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Calcineurin, protein phosphatase 2B (PP2B) or protein phosphatase 3 (PP3), is a calcium‐dependent serine/threonine protein phosphatase. Calcineurin is widely expressed in the kidney and regulates renal Na+ and K+ transport. In the thick ascending limb, calcineurin plays a role in inhibiting NKCC2 function by promoting the dephosphorylation of the cotransporter and an intracellular sorting receptor, called sorting‐related‐receptor‐with‐A‐type repeats (SORLA), is involved in modulating the effect of calcineurin on NKCC2. Calcineurin also participates in regulating thiazide‐sensitive NaCl‐cotransporter (NCC) in the distal convoluted tubule. The mechanisms by which calcineurin regulates NCC include directly dephosphorylation of NCC, regulating Kelch‐like‐3/CUL3 E3 ubiquitin–ligase complex, which is responsible for WNK (with‐no‐lysin‐kinases) ubiquitination, and inhibiting Kir4.1/Kir5.1, which determines NCC expression/activity. Finally, calcineurin is also involved in regulating ROMK (Kir1.1) channels in the cortical collecting duct and Cyp11 2 expression in adrenal zona glomerulosa. In summary, calcineurin is involved in the regulation of NKCC2, NCC, and inwardly rectifying K+ channels in the kidney, and it also plays a role in modulating aldosterone synthesis in adrenal gland, which regulates epithelial‐Na+‐channel expression/activity. Thus, application of calcineurin inhibitors (CNIs) is expected to abrupt calcineurin‐mediated regulation of transepithelial Na+ and K+ transport in the kidney. Consequently, CNIs cause hypertension, compromise renal K+ excretion, and induce hyperkalemia. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background The use of cyclosporin A (CsA) is hampered by the development of nephrotoxicity including hypertension, which is partially dependent on renal sodium retention. To address this issue, we have investigated in vivo sodium reabsorption in different nephron segments of CsA-treated rats through micropuncture study coupled to expression analyses of sodium transporters. To translate the findings in rats to human, kidney-transplanted patients having CsA treatment were enrolled in the study. Methods Adult male Sprague–Dawley rats were treated with CsA (15 mg/kg/day) for 21 days, followed by micropuncture study and expression analyses of sodium transporters. CsA-treated kidney-transplanted patients with resistant hypertension were challenged with 50 mg furosemide. Results CsA-treated rats developed hypertension associated with reduced glomerular filtration rate. In vivo microperfusion study demonstrated a significant decrease in rate of absolute fluid reabsorption in the proximal tubule but enhanced sodium reabsorption in the thick ascending limb of Henle's loop (TAL). Expression analyses of sodium transporters at the same nephron segments further revealed a reduction in Na+-H+ exchanger isoform 3 (NHE3) in the renal cortex, while TAL-specific, furosemide-sensitive Na+-K+-2Cl− cotransporter (NKCC2) and NHE3 were significantly upregulated in the inner stripe of outer medulla. CsA-treated patients had a larger excretion of urinary NKCC2 protein at basal condition, and higher diuretic response to furosemide, showing increased FeNa+, FeCl− and FeCa2+ compared with both healthy controls and FK506-treated transplanted patients. Conclusion Altogether, these findings suggest that up-regulation of NKCC2 along the TAL facilitates sodium retention and contributes to the development of CsA-induced hypertension. [ABSTRACT FROM AUTHOR]

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.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water. We previously identified transforming growth factor-beta as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-β receptor-1 led to active inhabitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5' AMP-activated kinase phosphorylation. The signals that undermine in vitro differentiation are complex, but susceptible to pharmacologic intervention. This achievement overcomes a major hurdle limiting the development of a bioreactor of cultured cells for renal replacement therapy that encompasses not only endocrine and metabolic functions but also transport and excretion. Impact statement Clinical tissue engineering requires functional fidelity of the cultured cell to its in vivo counterpart, but this has been elusive in renal tissue engineering. Typically, renal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this study, we build on our prior work by using small molecules to modulate pathways affected by substrate elasticity. In doing so, we are able to enhance differentiation of these cells on conventional noncompliant substrates and show transport. These results are fundamentally enabling a new generation of cell-based renal therapies.

The kidney regulates blood pressure through salt/water reabsorption affected by tubular sodium transporters. Expanding our prior research on placental cluster of differentiation 81 (CD81), this study explores the interaction of renal CD81 with sodium transporters in preeclampsia (PE). Effects of renal CD81 with sodium transporters were determined in lipopolysaccharide (LPS)‐induced PE rats and immortalized mouse renal distal convoluted tubule cells. Urinary exosomal CD81, sodium potassium 2 chloride cotransporter (NKCC2), and sodium chloride cotransporter (NCC) were measured in PE patients. LPS‐PE rats had hypertension from gestational days (GD) 6 to 18 and proteinuria from GD9 to GD18. Urinary CD81 in both groups tented to rise during pregnancy. Renal CD81, not sodium transporters, was higher in LPS‐PE than controls on GD14. On GD18, LPS‐PE rats exhibited higher CD81 in kidneys and urine exosomes, higher renal total and phosphorylated renal NKCC2 and NCC with elevated mRNAs, and lower ubiquitinated NCC than controls. CD81 was co‐immunoprecipitated with NKCC2 or NCC in kidney homogenates and co‐immunostained with NKCC2 or NCC in apical membranes of renal tubules. In plasma membrane fractions, LPS‐PE rats had greater amounts of CD81, NKCC2, and NCC than controls with enhanced co‐immunoprecipitations of CD81 with NKCC2 or NCC. In renal distal convoluted tubule cells, silencing CD81 with siRNA inhibited NCC and prevented LPS‐induced NCC elevation. Further, PE patients had higher CD81 in original urines, urine exosomes and higher NKCC2 and NCC in urine exosomes than controls. Thus, the upregulation of renal CD81 on NKCC2 and NCC may contribute to the sustained hypertension observed in LPS‐PE model. Urine CD81 with NKCC2 and NCC may be used as biomarkers for PE. [ABSTRACT FROM AUTHOR]

Background: Previous studies showed that miR-195a-5p was among the most abundant miRNAs expressed in the kidney.Methods: Lentivirus silencing of tumor necrosis factor-α (TNF) was performed in vivo and in vitro. Luciferase reporter assays confirmed that bumetanide-sensitive Na+-K+-2Cl- cotransporter isoform A (NKCC2A) mRNA is targeted and repressed by miR-195a-5p. Radiotelemetry was used to measure mean arterial pressure.Results: TNF upregulates mmu-miR-195a-5p, and -203 and downregulates mmu-miR-30c and -100 in the medullary thick ascending limb of male mice. miR-195a-5p was >3-fold higher in the renal outer medulla of mice given an intrarenal injection of murine recombinant TNF, whereas silencing TNF inhibited miR-195a-5p expression by ≈51%. Transient transfection of a miR-195a-5p mimic into medullary thick ascending limb cells suppressed NKCC2A mRNA by ≈83%, whereas transfection with Anti-miR-195a-5p increased NKCC2A mRNA. Silencing TNF in medullary thick ascending limb cells prevented increases in miR-195 induced by 400 mosmol/kg H2O medium, an effect reversed by transfection with a miR-195a-5p mimic. Expression of phosphorylated NKCC2 increased 1.5-fold in medullary thick ascending limb cells transfected with Anti-miR-195a-5p and a miR-195a-5p mimic prevented the increase, which was induced by silencing TNF in cells exposed to 400 mosmol/kg H2O medium after osmolality was increased by adding NaCl. Intrarenal injection of TNF suppressed NKCC2A mRNA, whereas injection of miR-195a-5p prevented the increase of NKCC2A mRNA abundance and phosphorylated NKCC2 expression when TNF was silenced. Intrarenal injection with miR-195a-5p markedly attenuated MAP after renal silencing of TNF in mice given 1% NaCl.Conclusions: The study identifies miR-195a-5p as a salt-sensitive and TNF-inducible miRNA that attenuates NaCl-mediated increases in blood pressure by inhibiting NKCC2A. [ABSTRACT FROM AUTHOR]

