NKCC1 Activity Research Articles

P097 SEXUAL DIMORPHISM IN A HYPERTENSIVE MODEL OF FRUCTOSE PLUS HIGH SALT DIET

High salt diet (HSD) and fructose are both consumed in excess and contributes to hypertension (HTN). Fructose is commonly used to sweeten soft drinks, and studies have linked soft drink consumption to salt-sensitive HTN. However, full mechanisms aren’t understood. Previous data indicates that an acute fructose-enriched diet (FED) increases Na/K/2Cl cotransporter (NKCC2) activity, promoting salt-sensitive HTN in rats; however, there is no research on long-term consumption. Here, our goal was to evaluate whether a FED, coupled to HSD, can induce HTN. Given the known sex-dependent differences in salt-sensitive HTN, we also hypothesized differences between male and female mice. Male and female C57BI/6 mice (10 weeks old, n=4-6) were treated with vehicle or fructose (10% in drinking water) plus HSD (4% in food) for 28 days. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography, and cardiac hypertrophy (morphometry). We performed an acute diuresis test using furosemide to inhibit NKCC2 (20 mg/kg bw, ip) at day 26, and then measured natriuresis (metabolic cages) for 2-hrs. All data are presented by mean standard deviation. Fructose+HSD increased SBP in male mice after 28 days (Veh=110.5±4.6 vs. Frucose+HSD=130.2±5.6 mmHg; p<0.01); in contrast, HTN was completely prevented in female mice (Veh=109.8±6.8 vs. Fructose+HSD=114.0±11.0 mmHg; ns). We did not observe changes in cardiac hypertrophy in either sex. We observed a significant decrease in natriuresis (∼20%) after furosemide injection in male mice (Veh=5.9±1.1 vs. Fructose+HSD=4.7±0.8 μEq/2 h/g bw; ns). Surprisingly, we observed an increase in natriuresis following furosemide injection in female mice (Veh=3.8±5.0 vs. Fructose+HSD= 7.7±2.7 μEq/2 h/g bw; p<0.01). We observed HTN in males following a FED combined with HSD, which was not associated with increased NKCC2 activation. In contrast, female mice showed a resistance to HTN, and yet an apparent increase in NKCC2 activity. This probably suggests a dysregulation of other transporters not evaluated in this work. Finally, we demonstrate that long-term FED, with HSD, induces HTN with a sexual dimorphic effect, apparently through differential renal sodium handling.

Aqueous extract of Descuraniae Semen attenuates lipopolysaccharide-induced inflammation by inhibiting ER stress and WNK4-SPAK-NKCC1 pathway.

Sepsis causes systemic inflammatory responses and acute lung injury (ALI). Despite modern treatments, sepsis-related ALI mortality remains high. Aqueous extract of Descuraniae Semen (AEDS) exerts anti-endoplasmic reticulum (ER) stress, antioxidant and anti-inflammatory effects. AEDS alleviates inflammation and oedema in ALI. Sodium-potassium-chloride co-transporter isoform 1 (NKCC1) is essential for regulating alveolar fluid and is important in ALI. The NKCC1 activity is regulated by upstream with-no-lysine kinase-4 (WNK4) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). This study aimed to investigate the effects of AEDS on lipopolysaccharide (LPS)-induced ALI model in A549 cells, considering the regulation of ER stress, WNK4-SPAK-NKCC1 cascades, inflammation and apoptosis. Cell viability was investigated by the CCK-8 assay. The expressions of the proteins were assessed by immunoblotting analysis assays. The levels of pro-inflammatory cytokines were determined by ELISA. The expression of cytoplasmic Ca2+ in A549 cells was determined using Fluo-4 AM. AEDS attenuates LPS-induced inflammation, which is associated with increased pro-inflammatory cytokine expression and activation of the WNK4-SPAK-NKCC1 pathway. AEDS inhibits the WNK4-SPAK-NKCC1 pathway by regulating of Bcl-2, IP3R and intracellular Ca2+. WNK4 expression levels are significantly higher in the WNK4-overexpressed transfected A549 cells and significantly decrease after AEDS treatment. AEDS attenuates LPS-induced inflammation by inhibiting the WNK4-SPAK-NKCC1 cascade. Therefore, AEDS is regarded as a potential therapeutic agent for ALI.

TRPV1-dependent NKCC1 activation in mouse lens involves integrin and the tubulin cytoskeleton.

Previously we showed hyperosmotic solution caused TRPV1-dependent NKCC1 activation in the lens by a mechanism that involved ERK1/2 signaling. In various tissues, integrins and the cytoskeletal network play a role in responses to osmotic stress. Here, we examined the association between integrins and TRPV1-dependent activation of NKCC1 in mouse lens epithelium. Wild-type (WT) lenses exposed to the integrin agonist leukadherin-1 (LA-1) for 10 min displayed a ~33% increase in the bumetanide-sensitive rate of Rb uptake indicating NKCC activation. Paclitaxel, a microtubule stabilizing agent, abolished the Rb uptake response. In primary cultured lens epithelium LA-1 caused a robust ERK1/2 activation response that was almost fully suppressed by paclitaxel. The TRPV1 agonist capsaicin caused a similar ERK1/2 activation response. Consistent with an association between integrins and TRPV1, the TRPV1 antagonist A889425 prevented the Rb uptake response to LA-1 as did the ERK inhibitor U0126. LA-1 did not increase Rb uptake by lenses from TRPV1 knockout mice. In cells exposed to a hyperosmotic stimulus, both the ERK1/2 activation and Rb uptake responses were prevented by paclitaxel. Taken together, the findings suggest TRPV1 activation is associated with integrins and the tubulin cytoskeleton. This aligned with the observation that LA-1 elicited a robust cytoplasmic calcium rise in cells from WT lenses but failed to increase calcium in cells from TRPV1 knockout lenses. The results are consistent with the notion that integrin activation by LA-1, or a hyperosmotic stimulus, causes TRPV1 channel opening and the consequent downstream activation of the ERK1/2 and NKCC1 responses.

