Osmoregulation requires brain expression of the renal Na-K-2Cl cotransporter NKCC2.
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ABSTRACT:
SUBMITTER: Konopacka A
PROVIDER: S-EPMC4380993 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The furosemide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca(2+)-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24D-GFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2-S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter.
| S-EPMC3975042 | biostudies-literature
Project description:The renal bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) is the major salt transport pathway in the apical membrane of the mammalian thick ascending limb. It is differentially spliced and the three major variants (A, B, and F) differ in their localization and transport characteristics. Most knowledge about its regulation comes from experiments in Xenopus oocytes as NKCC2 proved difficult to functionally express in a mammalian system. Here we report the cloning and functional expression of untagged and unmodified versions of the major splice variants from ferret kidney (fNKCC2A, -B, and -F) in human embryonic kidney (HEK) 293 cells. Many NKCC2 antibodies used in this study detected high molecular weight forms of the transfected proteins, probably NKCC2 dimers, but not the monomers. Interestingly, monomers were strongly detected by phosphospecific antibodies directed against phosphopeptides in the regulatory N terminus. Bumetanide-sensitive (86)Rb uptake was significantly higher in transfected HEK-293 cells and could be stimulated by incubating cells in a medium containing a low chloride concentration prior the uptake measurements. fNKCC2 was less sensitive to the reduction in chloride concentration than NKCC1. Using HEK-293 cells stably expressing fNKCC2A we also show that co-expression of variant NKCC2AF does not have the dominant-negative effect on NKCC2A activity that was seen in Xenopus oocytes, nor is it trafficked to the cell surface. In addition, fNKCC2AF is neither complex glycosylated nor phosphorylated in its N terminus regulatory region like other variants.
| S-EPMC2790964 | biostudies-literature
Project description:The effects of TNF gene deletion on renal Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) expression and activity were determined. Outer medulla from TNF(-/-) mice exhibited a twofold increase in total NKCC2 protein expression compared with wild-type (WT) mice. This increase was not observed in TNF(-/-) mice treated with recombinant human TNF (hTNF) for 7 days. Administration of hTNF had no effect on total NKCC2 expression in WT mice. A fourfold increase in NKCC2A mRNA accumulation was observed in outer medulla from TNF(-/-) compared with WT mice; NKCC2F and NKCC2B mRNA accumulation was similar between genotypes. The increase in NKCC2A mRNA accumulation was attenuated when TNF(-/-) mice were treated with hTNF. Bumetanide-sensitive O(2) consumption, an in vitro correlate of NKCC2 activity, was 2.8 ± 0.2 nmol·min(-1)·mg(-1) in medullary thick ascending limb tubules from WT, representing ∼40% of total O(2) consumption, whereas, in medullary thick ascending limb tubules from TNF(-/-) mice, it was 5.6 ± 0.3 nmol·min(-1)·mg(-1), representing ∼60% of total O(2) consumption. Administration of hTNF to TNF(-/-) mice restored the bumetanide-sensitive component to ∼30% of total O(2) consumption. Ambient urine osmolality was higher in TNF(-/-) compared with WT mice (2,072 ± 104 vs. 1,696 ± 153 mosmol/kgH(2)O, P < 0.05). The diluting ability of the kidney, assessed by measuring urine osmolality before and after 1 h of water loading also was greater in TNF(-/-) compared with WT mice (174 ± 38 and 465 ± 81 mosmol/kgH(2)O, respectively, P < 0.01). Collectively, these findings suggest that TNF plays a role as an endogenous inhibitor of NKCC2 expression and function.
| S-EPMC3129887 | biostudies-literature
Project description:The renal-specific NKCC2 (Na+-K+-2Cl- co-transporter 2) is regulated by changes in phosphorylation state, however, the phosphorylation sites and kinases responsible have not been fully elucidated. In the present study, we demonstrate that the metabolic sensing kinase AMPK (AMP-activated protein kinase) phosphorylates NKCC2 on Ser126 in vitro. Co-precipitation experiments indicated that there is a physical association between AMPK and the N-terminal cytoplasmic domain of NKCC2. Activation of AMPK in the MMDD1 (mouse macula densa-derived 1) cell line resulted in an increase in Ser126 phosphorylation in situ, suggesting that AMPK may phosphorylate NKCC2 in vivo. The functional significance of Ser126 phosphorylation was examined by mutating the serine residue to an alanine residue resulting in a marked reduction in co-transporter activity when exogenously expressed in Xenopus laevis oocytes under isotonic conditions. Under hypertonic conditions no significant change of activity was observed. Therefore the present study identifies a novel phosphorylation site that maintains NKCC2-mediated transport under isotonic or basal conditions. Moreover, the metabolic-sensing kinase, AMPK, is able to phosphorylate this site, potentially linking the cellular energy state with changes in co-transporter activity.
