NKCC2 Trafficking Research Articles

β3 Adrenergic Receptor Agonist Mirabegron Increases AQP2 and NKCC2 Urinary Excretion in OAB Patients: A Pleiotropic Effect of Interest for Patients with X-Linked Nephrogenic Diabetes Insipidus.

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.

CRISPR/Cas9-Mediated Suppression of the Focal Segmental Glomerulosclerosis Protein Actinin-4 (ACTN4) in Thick Ascending Limbs (TALs) Increases Sodium Chloride Reabsorption by Regulating NKCC2 Trafficking

CRISPR/Cas9-Mediated Suppression of the Focal Segmental Glomerulosclerosis Protein Actinin-4 (ACTN4) in Thick Ascending Limbs (TALs) Increases Sodium Chloride Reabsorption by Regulating NKCC2 Trafficking

The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking

NKCC2 is a Cl-dependent co-transporter that mediates all the NaCl absorption in the thick ascending limb (TAL). In humans, loss-of-function mutations in NKCC2 result in Bartter syndrome type 1, characterized by inability in concentrating urine, polyuria, and low blood pressure. Importantly, enhanced NKCC2 activity is associated with hypertension in humans and animal models. NKCC2 is present on the apical membrane and sub-apical space in the TAL and macula densa cells. We and others have shown that trafficking of NKCC2 to the apical membrane determines NKCC2-mediated NaCl absorption. These trafficking steps are controlled by signaling and protein-protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C-terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin-4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo-sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione-S-transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC-MS) or Western blot. GST-ALMS1 also pulled down ACTN4, identified by LC-MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co-localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p<0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p<0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron-specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p<0.05), but also decreased ACTN4 expression by 45±9% (p<0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.

Differential Effects of STCH and Stress-Inducible Hsp70 on the Stability and Maturation of NKCC2.

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.

The FSGS protein Actinin‐4 (ACTN4) is expressed in Thick Ascending Limbs and interacts with NKCC2 and ALMS1 to regulate NKCC2 trafficking.

Thick ascending limb (TAL) of the loop of Henle reabsorbs 30% NaCl via the apical Na/K/2Cl cotransporter (NKCC2). Maintenance of steady‐state surface NKCC2 levels is results from a balance between endocytosis, exocytosis and recycling. These trafficking steps are controlled by signaling and protein‐protein interactions. Very little is known about how NKCC2 endocytosis is controlled and only a handful of proteins that mediate NKCC2 internalization and trafficking are known. To identify new NKCC2 interacting partners we immunoprecipitated NKCC2 from TAL lysates using a C‐terminus antibody and identified interacting proteins by mass spectrometry. We identified Actinin‐4 (ACTN4), a protein studied in podocytes, that when mutated causes Focal segmental glomerulo‐sclerosis (FSGS) in humans. We hypothesized that in addition to podocytes, ACTN4 is expressed along the nephron and is part of protein complex that binds apical NKCC2 and other proteins such as Alstrom Syndrome 1 (ALMS1) and promotes its endocytosis. To begin studying these mechanisms we used a targeted proteomics screen to identify new binding partners for ACTN4 in the TAL. We confirmed that glutathione‐S‐transferase (GST) ACTN4 (full length) pulled down NKCC2 and ALMS1 from TAL lysates identified by mass spectrometry (LC‐MS) or Western blot. GST‐ALMS1 also pulled down ACTN4, identified by LC‐MS. Because there are no studies on the role of ACTN4 in NaCl reabsorption along the nephron, we studied its localization in the kidney. ACTN4 was abundant in glomeruli but also localized in cells along the nephron in a punctate vesicular pattern. ACTN4 was expressed in TALs and partially co‐localized with NKCC2 the subapical space. To study if ACTN4 mediates NKCC2 trafficking, we generated adenoviruses expressing ACTN4 shRNA. In vivo transduction of the rat renal medulla decreased ACTN4 expression in TALs by 66±9% (p&lt;0.04). Silencing ACTN4 in rat TALs increased the surface to total NKCC2 ratio by 49±16% (p&lt;0.01) but did not affect total NKCC2, suggesting that ACTN4 regulates NKCC2 trafficking. To study the relationship of ACTN4 with ALMS1 and NKCC2, we generated nephron‐specific ALMS1 knockout mice and measured surface NKCC2 and ACTN4. Deletion of ALMS1 increased surface to total NKCC2 ratio by 55±19% (p&lt;0.05), but also decreased ACTN4 expression by 45±9% (p&lt;0.05). We conclude that ACTN4 binds NKCC2 and ALMS1 and it is required for proper NKCC2 trafficking. It is likely that a large protein complex containing ACTN4 and ALMS1 mediates NKCC2 endocytosis to control renal salt reabsorption.Support or Funding InformationNIH

Trafficking and regulation of the NKCC2 cotransporter in the thick ascending limb.