We previously reported the novel finding that β3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of β3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the β3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease. [ABSTRACT FROM AUTHOR]

Calcineurin, protein phosphatase 2B (PP2B) or protein phosphatase 3 (PP3), is a calcium-dependent serine/threonine protein phosphatase. Calcineurin is widely expressed in the kidney and regulates renal Na + and K + transport. In the thick ascending limb, calcineurin plays a role in inhibiting NKCC2 function by promoting the dephosphorylation of the cotransporter and an intracellular sorting receptor, called sorting-related-receptor-with-A-type repeats (SORLA), is involved in modulating the effect of calcineurin on NKCC2. Calcineurin also participates in regulating thiazide-sensitive NaCl-cotransporter (NCC) in the distal convoluted tubule. The mechanisms by which calcineurin regulates NCC include directly dephosphorylation of NCC, regulating Kelch-like-3/CUL3 E3 ubiquitin-ligase complex, which is responsible for WNK (with-no-lysin-kinases) ubiquitination, and inhibiting Kir4.1/Kir5.1, which determines NCC expression/activity. Finally, calcineurin is also involved in regulating ROMK (Kir1.1) channels in the cortical collecting duct and Cyp11 2 expression in adrenal zona glomerulosa. In summary, calcineurin is involved in the regulation of NKCC2, NCC, and inwardly rectifying K + channels in the kidney, and it also plays a role in modulating aldosterone synthesis in adrenal gland, which regulates epithelial-Na + -channel expression/activity. Thus, application of calcineurin inhibitors (CNIs) is expected to abrupt calcineurin-mediated regulation of transepithelial Na + and K + transport in the kidney. Consequently, CNIs cause hypertension, compromise renal K + excretion, and induce hyperkalemia., (© 2024 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

We previously reported the novel finding that β3-AR is functionally expressed in the renal tubule and shares its cellular localization with the vasopressin receptor AVPR2, whose physiological stimulation triggers antidiuresis by increasing the plasma membrane expression of the water channel AQP2 and the NKCC2 symporter in renal cells. We also showed that pharmacologic stimulation of β3-AR is capable of triggering antidiuresis and correcting polyuria, in the knockout mice for the AVPR2 receptor, the animal model of human X-linked nephrogenic diabetes insipidus (XNDI), a rare genetic disease still missing a cure. Here, to demonstrate that the same response can be evoked in humans, we evaluated the effect of treatment with the β3-AR agonist mirabegron on AQP2 and NKCC2 trafficking, by evaluating their urinary excretion in a cohort of patients with overactive bladder syndrome, for the treatment of which the drug is already approved. Compared to baseline, treatment with mirabegron significantly increased AQP2 and NKCC2 excretion for the 12 weeks of treatment. This data is a step forward in corroborating the hypothesis that in patients with XNDI, treatment with mirabegron could bypass the inactivation of AVPR2, trigger antidiuresis and correct the dramatic polyuria which is the main hallmark of this disease.

Aim: The phosphorylation level of the furosemide‐sensitive Na+‐K+‐2Cl− cotransporter (NKCC2) in the thick ascending limb (TAL) is used as a surrogate marker for NKCC2 activation and TAL function. However, in mice, analyses of NKCC2 phosphorylation with antibodies against phosphorylated threonines 96 and 101 (anti‐pT96/pT101) give inconsistent results. We aimed (a) to elucidate these inconsistencies and (b) to develop a phosphoform‐specific antibody that ensures reliable detection of NKCC2 phosphorylation in mice. Methods: Genetic information, molecular biology, biochemical techniques and mouse phenotyping was used to study NKCC2 and kidney function in two commonly used mouse strains (ie 129Sv and in C57BL/6 mice). Moreover, a new phosphoform‐specific mouse NKCC2 antibody was developed and characterized. Results: Amino acids sequence alignment revealed that C57BL/6 mice have a strain‐specific five amino acids deletion (ΔF97‐T101) in NKCC2 that diminishes the detection of NKCC2 phosphorylation with previously developed pT96/pT101 NKCC2 antibodies. Instead, the antibodies cross‐react with the phosphorylated thiazide‐sensitive NaCl cotransporter (NCC), which can obscure interpretation of results. Interestingly, the deletion in NKCC2 does not impact on kidney function and/or expression of renal ion transport proteins as indicated by the analysis of the F2 generation of crossbred 129Sv and C57BL/6 mice. A newly developed pT96 NKCC2 antibody detects pNKCC2 in both mouse strains and shows no cross‐reactivity with phosphorylated NCC. Conclusion: Our work reveals a hitherto unappreciated, but essential, strain difference in the amino acids sequence of mouse NKCC2 that needs to be considered when analysing NKCC2 phosphorylation in mice. The new pNKCC2 antibody circumvents this technical caveat. [ABSTRACT FROM AUTHOR]

Mutations in Na‐K‐2Cl co‐transporter, NKCC2, lead to type I Bartter syndrome (BS1), a life‐threatening kidney disease. Yet, our knowledge of the molecular regulation of NKCC2 mutants remains poor. Here, we aimed to identify the molecular pathogenic mechanisms of one novel and three previously reported missense NKCC2 mutations. Co‐immunolocalization studies revealed that all NKCC2 variants are not functional because they are not expressed at the cell surface due to retention in the endoplasmic reticulum (ER). Cycloheximide chase assays together with treatment by protein degradation and mannose trimming inhibitors demonstrated that the defect in NKCC2 maturation arises from ER retention and associated degradation (ERAD). Small interfering RNA (siRNA) knock‐down experiments revealed that the ER lectin OS9 is involved in the ERAD of NKCC2 mutants. 4‐phenyl butyric acid (4‐PBA) treatment mimicked OS9 knock‐down effect on NKCC2 mutants by stabilizing their immature forms. Importantly, out of the four studied mutants, only one showed an increased protein maturation upon treatment with glycerol. In summary, our study reveals that BS1 is among diseases linked to the ERAD pathway. Moreover, our data open the possibility that maturation of some ER retained NKCC2 variants is correctable by chemical chaperones offering, therefore, promising avenues in elucidating the molecular pathways governing the ERAD of NKCC2 folding mutants. [ABSTRACT FROM AUTHOR]

BACKGROUND Genome-wide association studies have identified single nucleotide polymorphisms (SNPs) near the uromodulin gene (UMOD) affecting uromodulin excretion and blood pressure (BP). Uromodulin is almost exclusively expressed in the thick ascending limb (TAL) of the loop of Henle and its effect on BP appears to be mediated via the TAL sodium transporter, NKCC2. Loop-diuretics block NKCC2 but are not commonly used in hypertension management. Volume overload is one of the primary drivers for uncontrolled hypertension, so targeting loop-diuretics to individuals who are more likely to respond to this drug class, using the UMOD genotype, could be an efficient precision medicine strategy. METHODS The BHF UMOD Trial is a genotype-blinded, multicenter trial comparing BP response to torasemide between individuals possessing the AA genotype of the SNP rs13333226 and those possessing the G allele. 240 participants (≥18 years) with uncontrolled BP, on ≥1 antihypertensive agent for ≥3 months, will receive treatment with Torasemide, 5 mg daily for 16 weeks. Uncontrolled BP is average home systolic BP (SBP) >135 mmHg and/or diastolic BP >85 mmHg. The primary outcome is the change in 24-hour ambulatory SBP area under the curve between baseline and end of treatment. Sample size was calculated to detect a 4 mmHg difference between groups at 90% power. Approval by West of Scotland Research Ethics Committee 5 (16/WS/0160). RESULTS The study should conclude August 2021. CONCLUSIONS If our hypothesis is confirmed, a genotype-based treatment strategy for loop diuretics would help reduce the burden of uncontrolled hypertension. CLINICAL TRIALS REGISTRATION https://clinicaltrials.gov/ct2/show/NCT03354897. [ABSTRACT FROM AUTHOR]