| S-EPMC1925243 | biostudies-other
Project description:Oxidative stress-responsive kinase (OSR) 1 and sterile20-related, proline-, alanine-rich kinase (SPAK) are Ste20p-related protein kinases that bind to the sodium, potassium, two chloride cotransporter, NKCC. Here we present evidence that the protein kinase with no lysine [K] (WNK) 1 regulates OSR1, SPAK, and NKCC activities. OSR1 exists in a complex with WNK1 in cells, is activated by recombinant WNK1 in vitro, and is phosphorylated in a WNK1-dependent manner in cells. Depletion of WNK1 from HeLa cells by using small interfering RNA reduces OSR1 kinase activity. In addition, depletion of either WNK1 or OSR1 reduces NKCC activity, indicating that WNK1 and OSR1 are both required for NKCC function. OSR1 and SPAK are likely links between WNK1 and NKCC in a pathway that contributes to volume regulation and blood pressure homeostasis in mammals.
| S-EPMC1544143 | biostudies-literature
Project description:Acute administration of loop diuretics like furosemide leads to a stimulation of renin secretion, an effect thought to result from inhibition of Na-K-2Cl cotransporter (NKCC2)-mediated salt transport at the luminal surface of the macula densa (MD). However, loop diuretics also inhibit NKCC1, the second isoform of the Na-K-2Cl cotransporter, with similar potency. In the present study, we examined the influence of furosemide on renin secretion in NKCC1-deficient mice to distinguish between effects of the loop diuretic involving NKCC2 and, by implication, the MD pathway, and effects that might occur via inhibition of NKCC1. Baseline plasma renin concentration (PRC) was 1,212 +/- 211 in NKCC1+/+ (n = 13) and 3,851 +/- 579 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice (n = 14; P = 0.00024). Acute administration of furosemide (50 mg/kg i.p.) increased PRC significantly to 9,324 +/- 1,018 ng ANG I.ml(-1).h(-1) in NKCC1+/+ (n = 13; P < 0.0001 compared with basal) and to 14,188 +/- 2,274 ng ANG I.ml(-1).h(-1) in NKCC1-/- mice [n = 14; P = 0.0002 compared with basal; P = 0.034 compared with wild-type (WT) plus furosemide]. Renin mRNA expression was about threefold higher in NKCC1-/- compared with WT mice. There was considerable recruitment of granular cells to upstream regions of afferent arterioles in NKCC1-/- mice. Patch-clamp studies in single juxtaglomerular granular (JG) cells from WT mice showed an approximately 10% increase in membrane capacitance during incubation with furosemide (10(-4) M), indicating a direct effect of the loop diuretic on renin secretion. No effect of furosemide on membrane capacitance was observed in JG cells from NKCC1-deficient mice. Furosemide (10(-3) M) significantly stimulated renin release from primary cultures of JG cells from WT mice, whereas no response was observed in NKCC1-/- mice. Our data suggest that a functional NKCC1 suppresses basal renin release, at least in part, through a direct effect on JG cells.
| S-EPMC3153878 | biostudies-literature
Project description:Euryhaline fishes, such as the red drum (Sciaenops ocellatus), must quickly transition between hyperosmotic and hypoosmotic physiological strategies. When freshwater individuals transition to seawater they are exposed to increased diffusive water loss and ion gain. To maintain osmoregulatory balance these animals must drink and absorb seawater through the intestine, followed by ion excretion at the gills. The ability of fishes to transition between strategies can limit the magnitude of osmotic shock that can be tolerated. Here, we demonstrate that red drum can tolerate direct transfer from freshwater to full-strength seawater with marginal impacts on osmotic balance, as indicated by plasma and muscle ion concentration, as well as muscle water. Seawater transition is concurrent with a significant increase in intestinal fluid volume. Typical patterns of osmoregulatory plasticity were observed in the gill with increased expression of nkcc1 and cftr Expression changes in the anterior intestine were observed after 24 h for nkcc2 with smaller and later responses observed for slc26a3, slc26a6, and nbc Immunofluorescence staining demonstrated similar patterns of NKCC localization in freshwater and seawater intestines; however, reduced basolateral staining of V-type ATPase was observed in seawater. Electrophysiological preparations demonstrated that seawater fish had increased absorptive current in the anterior intestine, which was significantly reduced in the presence of 10 μmol/L bumetanide. Overall, these results suggest that nkcc2 plays a crucial role during hyperosmotic transitions, and may be a more important complement to the well-known bicarbonate secretion pathway than generally considered.