The kidney Na-K-2Cl cotransporter (NKCC2) is essential for urinary concentration and renal electrolyte handling. Loss of function mutations in the NKCC2 gene cause urinary salt and potassium wasting, whereas excessive NKCC2 function has been linked to high blood pressure. Loop diuretics, targeting the transporter, are instrumental for relieving edema or hypertension. This review focuses on intrinsic mechanisms regulating NKCC2 activity at the posttranslational level, namely its trafficking and phosphorylation. Protein networks mediating cellular turnover of NKCC2 have recently received major attention. Several key components of its apical trafficking were identified, including respective chaperones, SNARE protein family members and raft-associated proteins. NKCC2 internalization has been characterized qualitatively and quantitatively. Kinase and phosphatase pathways regulating NKCC2 activity have been clarified and links between NKCC2 phosphorylation and trafficking proposed. Constitutive and inducible NKCC2 trafficking and phosphorylation mechanisms have been specified with focus on endocrine control of thick ascending limb (TAL) function by vasopressin. Proper NKCC2 trafficking and phosphorylation are critical to the TAL function in the physiological context of urinary concentration and extracellular volume regulation. Clarification of the underlying mechanisms and respective protein networks may open new therapeutic perspectives for better management of renal electrolyte disorders and blood pressure control.

Abstract P229: cGMP Stimulates Ubiquitination and Degradation of NKCC2 in Thick Ascending Limbs

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter (NKCC2). Nitric oxide inhibits NaCl reabsorption in TALs by increasing the second messenger cyclic guanylate monophosphate (cGMP) and decreasing NKCC2 in the apical plasma membrane. Recently, we showed that internalized NKCC2 is constitutively degraded (0.33%/min). Protein degradation regulates channels and transporters activity along the nephron. However, whether NKCC2 is regulated by lysosomal degradation or ubiquitin-proteasomal system remains unknown. We hypothesized that internalized NKCC2 is degraded by the ubiquitin-proteasome system in a process stimulated by cGMP. TAL surface proteins were biotinylated and allowed to internalize. The biotin remaining in surface proteins was stripped away and only internalized NKCC2 measured by Western blot. cGMP enhanced the rate of disappearance of internalized NKCC2 by 83 % and this was blocked by a proteasomal (MG132) but not lysosomal (leupeptin) inhibitor (Control: 0.29 ± 0.04; cGMP: 0.53 ± 0.10 ; cGMP + MG132: 0.10 ± 0.10 ; cGMP + Leupeptin: 0.44 ± 0.06 %/min; p &lt; 0.05). In general, protein degradation by the proteasomal system requires ubiquitination of the targeted protein. We found that NKCC2 inmuno-precipitated with ubiquitin. Proteasome inhibition induced the accumulation of ubiquitin-NKCC2 and this was enhanced by cGMP (MG132: 59 ± 14%, MG132+cGMP: 111 ± 25%; p &lt;0.05). We concluded that internalized NKCC2 is degraded via the ubiquitin-proteasome pathway in a process stimulated by cGMP. cGMP-induced degradation of internalized NKCC2 may contribute to decreased NKCC2 trafficking to the apical membrane therefore decreasing NaCl reabsorption in TALs.