The Na+-K+-Cl- cotransporters play key physiological and pathophysiological roles by regulating the membrane potential of many cell types and the movement of fluid across a variety of epithelial or endothelial structures. As such, they should soon become invaluable targets for the treatment of various disorders including pain, epilepsy, brain edema, and hypertension. This review highlights the nature of these roles, the mechanisms at play, and the unresolved issues in the field. [ABSTRACT FROM AUTHOR]

Mutations in the apically located kidney Na-K-2Cl cotransporter NKCC2 cause type I Bartter syndrome, a life-threatening kidney disorder. We previously showed that transport from the ER represents the limiting phase in NKCC2 journey to the cell surface. Yet very little is known about the ER quality control components specific to NKCC2 and its disease-causing mutants. Here, we report the identification of Golgi alpha1, 2-mannosidase IA (ManIA) as a novel binding partner of the immature form of NKCC2. ManIA interaction with NKCC2 takes place mainly at the cis-Golgi network. ManIA coexpression decreased total NKCC2 protein abundance whereas ManIA knock-down produced the opposite effect. Importantly, ManIA coexpression had a more profound effect on NKCC2 folding mutants. Cycloheximide chase assay showed that in cells overexpressing ManIA, NKCC2 stability and maturation are heavily hampered. Deleting the cytoplasmic region of ManIA attenuated its interaction with NKCC2 and inhibited its effect on the maturation of the cotransporter. ManIA-induced reductions in NKCC2 expression were offset by the proteasome inhibitor MG132. Likewise, kifunensine treatment greatly reduced ManIA effect, strongly suggesting that mannose trimming is involved in the enhanced ERAD of the cotransporter. Moreover, depriving ManIA of its catalytic domain fully abolished its effect on NKCC2. In summary, our data demonstrate the presence of a ManIA-mediated ERAD pathway in renal cells promoting retention and degradation of misfolded NKCC2 proteins. They suggest a model whereby Golgi ManIA contributes to ERAD of NKCC2, by promoting the retention, recycling, and ERAD of misfolded proteins that initially escape protein quality control surveillance within the ER.

Extracellular fluid (ECF) potassium concentration ([K+]) is maintained by adaptations of kidney and skeletal muscle, responses heretofore studied separately. We aimed to determine how these organ systems work in concert to preserve ECF [K+] in male C57BL/6J mice fed a K+-deficient diet (0K) versus 1% K+ diet (1K) for 10 days (n = 5–6/group). During 0K feeding, plasma [K+] fell from 4.5 to 2 mM; hindlimb muscle (gastrocnemius and soleus) lost 28 mM K+ (from 115 ± 2 to 87 ± 2 mM) and gained 27 mM Na+ (from 27 ± 0.4 to 54 ± 2 mM). Doubling of muscle tissue [Na+] was not associated with inflammation, cytokine production or hypertension as reported by others. Muscle transporter adaptations in 0K- versus 1K-fed mice, assessed by immunoblot, included decreased sodium pump α2-β2 subunits, decreased K+-Cl− cotransporter isoform 3, and increased phosphorylated (p) Na+,K+,2Cl− cotransporter isoform 1 (NKCC1p), Ste20/SPS-1-related proline-alanine rich kinase (SPAKp), and oxidative stress-responsive kinase 1 (OSR1p) consistent with intracellular fluid (ICF) K+ loss and Na+ gain. Renal transporters’ adaptations, effecting a 98% reduction in K+ excretion, included two- to threefold increased phosphorylated Na+-Cl− cotransporter (NCCp), SPAKp, and OSR1p abundance, limiting Na+ delivery to epithelial Na+ channels where Na+ reabsorption drives K+ secretion; and renal K sensor Kir 4.1 abundance fell 25%. Mass balance estimations indicate that over 10 days of 0K feeding, mice lose ~48 μmol K+ into the urine and muscle shifts ~47 μmol K+ from ICF to ECF, illustrating the importance of the concerted responses during K+ deficiency. [ABSTRACT FROM AUTHOR]

Objective: Schistosoma mansoni infection is considered a public health problem. Glomerular involvement in schistosomiasis is a well-documented complication, especially in hepatosplenic schistosomiasis (HSS). However, renal tubular function is poorly understood. The aim of this study was to investigate, through urinary exosomes, tubular transporters functionally in HSS patients.Methods: Cross-sectional study of 20 HSS patients who had isolated exosomes from urine samples. Protease inhibitor was added in the urine samples who were immediately frozen at -80 °C for further exosomes isolation. After urine had thawed, urinary exosomes were obtained using extensive vortexing, centrifugation and ultracentrifugation steps of urine. Urinary transporters expression from exosomes was evaluated by western blot, including NHE3, AQP2 and NKCC2. Charge amounts for gel electrophoresis were adjusted by urinary creatinine concentration of each patient to avoid urinary concentration bias. All protein expression of HSS patients was relative to healthy controls.Results: The expression of aquaporin-2 (AQP2) was lower in HSS patients than in controls (46.8 ± 40.7 vs. 100 ± 70.2%, P = 0.03) and the expression of the NKCC2 co-transporter was higher (191.7 ± 248.6 vs. 100 ± 43.6%, P = 0.02).Conclusions: The decrease of AQP2 and the increase of NKCC2 expression in HSS patients seem to be involved with the inability of urinary concentration in these patients. These data show renal tubular abnormalities in HSS patients without manifest clinical disease. [ABSTRACT FROM AUTHOR]

Mutations in the Na-K-2Cl co-transporter NKCC2 lead to type I Bartter syndrome, a life-threatening kidney disease. We previously showed that export from the ER constitutes the limiting step in NKCC2 maturation and cell surface expression. Yet, the molecular mechanisms involved in this process remain obscure. Here, we report the identification of chaperone stress 70 protein (STCH) and the stress-inducible heat shock protein 70 (Hsp70), as two novel binding partners of the ER-resident form of NKCC2. STCH knock-down increased total NKCC2 expression whereas Hsp70 knock-down or its inhibition by YM-01 had the opposite effect. Accordingly, overexpressing of STCH and Hsp70 exerted opposite actions on total protein abundance of NKCC2 and its folding mutants. Cycloheximide chase assay showed that in cells over-expressing STCH, NKCC2 stability and maturation are heavily impaired. In contrast to STCH, Hsp70 co-expression increased NKCC2 maturation. Interestingly, treatment by protein degradation inhibitors revealed that in addition to the proteasome, the ER associated degradation (ERAD) of NKCC2 mediated by STCH, involves also the ER-to-lysosome-associated degradation pathway. In summary, our data are consistent with STCH and Hsp70 having differential and antagonistic effects with regard to NKCC2 biogenesis. These findings may have an impact on our understanding and potential treatment of diseases related to aberrant NKCC2 trafficking and expression.

