Kidney kinase network regulates renal ion cotransport (original) (raw)
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Pflügers Archiv : European journal of physiology, 2003
Reabsorption of phosphate in the proximal tubule is mainly mediated by the type IIa Na(+)/P(i) cotransporter (NaPi-IIa) and tightly regulated by a variety of factors including dietary phosphate intake and parathyroid hormone (PTH). PTH signals through both apical and basolateral PTH receptors and induces the rapid internalization and subsequent degradation of NaPi-IIa. At least two signalling cascades can be activated by PTH: the PLC/PKC and the cAMP/PKA pathways. Recent evidence from OK cell culture suggested the involvement of MAPK kinases in the PTH action. Here we used freshly isolated coronal mouse kidney slices and incubated them in a physiological buffer in the absence and presence of PTH with inhibitors and activators of the various signalling cascades to further study the events leading to internalization of NaPi-IIa. No alterations in the pattern of immunostaining for alpha-tubulin, actin and several brush border membrane proteins demonstrated intactness of the slices over...
Pflügers Archiv - European Journal of Physiology
Regulated Na+ transport in the distal nephron is of fundamental importance to fluid and electrolyte homeostasis. Further upstream, Na+ is the principal driver of secondary active transport of numerous organic and inorganic solutes. In the distal nephron, Na+ continues to play a central role in controlling the body levels and concentrations of a more select group of ions, including K+, Ca++, Mg++, Cl−, and HCO3−, as well as water. Also, of paramount importance are transport mechanisms aimed at controlling the total level of Na+ itself in the body, as well as its concentrations in intracellular and extracellular compartments. Over the last several decades, the transporters involved in moving Na+ in the distal nephron, and directly or indirectly coupling its movement to that of other ions have been identified, and their interrelationships brought into focus. Just as importantly, the signaling systems and their components—kinases, ubiquitin ligases, phosphatases, transcription factors, ...
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)
Novel aspects in regulated expression of the renal type IIa Na/Pi-cotransporter
Kidney International, 2004
Novel aspects in regulated expression of the renal type IIa Na/P i -cotransporter. Proximal tubular phosphate (P i ) reabsorption is a key element in overall phosphate homeostasis; physiologic/pathophysiologic alterations are related to the control of brush border membrane expression (regulated endocytosis) of the type IIa sodium (Na)/phosphate(P i )-cotransporter (NaPi-IIa). The carboxy terminus of NaPi-IIa contains sequences important for its apical delivery/expression; the last three amino acids are involved in PSD95/DglA/ZO-1 (PDZ) interactions involving NaPi-IIa, Na/H exchanger-regulatory factor 1 (NHERF1/2), and PDZK1/2 (apical scaffold). Regulated endocytosis of NaPi-IIa [e.g., parathyroid hormone (PTH)induced] is reduced in megalin-deficient mice; internalization occurs via clathrin-coated structures, early endosomes, and finally leads to lysosomal degradation. NaPi-IIa contains, in the third intracellular loop, a sequence motif required for internalization. Different hormonal [e.g., PTH, atrial natriuretic peptide (ANP), also nitric oxide (NO)] and nonhormonal factors activate a variety of intracellular signaling cascades [protein kinase A (PK-A), protein kinase C (PK-C), protein kinase G (PK-G), extracellular receptor kinase (ERK)-1/2] leading (by unknown mechanisms) to NaPi-IIa internalization. Different phosphatonins [e.g., fibroblast growth factor (FGF)-23, frizzled related protein (FRP)-4, matrix extracellularphosphoglycoprotein (MEPE)], associated with different pathophysiologic states of renal P i -handling, seem also to control apical expression of NaPi-IIa. Internalization of NaPi-IIa first requires its removal from the apical scaffold. This scaffold can also be considered as a regulatory scaffold containing also protein kinase A (PK-A)-anchoring proteins (AKAPs, ezrin) and the apical PTH receptor. The role of the different components of the regulatory scaffold in regulated endocytosis of NaPi-IIa is at present unknown.
