NKCC1 Activity Research Articles

Characterization of Na+-K+-2Cl- Cotransporter Activity in Rabbit Lacrimal Gland Duct Cells.

PurposeWe recently reported that isolated duct segments from rabbit lacrimal gland (LG) were able to secrete fluid in response to secretagogues, which were blocked completely by bumetanide. This suggests the functional involvement of Na+-K+-2Cl− cotransporter (NKCC1) in ductal fluid secretion. Therefore, the aim of this study was to investigate the activity profile of NKCC1 in isolated rabbit LG duct segments.MethodsInterlobular ducts were isolated from fresh rabbit LG tissue. Microfluorometry with the ammonium (NH4+)–pulse technique was used to elicit pH changes in duct cells, and the rate of bumetanide-sensitive cytosolic acidification after addition of NH4+ was used to quantify the activity of NKCC1.ResultsWhile basal activity of NKCC1 was undetectable, low cytosolic chloride (Cl−) level and hyperosmotic challenge (390 mOsm) were able to increase the activity of NKCC1. Carbachol (100 μM) had no significant effect on NKCC1 activity. Elevation of cytosolic calcium (Ca2+) level with Ca2+-ionophore (A 23187, 1 μM) did not cause any alteration in the activity of the cotransporter while direct activation of protein kinase C (phorbol myristate acetate, 100 nM) increased its activity slightly but in a significant manner. Addition of either forskolin (10 μM), cell-permeable cAMP analogue (8-bromo cAMP, 100 μM) or vasoactive intestinal peptide (200 nM) resulted in a significant increase in the activity of NKCC1.ConclusionsThese results highlight the functional involvement of NKCC1 in LG duct secretion. These findings may facilitate our understanding of LG function and may contribute to the development of targeted pharmacologic interventions in case of dry eye disease.

53. A Neuro-Specific Gene Therapy Approach to Treat Cognitive Impairment in Down Syndrome by RNA Interference

Down syndrome (DS) is a genetic disorder caused by the presence of a third copy of chromosome 21. DS affects multiple organs, resulting in characteristic facial features, muscular hypotonia, heart defects, brain development impairment, and varying degrees of intellectual disability. Trisomic mouse models of DS reproduce the main cognitive disabilities of the human syndrome. In particular, DS mice show structural and functional synaptic impairment as well as learning and memory deficits, largely determined by altered GABAergic transmission through chloride-permeable GABAa receptors (GABAaR). In particular, we have recently found that intracellular chloride accumulation shifts GABAAR-mediated signaling from inhibitory to excitatory in the adult brain of the Ts65Dn mouse model of DS. Accordingly, intracellular chloride accumulation was paralleled by increased expression of the chloride importer NKCC1 (Na-K-Cl cotransporter) in the brains of both trisomic mice and DS patients. Our findings on NKCC1 as a pivotal molecular target for the rescue of cognitive deficits in DS opens the possibility of a gene therapy approach to treat the disease. Here, to normalize NKCC1 expression and rescue synaptic dysfunctions as well as cognitive deficits in Ts65Dn mice we have developed and characterized a knock-down approach to normalize NKCC1 activity. Reducing the expression of the chloride importer NKCC1 by RNA interference restored GABAAR-mediated inhibition and also rescued the structural dendritic deficits found in trisomic neurons in vitro. Most importantly, focal administration of an AAV expressing a silencing RNA under the transcriptional control of a neuron-specific promoter in the hippocampus of Ts65Dn animals mediated NKCC1 knockdown in vivo and rescued behavioral performance on different learning and memory tests at levels undistinguishable from those of WT mice. Our findings demonstrate that NKCC1 overexpression drives excitatory GABAAR signaling in trisomic cells, leading to structural neuronal abnormalities and behavioral impairments in DS mice. Moreover, our study identifies a new gene therapy target for treatments aimed at rescuing cognitive disabilities in individuals with DS.

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<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<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< 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<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< 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.