| S-EPMC5358003 | biostudies-literature
Project description:The bumetanide (BMT)-sensitive Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) maintains cell volume homeostasis by increasing intracellular K+ and Cl- content via regulatory volume increase (RVI). Expression levels of NKCC1 positively correlate with the histological grade and severity of gliomas, the most common primary adult brain tumors, and up-regulated NKCC1 activity facilitates glioma cell migration and apoptotic resistance to the chemotherapeutic drug temozolomide (TMZ). However, the cellular mechanisms underlying NKCC1 functional up-regulation in glioma and in response to TMZ administration remain unknown.Expression of NKCC1 and its upstream kinases With-No-K (Lysine) kinase 1 (WNK1) and oxidative stress-responsive kinase-1 (OSR1) in different human glioma cell lines and glioma specimens were detected by western blotting and immunostaining. Live cell imaging and microchemotaxis assay were applied to record glioma cell movements under different treatment conditions. Fluorescence indicators were utilized to measure cell volume, intracellular K+ and Cl- content to reflect the activity of NKCC1 on ion transportation. Small interfering RNA (siRNA)-mediated knockdown of WNK1 or OSR1 was used to explore their roles in regulation of NKCC1 activity in glioma cells. Results of different treatment groups were compared by one-way ANOVA using the Bonferroni post-hoc test in the case of multiple comparisons.We show that compared to human neural stem cells and astrocytes, human glioma cells exhibit robust increases in the activation and phosphorylation of NKCC1 and its two upstream regulatory kinases, WNK1 and OSR1. siRNA-mediated knockdown of WNK1 or OSR1 reduces intracellular K+ and Cl- content and RVI in glioma cells by abolishing NKCC1 regulatory phospho-activation. Unexpectedly, TMZ activates the WNK1/OSR1/NKCC1 signaling pathway and enhances glioma migration. Pharmacological inhibition of NKCC1 with its potent inhibitor BMT or siRNA knockdown of WNK1 or OSR1 significantly decreases glioma cell migration after TMZ treatment.Together, our data show a novel role for the WNK1/OSR1/NKCC1 pathway in basal and TMZ-induced glioma migration, and suggest that glioma treatment with TMZ might be improved by drugs that inhibit elements of the WNK1/OSR1/NKCC1 signaling pathway.
| S-EPMC3936893 | biostudies-other
Project description:Na(+):K(+):2Cl(-) cotransporter 1 (NKCC1) has been implicated in mediating ischemia-, trauma- or ammonia-induced astrocyte swelling/brain edema in mammals. This study aimed to determine the effects of ammonia or terrestrial exposure on ammonia concentrations in the plasma and brain, and the mRNA expression and protein abundance of nkcc/Nkcc in the brain, of the swamp eel Monopterusalbus. Ammonia exposure led to a greater increase in the ammonia concentration in the brain of M. albus than terrestrial exposure. The brain ammonia concentration of M. albus reached 4.5 µmol g(-1) and 2.7 µmol g(-1) after 6 days of exposure to 50 mmol l(-1) NH4Cl and terrestrial conditions, respectively. The full cDNA coding sequence of nkcc1b from M. albus brain comprised 3276 bp and coded for 1092 amino acids with an estimated molecular mass of 119.6 kDa. A molecular characterization indicated that it could be activated through phosphorylation and/or glycosylation by osmotic and/or oxidative stresses. Ammonia exposure for 1 day or 6 days led to significant decreases in the nkcc1b mRNA expression and Nkcc1b protein abundance in the brain of M. albus. In comparison, a significant decrease in nkcc1b mRNA expression was observed in the brain of M. albus only after 6 days of terrestrial exposure, but both 1 day and 6 days of terrestrial exposure resulted in significant decreases in the protein abundance of Nkcc1b. These results are novel because it has been established in mammals that ammonia up-regulates NKCC1 expression in astrocytes and NKCC1 plays an important role in ammonia-induced astrocyte swelling and brain edema. By contrast, our results indicate for the first time that M. albus is able to down-regulate the mRNA and protein expression of nkcc1b/Nkcc1b in the brain when confronted with ammonia toxicity, which could be one of the contributing factors to its extraordinarily high brain ammonia tolerance.
| S-EPMC3777983 | biostudies-literature
Project description:Na+:K+:2Cl- cotransporters (NKCCs) belong to the SLC12A family of cation-coupled Cl- transporters. We investigated whether enamel-producing mouse ameloblasts express NKCCs. Transcripts for Nkcc1 were identified in the mouse dental epithelium by RT-qPCR and NKCC1 protein was immunolocalized in outer enamel epithelium and in the papillary layer but not the ameloblast layer. In incisors of Nkcc1-null mice late maturation ameloblasts were disorganized, shorter and the mineral density of the enamel was reduced by 10% compared to wild-type controls. Protein levels of gap junction protein connexin 43, Na+-dependent bicarbonate cotransporter e1 (NBCe1), and the Cl--dependent bicarbonate exchangers SLC26A3 and SLC26A6 were upregulated in Nkcc1-null enamel organs while the level of NCKX4/SLC24A4, the major K+, Na+ dependent Ca2+ transporter in maturation ameloblasts, was slightly downregulated. Whole-cell voltage clamp studies on rat ameloblast-like HAT-7 cells indicated that bumetanide increased ion-channel activity conducting outward currents. Bumetanide also reduced cell volume of HAT-7 cells. We concluded that non-ameloblast dental epithelium expresses NKCC1 to regulate cell volume in enamel organ and provide ameloblasts with Na+, K+ and Cl- ions required for the transport of mineral- and bicarbonate-ions into enamel. Absence of functional Nkcc1 likely is compensated by other types of ion channels and ion transporters. The increased amount of Cx43 in enamel organ cells in Nkcc1-null mice suggests that these cells display a higher number of gap junctions to increase intercellular communication.
| S-EPMC5702478 | biostudies-literature