ALMS1 (Alstrom Syndrome 1), a new interacting protein of NKCC2, regulates apical NKCC2 trafficking, urinary concentration and blood pressure

NaCl absorption by the Thick Ascending Limb (TAL) is mediated by the apical Na/K/2Cl cotransporter, NKCC2. Increased NKCC2 activity and apical trafficking are associated to hypertension. However, only few proteins are known to bind and regulate NKCC2 trafficking. A 150 amino acid region in the carboxyl terminus of NKCC2 (C‐NKCC2) was shown to be important for apical trafficking of NKCC2. We hypothesized that proteins which bind to this C‐terminus region in NKCC2 play a role in regulating NKCC2 trafficking in the TAL. To identify new TAL proteins that bind C‐NKCC2, we performed a proteomics‐based screening of TAL proteins which interacted with Glutathione‐S‐Transferase‐C‐NKCC2, and identified Alstrom syndrome 1 (ALMS1) as an interacting partner. ALMS1 has been linked to human hypertension and renal function in Genome Wide Association Studies. Thus, we hypothesized that ALMS1 is involved in NKCC2 trafficking in the TAL, sodium reabsorption and blood pressure regulation. First, we confirmed that ALMS1 is expressed in TALs by Western blot and immuno‐labeling of isolated perfused TALs. To study the role of ALMS1 we obtained ALMS1 KO rats in collaboration with the Genome Editing Rat Resource Consortium. To study the effect of ALMS1 deletion on NKCC2 trafficking we isolated outer medullary TALs and measured surface and total NKCC2 expression. In TALs from ALMS1 KO, the percentage of total NKCC2 at the surface was higher compared to WT (13.8 ± 1.2% vs 9.1 ± 1.0%, p&lt;0.05, n=6). Total NKCC2 expression was not different between strains. Urine osmolality was 45% higher in ALMS1 KO rats (2800±37 vs. 1927±167 mOsm/kg H2O, p&lt;0.001, n=5), whereas urinary volume was lower in ALMS1 KO (ALMS1: 10.1±0.5 vs. WT: 14.4± 1.7 ml/day, p&lt; 0.05, n=5). Water intake was also lower in ALMS1 KO rats (18.4±1.1 vs. 28.2±1.5 ml/day, p&lt;0.001, n=5). At three months of age, ALMS1 KO rats had higher mean arterial pressure (ALMS1: 141±5 vs WT: 99 ±6 mmHg, p&lt; 0.001). Combined, these data indicate that ALMS1 binds and regulates NKCC2 trafficking and suggest that higher urine concentration is in part due to increased NKCC2 activity in the TAL of ALMS1 KO rats. We conclude that ALMS1 plays a role in NKCC2 trafficking and regulates blood pressure. The mechanism causing hypertension in ALMS1 KO rats may involve an increase in NKCC2 activity and higher sodium reabsorption by the TAL.

Abstract 104: Targeting the Alms1 Gene in Rats Causes Hypertension, Obesity and Enhanced NKCC2 Trafficking

Enhanced NaCl reabsorption by the thick ascending limb (TAL) is associated with salt sensitive hypertension in rodents and humans. NaCl absorption by the TAL depends on the apical Na/K/2Cl cotransporter - NKCC2. NKCC2 activity is regulated in part by protein-protein interactions with its carboxyl terminus that controls its trafficking to the apical membrane. We hypothesized that the proteins binding to this region of NKCC2 may be involved in NKCC2 regulation and trafficking. To identify new TAL proteins that bind NKCC2, we performed a proteomics screen between the NKCC2 carboxyl terminus and TAL proteins. We identified ALMS1 (Alstrom syndrome 1) as a specific interacting partner for this NKCC2 region and confirmed that ALMS1 is expressed in TALs. Little is known about ALMS1 in renal function. Mutation of this gene causes severe metabolic syndrome in humans and is associated with hypertension. To study ALMS1 function we obtained ALMS1 knockout (KO) rats in collaboration with the rat genome editing consortium. We found that ALMS1 KO rats fed a normal salt diet have higher systolic blood pressure (151± 5 mmHg) compared to wild type littermates (125± 4 mmHg, p&lt; 0.02). We also observed an increase in body weight in ALMS1 KO at all ages (WT: 344± 4 g vs KO: 429±11 g, at 11 weeks, p&lt;0.05). ALMS1 KO rats showed large intra-abdominal and sub-cutaneous fat deposits. We then obtained TALs and measured surface and total NKCC2 expression. In TALs from ALMS1 KO, the percentage of total NKCC2 at the surface was higher compared to WT (27±4 vs 14±2 %, p&lt;0.05, n=6). Total NKCC2 expression was not significantly different between ALMS1 KO and WT rats. We conclude that ALMS1 is important for blood pressure regulation. The mechanism for hypertension in ALMS1 KO is unclear but may involve an increase in NKCC2 trafficking to the apical membrane and activity. ALMS1 has been associated with poor kidney function, hypertension, and type 2 diabetes in humans. Understanding the function of ALMS1 could develop new avenues for research into these diseases.