Romk1 Knockout Mice Do Not Produce Bartter Phenotype but Exhibit Impaired K Excretion

Romk knock-out mice show a similar phenotype to Bartter syndrome of salt wasting and dehydration due to reduced Na-K-2Cl-cotransporter activity. At least three ROMK isoforms have been identified in the kidney; however, unique functions of any of the isoforms in nephron segments are still poorly understood. We have generated a mouse deficient only in Romk1 by selective deletion of the Romk1-specific first exon using an ES cell Cre-LoxP strategy and examined the renal phenotypes, ion transporter expression, ROMK channel activity, and localization under normal and high K intake. Unlike Romk(-/-) mice, there was no Bartter phenotype with reduced NKCC2 activity and increased NCC expression in Romk1(-/-) mice. The small conductance K channel (SK) activity showed no difference of channel properties or gating in the collecting tubule between Romk1(+/+) and Romk1(-/-) mice. High K intake increased SK channel number per patch and increased the ROMK channel intensity in the apical membrane of the collecting tubule in Romk1(+/+), but such regulation by high K intake was diminished with significant hyperkalemia in Romk1(-/-) mice. We conclude that 1) animal knockouts of ROMK1 do not produce Bartter phenotype. 2) There is no functional linking of ROMK1 and NKCC2 in the TAL. 3) ROMK1 is critical in response to high K intake-stimulated K(+) secretion in the collecting tubule.

Everything we always wanted to know about furosemide but were afraid to ask.

Furosemide is a widely used, potent natriuretic drug, which inhibits the Na(+)-K(+)-2Cl(-) cotransporter (NKCC)-2 in the ascending limb of the loop of Henle applied to reduce extracellular fluid volume expansion in heart and kidney disease. Undesirable consequences of furosemide, such as worsening of kidney function and unpredictable effects on sodium balance, led to this critical evaluation of how inhibition of NKCC affects renal and cardiovascular physiology. This evaluation reveals important knowledge gaps, involving furosemide as a drug, the function of NKCC2 (and NKCC1), and renal and systemic indirect effects of NKCC inhibition. Regarding renal effects, renal blood flow and glomerular filtration rate could become compromised by activation of tubuloglomerular feedback or by renin release, particularly if renal function is already compromised. Modulation of the intrarenal renin angiotensin system, however, is ill-defined. Regarding systemic effects, vasodilation followed by nonspecific NKCC inhibition and changes in venous compliance are not well understood. Repetitive administration of furosemide induces short-term (braking phenomenon, acute diuretic resistance) and long-term (chronic diuretic resistance) adaptations, of which the mechanisms are not well known. Modulation of NKCC2 expression and activity in kidney and heart failure is ill-defined. Lastly, furosemide's effects on cutaneous sodium stores and on uric acid levels could be beneficial or detrimental. Concluding, a considerable knowledge gap is identified regarding a potent drug with a relatively specific renal target, NKCC2, and renal and systemic actions. Resolving these questions would increase the understanding of NKCCs and their actions and improve rational use of furosemide in pathophysiology of fluid volume expansion.

Angiotensin II-mediated hypertension impairs nitric oxide-induced NKCC2 inhibition in thick ascending limbs.

Δ-41 ± 10%). NO increased cGMP by 2.08 ± 0.36 fmol/μg protein in THALs from vehicle-treated rats but only 1.06 ± 0.25 fmol/μg protein in ANG II-hypertensive rats (P < 0.04). Vardenafil (25 nM), a PDE5 inhibitor, restored NO's ability to inhibit NKCC2 activity in THALs from ANG II-hypertensive rats (Δ: -60 ± 9%, P < 0.003). Similarly, NO's stimulation of cGMP was also restored by vardenafil (vehicle-treated: 1.89 ± 0.71 vs. ANG II-hypertensive: 2.02 ± 0.32 fmol/μg protein). PDE5 expression did not differ between vehicle-treated and ANG II-hypertensive rats. We conclude that NO-induced inhibition of NKCC2 and increases in cGMP are blunted in ANG II-hypertensive rats due to PDE5 activation. Defects in the response of THALs to NO may enhance NaCl retention in ANG II-induced hypertension.