Protein Quality Control of NKCC2 in Bartter Syndrome and Blood Pressure Regulation.

Mutations in NKCC2 generate antenatal Bartter syndrome type 1 (type 1 BS), a life-threatening salt-losing nephropathy characterized by arterial hypotension, as well as electrolyte abnormalities. In contrast to the genetic inactivation of NKCC2, inappropriate increased NKCC2 activity has been associated with salt-sensitive hypertension. Given the importance of NKCC2 in salt-sensitive hypertension and the pathophysiology of prenatal BS, studying the molecular regulation of this Na-K-2Cl cotransporter has attracted great interest. Therefore, several studies have addressed various aspects of NKCC2 regulation, such as phosphorylation and post-Golgi trafficking. However, the regulation of this cotransporter at the pre-Golgi level remained unknown for years. Similar to several transmembrane proteins, export from the ER appears to be the rate-limiting step in the cotransporter's maturation and trafficking to the plasma membrane. The most compelling evidence comes from patients with type 5 BS, the most severe form of prenatal BS, in whom NKCC2 is not detectable in the apical membrane of thick ascending limb (TAL) cells due to ER retention and ER-associated degradation (ERAD) mechanisms. In addition, type 1 BS is one of the diseases linked to ERAD pathways. In recent years, several molecular determinants of NKCC2 export from the ER and protein quality control have been identified. The aim of this review is therefore to summarize recent data regarding the protein quality control of NKCC2 and to discuss their potential implications in BS and blood pressure regulation.

Thick Ascending Limbs (TALs)-specific deletion of ALMS1 (Alstrom syndrome 1) increases surface NKCC2 expression and NaCl reabsorption in the TAL

The regulation of NKCC2 mediated NaCl reabsorption in Thick Ascending Libs (TALs) is essential to salt homeostasis and blood pressure regulation. Previously, our lab showed that NKCC2 activity is regulated by membrane traffcking and inhibiting endocytosis enhanced surface NKCC2 expression and NaCl reabsorption. We also found that ALMS1, a protein mutated in Alström syndrome, is expressed in TALs. Whole animal gene deletion of ALMS1 caused hypertension, and inhibits NKCC2 endocytosis, increasing its surface expression and renal NaCl reabsorption. Little is known about additional functions of ALMS1, however, global gene deletion of ALMS1 induces obesity, and that, in turn may affect TAL and NKCC2 function. We hypothesized that ALMS1 is a part of protein complex that binds apical NKCC2 in TALs and promotes its endocytosis. To dissect the specific role of ALMS1 in TALs, without interference of systemic effects, we studied surface NKCC2 expression and TAL mediated Na transport after inducible deletion of ALMS1 in TALs. To do this, we generated a loxP flanked exon 7 ALMS1 transgenic mouse line, that we crossed with TAL specific inducible Umod-ERT2 Cre mice (Umod-ALMS1 KO). 3 weeks after inducing Cre expression with tamoxifen, ALMS1 expression in TAL suspensions decreased by 85% (p<0.01) (n=5). In Umod-ALMS1 KO TALs the surface to total NKCC2 ratio, was increased by 37±15% compared to control floxed ALMS1 (p<0.01) (n=5). Recombinant ALMS1 (GST-Carboxyl terminus-ALMS1), or immunoprecipitation of ALMS1, pulled down NKCC2 from TAL lysates (n=3), suggesting that the mechanism by which ALMS1 regulates surface NKCC2 involves protein-protein interactions. Three weeks after inducing ALMS1 deletion, we measured bumetanide-induced natriuresis (4h) as an index of NKCC2-mediated NaCl absorption. We found that bumetanide induced UNa excretion was enhanced in Umod-ALMS1 KO, compared to control (control: 110±7 vs Umod-ALMS1 KO: 173±26 μmols Na/4h, p<0.01). We conclude that ALMS1, binds NKCC2 to regulate its surface expression. Depletion of ALMS1, specifically in the TAL enhances surface NKCC2 and TAL NaCl reabsorption. Our data indicate that regulation of the ALMS1-NKCC2 interaction in the TAL is important for renal NaCl reabsorption. Decreased ALMS1 function or expression increased TAL NaCl reabsorption and this may contribute to the hypertension and kidney disease that is associated with SNPs in the ALMS1 gene in the general population, or in patients with Alstrom Syndrome. NIH R01, Henry Ford Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Calcineurin mediates the inhibitory effects of TNF on NKCC2 phosphorylation