Phosphorylation Regulates NCC Stability and Transporter Activity In Vivo
Journal of the American Society of Nephrology, 2013
A T60M mutation in the thiazide-sensitive sodium chloride cotransporter (NCC) is common in patients with Gitelman's syndrome (GS). This mutation prevents Ste20-related proline and alanine-rich kinase (SPAK)/ oxidative stress responsive kinase-1 (OSR1)-mediated phosphorylation of NCC and alters NCC transporter activity in vitro. Here, we examined the physiologic effects of NCC phosphorylation in vivo using a novel Ncc T58M (human T60M) knock-in mouse model. Ncc T58M/T58M mice exhibited typical features of GS with a blunted response to thiazide diuretics. Despite expressing normal levels of Ncc mRNA, these mice had lower levels of total Ncc and p-Ncc protein that did not change with a low-salt diet that increased p-Spak. In contrast to wild-type Ncc, which localized to the apical membrane of distal convoluted tubule cells, T58M Ncc localized primarily to the cytosolic region and caused an increase in late distal convoluted tubule volume. In MDCK cells, exogenous expression of phosphorylation-defective NCC mutants reduced total protein expression levels and membrane stability. Furthermore, our analysis found diminished total urine NCC excretion in a cohort of GS patients with homozygous NCC T60M mutations. When Wnk4 D561A/+ mice, a model of pseudohypoaldosteronism type II expressing an activated Spak/ Osr1-Ncc, were crossed with Ncc T58M/T58M mice, total Ncc and p-Ncc protein levels decreased and the GS phenotype persisted over the hypertensive phenotype. Overall, these data suggest that SPAK-mediated phosphorylation of NCC at T60 regulates NCC stability and function, and defective phosphorylation at this residue corrects the phenotype of pseudohypoaldosteronism type II. represent the fractional excretion of Na + , K + , and Mg 2+ , respectively. a P,0.05 when homozygous versus WT.
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...
Pfl�gers Archiv - European Journal of Physiology, 2004
Sodium-dependent phosphate cotransport in renal proximal tubules (PTs) is heterogeneous with respect to proximal tubular segmentation (S1 vs. S3) and nephron generation (superficial vs. juxtamedullary). In the present study, S1 and S3 segments of superficial and juxtamedullary nephrons were laser-microdissected and mRNA and protein expression of the Na/Pi-cotransporters NaPi-IIa and NaPi-IIc and the PDZ proteins NHERF-1 and PDZK1 determined. Expression of NaPi-IIa mRNA decreased axially in juxtamedullary nephrons. There was no effect of dietary Pi content on NaPi-lla mRNA expression in any proximal tubular segment. The abundance of the NaPi-IIa cotransporter in the brush-border membrane showed interand intranephron heterogeneity and increased in response to a low-Pi diet (5 days), suggesting that up-regulation of NaPi-lla occurs via post-transcriptional mechanisms. In contrast, NaPi-IIc mRNA and protein was up-regulated by the low-Pi diet in all nephron generations analysed. NHERF-1 and PDZK1, at both mRNA and protein levels, were distributed evenly along the PTs and did not change after a low-Pi diet.
The Journal of Membrane Biology, 1985
86Rb uptake into LLC-PK~ cells (an established renal epithelial cell line) was found to be comprised of an active ouabain-sensitive component, a loop diuretic-sensitive component which was passive and strictly dependent upon the presence of extracellular Na + and C1 for activity, and a "leak" component. The diuretic-sensitive component of influx was investigated further in apical membrane vesicles derived from these cells. A large fraction of 86Rb, 22Na and 36C1 flux into these vesicles was sensitive to inhibition by furosemide and dependent upon the presence of the other two co-ions, in keeping with the presence of a loop diuretic-sensitive Na + : K + : CI cotransport system. The kinetic parameters for Na + and K + interaction have been analyzed under initial linear zero trans conditions. The following values were obtained: KmNa + = 0.42 + 0.05 mmol/liter, Vm~x -303 -24 pmol/mg/6 sec; KINK+ = 11.9 • 1.0 mmol/liter, VmaxK+ = 307 • 27 pmol/mg/6 sec. For C1-interaction evidence for two cooperative binding sites with different affinities and different specificities were obtained. Thus, a stoichiometry of 1Na + : 1K § : 2C1 can be calculated. It is concluded that the apical membrane of LLC-PK~ cells contains a Na § : K § : 2C1 cotransport system with properties similar to those described for the thick ascending limb of the loop of Henle.
American Journal of Physiology Renal Physiology, 2012
Studying the molecular regulation of the thiazide-sensitive Na ϩ-Cl Ϫ 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 ϩ 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 Ϯ 33 ⍀•cm 2. The monolayers also established opposing transcellular concentration gradients of Na ϩ and K ϩ. Radioactive 22 Na ϩ flux experiments showed a net apical-to-basolateral thiazidesensitive Na ϩ transport across the monolayers. Both hypotonic lowchloride medium and 1 M angiotensin II increased this 22 Na ϩ transport significantly by four times, which could be totally blocked by 100 M hydrochlorothiazide. Angiotensin II-stimulated 22 Na ϩ 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 ϩ-Cl Ϫ transport. NCC; DCT; COPAS THE NA ϩ-CL Ϫ COTRANSPORTER (NCC) plays a pivotal role in the regulation of salt homeostasis by the kidney (29) and constitutes the rate-limiting step of Na ϩ reabsorption across the early part of the distal convoluted tubule (DCT1) (10). NCC downor 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)