Interleukin-1 Receptor Activation Potentiates Salt Reabsorption in Angiotensin II-Induced Hypertension via the NKCC2 Co-transporter in the Nephron

Hypertension is among the most prevalent and catastrophic chronic diseases worldwide. While the efficacy of renin angiotensin system (RAS) blockade in lowering blood pressure illustrates that the RAS is broadly activated in human hypertension, the frequent failure of RAS inhibition to prevent or reverse hypertensive organ damage highlights the need for novel therapies to combat RAS-dependent hypertension. We previously discovered elevated levels of the macrophage cytokine IL-1 in the kidney in a murine model of RAS-mediated hypertension. Here we report that IL-1 receptor (IL-1R1) deficiency or blockade limits blood pressure elevation in this model by mitigating sodium reabsorption via the NKCC2 co-transporter in the nephron. In this setting, IL-1R1 activation prevents intra-renal myeloid cells from maturing into Ly6C(+)Ly6G(-) macrophages that elaborate nitric oxide, a natriuretic hormone that suppresses NKCC2 activity. By revealing how the innate immune system regulates tubular sodium transport, these experiments should lead to new immunomodulatory anti-hypertensive therapies.

Effect of vasopressin on Na+–K+–2Cl− cotransporter (NKCC) and the signaling mechanisms on the murine late distal colon

It has been demonstrated that the antidiuretic hormone vasopressin is able to regulate the expression of Na–K–Cl cotransporters (NKCC1 and NKCC2) in the kidney. The present study investigated the effects of long- and short-term administration of vasopressin on NKCC and the possible signaling mechanism of vasopressin in the mouse distal colon using the siRNA, real-time PCR, western blotting and Ussing chambers method. The results showed the presence of NKCC2 expression in the colon, which was verified with a siRNA technique. The mRNA and protein expression level of NKCC2 significantly increased by about 40% and 90% respectively in response to restricting water intake to 1ml/day/20g for 7 days. In contrast, the NKCC1 expression level was unchanged in the colon. To determine the short-term activation of NKCC2 by vasopressin in vitro, we found that the administration of vasopressin caused a 3-fold increase in mouse colon NKCC2 phosphorylation, which was detected with phosphospecific antibody R5. In addition, the Ussing chamber results showed that NKCC2, cAMP and Ca2+ signaling pathway may be involved in the vasopressin-induced response. Further, adenylate cyclase inhibitor MDL-12330A and PKA inhibitor H89 and Ca2+ chelator BAPTA-AM reversed the vasopressin induced NKCC2 phosphorylation level increase by about 35%, 28% and 42% respectively suggesting vasopressin stimulate NKCC2 phosphorylation increase mediated by cAMP-PKA and Ca2+ signaling in the colon. Collectively, these data suggest that the expression and phosphorylation of NKCC2 are increased in the colon by vasopressin stimulation, in association with enhanced activity of the vasopressin/cAMP and Ca2+ pathways.

