-Adducin Stimulates the Thiazide-sensitive NaCl Cotransporter (original) (raw)
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Regulation of the renal Na+-Cl−cotransporter by phosphorylation and ubiquitylation
American Journal of Physiology-Renal Physiology, 2012
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.
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 ...
American Journal of Physiology-Renal Physiology, 2009
The thiazide-sensitive Na+-Cl−cotransporter is the major salt reabsorption pathway in the distal convoluted tubule, which is located just after the macula densa at the beginning of the aldosterone-sensitive nephron. This cotransporter was identified at the molecular level in the early 1990s by the pioneering work of Steven C. Hebert and coworkers, opening the molecular area, not only for the Na+-Cl−cotransporter but also for the family of electroneutral cation-coupled chloride cotransporters that includes the loop diuretic-sensitive Na+-K+-2Cl−cotransporter of the thick ascending limb of Henle's loop. This work honoring the memory of Steve Hebert presents a brief review of our current knowledge about salt and water homeostasis generated as a consequence of cloning the cotransporter, with particular emphasis on the molecular biology, physiological properties, human disease due to decreased or increased activity of the cotransporter, and regulation of the cotransporter by a family...
Physiology and pathophysiology of the renal Na-K-2Cl cotransporter (NKCC2
is located in the apical membrane of the epithelial cells of the thick ascending limb of the loop of Henle (TAL). NKCC2 facilitates 20-25% of the reuptake of the total filtered NaCl load. NKCC2 is therefore one of the transport proteins with the highest overall reabsorptive capacity in the kidney. Consequently, even subtle changes in NKCC2 transport activity considerably alter the renal reabsorptive capacity for NaCl and eventually lead to perturbations of the salt and water homoeostasis. In addition to facilitating the bulk reabsorption of NaCl in the TAL, NKCC2 transport activity in the macula densa cells of the TAL constitutes the initial step of the tubular-vascular communication within the juxtaglomerular apparatus (JGA); this communications allows the TAL to modulate the preglomerular resistance of the afferent arteriole and the renin secretion from the granular cells of the JGA. This review provides an overview of our current knowledge with respect to the general functions of NKCC2, the modulation of its transport activity by different regulatory mechanisms, and new developments in the pathophysiology of NKCC2-dependent renal NaCl transport. differential splicing; macula densa; NKCC2; Slc12a1; thick ascending limb HUMAN KIDNEYS FILTER APPROXIMATELY 1.5 kg of NaCl and 180 liters of water each day. The bulk of the filtered load is reabsorbed along the tubular system and the collecting ducts, which results in the formation of 1.5 liters of urine/day. In total, 20-25% of the filtered NaCl is reabsorbed along the thick ascending limb of the loop of Henle (TAL), whereas virtually no water is reabsorbed in this portion of the nephron due to the lack of paracellular and transcellular water permeability. The major apical entry pathway for NaCl is provided by the Na-K-2Cl cotransporter, NKCC2 (BSC1, bumetanide-sensitive cotrans-porter 1), which accounts for 80% of the total salt reabsorption of the TAL (52, 64). The driving force for NKCC2-dependent salt transport is provided by the activity of the basolateral Na-K-ATPase; basolateral chloride conductivity (clcnkb channels) and apical K recycling via ROMK channels complete the net trans-cellular transport of NaCl in the TAL (53). NKCC2 is encoded by a single gene, but differential splicing of its pre-mRNA gives rise to several splice isoforms, which differ markedly in their transport characteristics and in their localization along the TAL (46, 63). Three different isoforms, NKCC2B, NKCC2A, and NKCC2F, differ in the variable exon 4, which encodes the amino acids of the second transmembrane domain and parts of the adjacent intracellular loop of the cotransporter (96, 114). This specific portion of NKCC2 has been show to be crucial for chloride binding. In addition to these three full-length isoforms, truncated variants of NKCC2 have been reported; these variants differ in the C-terminal portion of the protein and increase the total number of NKCC2 isoforms to at least six (96, 114). In addition, isoforms with tandem repeats of exon 4, such as exon 4A/4F, 4B/4F, and 4B/4A, have been described (20, 43, 67, 161). Because of the high overall salt transport capacity of NKCC2 and its crucial role in the urinary concentrating mechanism , even subtle modulations in NKCC2 transport activity result in considerable changes in renal salt reabsorptive capacity (103). Thus inhibitors of NKCC2, known as loop diuretics, constitute the most potent class of diuretics. The profound impact of NKCC2 inhibition on renal salt reabsorption is further enhanced by the limited transport capacity of the portions of the nephron downstream of the TAL, such as the distal convoluted tubule or the collecting duct. The large amount of salt reabsorption mediated by NKCC2 is most likely responsible for the evolution of a complex regulatory network in the TAL; this network modulates NKCC2 expression, differential splicing of its pre-mRNA, surface trafficking, specific transport activity, and, ultimately, the TAL salt-reabsorptive capacity. The regulatory network that controls NKCC2 expression and activity comprises systemic hormones (such as angiotensin II, catecholamines, and vasopressin) and local paracrine/autocrine factors (such as nitric oxide), all of which modulate the complex intracellular signaling network of TAL epithelial cells (61). The NKCC2-dependent salt retrieval in the TAL substantially contributes to the overall salt-reabsorptive capacity of the kidney; in addition, NKCC2 transport activity initiates tubular-vascular cross talk within the kidney (81, 126). Thus NKCC2-dependent salt transport is the initial step that links the tubular chloride concentration at the macula densa (MD) to the control of the tone of the afferent arteriole and eventually the glomer-ular filtration rate (GFR) of the respective nephron (129). This mechanism is known as tubuloglomerular feedback (TGF)
Journal of Biological Chemistry, 2006
The renal Na ؉ :Cl ؊ cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl ؊ influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl ؊ efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr 53 and Thr 58 . Elimination of these threonines together with serine residue Ser 71 completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.
Revisiting the NaCl cotransporter regulation by With No-lysine Kinases
American journal of physiology. Cell physiology, 2015
The renal thiazide-sensitive Na(+):Cl(-) cotransporter (NCC) is the salt transporter in the distal convoluted tubule. Its activity is fundamental for defining blood pressure levels. Decreased NCC activity is associated with salt remediable arterial hypotension with hypokalemia (Gitelman disease), while increased activity results in salt sensitive arterial hypertension with hyperkalemia (Pseudohypoaldosteronism type II; PHAII). The discovery of four different genes causing PHAII revealed a complex multi protein system that regulates the activity of NCC. Two genes encode for With-No-Lysine (K) kinases WNK1 and WNK4, while two encode for kelch-like 3 (KLHL3) and cullin 3 (CUL3) proteins that form a RING type E3-ubiquiting ligase complex. Extensive research has shown that WNK1 and WNK4 are the targets for the KLHL3-CUL3 complex and that WNKs modulate the activity of NCC by means of intermediary Ste20-type kinases known as SPAK or OSR1. The understanding of the effect of WNKs on NCC is a...
Renal Function in Mice with Targeted Disruption of the A Isoform of the Na-K-2Cl Co-Transporter
Journal of the American Society of Nephrology, 2007
Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A-/- mice were generated by homologous recombination. NKCC2A-/- mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A-/- compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high- and a low-salt diet on plasma renin concentration were similar in NKCC2A+/+ and -/- mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A-/- mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A-/- mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A-/- compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high- and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range.
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.
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.