• MTX produces a diminish on water reabsorption through an increase of AQP2 renal expression. • MTX increases electrolytes reabsorption by upregulation of NKCC2 renal expression. • AQP2 and NKCC2 urinary excretion may be useful biomarkers to diagnose MTX-induced nephrotoxicity. Methotrexate (MTX) is a derivate of folic acid, commonly used as an anchor drug for the treatment and management of malignant diseases and autoimmune disorders. However, nephrotoxicity is an important drawback of MTX therapy. Unfortunately, there are not enough studies reporting the nature of the renal failure induced by MTX. Thus, the aim of this study was to evaluate the time course of renal handling of water and electrolytes in male Wistar rats, after the exposure to a unique dose of MTX (80 mg/kg b.w.). Experiments were carried out at day 2, day 4, day 8 and day 14 after MTX administration. Several parameters of kidney function related to water and electrolytes handling were evaluated. Renal expression and urinary excretion of aquaporin-2 (AQP2) and Na-K-2Cl-cotransporter (NKCC2) were determined by Western blotting. MTX produced alterations on water handling on the second day after treatment, showing a significant increase in solute free water reabsorption which might be mediated by the increased expression of AQP2 in apical membranes. On the other hand, MTX produced alterations on electrolytes handling on the fourth day after treatment, showing a significant decrease of sodium chloride excretion, mediated at least in part, by the increase renal expression of NKCC2. These results provide valuable information to clinical practice in order to be able to find therapeutic targets that diminish adverse effects and health deterioration. Moreover, MTX treatment altered AQP2 and NKCC2 urinary excretion allowing postulating these transporters as potential biomarkers of MTX induced nephrotoxicity. [ABSTRACT FROM AUTHOR]

Key points: Na+–K+–Cl− cotransporter type 2 (NKCC2) is a 27‐exon membrane protein that is expressed in the thick ascending limb (TAL) of Henle where it is involved in reabsorption of the ultrafiltered NaCl load.It comes as three splice variants that are identical to each other except for the residue composition of exon 4 and that differ in their transport characteristics, functional roles and distributions along the TAL.In this report, it is shown that the variants also differ in their trafficking properties and that two residues in exon 4 play a key role in this regard. One of these residues was also shown to sustain carrier internalization.Through these results, a novel function for the alternatively spliced exon of NKCC2 has been identified and a domain that is involved in carrier trafficking has been uncovered for the first time in a cation–Cl− cotransporter family member. Na+–K+–Cl− cotransporter type 2 (NKCC2) is a 12‐transmembrane (TM) domain cell surface glycoprotein that is expressed in the thick ascending limb (TAL) of Henle and stimulated during cell shrinkage. It comes as three splice variants (A, B and F) that are identical to each other except for TM2 and the following connecting segment (CS2). Yet, these variants do not share the same localization, transport characteristics and physiological roles along the TAL. We have recently found that while cell shrinkage could exert its activating effect by increasing NKCC2 expression at the cell surface, the variants also responded differentially to this stimulus. In the current work, a mutagenic approach was exploited to determine whether CS2 could play a role in carrier trafficking and identify the residues potentially involved. We found that when the residue of position 238 in NKCC2A (F) and NKCC2B (Y) was replaced by the corresponding residue in NKCC2F (V), carrier activity increased by over 3‐fold and endocytosis decreased concomitantly. We also found that when the residue of position 230 in NKCC2F (M) was replaced by the one in NKCC2B (T), carrier activity and affinity for ions both increased substantially whereas expression at the membrane decreased. Taken together, these results suggest that CS2 is involved in carrier trafficking and that two of its residues, those of positions 238 and 230, are part of an internalization motif. They also indicate that the divergent residue of position 230 plays the dual role of specifying ion affinity and sustaining carrier internalization. Key points: Na+–K+–Cl− cotransporter type 2 (NKCC2) is a 27‐exon membrane protein that is expressed in the thick ascending limb (TAL) of Henle where it is involved in reabsorption of the ultrafiltered NaCl load.It comes as three splice variants that are identical to each other except for the residue composition of exon 4 and that differ in their transport characteristics, functional roles and distributions along the TAL.In this report, it is shown that the variants also differ in their trafficking properties and that two residues in exon 4 play a key role in this regard. One of these residues was also shown to sustain carrier internalization.Through these results, a novel function for the alternatively spliced exon of NKCC2 has been identified and a domain that is involved in carrier trafficking has been uncovered for the first time in a cation–Cl− cotransporter family member. [ABSTRACT FROM AUTHOR]

Cellular chloride transport has a fundamental role in cell volume regulation and renal salt handling. Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Thus, we identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume. • Protein kinase SYK phosphorylates NKCC2 and KCC3 at an N-terminal tyrosine residue. • Cellular SYK activity modulates the expression of NKCC2 and KCC3 at the cell surface. • SYK activity modulates cell surface expression of NKCC2 and KCC3 in opposite ways. • SYK-modulated cell surface expression of NKCC2 contributes to cell-volume regulation. [ABSTRACT FROM AUTHOR]

Na+-K+-Cl- cotransporter type 2 (NKCC2) is confined to the apical membrane of the thick ascending limb of Henle, where it reabsorbs a substantial fraction of the ultrafiltered NaCl load. It is expressed along this nephron segment as three main splice variants (called NKCC2A, NKCC2B, and NKCC2F) that differ in residue composition along their second transmembrane domain and first intracellular cytosolic connecting segment (CS2). NKCC2 is known to be activated by cell shrinkage and intracellular [Cl-] reduction. Although the with no lysine (WNK) kinases could play a role in this response, the mechanisms involved are ill defined, and the possibility of variant-specific responses has not been tested thus far. In this study, we have used the Xenopus laevis oocyte expression system to gain further insight in these regards. We have found for the first time that cell shrinkage could stimulate NKCC2A- and NKCC2B-mediated ion transport by increasing carrier abundance at the cell surface and that this response was achieved (at least in part) by the enzymatic function of a WNK kinase. Interestingly, we have also found that the activity and cell surface abundance of NKCC2F were less affected by cell shrinkage compared with the other variants and that ion transport by certain variants could be stimulated through WNK kinase expression in the absence of carrier redistribution. Taken together, these results suggest that the WNK kinase-dependent pathway can affect both the trafficking as well as intrinsic activity of NKCC2 and that CS2 plays an important role in carrier regulation. [ABSTRACT FROM AUTHOR]

The aim of this mini review is to provide an overview regarding the role of inwardly rectifying potassium channel 4.1 (Kir4.1)/Kir5.1 in regulating renal K excretion. Deletion of Kir4.1 in the kidney inhibited thiazide-sensitive NaCl cotransporter (NCC) activity in the distal convoluted tubule (DCT) and slightly suppressed Na-K-2Cl cotransporter (NKCC2) function in the thick ascending limb (TAL). Moreover, increased dietary K intake inhibited, whereas decreased dietary K intake stimulated, the basolateral potassium channel (a Kir4.1/Kir5.1 heterotetramer) in the DCT. The alteration of basolateral potassium conductance is essential for the effect of dietary K intake on NCC because deletion of Kir4.1 in the DCT abolished the effect of dietary K intake on NCC. Since potassium intake-mediated regulation of NCC plays a key role in regulating renal K excretion and potassium homeostasis, the deletion of Kir4.1 caused severe hypokalemia and metabolic alkalosis under control conditions and even during increased dietary K intake. Finally, recent studies have suggested that the angiotensin II type 2 receptor (AT2R) and bradykinin-B2 receptor (BK2R) are involved in mediating the effect of high dietary K intake on Kir4.1/Kir5.1 in the DCT. [ABSTRACT FROM AUTHOR]