We previously showed that TNF inhibits NKCC2 activity and phosphorylation along the TAL, however, the mechanism has not yet been defined. As calcineurin (CN) activity in synoviocytes was increased by proinflammatory cytokines such as IL-1 and TNF, we tested the hypothesis that induction of CN activity and expression of CN isoforms is part of a pathway by which TNF inhibits NKCC2 phosphorylation. In this study, we found that CN activity increased by approximately 2-fold in primary cultures of medullary thick ascending limb (mTAL) cells that were maintained at 37oC/5%CO2 then challenged with mouse recombinant TNF (1 nM) for 60 min. Similarly, TNF induced a 3-fold increase in CN activity when mTAL cells were exposed to hypertonic media (400 mOsm/Kg H2O), an effect that was prevented by cyclosporine A (CsA). In contrast, silencing TNF production in mTAL cells using lentivirus (U6-TNF-ex4) significantly reduced CN activity by approximately 56% in cells exposed to 400 mOsm/Kg H2O for 1 h. CN activity also was inhibited in cells pretreated with CsA for 30 min followed by incubation in 400 mOsm/Kg H2O. Our data indicate that NKCC2 phosphorylation was significantly decreased in cultured mTAL cells challenged with TNF (1 nM) for 4 hr while inhibition of CN activity with CsA increased pNKCC2 expression. Analysis of qRT-PCR experiments showed that mTAL cells express the calcineurin A subunit (CNA) isoforms a (Sequence ID: NM_001293622.1) and b (sequence ID: NM_001310426.1). However, only the mRNA abundance of the CNAb isoform increased when mTAL cells were incubated with TNF (1 nM) for 4 hr under NS or HS (400 mOsm/Kg H2O) conditions as TNF had no effect on the a isoform of CNA. In vivo, both TNF and CNAb expression increased in outer medulla (OM) from mice given 1% NaCl in the drinking water (HS) for 7 days while intrarenal silencing of TNF with the U6-TNF-ex4 lentivirus reduced expression of CNAb under NS or HS conditions. Similarly, intrarenal injection of a lentivirus construct that specifically silenced CNAb in the kidney (U6-CNAb-ex6) reduced expression of CNAb in mice ingesting HS for 7 days. Interestingly, co-administration of U6-CNAb-ex6 with U6-TNF-ex4 increased pNKCC2 expression by approximately 63% in the OM from mice compared with silencing of TNF alone. Collectively, these data are the first to demonstrate that TNF increases CN activity as well as specific isoform expression in the kidney. Since NKCC2 has been identified as a target of the CNAb isoform, which is increased by TNF, these data demonstrate that a calcineurin-dependent signaling pathway is part of the mechanism by which TNF inhibits the phosphorylation of NKCC2. NIH grants R01 HL133077 and HL153525. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Renal upregulation of NCC counteracts empagliflozin-mediated NHE3 inhibition in normotensive but not in hypertensive male rat.

Sodium-glucose cotransporter-2 inhibitors (SGLT2i) reduce blood pressure (BP) in patients with hypertension, yet the precise molecular mechanisms remain elusive. SGLT2i inhibits proximal tubule (PT) NHE3-mediated sodium reabsorption in normotensive rodents, yet no hypotensive effect is observed under this scenario. This study examined the effect of empagliflozin (EMPA) on renal tubular sodium transport in normotensive and spontaneously hypertensive rats (SHRs). It also tested the hypothesis that EMPA-mediated PT NHE3 inhibition in normotensive rats is associated with upregulation of distal nephron apical sodium transporters. EMPA administration for 14 days reduced BP in 12-wk-old SHRs but not in age-matched Wistar rats. PT NHE3 activity was inhibited by EMPA treatment in both Wistar and SHRs. In Wistar rats, EMPA increased NCC activity, mRNA expression, protein abundance, and phosphorylation levels, but not in SHRs. SHRs showed higher NKCC2 activity and an abundance of cleaved ENaC α and γ subunits compared with Wistar rats, none of which were affected by EMPA. Another set of male Wistar rats was treated with EMPA, the NCC inhibitor hydrochlorothiazide (HCTZ), and EMPA combined with HCTZ or vehicle for 14 days. In these rats, BP reduction was observed only with combined EMPA and HCTZ treatment, not with either drug alone. These findings suggest that NCC upregulation counteracts EMPA-mediated inhibition of PT NHE3 in male normotensive rats, maintaining their baseline BP. Moreover, the reduction of NHE3 activity without further upregulation of major apical sodium transporters beyond the PT may contribute to the BP-lowering effect of SGLT2i in experimental models and patients with hypertension.NEW & NOTEWORTHY This study suggests that reduced NHE3-mediated sodium reabsorption in the renal proximal tubule may account, at least in part, for the BP-lowering effect of SGLT2 inhibitors in the setting of hypertension. It also demonstrates that chronic treatment with SGLT2 inhibitors upregulates NCC activity, phosphorylation, and expression in the distal tubule of normotensive but not hypertensive rats. SGLT2 inhibitor-mediated upregulation of NCC seems crucial to counteract proximal tubule natriuresis in subjects with normal BP.

The epithelial polarity axis controls the resting membrane potential and Cl- co-transport in breast glandular structures.

