The Thiazide-Sensitive NaCl Cotransporter is an Aldosterone-Induced Protein (original) (raw)
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Mini-review: regulation of the renal NaCl cotransporter by hormones
American Journal of Physiology-Renal Physiology, 2015
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.
The Sodium Chloride Cotransporter (NCC) and Epithelial Sodium Channel (ENaC) Associate
The Biochemical journal, 2016
The thiazide-sensitive sodium chloride cotransporter (NCC) and the Epithelial Sodium Channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are co-expressed, the second part of the Distal Convoluted Tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and co-expression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by Blue Native PAGE. Extensive co-immunoprecipitation experiments demonstrated a consistent interaction of NCC with alpha and ga...
A primary culture of distal convoluted tubules expressing functional thiazide-sensitive NaCl transport.Studying the molecular regulation of the thiazide-sensitive Na-Cl cotrans-porter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 33 ·cm 2. The monolayers also established opposing transcellular concentration gradients of Na and K. Radioactive 22 Na flux experiments showed a net apical-to-basolateral thiazide-sensitive Na transport across the monolayers. Both hypotonic low-chloride medium and 1 M angiotensin II increased this 22 Na transport significantly by four times, which could be totally blocked by 100 M hydrochlorothiazide. Angiotensin II-stimulated 22 Na transport was also inhibited by 1 M losartan. Furthermore, NCC present in the DCT monolayers was detected by immunoblot and immunocytochemistry studies. In conclusion, a murine primary DCT culture was established which expresses functional thiazide-sensitive Na-Cl transport. NCC; DCT; COPAS THE NA-CL COTRANSPORTER (NCC) plays a pivotal role in the regulation of salt homeostasis by the kidney (29) and constitutes the rate-limiting step of Na reabsorption across the early part of the distal convoluted tubule (DCT1) (10). NCC down-or upregulation, therefore, modifies renal NaCl reabsorption (3, 31). Diuretics, such as hydrochlorothiazide, inhibit NCC and induce NaCl wasting (31). Diseases are associated with mutations in NCC or the genes it interacts with (9). Loss-of-function mutations in NCC cause Gitelman's syndrome, which is characterized by hypotension, renal salt wasting, hypokalemia, hypocalciuria, and hypomagnesemia (14). On the other hand, missense mutations in the With-no-lysine kinase 4 (WNK4) gene, which normally inhibits the activity of NCC (20), results in the increased activity of NCC and leads to pseudohypoaldo-steronism type II (PHAII), also known as Gordon's syndrome (15, 19). PHAII is the mirror image of Gitelman syndrome and is characterized by hypertension, hyperkalemia, metabolic ac-idosis, and hypercalciuria (15). Elucidating the molecular mechanisms that underlie the regulation of NCC is complicated by the lack of a native cell model exhibiting transcellular thiazide-sensitive Na transport. Few cell models exist, but not one displays true distal convoluted tubule (DCT) characteristics. Heterologous expression systems, such as NCC-transfected HEK293 cells and Xenopus laevis oocytes have been used for 22 Na uptake experiments (24, 26), while isolated mDCT cells [which contain cells from the thick ascending limb (TAL) and are therefore not pure cultures] and mpkDCT cells have been used to study phosphorylated signaling pathways involved in NCC regulation (8, 16). In these cells, the different regulators of NCC, angiotensin II, and hypotonic low-chloride medium, have been shown to phosphorylate and activate NCC (25, 27, 28, 30, 32). However, mpkDCT and mDCT cells lack thiazide-sensitive transcellular Na transport (8, 16). This could be explained by the fact that immortalized cells or secondary cell cultures can lose their phenotype after a few passages. The aim of the present study was, therefore, to establish a primary cell culture of mouse DCT that shows unidirectional thiazide-sensitive Na transport. We combined the use of enhanced green-fluorescent protein parvalbumin (PV) trans-genic mice (eGFP-PV-mice) (21) with the Complex Object Parametric Analyzer and Sorter (COPAS) (7). PV is a Ca 2-binding protein that, in the kidney, is mainly expressed in the DCT. Belge et al. (1) showed that PV colocalizes with NCC. PV is, therefore, a relevant indicator of the presence of NCC in isolated tubular segments. The COPAS is a large-particle-based flow cytometer that can sort and collect cell clusters as a function of size and fluorescent intensity. Recently, Miller et al. (23) showed that it is a fast and viable automated method to isolate tubular segments from the collecting duct. Subsequently , the 22 Na transport regulated by NCC was investigated across the established primary monolayers. To our knowledge, we are the first to measure unidirectional thiazide-sensitive Na transport across native DCT monolayers that can be regulated by physiological maneuvers.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2013
The mechanisms by which aldosterone increases Na + , K + ATPase and sodium channel activity in cortical collecting duct and distal nephron have been extensively studied. Recent investigations demonstrate that aldosterone increases Na-H exchanger-3 (NHE-3) activity, bicarbonate transport, and H + ATPase in proximal tubules. However, the role of aldosterone in regulation of Na + , K + ATPase in proximal tubules is unknown. We hypothesize that aldosterone increases Na + , K + ATPase activity in proximal tubules through activation of the mineralocorticoid receptor (MR). Immunohistochemistry of kidney sections from human, rat, and mouse kidneys revealed that the MR is expressed in the cytosol of tubules staining positively for Lotus tetragonolobus agglutinin and type IIa sodium-phosphate cotransporter (NpT2a), confirming proximal tubule localization. Adrenalectomy in Sprague-Dawley rats decreased expression of MR, ENaC α, Na + , K + ATPase α1, and NHE-1 in all tubules, while supplementation with aldosterone restored expression of above proteins. In human kidney proximal tubule (HKC11) cells, treatment with aldosterone resulted in translocation of MR to the nucleus and phosphorylation of SGK-1. Treatment with aldosterone also increased Na + , K + ATPase-mediated 86 Rb uptake and expression of Na + , K + ATPase α1 subunits in HKC11 cells. The effects of aldosterone on Na + , K + ATPase-mediated 86 Rb uptake were prevented by spironolactone, a competitive inhibitor of aldosterone for the MR, and partially by Mifepristone, a glucocorticoid receptor (GR) inhibitor. These results suggest that aldosterone regulates Na + , K + ATPase in renal proximal tubule cells through an MR-dependent mechanism.