K+ loading inhibits NKCC2 (Na-K-Cl cotransporter) and NCC (Na-Cl cotransporter) in the early distal tubules, resulting in Na+ delivery to the late distal convoluted tubules (DCTs). In the DCTs, Na+ entry through ENaC (epithelial Na channel) drives K+ secretion through ROMK (renal outer medullary potassium channel). WNK4 (with-no-lysine 4) regulates the NCC/NKCC2 through SAPK (Ste20-related proline-alanine-rich kinase)/OSR1 (oxidative stress responsive). K+ loading increases intracellular Cl−, which binds to the WNK4, thereby inhibiting autophosphorylation and downstream signals. Acute K+ loading-deactivated NCC was not observed in Cl−-insensitive WNK4 mice, indicating that WNK4 was involved in K+ loading-inhibited NCC activity. However, chronic K+ loading deactivated NCC in Cl−-insensitive WNK4 mice, indicating that other mechanisms may be involved. We previously reported that mammalian Ste20-like protein kinase 3 (MST3/STK24) was expressed mainly in the medullary TAL (thick ascending tubule) and at lower levels in the DCTs. MST3−/− mice exhibited higher ENaC activity, causing hypernatremia and hypertension. To investigate MST3 function in maintaining Na+/K+ homeostasis in kidneys, mice were fed diets containing various concentrations of Na+ and K+. The 2% KCl diets induced less MST3 expression in MST3−/− mice than that in wild-type (WT) mice. The MST3−/− mice had higher WNK4, NKCC2-S130 phosphorylation, and ENaC expression, resulting in lower urinary Na+ and K+ excretion than those of WT mice. Lower urinary Na+ excretion was associated with elevated plasma [Na+] and hypertension. These results suggest that MST3 maintains Na+/K+ homeostasis in response to K+ loading by regulation of WNK4 expression and NKCC2 and ENaC activity.

The kidney is essential for systemic calcium homeostasis. Urinary calcium excretion can be viewed as an integrative renal response to endocrine and local stimuli. The extracellular calcium-sensing receptor (CaSR) elicits a number of adaptive reactions to increased plasma Ca2+ levels including the control of parathyroid hormone release and regulation of the renal calcium handling. Calcium reabsorption in the distal nephron of the kidney is functionally coupled to sodium transport. Apart from Ca2+ transport systems, CaSR signaling affects relevant distal Na+-(K+)-2Cl− cotransporters, NKCC2 and NCC. NKCC2 and NCC are activated by a kinase cascade comprising with-no-lysine [K] kinases (WNKs) and two homologous Ste20-related kinases, SPAK and OSR1. Gain-of-function mutations within the WNK-SPAK/OSR1-NKCC2/NCC pathway lead to renal salt retention and hypertension, whereas loss-of-function mutations have been associated with salt-losing tubulopathies such as Bartter or Gitelman syndromes. A Bartter-like syndrome has been also described in patients carrying gain-of-function mutations in the CaSR gene. Recent work suggested that CaSR signals via the WNK-SPAK/OSR1 cascade to modulate salt reabsorption along the distal nephron. The review presented here summarizes the latest progress in understanding of functional interactions between CaSR and WNKs and their potential impact on the renal salt handling and blood pressure.

Countercurrent multiplication (CCM) is widely accepted as the mechanism for the generation of the corticopapillary osmotic gradient in the outer medulla of mammalian kidneys. However, several issues in the literature cause the current explanations of CCM to be inefficient and incomplete. As a result, it is challenging to clearly explain CCM in physiology education. The goal of this article is to share a modified version of CCM with more understandable explanation in the hopes of motivating peer discussion, further improvement, and future research. To reach this goal, the logical processes leading to CCM are first analyzed, which results in a set of formulas that serve as the principles governing CCM. Next, the cessation of CCM is addressed to provide a complete picture of the modified version of CCM. Throughout these two steps, the issues mentioned above are identified and addressed so that how the modified version of CCM eliminates these issues becomes clear. The formulas mentioned above are provided in the Tables S1, S2, and S3 (all Supplemental material is available in the Supplemental Excel File at https://doi.org/10.6084/m9.figshare.23515614) to explain how the interstitial and intrathick ascending limb osmotic concentration (OC) values used in the figures in this article are simulated and how alternative OC values can be generated from Tables S1 and S2 to illustrate CCM. NEW & NOTEWORTHY Countercurrent multiplication is widely accepted as the mechanism for the generation of the corticopapillary osmotic gradient in the outer medulla of mammalian kidneys, but the current explanations of it in textbooks and the literature are inefficient and incomplete, leading to confusion for students. This article shares a modified version of countercurrent multiplication with more understandable explanation as a way of motivating peer discussion, further improvement, and future research.

With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4-/- mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide- sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that wnk4-/- mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4-/- mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4-/- mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo. [ABSTRACT FROM AUTHOR]

Changes in the expression of Na transport proteins were measured in the kidneys of mice with increased dietary K intake for 1 wk. The epithelial Na channel (ENaC) was upregulated, with enhanced expression of full-length and cleaved forms of α-ENaC and cleaved α-ENaC. At the same time, the amount of the NaCl cotransporter NCC and its phosphorylated form decreased by **50% and **80%, respectively. The expression of the phosphorylated form of the Na-K-2Cl cotransporter NKCC2 also decreased, despite an increase in overall protein content. The effect was stronger in males (80%) than in females (40%). This implies that less Na+ is reabsorbed in the thick ascending limb of Henle's loop and distal convoluted tubule along with Cl-, whereas more is reabsorbed in the aldosterone-sensitive distal nephron in exchange for secreted K+. The abundance of the proximal tubule Na/H exchanger NHE3 decreased by ~40%, with similar effects in males and females. Time-course studies indicated that NCC and NHE3 proteins decreased progressively over 7 days on a high-K diet. Expression of mRNA encoding these proteins increased, implying that the decreased protein levels resulted from decreased rates of synthesis or increased rates of degradation. The potential importance of changes in NHE3, NKCC2, and NCC in promoting K+ excretion was assessed with a mathematical model. Simulations indicated that decreased NHE3 produced the largest effect. Regulation of proximal tubule Na+ transport may play a significant role in achieving K homeostasis. [ABSTRACT FROM AUTHOR]

Expression of Tamm-Horsfall protein (THP or uromodulin) is highly restricted to the kidney thick ascending limb (TAL) of loop of Henle. Despite the unique location and recent association of THP gene mutations with hereditary uromodulin-associated kidney disease and THP single nucleotide polymorphisms with chronic kidney disease and hypertension, the physiological function(s) of THP and its pathological involvement remain incompletely understood. By studying age-dependent changes of THP knockout (KO) mice, we show here that young KO mice had significant salt and water wasting but were partially responsive to furosemide, due to decreased luminal translocation of Na-K-Cl cotransporter 2 (NKCC2) in the TAL. Aged THP KO mice were, however, markedly oliguric and unresponsive to furosemide, and their NKCC2 was localized primarily in the cytoplasm as evidenced by lipid raft floatation assay, cell fractionation, and confocal and immunoelectron microscopy. These aged KO mice responded to metolazone and acetazolamide, known to target distal and proximal tubules, respectively. They also had marked upregulation of renin in juxtaglomerular apparatus and serum, and they were hypertensive. Finally, the aged THP KO mice had significant upregulation of Na-coupled urate transporters Slc5a8 and Slc22a12 as well as sodiumhydrogen exchanger 3 (NHE3) in the proximal tubule and elevated serum uric acid and allantoin. Collectively, our results suggest that THP deficiency can cause progressive disturbances in renal functions via initially NKCC2 dysfunction and later compensatory responses, resulting in prolonged activation of the renin-angiotensin-aldosterone axis and hyperuricemia. [ABSTRACT FROM AUTHOR]