The membrane potential (MP) controls cell homeostasis by directing molecule transport and gene expression. How the MP is set upon epithelial differentiation is unknown. Given that tissue architecture also controls homeostasis, we investigated the relationship between basoapical polarity and resting MP in three-dimensional culture of the HMT-3522 breast cancer progression. A microelectrode technique to measure MP and input resistance reveals that the MP is raised by gap junction intercellular communication (GJIC), which directs tight-junction mediated apical polarity, and is decreased by the Na+/K+/2Cl- (NKCC, encoded by SLC12A1 and SLC12A2) co-transporter, active in multicellular structures displaying basal polarity. In the tumor counterpart, the MP is reduced. Cancer cells display diminished GJIC and do not respond to furosemide, implying loss of NKCC activity. Induced differentiation of cancer cells into basally polarized multicellular structures restores widespread GJIC and NKCC responses, but these structures display the lowest MP. The absence of apical polarity, necessary for cancer onset, in the non-neoplastic epithelium is also associated with the lowest MP under active Cl- transport. We propose that the loss of apical polarity in the breast epithelium destabilizes cellular homeostasis in part by lowering the MP.

Abstract 045: High Fat Diet Increases Blood Pressure In Sprague Dawley Rats And Induces Salt Sensitive Hypertension: Role Of NKCC2 In The Thick Ascending Limb (TAL)

The Western Diet is characterized by elevated added sugars, fat, and salt and is known to promote hypertension. Chronic high fat intake induces obesity in rodents but the effects on blood pressure are variable between rat/mouse strains. The early effects of a high fat diet, prior to the onset of obesity, in kidney function, signaling and NaCl reabsorption have been poorly studied. In the kidneys, the thick ascending limb (TAL) absorbs 25% of filtered NaCl through the apical Na/K/2Cl cotransporter NKCC2. NKCC2 is activated by phosphorylation at residues Thr 96,101 , or Ser 126 or by increasing its trafficking to the apical surface. The effects of a high fat diet on NKCC2 activity and blood pressure have been poorly studied in the Sprague Dawley (SD) rat, a widely used rodent model. We hypothesized that a high fat diet (60%kCal saturated fat, HF) activates NKCC2 and induces salt sensitive hypertension. We first tested the effects HF on blood pressure (BP). SD rats (Charles Rivers) were trained for tail cuff SBP and placed on HF or control diet for 4 weeks. After 2 weeks, HF diet increased SBP by 9±4 mmHg and at 4 weeks by 13±4 mmHg (p<0.05). We then added high salt diet (4% Na, HS, n=7) which further increased SBP by 19±5 mmHg in HF rats reaching 151±5 mmHg after 4 weeks. SBP did not change in rats fed HS or HF alone (HS: 121±4, HF: 132±4 mmHg, p<0.05 vs HF+HS). In isolated TALs, 2 weeks of HF did not change total NKCC2 expression or P-Thr 96,101 , but enhanced P-Ser 126 NKCC2 (78±26%, p<0.05) and increased surface/intracellular NKCC2 ratio by 22±8% (p<0.03). After 8 weeks of HF diet, surface to intracellular NKCC2 was higher in HF (+54±9% from HS, p<0.05) and HF+HS rats (+40±18% of HS) compared to those fed HS alone. Body weight gain was similar in groups after 2 weeks, but after 8 weeks, rats on HF or HF+HS gained more weight than HS alone (p<0.05). Blood glucose was not different between groups. Our data suggest that 2 weeks of a HF diet induces early signaling in TALs, prior to obesity, that may contribute to increase BP or induce salt sensitivity in normal rats.

Dysregulation of the WNK4-SPAK/OxSR1 pathway has a mild effect on NKCC2 activity

Background: The furosemide-sensitive Na + -K+ - 2Cl − cotransporter (NKCC2) reabsorbs 20% of filtered Na+ along the thick ascending limb (TAL), while and thiazide-sensitive NaCl cotransporter (NCC) reabsorbs 5-10% along the distal convoluted tubule (DCT). The WNK4-SPAK/OxSR1 pathway mediates NKCC2 phosphorylation at threonines 96 and 101 (pT96/pT101) and NCC phosphorylation at threonine 53 (pT53). This activates both cotransporters. A recent study reported that the commonly used pT96/pT101 pNKCC2 antibody cross-reacts with pT53 NCC in mice on the C57BL/6 background due to a 5 amino acid deletion, calling into question some previous findings regarding WNK4-SPAK/OxSR1 regulation of NKCC2. Although WNK4 is highly expressed along both TAL and DCT, dysregulation of WNK4-SPAK/OxSR1 pathway specifically causes Familial Hyperkalemic Hypertension (FHHt), which is exquisitely sensitive to thiazide diuretics, and thus a disease of NCC dysregulation. Using a new pNKCC2 antibody specific in C57BL/6 mice, we tested the hypothesis that the WNK4-SPAK/OxSR1 pathway more strongly activates NCC than it does NKCC2. Methods: A new pT96 NKCC2 antibody was validated in C57BL/6, 129Sv, and Slc12a3 −/− mice. The abundances of pT96 NKCC2 and pT53 NCC were evaluated in Wnk4 −/− , Oxsr1 −/− , Spak −/− , Oxsr1 −/− / Spak −/− mice, and several FHHt model, Cul3 +/−/Δ9 , Klhl3 −/− , and Klhl3 R528H/R528H mice. NKCC2 activity was confirmed by furosemide response test with thiazide pre-treatment in Klhl3 −/− mice. Both male and female were used and all genetically modified strains were on the C57BL/6 background. Results: The pT96 NKCC2 antibody detected pNKCC2 in C57BL/6 mice but not 129Sv mice, and did not cross-react with phosphorylated NCC. pT53 NCC was almost absent but pT96 NKCC2 was only slightly lower in Wnk4 −/− mice. pT53 NCC was almost absent with Spak deletion ( Spak −/− and Oxsr1 −/− / Spak −/− mice) but pT96 NKCC2 abundance did not differ from controls. Oxsr1 deletion ( Oxsr1 −/− and Oxsr1 −/− / Spak −/− mice) led to a modest lowering of pT96 NKCC2/total NKCC2 ratio but not pT96 NKCC2 abundance. Interestingly, immunofluorescence revealed that WNK4 expression was increased along not only DCT but also cortical TAL in Klhl3 −/− mice, but pT96 NKCC2 abundance was not changed. pT53 NCC abundance, but not pT96 NKCC2 abundance, was higher in two other FHHt mouse models with higher WNK4 ( Cul3 +/−/Δ9 and Klhl3 R528H/R528H mice). Consistent with no difference in pT96 NKCC2 abundance, urine Na + excretion after furosemide injection following thiazide treatment was similar between Klhl3 −/− and control mice. Conclusions: NCC is strongly phosphorylated by the WNK4-SPAK pathway, whereas NKCC2 is only mildly phosphorylated by the WNK4-OxSR1 pathway, suggesting another kinase can phosphorylate NKCC2. In FHHt models with Cul3 or Klhl3 mutations, NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide-sensitivity of FHHt. Y.M. received a postdoctoral award from the Uehara Foundation; J.A.M. is funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK098141; M.C.B. is funded by CONACyT, Mexico, Grant 101720. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CAMP stimulated NKCC2 phosphorylation at Thr-96,101 is in part independent from SPAK: a Role for TNIK