Fructose stimulates phosphorylation and trafficking of the Na/K/2Cl cotransporter in rat thick ascending limbs (1109.2)

High fructose consumption affects renal salt handling and is linked to salt‐sensitive hypertension in humans and rodents. The thick ascending limb (TAL) reabsorbs 25% of filtered NaCl via NKCC2. However, the effect of fructose on NKCC2 and TAL NaCl absorption is unknown. We hypothesized that acute and chronic fructose treatment stimulate NKCC2 activity and trafficking in TALs. Acute fructose treatment (20 min) enhanced surface NKCC2 expression (Fructose 1mM= 22 ± 5%, 5mM= 49 ± 10%, 10mM= 101 ± 59%; p&lt;0.05), but did not increase NKCC2 phosphorylation at Ser126, Thre96/101, nor total NKCC2 expression. Fructose (5mM) increased NaCl transport‐related TAL oxygen consumption by 11 ± 3% (p&lt;0.05), and this effect was absent when NKCC2 was blocked by furosemide. In contrast, glucose (5 mM) did not affect surface NKCC2 nor TAL oxygen consumption. When Sprague‐Dawley rats were fed 20% fructose for 1 week, surface‐to‐intracellular NKCC2 increased by 61 ± 23% (p&lt;0.05), Thre96,101 phosphorylation increased by 8 ± 3 fold (p&lt;0.05) but total NKCC2 expression was reduced by 40 ± 9% (p&lt;0.05). We concluded that acute fructose treatment increases surface NKCC2 expression and NaCl transport by TALs. One week on a fructose enriched diet increased NKCC2 trafficking and phosphorylation at Thre96/101. Our data suggest that fructose but not glucose may contribute to salt‐sensitive hypertension by stimulating NKCC2 and NaCl absorption.Grant Funding Source: 1P01HL090550

Annexin A2 mediates apical trafficking of renal Na‐K‐2Cl cotransporter (892.22)

Transport activity of the thick ascending limb (TAL) Na‐K‐2Cl‐cotransporter (NKCC2) critically depends on its association with lipid raft membrane microdomains (LR). The mechanisms involved in the recruitment of NKCC2 into LR have not been elucidated so far. In the present study we hypothesized that the lipid raft scaffolding protein annexin A2 serves as an essential mediator in this process. Detergent‐resistant LR were isolated from rat renal outer medullary tissue samples using discontinuous sucrose density gradient and ultracentrifugation (floating assay). Interaction between NKCC2 and Anxa2 was analyzed by immunoprecipitation, GST pull down, and peptide spot assays. Distribution of the products in the apical TAL membrane was studied by immunofluorescence and immunogold electron microscopy. Trafficking was studied in AVP‐deficient rats and cultured TAL cells using the AVP analogue desmopressin (dDAVP) or low chloride hypotonic stress. Functional relevance of Anxa2 for NKCC2 trafficking was studied using RNAi‐mediated knockdown strategies. Floating assay, immunoprecipitation and colocalization studies confirmed the parallel presence of NKCC2 and Anxa2 in apical membrane LR of the TAL. Binding assays approved physical interaction of Anxa2 and the non‐phosphorylated N‐terminus of NKCC2. Moreover, dDAVP and low chloride hypotonic stress caused parallel trafficking of NKCC2 and Anxa2 in rats and in cultured TAL cells and this response was inhibited by RNAi‐mediated knockdown of Anxa2. In conclusion our data demonstrate that Anxa2 is an essential component of the NKCC2 trafficking protein complex and mediates its recruitment into LR microdomains.