Objectives Endolymphatic sac (ELS) pathophysiology in Ménière's disease (MD) remains poorly understood. We identified from the literature a group of proteins expressed on the ELS and involved in endolymph volume regulation: aquaporin-2 (AQP2), vasopressin receptor V2R, sodium potassium chloride cotransporter 2 (NKCC2), and transient receptor potential cation channel V4 (TRPV4). Our objective was to determine whether their ELS expression was altered in MD, to better understand the pathophysiology of endolymphatic hydrops. Study Design Prospective case-control study. Setting Tertiary care center. Subjects Twenty-four patients with definite MD undergoing endolymphatic duct blockage surgery were recruited, as well as 23 controls with no history of MD undergoing surgery for vestibular schwannoma (VS). Methods ELS biopsies and blood samples for plasma arginine vasopressin (AVP) were obtained. Immunohistochemistry for AQP2, V2R, NKCC2, and TRPV4 was performed. Slides were scanned digitally for highly sensitive pixel density analysis by specialized software (VIS; Visiopharm). Results Global scores generated by the software represent total and relative protein expression density of 3 staining intensity levels, exclusively on ELS epithelium. AQP2 expression density was significantly elevated in MD compared to VS ( P = .003). There was no significant difference in plasma AVP, V2R, NKCC2, and TRPV4 expression. Conclusion This original study evaluates simultaneous in situ expression of AQP2, V2R, NKCC2, and TRPV4 on the human ELS in MD, with a control group. Our results show only AQP2 upregulation on the ELS of patients with MD. We suggest a constitutively increased expression of AQP2 in MD, independent of its regulatory axis (AVP-V2R). Acquired regulator sequence mutations could support this model. [ABSTRACT FROM AUTHOR]

Geranium seemannii Peyr is a perennial plant endemic to central Mexico that has been widely used for its diuretic effect, but the responsible compound of this effect is unknown as well as the mechanism by which the diuretic effect is achieved. Geraniin is one of the compounds isolated from this kind of geranium. This study was designed to determinate whether geraniin possesses diuretic activity and to elucidate the mechanism of action. Geraniin was extracted and purified from Geranium seemannii Peyr. Male Wistar rats were divided into four groups: 1) Control, 2) 75 mg/kg of geraniin, 3) 20 mg/kg of furosemide, and 4) 10 mg/kg of hydrochlorothiazide. Each treatment was administered by gavage every 24 h for 7 days. The urinary excretion of electrolytes and the fractional excretion of sodium (FENa) were determined. To uncover the molecular target of geraniin, Xenopus laevis oocytes were microinjected with cRNAs encoding the Na+-Cl- cotransporter (NCC) and the Na+-K+-2Cl- cotransporter NKCC2 to functionally express these cotransporters. Geraniin significantly increased diuresis, natriuresis, and calciuresis to a similar extent as was observed in the furosemide-treated rats. Consistent with the furosemide-like effect, in X. laevis oocytes, geraniin significantly reduced the activity of NKCC2, with no effect on NCC activity. In contrast to furosemide, the effect of geraniin on NKCC2 was irreversible, apparently due to its inhibitory effect on heat shock protein 90. Our observations suggest that geraniin could have a potential role in the treatment of hypertension or edematous states. [ABSTRACT FROM AUTHOR]

Acute renal failure is a condition in which kidneys are unable to eliminate all the waste from the body. Renal failure results in acid-base imbalance and electrolyte homeostasis. Na+-K+-2Cl-Cotransporter (NKCC2), Na+-Cl-Cotransporter (NCC) and Na+-K+-ATPase Cotransporter (Na+-K+-ATPase) are the important water and sodium channels proteins in the kidney. The aim of this study was to determine the gene expression level in the kidney of dogs suffering with the acute renal failure. A total four dogs with acute renal failure and four healthy dogs were used in this study. The biochemical analysis was done via semi-automatic biochemical machine, while the urine and urinary sediment examination was observed via microscopic study. The acute renal failure was diagnosed and confirmed via necropsy and histopathological examinations and finally the protein expression was analyzed by immunohistochemistry. Results showed that the concentration of sodium, potassium, creatinine and urea nitrogen were significantly different from control group. The level of pH and specific gravity (SG) were decreased with urobilinogen (URO) and were increased during acute renal failure. The urinary sedimentation results revealed the presence of RBCs and WBCs in urine samples. The kidney volume was increased with renal capsule due to hyperplasia and renal pelvis expansion. The hydronephrosis was also observed along with hemorrhages, shrinkage, necrosis, and inflammation in acute renal failure groups by histopathology. The immunohistochemical results revealed that the NKCC2, NCC and Na+-K+-ATPase were highly expressed in acute renal failure dogs. Therefore, it was concluded that sodium channels proteins possibly play an important role in dog's acute renal failure cases. [ABSTRACT FROM AUTHOR]

Sodium and its associated anions are the major determinant of extracellular fluid volume, and the reabsorption of Na+ by the kidney plays a crucial role in long-term blood pressure control. The goal of this study was to investigate the extent to which inhibitors of transepithelial N+ transport (TNa) along the nephron alter urinary solute excretion and TNa efficiency and how those effects may vary along different nephron segments. To accomplish that goal, we used the multinephron model developed in the companion study (28). That model represents detailed transcellular and paracellular transport processes along the nephrons of a rat kidney. We simulated the inhibition of the Na+/H+ exchanger (NHE3), the bumetanide-sensitive Na+-K+-2Cl- transporter (NKCC2), the Na+-Cl- cotransporter (NCC), and the amiloride-sensitive Na+ channel (ENaC). Under baseline conditions, NHE3, NKCC2, NCC, and ENaC reabsorb 36, 22, 4, and 7%, respectively, of filtered Na+. The model predicted that inhibition of NHE3 substantially reduced proximal tubule TNa and oxygen consumption (QO2). Whole-kidney TNa efficiency, as reflected by the number of moles of Na+ reabsorbed per moles of O2 consumed (denoted by the ratio TNa/QO2), decreased by ~20% with 80% inhibition of NHE3. NKCC2 inhibition simulations predicted a substantial reduction in thick ascending limb TNa and QO2; however, the effect on whole-kidney TNa/QO2 was minor. Tubular K+transport was also substantially impaired, resulting in elevated urinary K+ excretion. The most notable effect of NCC inhibition was to increase the excretion of Na+, K+, and Cl- its impact on whole-kidney TNa and its efficiency was minor. Inhibition of ENaC was predicted to have opposite effects on the excretion of Na+ (increased) and K+ (decreased) and to have only a minor impact on whole-kidney TNa and TNa/QO2. Overall, model predictions agree well with measured changes in Na+ and K+ excretion in response to diuretics and Na+ transporter mutations. [ABSTRACT FROM AUTHOR]

Activation of the thick ascending limb (TAL) Na+-K+-2Cl- cotransporter (NKCC2) by the antidiuretic hormone arginine vasopressin (AVP) is an essential mechanism of renal urine concentration and contributes to extracellular fluid and electrolyte homeostasis. AVP effects in the kidney are modulated by locally and/or by systemically produced epoxyeicosatrienoic acid derivates (EET). The relation between AVP and EET metabolism has not been determined. Here, we show that chronic treatment of AVP-deficient Brattleboro rats with the AVP V2 receptor analog desmopressin (dDAVP; 5 ng/h, 3 days) significantly lowered renal EET levels (-56 ± 3% for 5,6-EET, -50 ± 3.4% for 11,12-EET, and -60 ± 3.7% for 14,15-EET). The abundance of the principal EET-degrading enzyme soluble epoxide hydrolase (sEH) was increased at the mRNA (+160 ± 37%) and protein levels (+120 ± 26%). Immunohistochemistry revealed dDAVP-mediated induction of sEH in connecting tubules and cortical and medullary collecting ducts, suggesting a role of these segments in the regulation of local interstitial EET signals. Incubation of murine kidney cell suspensions with 1 μM 14,15- EET for 30 min reduced phosphorylation of NKCC2 at the AVPsensitive threonine residues T96 and T101 (-66 ± 5%; P < 0.05), while 14,15-DHET had no effect. Concomitantly, isolated perfused cortical thick ascending limb pretreated with 14,15-EET showed a 30% lower transport current under high and a 70% lower transport current under low symmetric chloride concentrations. In summary, we have shown that activation of AVP signaling stimulates renal sEH biosynthesis and enzyme activity. The resulting reduction of EET tissue levels may be instrumental for increased NKCC2 transport activity during AVP-induced antidiuresis. [ABSTRACT FROM AUTHOR]

