) did not seem to involve a decrease in NCC protein abundance or cellular location. Decreased NCC activity may have contributed to the maintenance of normotension in RGZ-treated obese rats.

The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon

The kidneys “escape” from the Na-retaining effects of aldosterone when circulating levels of aldosterone are inappropriately elevated in the setting of normal or expanded extracellular fluid volume, e.g., in primary aldosteronism. Using a targeted proteomics approach, we screened renal protein extracts with rabbit polyclonal antibodies directed to each of the major Na transporters expressed along the nephron to determine whether escape from aldosterone-mediated Na retention is associated with decreased abundance of one or more of renal Na transporters. The analysis revealed that the renal abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) was profoundly and selectively decreased. None of the other apical solute-coupled Na transporters displayed decreases in abundance, nor were the total abundances of the three ENaC subunits significantly altered. Immunocytochemistry showed a strong decrease in NCC labeling in distal convoluted tubules of aldosterone-escape rats with no change in the cellular distribution of NCC. Ribonuclease protection assays (RPAs) revealed that the decrease in NCC protein abundance was not associated with altered NCC mRNA abundance. Thus, the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule appears to be the chief molecular target for regulatory processes responsible for mineralocorticoid escape, decreasing in abundance via a posttranscriptional mechanism.

The renal thiazide-sensitive Na-Cl cotransporter as mediator of the aldosterone-escape phenomenon.

The kidneys "escape" from the Na-retaining effects of aldosterone when circulating levels of aldosterone are inappropriately elevated in the setting of normal or expanded extracellular fluid volume, e.g., in primary aldosteronism. Using a targeted proteomics approach, we screened renal protein extracts with rabbit polyclonal antibodies directed to each of the major Na transporters expressed along the nephron to determine whether escape from aldosterone-mediated Na retention is associated with decreased abundance of one or more of renal Na transporters. The analysis revealed that the renal abundance of the thiazide-sensitive Na-Cl cotransporter (NCC) was profoundly and selectively decreased. None of the other apical solute-coupled Na transporters displayed decreases in abundance, nor were the total abundances of the three ENaC subunits significantly altered. Immunocytochemistry showed a strong decrease in NCC labeling in distal convoluted tubules of aldosterone-escape rats with no change in the cellular distribution of NCC. Ribonuclease protection assays (RPAs) revealed that the decrease in NCC protein abundance was not associated with altered NCC mRNA abundance. Thus, the thiazide-sensitive Na-Cl cotransporter of the distal convoluted tubule appears to be the chief molecular target for regulatory processes responsible for mineralocorticoid escape, decreasing in abundance via a posttranscriptional mechanism.

Gitelman's syndrome (Bartter's variant) maps to the thiazide-sensitive cotransporter gene locus on chromosome 16q13 in a large kindred.

A defect in distal renal tubular sodium chloride handling is thought to be responsible for the clinical phenotype of Gitelman's syndrome, a variant of Bartter's syndrome. To study the possible involvement of the renal thiazide-sensitive NaCl cotransporter gene in the syndrome, a linkage analysis study in the largest reported kindred with the syndrome was performed. A human homolog of rat thiazide-sensitive cotransporter was cloned and mapped to chromosome 16q13 by fluorescent in situ hybridization. All 17 family members in two generations were genotyped at loci in this region. There were no recombinants observed between the Gitelman's syndrome phenotype and inheritance of D16S408 alleles, yielding a lod score of 3.88 at Q = 0. By contrast, recombinants were observed between Gitelman's syndrome and the flanking markers D16S419 and D16S400, localizing the responsible gene in this family to a 15 centimorgan region on chromosome 16q. These genetic data, together with current understanding of the molecular physiology of the thiazide-sensitive cotransporter, are strong evidence that the latter is defective in this kindred with Gitelman's syndrome.

Gitelman's variant of Barter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na–Cl cotransporter

Maintenance of fluid and electrolyte homeostasis is critical for normal neuromuscular function. Bartter's syndrome is an autosomal recessive disease characterized by diverse abnormalities in electrolyte homeostasis including hypokalaemic metabolic alkalosis; Gitelman's syndrome represents the predominant subset of Bartter's patients having hypomagnesemia and hypocalciuria. We now demonstrate complete linkage of Gitelman's syndrome to the locus encoding the renal thiazide-sensitive Na-Cl cotransporter, and identify a wide variety of non-conservative mutations, consistent with loss of function alleles, in affected subjects. These findings demonstrate the molecular basis of Gitelman's syndrome. We speculate that these mutant alleles lead to reduced sodium chloride reabsorption in the more common heterozygotes, potentially protecting against development of hypertension.