Abnormally enhanced NaCl reabsorption by the thick ascending limb of the loop of Henle (TAL) contributes to the development of salt-sensitivity. NaCl reabsorption by the TAL is mediated by the apical Na/K/2Cl cotransporter NKCC2. NKCC2 activity can be stimulated by phosphorylation at Thr 96,101 by upstream kinases SPAK or OSR1. We found that Thr 96,101 phosphorylation is enhanced in TALs of Dahl Salt Sensitive rats (Dahl SS) on normal or high salt diet. Genetic deletion of SPAK in Dahl SS lowers NKCC2 phosphorylation by 40% and blunts salt-sensitive hypertension but does not completely restore these to baseline. Other kinases may be involved in NKCC2 phosphorylation. Using a targeted proteomics approach, we identified TNIK (Traf2 and NCK interacting kinase) as a kinase that binds and phosphorylates Thr 96,101 in NKCC2 in rats and mice (manuscript under revision). In addition, we and others found that cAMP is a potent stimulus for NKCC2 Thr-96,101 phosphorylation. We hypothesize that TNIK is in part responsible for cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. First, we tested the ability of a novel TNIK inhibitor (NCB-0846) to decrease baseline NKCC2 phosphorylation in Dahl SS rats. Treating TALs from Dahl SS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr 96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.0001, n=3). Next, we measured NKCC2 phosphorylation in rats with genetic deletion of SPAK in a Dahl SS background. In SPAK KO rats, the cAMP analogue db-cAMP (500 μM) increased NKCC2 phosphorylation at Thr 96,101 by 626 ± 46% (p<0.0001, n=3) and the TNIK inhibitor NCB-0846, blunted cAMP-stimulated NKCC2 phosphorylation by 56 ± 4.3% (p=0.0013, n=3). There was no significant decrease in phosphorylation at phospho-Ser 126 NKCC2 (cAMP=46 ± 7-fold vs cAMP+NCB= 31±5-fold, n.s). These data indicate that in the absence of SPAK, TNIK mediates cAMP-stimulated NKCC2 phosphorylation at Thr-96,101. To further support this, we studied TALs from whole animal TNIK knock-out mice (TNIK KO). db-cAMP (500 μM) increased NKCC2 phosphorylation by 1330 ± 268% in wild-type mice (p<0.01) whereas cAMP-stimulated NKCC2 phosphorylation was blunted by 49 ± 15% in TNIK KO TALs (p<0.05, n=3). No significant difference was found in cAMP-stimulated NKCC2-Ser 126 phosphorylation in TNIK KO (WT=23 ± 12-fold vs TNIK KO=15 ± 4-fold, n=3). We conclude that enhanced NKCC2 phosphorylation at baseline in Dahl SS TALs is in part caused by TNIK and that cAMP-stimulated NKCC2 Thr-96,101 phosphorylation in TALs is in part independent from SPAK and involves TNIK. Our data point to potential role for TNIK in renal ion transport, blood pressure regulation and urine concentration. AHA15GRNT25710369 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Insulin-regulated aminopeptidase (IRAP) is required for appropriate dilution of urine after an acute water load