The antidiuretic hormone vasopressin (AVP) regulates renal salt and water reabsorption along the distal nephron and collecting duct system. These effects are mediated by vasopressin 2 receptors (V2R) and release of intracellular Gsmediated cAMP to activate epithelial transport proteins. Inactivating mutations in the V2R gene lead to the X-linked form of nephrogenic diabetes insipidus (NDI), which has chiefly been related with impaired aquaporin 2-mediated water reabsorption in the collecting ducts. Previous work also suggested the AVP-V2R-mediated activation of Na+-K+-2Cl--cotransporters (NKCC2) along the thick ascending limb (TAL) in the context of urine concentration, but its individual contribution to NDI or, more generally, to overall renal function was unclear. We hypothesized that V2R-mediated effects in TAL essentially determine its reabsorptive function. To test this, we reevaluated V2R expression. Basolateral membranes of medullary and cortical TAL were clearly stained, whereas cells of the macula densa were unreactive. A dominant-negative, NDI-causing truncated V2R mutant (Ni3-Glu242stop) was then introduced into the rat genome under control of the Tamm-Horsfall protein promoter to cause a tissuespecific AVP-signaling defect exclusively in TAL. Resulting Ni3-V2R transgenic rats revealed decreased basolateral but increased intracellular V2R signal in TAL epithelia, suggesting impaired trafficking of the receptor. Rats displayed significant baseline polyuria, failure to concentrate the urine in response to water deprivation, and hypercalciuria. NKCC2 abundance, phosphorylation, and surface expression were markedly decreased. In summary, these data indicate that suppression of AVP-V2R signaling in TAL causes major impairment in renal fluid and electrolyte handling. Our results may have clinical implications. [ABSTRACT FROM AUTHOR]

Angiotensin II (ANG II) is secreted by the proximal tubule resulting in a luminal concentration that is 100- to 1,000-fold greater than that in the blood. Luminal ANG II has been shown to stimulate sodium transport in the proximal tubule and distal nephron. Surprisingly, luminal ANG II inhibits NaCl transport in the medullary thick ascending limb (mTAL), a nephron segment responsible for a significant amount of NaCl absorption from the glomerular ultrafiltrate. We confirmed that addition of 10-8 M ANG II to the lumen inhibited mTAL chloride transport (220 ± 19 to 165 ± 25 pmol·mm-1·min-1, P < 0.01) and examined whether an interaction with basolateral norepinephrine existed to simulate the in vivo condition of an innervated tubule. We found that in the presence of a 10-6 Mnorepinephrine bath, luminal ANG II stimulated mTAL chloride transport from 298 ± 18 to 364 ± 42 pmol·mm-1·min-1 (P < 0.05). Stimulation of chloride transport by luminal ANG II was also observed with 10-3 M bath dibutyryl cAMP in the bathing solution and bath isoproterenol. A bath of 10-5 H-89 blocked the stimulation of chloride transport by norepinephrine and prevented the effect of luminal ANG II to either stimulate or inhibit chloride transport. Bath phentolamine, an α-adrenergic agonist, also prevented the decrease in mTAL chloride transport by luminal ANG II. Thus luminal ANG II increases chloride transport with basolateral norepinephrine; an effect likely mediated by stimulation of cAMP. Alpha-1 adrenergic stimulation prevents the inhibition of chloride transport by luminal ANG II. [ABSTRACT FROM AUTHOR]

Mammalian with-no-lysine [K] (WNK) kinases are a family of four serine-threonine protein kinases, WNK1-4. Mutations of WNK1 and WNK4 in humans cause pseudohypoaldosteronism type II (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. Increased Na reabsorption through Na-Cl cotransporter (NCC) in the distal convoluted tubule plays an important role in the pathogenesis of hypertension in patients with PHA2. However, how WNK1 and WNK4 regulate NCC and how mutations of WNKs cause activation of NCC have been controversial. Here, we review current state of literature supporting a compelling model that WNK1 and WNK4 both contribute to stimulation of NCC. The precise combined effects of WNK1 and WNK4 on NCC remain unclear but likely are positive rather than antagonistic. The recent discovery that WNK kinases may function as an intracellular chloride sensor adds a new dimension to the physiological role of WNK kinases. Intracellular chloride-dependent regulation of WNK's may underlie the mechanism of regulation of NCC by extracellular K. Definite answer yet will require future investigation by tubular perfusion in mice with altered WNK kinase expression. [ABSTRACT FROM AUTHOR]

Consumption of fructose has increased during the last 50 years. Excessive fructose consumption has a detrimental effect on mammalian health but the mechanisms remain unclear. In humans, a direct relationship exists between dietary intake of added sugars and increased risk for cardiovascular disease mortality (52). While the causes for this are unclear, we recently showed that fructose provided in the drinking water induces a salt-dependent increase in blood pressure in Sprague-Dawley rats in a matter of days (6). However, little is known about the effects of fructose in renal salt handling and whether combined intake of high fructose and salt can lead to salt-sensitive hypertension before the development of metabolic abnormalities. The long-term (more than 4 wk) adverse effects of fructose intake on renal function are not just due to fructose but are also secondary to alterations in metabolism which may have an impact on renal function. This minireview focuses on the acute effect of fructose intake and its effect on salt regulation, as they affect blood pressure. [ABSTRACT FROM AUTHOR]

The renal phenotype of EAST syndrome, a disease caused by the loss-of-function-mutations of Kcnj10 (Kir4.1), is a reminiscence of Gitelman's syndrome characterized by the defective function in the distal convoluted tubule (DCT). The aim of the present study is to test whether antidiuretic hormone (vasopressin)-induced stimulation of the Na + -activated 80–150 pS K + channel is responsible for compensating the lost function of Kcnj10 in the thick ascending limb (TAL) of subjects with EAST syndrome. Immunostaining and western blot showed that the expression of aquaporin 2 (AQP2) was significantly higher in Kcnj10 − / − mice than those of WT littermates, suggesting that the disruption of Kcnj10 stimulates vasopressin response in the kidney. The role of vasopressin in stimulating the basolateral K + conductance of the TAL was strongly indicated by the finding that the application of arginine-vasopressin (AVP) hyperpolarized the membrane in the TAL of Kcnj10 − / − mice. Application of AVP significantly stimulated the 80–150 pS K + channel in the TAL and this effect was blocked by tolvaptan (V2 receptor antagonist) or by inhibiting PKA. Moreover, the water restriction for 24 h significantly increased the probability of finding the 80–150 pS K + channel and the K + channel open probability in the TAL. The application of a membrane permeable cAMP analog also mimicked the effect of AVP and activated this K + channel, suggesting that cAMP–PKA pathway stimulates the 80–150 pS K + channels. The role of the basolateral K + conductance in maintaining transcellular Cl − transport is further suggested by the finding that the inhibition of basolateral K + channels significantly diminished the AVP-induced stimulation of the basolateral 10 pS Cl − channels. We conclude that vasopressin stimulates the 80–150 pS K + channel in the TAL via a cAMP-dependent mechanism. The vasopressin-induced stimulation of K + channels is responsible for compensating lost function of Kcnj10 thereby rescuing the basolateral K + conductance which is essential for the transport function in the TAL. [ABSTRACT FROM AUTHOR]