Channels (Austin, Tex.), 2017
Renal sodium reabsorption depends on the activity of the Na(+),K(+)-ATPase α/β heterodimer. Four α (α1-4) and three β (β1-3) subunit isoforms have been described. It is accepted that renal tubule cells express α1/β1 dimers. Aldosterone stimulates Na(+),K(+)-ATPase activity and may modulate α1/β1 expression. However, some studies suggest the presence of β3 in the kidney. We hypothesized that the β3 isoform of the Na(+),K(+)-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β3 is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β3. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.
American journal of physiology. Renal physiology, 2015
The thiazide-sensitive NaCl cotransporter (NCC) is an important pharmacological target in the treatment of hypertension. Human SLC12A3 gene, encoding NCC, gives rise to three isoforms. Only the 3(rd) isoform has been extensively investigated. The aim of the present study was, therefore, to establish the abundance and localization of the almost identical isoforms 1 and 2 (NCC1/2) in the human kidney and to determine their functional properties and regulation in physiological conditions. Immunohistochemical analysis of NCC1/2 in the human kidney revealed that NCC1/2 localizes to the apical plasma membrane of the distal convoluted tubule. Importantly, NCC1/2 mRNA constitutes approximately 44% of all NCC isoforms in the human kidney. Functional analysis performed in the Xenopus laevis oocyte revealed that thiazide-sensitive (22)Na(+) transport of NCC1 was significantly increased in comparison to NCC3. Mimicking a constitutively active phosphorylation site at residue 811 (S811D) in NCC1 ...
Frontiers in Physiology
Chronic glucocorticoid infusion impairs NCC activity and induces a non-dipping profile in mice, suggesting that glucocorticoids are essential for daily blood pressure variations. In this paper, we studied mice lacking the renal tubular glucocorticoid receptor (GR) in adulthood (GR knockouts, Nr3c1 Pax8/LC1 ). Upon standard salt diet, Nr3c1 Pax8/LC1 mice grow normally, but show reduced NCC activity despite normal plasma aldosterone levels. Following diet switch to low sodium, Nr3c1 Pax8/LC1 mice exhibit a transient but significant reduction in the activity of NCC and expression of NHE3 and NKCC2 accompanied by significant increased Spak activity. This is followed by transiently increased urinary sodium excretion and higher plasma aldosterone concentrations. Plasma corticosterone levels and 11βHSD2 mRNA expression and activity in the whole kidney remain unchanged. High salt diet does not affect whole body Na + and/or K + balance and NCC activity is not reduced, but leads to a significant increase in diastolic blood pressure dipping in Nr3c1 Pax8/LC1 mice. When high sodium treatment is followed by 48 h of darkness, NCC abundance is reduced in knockout mice although activity is not different. Our data show that upon Na + restriction renal tubular GR-deficiency transiently affects Na + handling and transport pathways. Overall, upon standard, low Na + and high Na + diet exposure Na + and K + balance is maintained as evidenced by normal plasma and urinary Na + and K + and aldosterone concentrations.
AJP: Renal Physiology, 2012
Studying the molecular regulation of the thiazide-sensitive Na+-Cl− cotransporter (NCC) is important for understanding how the kidney contributes to blood pressure regulation. Until now, a native mammalian cell model to investigate this transporter remained unknown. Our aim here is to establish, for the first time, a primary distal convoluted tubule (DCT) cell culture exhibiting transcellular thiazide-sensitive Na+ transport. Because parvalbumin (PV) is primarily expressed in the DCT, where it colocalizes with NCC, kidneys from mice expressing enhanced green-fluorescent protein (eGFP) under the PV gene promoter (PV-eGFP-mice) were employed. The Complex Object Parametric Analyzer and Sorter (COPAS) was used to sort fluorescent PV-positive tubules from these kidneys, which were then seeded onto permeable supports. After 6 days, DCT cell monolayers developed transepithelial resistance values of 630 ± 33 Ω·cm2. The monolayers also established opposing transcellular concentration gradien...