ATPS-63OSMOTIC SWELLING REGULATES TUMOR GROWTH AND DRUG UPTAKE IN HUMAN GLIOBLASTOMA

BACKGROUND: High interstitial fluid pressure (IFP) represents a barrier for drug uptake in human GBM, the most common primary brain tumor. Fluid accumulation and high cell density compress tumors and promote osmotic swelling of tumor cells. Although studies have clarified the role of tumor vasculature, it remains unclear whether osmotic swelling of cancer cells regulates tumor growth and drug uptake. METHODS: To address this, we used human GBM tumorspheres and xenografts. We developed methodology to measure IFP in GBM xenografts and mechanical compression in 3D-cultures. Fluorescent-based sensors were used to measure cell volume and chloride levels. To reduce IFP and osmotic swelling in GBM cells, we established exogenous or endogenous induction of antisecretory factor (AF), known to be safe in patients, reduces elevated intracranial pressure in rodents, and lowers IFP in subcutaneous solid tumors. RESULTS: Intriguingly, our data demonstrate that elevated pressure drives proliferation of GBM tumorspheres. Transcriptional profiling showed that increased compression regulates genes involved in translation and ion transport. AF peptide completely blocked compression-induced proliferation and transcriptional changes. We found that AF targeted the sodium-potassium-chloride channel (NKCC1), more potently than the NKCC1 inhibitor bumetanide, and prevented restoration of cell volume and chloride permeability under hyperosmotic conditions. AF therapy effectively reduced tumor growth, increased drug uptake, and extended survival in GBM xenografts, effects that were mediated through lowering of IFP, reduced cell volume, and inhibition of NKCC1 activity. CONCLUSIONS: We find that elevated IFP in human GBMs maintains osmotic swelling in tumor cells as a second barrier for drug uptake. Our results further show that elevated pressure is a driver of proliferation, survival, and translational control. AF therapy represents a novel approach to inhibit NKCC1 activity and osmotic swelling in human GBMs, leading to reduced tumor growth and increased drug uptake, ultimately improving the outcome for this disease.

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons.

The equilibrium potential for GABA-A receptor mediated currents (EGABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K+/Cl- co-transporter (KCC2) but a relatively high expression of Na+-K+-Cl- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in EGABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on EGABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus EGABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in EGABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in EGABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in EGABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.

Abstract 142: A Fructose-but Not a Glucose-enriched Diet, Induces a Salt-dependent Increase in Blood Pressure and Enhances NKCC2 Phosphorylation in Normal Rats

Human consumption of fructose as a sweetener has increased in the past 30 years. High fructose intake has been implicated in the development of hypertension, diabetes, and obesity. In the US, the upper 10th percentile of the population consumes up to 40% of their caloric intake from added sugars, in which fructose represents half of these. Fructose metabolism is strikingly different from that of glucose. Yet, the effect of a fructose or glucose-enriched diet in salt handling by the kidney, affecting blood pressure, and its interaction with high salt intake has been poorly studied. In genetic models of salt-sensitive hypertension, the activity of the Na + /K + /2Cl - cotransporter (NKCC2) in the thick ascending limb (TAL) is abnormally enhanced. We hypothesized that chronic fructose in drinking water induces a salt-dependent increase in blood pressure and stimulates NKCC2 during high salt intake in normal rats. Sprague-Dawley rats were given 20% fructose or 20% glucose in drinking water for 1 week after which a high salt (HS) diet (4% Na + in chow) was started for 3 weeks. When we measured systolic blood pressure (SBP) by tail cuff plethysmography in fructose-fed and glucose-fed rats on a HS diet, only the fructose-fed rats had an increased SBP from 120±10 to 132±6 mmHg on day 7 of HS (p&lt;0.01). SBP continued to increase up to 144±18 mmHg after 3 weeks (p&lt;0.01 vs glucose). Fructose or glucose alone did not increase SBP after 4 weeks. We then repeated the protocol using radiotelemetry to monitor the blood pressure (BP). In rats fed fructose, by day 5 of HS the SBP increased by 12±3 mmHg (p&lt;0.02) and SBP remained elevated for 3 weeks (delta: 10±2.5 mmHg, n=3). In rats fed glucose, a HS diet did not significantly change SBP for 3 weeks (n=5). Moreover, NKCC2 activity in the TAL is enhanced by phosphorylation at Thr96, 101. We found that NKCC2 phosphorylation was higher in rats fed fructose plus HS (p&lt;0.02) but not in rats fed glucose plus HS for 3 weeks (HS: 100, fructose+HS: 250±40%, glucose+HS: 95±10%). Therefore, we conclude that a high fructose (but not a glucose) diet in normal rats induces a salt-dependent increase in BP independently from caloric intake. Thus, the increase in BP may in part be due to the stimulation of NKCC2 phosphorylation in the TAL by fructose.