Study Objective: Understand the response to acute water load in mice lacking the insulin-regulated aminopeptidase (IRAP). Hypothesis: IRAP degrades vasopressin in vivo, therefore, mice lacking IRAP would have impaired ability to dilute urine and to excrete water. Methods: Age matched 8–10-week-old IRAP WT and KO male mice were used for all experiments. Immunofluorescence and immunoblotting were performed on kidneys at baseline and after 1-hr acute water load (2ml sterile water/IP). Urine/serum electrolytes and urine osmolality were measured before and after water load. Urine was collected from IRAP WT and KO mice before and after 1-hr administration of OPC 31260 (10mg/kg/IP) and furosemide (2.5mg/ml/IP) for urine osmolality and electrolyte measurements. Results: IRAP is expressed in the glomerulus, thick ascending loop of Henle, distal tubule, connecting and collecting ducts. At baseline, IRAP WT and KO mice have similar systolic blood pressure (122 ± 6.6 vs 128 ± 1.9 mmHg), glomerular filtration rate (251 ± 20 vs 254 ± 16 uL/min), and serum Na + levels (142 ± 1.9 vs 143 ± 2.6 mmol/L), but KO mice have reduced proteinuria (3.3 ± 0.57 vs 4.5 ± 1.6 mg/mg). At baseline, IRAP KO have elevated serum copeptin levels (111 ± 43 vs 53 ± 31 pg/mL) and urine osmolality compared to respective IRAP WT (2506 ± 310 vs 1007 ± 212 mOsm/L). Urine osmolality decreased in IRAP KO after administration of vasopressin type 2 receptor antagonist, OPC 31260 (473 ± 341 vs 2479 ± 215 mOsm/L) and loop diuretic, furosemide (517 ± 96 vs 2750 ± 414 mOsm/L). After an acute water load, IRAP KO have lower serum Na + levels (127 ± 3.8 vs 134 ±3.1 mmol/L) and increased urine osmolality compared to IRAP WT (2741 ± 400 vs 577 ± 274 mOsm/L). Additionally, after an acute water load, IRAP KO mice also have higher protein abundance of the Na + /K + /2Cl - (NKCC2) co-transporter (0.36 ± 0.04 vs 0.20 ± 0.03 AU) and phosphorylation of water channel, AQP2 (0.032 ± 0.005 vs 0.025 ± 0.003 AU). Conclusions: IRAP is required to dilute urine and to increase water excretion in response to an acute water load due to persistent activation of NKCC2 and AQP2. Funding Sources: Harold Amos Medical Faculty Development Award - Robert Wood Johnson Foundation; Vanderbilt Center for Kidney Disease This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MTOR signaling in renal ion transport.

The mammalian target of rapamycin (mTOR) signaling pathway is crucial in maintaining cell growth and metabolism. The mTOR protein kinase constitutes the catalytic subunit of two multimeric protein complexes called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). As such, this pathway is indispensable for many organs, including the kidney. Since its discovery, mTOR has been associated with major renal disorders such as acute kidney injury, chronic kidney disease, and polycystic kidney disease. On top of that, emerging studies using pharmacological interventions and genetic disease models have unveiled mTOR role in renal tubular ion handling. Along the tubule, mTORC1 and mTORC2 subunits are ubiquitously expressed at mRNA level. Nevertheless, at the protein level, current studies suggest that a tubular segment-specific balance between mTORC1 and mTORC2 exists. In the proximal tubule, mTORC1 regulates nutrients transports through various transporters located in this segment. On the other hand, in the thick ascending limb of the loop of Henle, both complexes play a role in regulating NKCC2 expression and activity. Lastly, in the principal cells of the collecting duct, mTORC2 determines Na+ reabsorption and K+ excretion by regulating of SGK1 activation. Altogether, these studies establish the relevance of the mTOR signaling pathway in the pathophysiology of tubular solute transport. Despite extensive studies on the effectors of mTOR, the upstream activators of mTOR signaling remain elusive in most nephron segments. Further understanding of the role of growth factor signaling and nutrient sensing is essential to establish the exact role of mTOR in kidney physiology.

Choroid plexus-targeted NKCC1 overexpression to treat post-hemorrhagic hydrocephalus

Post-hemorrhagic hydrocephalus (PHH) refers to a life-threatening accumulation of cerebrospinal fluid (CSF) that occurs following intraventricular hemorrhage (IVH). An incomplete understanding of this variably progressive condition has hampered the development of new therapies beyond serial neurosurgical interventions. Here, we show a key role for the bidirectional Na-K-Cl cotransporter, NKCC1, in the choroid plexus (ChP) to mitigate PHH. Mimicking IVH with intraventricular blood led to increased CSF [K+] and triggered cytosolic calcium activity in ChP epithelial cells, which was followed by NKCC1 activation. ChP-targeted adeno-associated viral (AAV)-NKCC1 prevented blood-induced ventriculomegaly and led to persistently increased CSF clearance capacity. These data demonstrate that intraventricular blood triggered a trans-choroidal, NKCC1-dependent CSF clearance mechanism. Inactive, phosphodeficient AAV-NKCC1-NT51 failed to mitigate ventriculomegaly. Excessive CSF [K+] fluctuations correlated with permanent shunting outcome in humans following hemorrhagic stroke, suggesting targeted gene therapy as a potential treatment to mitigate intracranial fluid accumulation following hemorrhage.

Ubiquitination of NKCC2 by the cullin-RING E3 ubiquitin ligase family in the thick ascending limb of the loop of Henle.