Annexin A2 Mediates Apical Trafficking of Renal Na+-K+-2Cl− Cotransporter

The furosemide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is responsible for urine concentration and helps maintain systemic salt homeostasis. Its activity depends on trafficking to, and insertion into, the apical membrane, as well as on phosphorylation of conserved N-terminal serine and threonine residues. Vasopressin (AVP) signaling via PKA and other kinases activates NKCC2. Association of NKCC2 with lipid rafts facilitates its AVP-induced apical translocation and activation at the surface. Lipid raft microdomains typically serve as platforms for membrane proteins to facilitate their interactions with other proteins, but little is known about partners that interact with NKCC2. Yeast two-hybrid screening identified an interaction between NKCC2 and the cytosolic protein, annexin A2 (AnxA2). Annexins mediate lipid raft-dependent trafficking of transmembrane proteins, including the AVP-regulated water channel, aquaporin 2. Here, we demonstrate that AnxA2, which binds to phospholipids in a Ca(2+)-dependent manner and may organize microdomains, is codistributed with NKCC2 to promote its apical translocation in response to AVP stimulation and low chloride hypotonic stress. NKCC2 and AnxA2 interact in a phosphorylation-dependent manner. Phosphomimetic AnxA2 carrying a mutant phosphoacceptor (AnxA2-Y24D-GFP) enhanced surface expression and raft association of NKCC2 by 5-fold upon low chloride hypotonic stimulation, whereas AnxA2-Y24A-GFP and PKC-dependent AnxA2-S26D-GFP did not. As the AnxA2 effect involved only nonphosphorylated NKCC2, it appears to affect NKCC2 trafficking. Overexpression or knockdown experiments further supported the role of AnxA2 in the apical translocation and surface expression of NKCC2. In summary, this study identifies AnxA2 as a lipid raft-associated trafficking factor for NKCC2 and provides mechanistic insight into the regulation of this essential cotransporter.

Localization and vasopressin regulation of the Na⁺-K⁺-2Cl⁻ cotransporter in the distal colonic epithelium.

To investigate whether Na(+)-K(+)-2Cl(-) cotransporter (NKCC2) is expressed in the mouse distal colonic epithelia and whether it is regulated by vasopressin in the colon. The mRNA expression of NKCC2 in the mouse colonic mucosa was examined by reverse transcription-polymerase chain reaction. NKCC trafficking in the colon stimulated by 1-D-amino(8-D-arginine)-vasopressin (dDAVP) infusion (10 ng/mouse, intraperitoneal injection ) within 15 min, 30 min and 1h was investigated by laser confocal scanning microscopy. Total and membrane NKCC2 expression in the colonic mucosa from control and dDAVP-treated mice was detected by Western blotting. Short circuit current method was performed to determine regulation of NKCC2 by vasopressin in the colon. NKCC2 was predominantly located in the apical region of the surface of the distal colonic epithelia; by comparison, a large amount of NKCC1 was distributed in the basolateral membrane of the lower crypt epithelia of the mouse distal colon. Short-term treatment with dDAVP, a V2-type receptor-specific vasopressin analog, induced NKCC2 re-distribution, i.e., NKCC2 traffics to the apical membrane after dDAVP stimulation. In contrast, no obvious NKCC1 membrane translocation was observed. Western blotting results confirmed that membrane NKCC2 had significantly higher abundance in the dDAVP-treated mouse colonic mucosa relative to that in the untreated control, which is consistent with our immunostaining data. Moreover, the short-circuit current method combined with a NKCC2 inhibitor demonstrated that NKCC2 was also activated by serosal vasopressin in isolated distal colonic mucosa. Our results provide direct evidence that vasopressin also plays an important role in the colonic epithelia by stimulating NKCC2 trafficking to the apical membrane and inducing NKCC2-mediated ion transport.

Abstract 639: Fructose Potently Stimulates NKCC2 Trafficking in rat Thick Ascending Limbs

