Regulation of the renal Na+-Cl−cotransporter by phosphorylation and ubiquitylation (original) (raw)
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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 ...
Regulatory control of the Na–Cl co-transporter NCC and its therapeutic potential for hypertension
Acta Pharmaceutica Sinica B, 2020
Hypertension is the largest risk factor for cardiovascular disease, the leading cause of mortality worldwide. As blood pressure regulation is influenced by multiple physiological systems, hypertension cannot be attributed to a single identifiable etiology. Three decades of research into Mendelian forms of hypertension implicate alterations in the renal tubular sodium handling, particularly the distal convoluted tubule (DCT)-native, thiazide-sensitive Na-Cl cotransporter (NCC). Altered function of the NCC has shown to have profound effects on blood
Scientific reports, 2017
The renal sodium chloride cotransporter, NCC, in the distal convoluted tubule is important for maintaining body Na and K homeostasis. Endogenous NCC is highly ubiquitylated, but the role of individual ubiquitylation sites is not established. Here, we assessed the role of 10 ubiquitylation sites for NCC function. Transient transfections of HEK293 cells with human wildtype (WT) NCC or various K to R mutants identified greater membrane abundance for K706R, K828R and K909R mutants. Relative to WT-NCC, stable tetracycline inducible MDCKI cell lines expressing K706R, K828R and K909R mutants had significantly higher total and phosphorylated NCC levels at the apical plasma membrane under basal conditions. Low chloride stimulation increased membrane abundance of all mutants to similar or greater levels than WT-NCC. Under basal conditions K828R and K909R mutants had less ubiquitylated NCC in the plasma membrane, and all mutants displayed reduced NCC ubiquitylation following low chloride stimu...
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2018
The NaCl cotransporter (NCC) is essential for electrolyte homeostasis and control of blood pressure. The human SLC12A3 gene, which encodes NCC, gives rise to 3 isoforms, of which only the shortest isoform [NaCl cotransporter isoform 3 (NCC)] has been studied extensively. All NCC isoforms share key phosphorylation sites at T55 and T60 that are essential mediators of NCC function. Recently, a novel phosphorylation site at S811 was identified in isoforms 1 and 2 [NaCl cotransporter splice variant (NCC)], which are only present in humans and higher primates. The aim of the current study, therefore, is to investigate the role of S811 phosphorylation in the regulation of NCC by a combination of biochemical and fluorescent microscopy analyses. We demonstrate that hypotonic low-chloride buffer increases S811 phosphorylation, whereas phosphorylation-deficient S811A mutant hinders phosphorylation at T55 and T60 in NCC and NCC. NCC S811A impairs NCC activity in a dominant-negative fashion, alt...
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...
Physiological reports, 2014
Na,K-ATPase generates the driving force for sodium reabsorption in the kidney. Na,K-ATPase functional properties are regulated by small proteins belonging to the FXYD family. In kidney FXYD2 is the most abundant: it is an inhibitory subunit expressed in almost every nephron segment. Its absence should increase sodium pump activity and promote Na(+) retention, however, no obvious renal phenotype was detected in mice with global deletion of FXYD2 (Arystarkhova et al. 2013). Here, increased total cortical Na,K-ATPase activity was documented in the Fxyd2(-/-) mouse, without increased α1β1 subunit expression. We tested the hypothesis that adaptations occur in distal convoluted tubule (DCT), a major site of sodium adjustments. Na,K-ATPase immunoreactivity in DCT was unchanged, and there was no DCT hypoplasia. There was a marked activation of thiazide-sensitive sodium chloride cotransporter (NCC; Slc12a3) in DCT, predicted to increase Na(+) reabsorption in this segment. Specifically, NCC t...
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.
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)