The Na+/K+/2Cl- cotransporter (NKCC2) in the thick ascending limb of the loop of Henle (TAL) mediates NaCl reabsorption. cGMP, the second messenger of nitric oxide and atrial natriuretic peptide, inhibits NKCC2 activity by stimulating NKCC2 ubiquitination and decreasing surface NKCC2 levels. Among the E3 ubiquitin ligase families, the cullin-RING E3 ubiquitin ligase (CRL) family is the largest. Cullins are molecular scaffold proteins that recruit multiple subunits to form the CRL complex. We hypothesized that a CRL complex mediates the cGMP-dependent increase in NKCC2 ubiquitination in TALs. Cullin-1, cullin-2, cullin-3, cullin-4A, and cullin-5 were expressed at the protein level, whereas the other members of the cullin family were expressed at the mRNA level, in rat TALs. CRL complex activity is regulated by neuronal precursor cell-expressed developmentally downregulated protein 8 (Nedd8) to cullins, a process called neddylation. Inhibition of cullin neddylation blunted the cGMP-dependent increase in ubiquitinated NKCC2 while increasing the expression of cullin-1 by threefold, but this effect was not seen with other cullins. CRL complex activity is also regulated by cullin-associated Nedd8-dissociated 1 (CAND1). CAND1 binds to cullins and promotes the exchange of substrate-recognition proteins to target different proteins for ubiquitination. CAND1 inhibition exacerbated the cGMP-dependent increase in NKCC2 ubiquitination and decreased surface NKCC2 expression. Finally, cGMP increased neddylation of cullins. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by a CRL complex. To the best of our knowledge, this is the first evidence that a CRL complex mediates NKCC2 ubiquitination in native TALs.NEW & NOTEWORTHY The Na+/K+/2Cl- cotransporter (NKCC2) reabsorbs NaCl by the thick ascending limb. Nitric oxide and atrial natriuretic peptide decrease NaCl reabsorption in thick ascending limbs by increasing the second messenger cGMP. The present findings indicate that cGMP increases NKCC2 ubiquitination via a cullin-RING ligase complex and regulates in part surface NKCC2 levels. Identifying the E3 ubiquitin ligases that regulate NKCC2 expression and activity may provide new targets for the development of specific loop diuretics.

Regulation of NKCC2 ubiquitination by Neddylation of Cullin‐Ring Ligases in Thick Ascending Limb

Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan. Wayne State University, Physiology department, Detroit, Michigan.The Na/K/2Cl cotransporter (NKCC2) reabsorbs ~30% of the filtered NaCl by the glomeruli. Nitric Oxide and atrial natriuretic peptide decrease NaCl reabsorption in thick ascending limbs (TALs) by increasing the second messenger cyclic guanosine monophosphate (cGMP). cGMP decreases surface NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. The Cullin‐RING Ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for ~20% of protein ubiquitination in mammals. Since the CRLs requires neddylation for its activation, we hypothesized that neddylation of CRLs is required for the cGMP‐dependent increase on NKCC2 ubiquitination in TALs. To test our hypothesis, we pulled down ubiquitinated proteins and biotinylated surface proteins from Sprague‐Dawley (SD) rats and blotted for NKCC2. First, we look at the expression of members of the cullin family and found expression of cullin‐1, ‐2, ‐3, ‐4 and ‐5 in TALs from SD rats. Since activity of CRLs is dynamically regulated by neddylation, we blocked cullin neddylation using (MLN4924, 1 µM) and studied whether cGMP increases NKCC2 ubiquitination. To measure ubiquitination of NKCC2, TALs suspensions were treated with proteasomal inhibitor (MG132, 20 µM). TALs was divided in 4 aliquots and incubated at 37°C with vehicle or MLN4924 for 10 minutes and treated with vehicle or db‐cGMP 500 µM at 37°C for an extra 50 minutes. We found that blockade of cullin neddylation, inhibits the cGMP‐dependent increase in ubiquitinated NKCC2 (baseline: 100%; db‐cGMP 500 µM: 162.5 ± 10.3%; MLN4924: 75.1 ± 20.2%; MLN4924 + db‐cGMP 500 µM: 96.8 ± 19.6 %, p<0.05). As expected, neddylation of cullins was blunted in MLN4924 treated samples (baseline: 100%; db‐cGMP: 120.3 ± 24.4%; MLN4924: 11.0 ± 3.5%; MLN4924 + db‐cGMP: 11.4 ± 5.8 %, p<0.05). These data indicate that neddylation of the CRLs mediates the cGMP‐dependent increase in NKCC2 neddylation. NKCC2 activity is directly related to surface NKCC2 expression. Therefore, we study whether blockade of cullin neddylation prevents the cGMP‐dependent decrease in surface NKCC2 expression. TALs suspensions were divided in 4 aliquots. TALs were incubated at 37°C with vehicle or MLN4924 for 10 minutes and then treated with vehicle or db‐cGMP 500 µM at 37°C for an extra 20 minutes. We found that blockade of cullin neddylation, inhibits the cGMP‐dependent decrease in surface NKCC2 expression. (baseline: 100%; db‐cGMP: 81.0 ± 3.4%; MLN4924: 96.4 ± 2.2%; MLN4924 + db‐cGMP: 99.8 ± 9.3 %, p<0.04). These data indicate that cullin neddylation is required for the cGMP‐dependent decrease in surface NKCC2 expression. To our knowledge, this is the first evidence pointing to the E3 ubiquitin ligase complex involved in the ubiquitination of NKCC2 in native TALs. Identifying the E3 ubiquitin ligases that regulates NKCC2 activity may provide new targets for the development of specific loops diuretics.