Fructose consumption has increased dramatically in the last 30 years and is directly linked to the epidemic of obesity, type-2 diabetes and also hypertension. A moderately high fructose intake induces salt-sensitive hypertension and enhances urinary excretion of fructose. Despite the potential implications of fructose in renal salt handling, very little is known about its effect along the nephron. The thick ascending limb (TAL) reabsorbs 25% of filtered NaCl, primarily via the apical Na/K/2Cl cotransporter (NKCC2). Enhanced NKCC2 activity and trafficking is related to salt-sensitive hypertension in genetic animal models. Thus, we hypothesized that fructose acutely stimulate NKCC2 trafficking to the apical surface of TALs. To test this hypothesis we measured membrane and total NKCC2 expression as well as phosphorylation of NKCC2 at Ser126 and Thre96,101 in suspensions of rat TALs. We found that acute treatment (20 minutes) of TALs with fructose potently enhanced apical surface NKCC2 in a concentration-dependent manner (Baseline: 100%; fructose 1 mM= 122 ± 5%, fructose 5 mM= 49 ± 10%, fructose 10 mM = 201 ± 59%; p &lt;0.05). Acute fructose treatment did not increase NKCC2 phosphorylation at Ser126 or Thre96,101 and did not affect total NKCC2 expression. To test if the effect of fructose on apical surface NKCC2 was specific to this hexose, we studied the effect of glucose and fructose 1,6 bisphosphate (1,6BP). We found that 20 minute-incubation with glucose (5 mM) and fructose 1,6BP (5 mM) did not affect baseline surface NKCC2 levels (baseline = 100, glucose (5 mM)= 101 ± 7%, fructose 1,6BP (5 mM)= 92 ± 6%, not significant). We concluded that acute fructose treatment, at physiologically relevant concentrations, increases membrane NKCC2 expression without enhancing its phosphorylation. Compared to β-adrenergic receptor stimulation and vasopressin (which enhance apical surface NKCC2 in TALs by 25-40%, fructose (but not glucose) is a very potent agonist of NKCC2 trafficking to the apical membrane of rat TALs. Our data suggest that fructose-stimulated TAL NaCl reabsorption could contribute to the salt-sensitive hypertension caused by a high fructose diet.

VAMP3 mediates NKCC2 trafficking in thick ascending limbs and is required for normal renal function and blood pressure

In the thick ascending limb of Henle's loop (TAL), trafficking of NKCC2 into the apical membrane is essential for NaCl absorption and blood pressure control. The vesicle associated membrane protein 3 (VAMP3), a membrane fusion protein of the SNARE family, is expressed in the kidney. VAMP3 is localized apically in TALs and co‐immunoprecipitates with NKCC2. The role of VAMP3 on Na excretion and NKCC2 trafficking is unknown. We hypothesized that VAMP3 mediates NKCC2 exocytic delivery and maintains normal TAL function and renal Na excretion. We used adenovirus‐mediated in vivo gene silencing to decrease VAMP3 expression in rat outer medullas and measured NKCC2 exocytic delivery by surface biotinylation in TAL suspensions. Silencing VAMP3 by 68 ± 10% did not affect VAMP2/7/8 expression in TALs but blocked NKCC2 exocytic delivery rate by 91 ± 6% (p&lt;0.05) and decreased total NKCC2 expression by 59 ± 8% (p&lt;0.05). We studied the role of VAMP3 in urinary Na (UNa) excretion and blood pressure in VAMP3 knockout (KO) mice. VAMP3 KOs exhibited reduced NKCC2 expression by 44 ± 12% (p&lt;0.05), elevated UNa excretion (wild type: 251 ± 11 vs VAMP3 KO: 294 ± 13 μmol/day, p&lt;0.05) and lower systolic arterial pressure (WT: 114 ± 2 vs VAMP3 KO: 95 ± 2 mmHg, p&lt;0.05). We conclude that VAMP3 mediates NKCC2 trafficking to the apical membrane and maintains NKCC2 expression. VAMP3 is required for normal Na excretion and blood pressure control. Funding: NIH, AHA

Enhanced sensitivity of NKCC2 to β‐adrenergic receptor stimulation in TALs from Dahl salt sensitive (DSS) rats