The products of the Slc12a1 and Slc12a2 genes, commonly known as Na+-dependent K+2Cl- K co-transporters NKCC2 and NKCC1, respectively, are the targets for the diuretic bumetanide. NKCCs are implicated in the regulation of intracellular chloride concentration ([Cl-]i) in pancreatic β-cells, and as such, they may play a role in glucose-stimulated plasma membrane depolarization and insulin secretion. Unexpectedly, permanent elimination of NKCC1 does not preclude insulin secretion, an event potentially linked to the homeostatic regulation of additional Cl- transporters expressed in β-cells. In this report we provide evidence for such a mechanism. Mice lacking a single allele of Slc12a2 exhibit lower fasting glycemia, increased acute insulin response (AIR) and lower blood glucose levels 15-30 min after a glucose load when compared to mice harboring both alleles of the gene. Furthermore, heterozygous expression or complete absence of Slc12a2 associates with increased NKCC2 protein expression in rodent pancreatic β-cells. This has been confirmed by using chronic pharmacological down-regulation of NKCC1 with bumetanide in the mouse MIN6 β-cell line or permanent molecular silencing of NKCC1 in COS7 cells, which results in increased NKCC2 expression. Furthermore, MIN6 cells chronically pretreated with bumetanide exhibit increased initial rates of Cl- uptake while preserving glucose-stimulated insulin secretion. Together, our results suggest that NKCCs are involved in insulin secretion and that a single Slc12a2 allele may protect β-cells from failure due to increased homeostatic expression of Slc12a1. [ABSTRACT FROM AUTHOR]

Sodium chloride reabsorption in the thick ascending limb of the loop of Henle is mediated by the Na+-K+-2Cl− cotransporter ( NKCC2). The loop diuretic furosemide is a potent inhibitor of NKCC2. However, less is known about the mechanism regulating the electrolyte transporter. Considering the well-established effects of nitric oxide on NKCC2 activity, c GMP is likely involved in this regulation. c GMP-dependent protein kinase I (c GKI; PKGI) is a c GMP target protein that phosphorylates different substrates after activation through c GMP. We investigated the potential correlation between the c GMP/c GKI pathway and NKCC2 regulation. We treated wild-type (wt) and c GKIα-rescue mice with furosemide. c GKIα-rescue mice expressed c GKIα only under the control of the smooth muscle-specific transgelin ( SM22) promoter in a c GKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in c GKIα-rescue mice compared to that in wt mice. We analyzed the phosphorylation of NKCC2 by western blotting and immunostaining using the phosphospecific antibody R5. The administration of furosemide significantly increased the phosphorylated NKCC2 signal in wt but not in c GKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether c GKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by c GKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated c GKI, leading to NKCC2 phosphorylation and membrane translocation. This c GKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2. [ABSTRACT FROM AUTHOR]

In Sprague Dawley rats, 2-week angiotensin II (Ang II) infusion increases Na+ transporter abundance and activation from cortical thick ascending loop of Henle ( TALH) to medullary collecting duct ( CD) and raises blood pressure associated with a pressure natriuresis, accompanied by depressed Na+ transporter abundance and activation from proximal tubule ( PT) through medullary TALH. This study tests the hypothesis that early during Ang II infusion, before blood pressure raises, Na+ transporters' abundance and activation increase all along the nephron. Male Sprague Dawley rats were infused via osmotic minipumps with a subpressor dose of Ang II (200 ng/kg/min) or vehicle for 3 days. Overnight urine was collected in metabolic cages and sodium transporters' abundance and phosphorylation were determined by immunoblotting homogenates of renal cortex and medulla. There were no significant differences in body weight gain, overnight urine volume, urinary Na+ and K+ excretion, or rate of excretion of a saline challenge between Ang II and vehicle infused rats. The 3-day nonpressor Ang II infusion significantly increased the abundance of PT Na+/H+ exchanger 3 ( NHE3), cortical TALH Na-K-2Cl cotransporter 2 ( NKCC2), distal convoluted tubule ( DCT) Na-Cl cotransporter ( NCC), and cortical CD ENaC subunits. Additionally, phosphorylation of cortical NKCC2, NCC, and STE20/ SPS1-related proline-alanine-rich kinase ( SPAK) were increased; medullary NKCC2 and SPAK were not altered. In conclusion, 3-day Ang II infusion provokes PT NHE3 accumulation as well as NKCC2, NCC, and SPAK accumulation and activation in a prehypertensive phase before evidence for intrarenal angiotensinogen accumulation. [ABSTRACT FROM AUTHOR]

The aim of the present study is to examine the role of Kcnj10 (Kir.4.1) in contributing to the basolateral K conductance in the cortical thick ascending limb (cTAL) using Kcnj10+/+ wild-type (WT) and Kcnj10-/- knockout (KO) mice. The patch-clamp experiments detected a 40- and an 80-pS K channel in the basolateral membrane of the cTAL. Moreover, the probability of finding the 40-pS K was significantly higher in the late part of the cTAL close to the distal convoluted tubule than those in the initial part. Immunostaining showed that Kcnj10 staining was detected in the basolateral membrane of the cTAL but the expression was not uniformly distributed. The disruption of Kcnj10 completely eliminated the 40-pS K channel but not the 80-pS K channel, suggesting the role of Kcnj10 in forming the 40-pS K channel of the cTAL. Also, the disruption of Kcnj10 increased the probability of finding the 80-pS K channel in the cTAL, especially in the late part of the cTAL. Because the channel open probability of the 80-pS K channel in KO was similar to those of WT mice, the increase in the 80-pS K channel may be achieved by increasing K channel number. The whole cell recording further showed that K reversal potential measured with 5 mM K in the bath and 140 mM K in the pipette was the same in the WT and KO mice. Moreover, Western blot and immunostaining showed that the disruption of Kcnj10 did not affect the expression of Na-K-Cl cotransporter 2 (NKCC2). We conclude that Kir.4.1 is expressed in the basolateral membrane of cTAL and that the disruption of Kir.4.1 has no significant effect on the membrane potential of the cTAL and NKCC2 expression. [ABSTRACT FROM AUTHOR]

Background. Gitelman syndrome (GS) and Bartter syndrome (BS) are hereditary salt-losing tubulopathies (SLTs) resulting from defects of renal proteins involved in electrolyte reabsorption, as for sodium-chloride cotransporter (NCC) and furosemide-sensitive sodium-potassium-chloride cotransporter (NKCC2) cotransporters, affected in GS and BS Type 1 patients, respectively. Currently, definitive diagnosis is obtained through expensive and time-consuming genetic testing. Urinary exosomes (UE), nanovesicles released by every epithelial cell facing the urinary space, represent an ideal source of markers for renal dysfunction and injury, because UE molecular composition stands for the cell of origin. On these assumptions, the aim of this work is to evaluate the relevance of UE for the diagnosis of SLTs. Methods. UE were purified from second morning urines collected from 32 patients with genetically proven SLTs (GS, BS1, BS2 and BS3 patients), 4 with unclassified SLTs and 22 control subjects (age and sex matched). The levels of NCC and NKCC2 were evaluated in UE by SDS-PAGE/western blotting with specific antibodies. Results. Due to their location on the luminal side of tubular cells, NCC and NKCC2 are well represented in UE proteome. The NCC signal is significantly decreased/absent in UE of Gitelman patients compared with control subjects (Mann-Whitney t-test, P < 0.001) and, similarly, the NKCC2 in those of Bartter type 1 (P < 0.001). The difference in the levels of the two proteins allows recognition of Gitelman and Bartter type 1 patients from controls and, combined with clinical data, from other Bartter patients. Moreover, the receiver operating characteristic curve analysis using UE NCC densitometric values showed a good discriminating power of the test comparing GS patients versus controls and BS patients (area under the curve value = 0.92; sensitivity 84.2% and specificity 88.6%). Conclusions. UE phenotyping may be useful in the diagnosis of GS and BS, thus providing an alternative/complementary, urine-based diagnostic tool for SLT patient recognition and a diagnostic guidance in complex cases. [ABSTRACT FROM AUTHOR]