The opposing chloride cotransporters KCC and NKCC control locomotor activity in constant light and during long days

Cation chloride cotransporters (CCCs) regulate intracellular chloride ion concentration ([Cl-]i) within neurons, which can reverse the direction of the neuronal response to the neurotransmitter GABA.1 Na+ K+ Cl- (NKCC) and K+ Cl- (KCC) cotransporters transport Cl- into or out of the cell, respectively. When NKCC activity dominates, the resulting high [Cl-]i can lead to an excitatory and depolarizing response of the neuron upon GABAA receptor opening, while KCC dominance has the opposite effect.1 This inhibitory-to-excitatory GABA switch has been linked to seasonal adaption of circadian clock function to changing day length,2-4 and its dysregulation is associated with neurodevelopmental disorders such as epilepsy.5-8 In Drosophila melanogaster, constant light normally disrupts circadian clock function and leads to arrhythmic behavior.9 Here, we demonstrate a function for CCCs in regulating Drosophila locomotor activity and GABA responses in circadian clock neurons because alteration of CCC expression in circadian clock neurons elicits rhythmic behavior in constant light. We observed the same effects after downregulation of the Wnk and Fray kinases, which modulate CCC activity in a [Cl-]i-dependent manner. Patch-clamp recordings from the large LNv clock neurons show that downregulation of KCC results in a more positive GABA reversal potential, while KCC overexpression has the opposite effect. Finally, KCC and NKCC downregulation reduces or increases morning behavioral activity during long photoperiods, respectively. In summary, our results support a model in which the regulation of [Cl-]i by a KCC/NKCC/Wnk/Fray feedback loop determines the response of clock neurons to GABA, which is important for adjusting behavioral activity to constant light and long-day conditions.

Ion Transport Regulation by TRPV4 and TRPV1 in Lens and Ciliary Epithelium.

Aside from a monolayer of epithelium at the anterior surface, the lens is formed by tightly compressed multilayers of fiber cells, most of which are highly differentiated and have a limited capacity for ion transport. Only the anterior monolayer of epithelial cells has high Na, K-ATPase activity. Because the cells are extensively coupled, the lens resembles a syncytium and sodium-potassium homeostasis of the entire structure is largely dependent on ion transport by the epithelium. Here we describe recent studies that suggest TRPV4 and TRPV1 ion channels activate signaling pathways that play an important role in matching epithelial ion transport activity with needs of the lens cell mass. A TRPV4 feedback loop senses swelling in the fiber mass and increases Na, K-ATPase activity to compensate. TRPV4 channel activation in the epithelium triggers opening of connexin hemichannels, allowing the release of ATP that stimulates purinergic receptors in the epithelium and results in the activation of Src family tyrosine kinases (SFKs) and SFK-dependent increase of Na, K-ATPase activity. A separate TRPV1 feedback loop senses shrinkage in the fiber mass and increases NKCC1 activity to compensate. TRPV1 activation causes calcium-dependent activation of a signaling cascade in the lens epithelium that involves PI3 kinase, ERK, Akt and WNK. TRPV4 and TRPV1 channels are also evident in the ciliary body where Na, K-ATPase is localized on one side of a bilayer in which two different cell types, non-pigmented and pigmented ciliary epithelium, function in a coordinated manner to secrete aqueous humor. TRPV4 and TRPV1 may have a role in maintenance of cell volume homeostasis as ions and water move through the bilayer.

Roles of adenosine monophosphate-activated protein kinase (AMPK) in the kidney.

Roles of adenosine monophosphate-activated protein kinase (AMPK) in the kidney.

Elevated CSF inflammatory markers in patients with idiopathic normal pressure hydrocephalus do not promote NKCC1 hyperactivity in rat choroid plexus

BackgroundIdiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible neurological condition of unresolved etiology characterized by a clinical triad of symptoms; gait disturbances, urinary incontinence, and cognitive deterioration. In the present study, we aimed to elucidate the molecular coupling between inflammatory markers and development of iNPH and determine whether inflammation-induced hyperactivity of the choroidal Na+/K+/2Cl− cotransporter (NKCC1) that is involved in cerebrospinal fluid (CSF) secretion could contribute to the iNPH pathogenesis.MethodsLumbar CSF samples from 20 iNPH patients (10 with clinical improvement upon CSF shunting, 10 without clinical improvement) and 20 elderly control subjects were analyzed with the novel proximity extension assay technique for presence of 92 different inflammatory markers. RNA-sequencing was employed to delineate choroidal abundance of the receptors for the inflammatory markers found elevated in the CSF from iNPH patients. The ability of the elevated inflammatory markers to modulate choroidal NKCC1 activity was determined by addition of combinations of rat version of these in ex vivo experiments on rat choroid plexus.Results11 inflammatory markers were significantly elevated in the CSF from iNPH patients compared to elderly control subjects: CCL28, CCL23, CCL3, OPG, CXCL1, IL-18, IL-8, OSM, 4E-BP1, CXCL6, and Flt3L. One inflammatory marker, CDCP1, was significantly decreased in iNPH patients compared to control subjects. None of the inflammatory markers differed significantly when comparing iNPH patients with and without clinical improvement upon CSF shunting. All receptors for the elevated inflammatory markers were expressed in the rat and human choroid plexus, except CCR4 and CXCR1, which were absent from the rat choroid plexus. None of the elevated inflammatory markers found in the CSF from iNPH patients modulated the choroidal NKCC1 activity in ex vivo experiments on rat choroid plexus.ConclusionThe CSF from iNPH patients contains elevated levels of a subset of inflammatory markers. Although the corresponding inflammatory receptors are, in general, expressed in the choroid plexus of rats and humans, their activation did not modulate the NKCC1-mediated fraction of choroidal CSF secretion ex vivo. The molecular mechanisms underlying ventriculomegaly in iNPH, and the possible connection to inflammation, therefore remains to be elucidated.