Abnormally enhanced NKCC2 activity and trafficking in the thick ascending limb (TAL) are related to hypertension in DSS rats. In vivo, DSS rats exhibit higher renal sympathetic nerve activity than Dahl salt resistant (DSR) and greater sympathetic‐induced renal Na reabsorption. In Sprague Dawley rats, β‐adrenergic receptor (β‐AR) stimulation enhances surface NKCC2 levels via cAMP and PKA. We hypothesized that the response to β‐AR stimulation is enhanced in DSS rats prior to the development of hypertension resulting in higher surface NKCC2 expression in TALs. We obtained TALs from DSS and DSR rats fed normal salt and measured surface NKCC2 and cAMP. The β‐AR agonist isoproterenol (ISO, 1μM) slightly enhanced surface NKCC2 in DSR (13 ± 9%), but significantly enhanced it in DSS rats (70 ± 24 %, p&lt;0.05). We next studied if this difference was due to higher cAMP production. ISO‐stimulated cAMP production was similar in TALs from DSS and DSR rats (DSR: 106 ± 22, DSS: 143 ± 18 % increase from baseline). To test whether the higher response to ISO in DSS was due to enhanced cAMP‐induced signaling we measured cAMP‐stimulated NKCC2 phosphorylation at Ser126. db‐cAMP (25 μM) stimulated Ser126 phosphorylation in DSS from 1.2 ± 0.3 to 10.5 ± 3.5 a.u, and from 0.6 ± 0.3 to 1.9 ± 0.6 a.u in DSR TALs (p&lt;0.05). We concluded that β‐AR stimulation of NKCC2 trafficking is enhanced in TALs from DSS rats by a mechanism that may involve enhanced cAMP signaling.

Enhanced urinary sodium and potassium excretion in VAMP3 knockout mice

Several members of the vesicle associated membrane proteins (VAMP) family are expressed in the kidney. VAMP3 is expressed in the apical membrane of the thick ascending limb (TAL) where it mediates NKCC2 trafficking to the apical membrane. Other transporters along the nephron segment may also be regulated by VAMP3 thereby influencing renal function. We hypothesized that VAMP3 is involved in maintaining renal Na excretion and thus blood pressure. To test our hypothesis we obtained homozygous VAMP3 knockout mice (KO) and measured systolic blood pressure (SBP) and urinary parameters using c57BL6 mice (WT) as controls. Urinary Na excretion was higher (325±25 vs 248±20 μmoles/day) as was K excretion (785±76 vs 645±27 μmoles/day) and urine volume (2.56±0.16 vs 1.95±0.19 ml/day) without a change in urinary creatinine. Body weight tended to be higher but not significantly so (27.2±0.6 vs 26.3 ±0.2 gram). After decreasing Na in the diet from 0.4 to 0.02% for 24 hours, Na excretion decreased in both strains. However, it remained elevated in KO (61±14 vs 38±3 μmoles/day). Baseline SBP was lower in KO mice compared to WT (KO: 101±3 vs WT: 115±3 mmHg). Feeding mice a low salt diet for 7 days did not further decrease SBP in VAMP3 KO (96±4 mmHg) but it decreased in WT (99±3 mmHg). Taken together our data suggest that VAMP3 maintains normal renal Na excretion and is involved in blood pressure homeostasis. This is the first phenotype observed in VAMP3 KO mice.

Multiple Evolutionarily Conserved Di-leucine Like Motifs in the Carboxyl Terminus Control the Anterograde Trafficking of NKCC2

Mutations in the apical Na-K-2Cl co-transporter, NKCC2, cause type I Bartter syndrome, a life-threatening kidney disease. Yet the mechanisms underlying the regulation of NKCC2 trafficking in renal cells are scarcely known. We previously showed that naturally occurring mutations depriving NKCC2 of its distal COOH-terminal tail and interfering with the (1081)LLV(1083) motif result in defects in the ER exit of the co-transporter. Here we show that this motif is necessary but not sufficient for anterograde trafficking of NKCC2. Indeed, we have identified two additional hydrophobic motifs, (1038)LL(1039) and (1048)LI(1049), that are required for ER exit and surface expression of the co-transporter. Double mutations of (1038)LL(1039) or (1048)LI(1049) to di-alanines disrupted glycosylation and cell surface expression of NKCC2, independently of the expression system. Pulse-chase analysis demonstrated that the absence of the terminally glycosylated form of NKCC2 was not due to reduced synthesis or increased rates of degradation of mutant co-transporters, but was instead caused by defects in maturation. Co-immunolocalization experiments revealed that (1038)AA(1039) and (1048)AA(1049) were trapped mainly in the ER as indicated by extensive co-localization with the ER marker calnexin. Remarkably, among several analyzed motifs present in the NKCC2 COOH terminus, only those required for ER exit and surface expression of NKCC2 are evolutionarily conserved in all members of the SLC12A family, a group of cation-chloride co-transporters that are targets of therapeutic drugs and mutated in several human diseases. Based upon these data, we propose abnormal anterograde trafficking as a common mechanism associated with mutations depriving NKCC2, and also all other members of the SLC12A family, of their COOH terminus.

Hyperphosphorylation of Na-K-2Cl Cotransporter in Thick Ascending Limbs of Dahl Salt-Sensitive Rats

Salt-sensitive hypertension involves a renal defect preventing the kidney from eliminating excess NaCl. The thick ascending limb of Henle loop reabsorbs ≈ 30% of filtered NaCl via the apical Na-K-2Cl cotransporter (NKCC2). Higher NKCC2 activity and Cl reabsorption have been reported in the thick ascending limbs from Dahl salt-sensitive rats (DSS) fed normal salt. NKCC2 activity is primarily regulated by protein trafficking and phosphorylation at Thr(96)/Thr(101) via STE20- and SPS1-related proline and alanine-rich kinases and oxidative stress-responsive kinase 1. However, the mechanism for enhanced NKCC2 activity in DSS is unclear. We hypothesized that DSS exhibit enhanced NKCC2 trafficking and higher NKCC2 phosphorylation compared with Dahl salt-resistant rats on normal salt diet. We measured steady state surface NKCC2 expression and phosphorylation at Thr(96) and Thr(101) by surface biotinylation and Western blot. In DSS, the surface:total NKCC2 ratio was enhanced by 25% compared with Dahl salt-resistant rats (P<0.05) despite lower NKCC2 expression. Total NKCC2 phosphorylation at Thr(96) and Thr(101) was enhanced ≈5-fold in DSS thick ascending limbs. Moreover, total STE20- and SPS1-related proline and alanine-rich kinases expression, kidney-specific STE20- and SPS1-related proline and alanine-rich kinases, and oxidative stress-responsive kinase 1 were not different between strains, although STE20- and SPS1-related proline and alanine-rich kinases/oxidative stress-responsive kinase 1 phosphorylation was enhanced by 60% (P<0.05) in DSS rats, suggesting increased activity. We concluded that phosphorylation of NKCC2 Thr(96) and Thr(101) and surface:total NKCC2 ratio are enhanced in DSS rats. These differences in NKCC2 may be, in part, responsible for higher NKCC2 activity and abnormally enhanced thick ascending limb NaCl reabsorption in DSS rats.

β-Adrenergic receptor stimulation increases surface NKCC2 expression in rat thick ascending limbs in a process inhibited by phosphodiesterase 4

The thick ascending limb of the loop of Henle (THAL) reabsorbs ∼30% of the filtered NaCl in a process mediated by the apical Na-K-2Cl cotransporter NKCC2. Stimulation of β-adrenergic receptors in the THAL enhances NaCl reabsorption and increases intracellular cAMP. We found that intracellular cAMP stimulates NKCC2 trafficking to the apical membrane via protein kinase A (PKA). Several cAMP-specific phosphodiesterases (PDE) have been identified in rat THALs, and PDE4 decreases cAMP generated by β-adrenergic stimulation in other cells. However, it is not known whether β-adrenergic receptors activation stimulates NKCC2 trafficking. Thus we hypothesized that β-adrenergic receptor stimulation enhances THAL apical membrane NKCC2 expression via the PKA pathway and PDE4 blunts this effect. THAL suspensions were obtained from Sprague-Dawley rats, and surface NKCC2 expression was measured by surface biotinylation and Western blot. Incubation of THALs with the β-adrenergic receptor agonist isoproterenol at 0.5 and 1.0 μM increased surface NKCC2 by 17 ± 1 and 29 ± 5% respectively (P < 0.05). Preventing cAMP degradation with 3-isobutyl-methylxanthine (IBMX; a nonselective phosphodiesterase inhibitor) enhanced isoproterenol-stimulated surface NKCC2 expression to 51 ± 7% (P < 0.05 vs. isoproterenol). The β-adrenergic receptor antagonist propranolol or the PKA inhibitor H-89 completely blocked isoproterenol + IBMX-induced increase on surface NKCC2, while propranolol or H-89 alone had no effect. Selective inhibition of PDE4 with rolipram (20 μM) potentiated the effect of isoproterenol on surface NKCC2 and increased cAMP levels. We concluded that β-adrenergic receptor stimulation enhances surface NKCC2 expression in the THALs via PKA and PDE4 blunts this effect.