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.

Regulation of NKCC2 splicing and phosphorylation.

Transepithelial salt transport in the thick ascending limb of Henle's loop (TAL) crucially depends on the activity of the Na/K/2Cl cotransporter NKCC2. The pharmacologic blockade of NKCC2 leads to pronounced natriuresis and diuresis, which indicate key roles for NKCC2 in renal salt retrieval. The inadequate regulation of NKCC2 and the loss of NKCC2 function are associated with the disruption of salt and water homoeostasis. This review provides a specific overview of our current knowledge with respect to the regulation of NKCC2 by differential splicing and phosphorylation. Several mechanisms have evolved to adapt NKCC2 transport to reabsorptive needs. These mechanisms include the regulation of NKCC2 gene expression, the differential splicing of the NKCC2 pre-mRNA, the membrane trafficking, and the modulation of the specific transport activity. Substantial progress has been made over the past few years in deciphering the function of kinases in the regulatory network controlling NKCC2 activity and in elucidating the underlying mechanism and the functional consequences of the regulated differential splicing of the NKCC2 pre-mRNA. NKCC2 differential splicing and phosphorylation are critically involved in the modulation of the thick ascending limb of Henle's loop reabsorptive capacity and, consequently, in salt homoeostasis, volume regulation, and blood pressure control.

Regulation of the Na(+)-K(+)-2Cl(-) cotransporter by cGMP/cGMP-dependent protein kinase I after furosemide administration.

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, cGMP is likely involved in this regulation. cGMP-dependent protein kinase I (cGKI; PKGI) is a cGMP target protein that phosphorylates different substrates after activation through cGMP. We investigated the potential correlation between the cGMP/cGKI pathway and NKCC2 regulation. We treated wild-type (wt) and cGKIα-rescue mice with furosemide. cGKIα-rescue mice expressed cGKIα only under the control of the smooth muscle-specific transgelin (SM22) promoter in a cGKI deficient background. Furosemide treatment increased the urine excretion of sodium and chloride in cGKIα-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 cGKIα-rescue mice. NKCC2 activation led to its phosphorylation and membrane translocation. To examine whether cGKI was involved in this process, we analyzed vasodilator-stimulated phosphoprotein, which is phosphorylated by cGKI. Furosemide injection resulted in increased vasodilator-stimulated phosphoprotein phosphorylation in wt mice. We hypothesize that furosemide administration activated cGKI, leading to NKCC2 phosphorylation and membrane translocation. This cGKI-mediated pathway could be a mechanism to compensate for the inhibitory effect of furosemide on NKCC2.

NKCC1 downregulation induces hyperpolarizing shift of GABA responsiveness at near term fetal stages in rat cultured dorsal root ganglion neurons.

BackgroundGABAA receptor-mediated neurotransmission is greatly influenced by cation-chloride cotransporter activity during developmental stages. In embryonic neurons Na–K–2Cl (NKCC1) cotransporters mediate active chloride uptake, thus increasing the intracellular chloride concentration associated with GABA-induced depolarization. At fetal stages near term, oxytocin-induced NKCC1 downregulation has been implicated in the developmental shift from depolarizing to hyperpolarizing GABA action. Mature dorsal root ganglion neurons (DRGN), however, express high NKCC1 levels and maintain high intracellular chloride levels with consequent GABA-induced depolarization.ResultsGramicidin-perforated patch-clamp recordings were used to assess the developmental change in chloride homeostasis in rat cultured small DRGN at the embryonic day 16 (E16) and 19 (E19). The results were compared to data previously obtained in fetal DRGN at E14 and in mature cells. A significant NKCC1 downregulation, leading to reduction in excitatory GABAergic transmission, was observed at E16 and E19.ConclusionThese results indicate that NKCC1 activity transiently decreases in DRGN at fetal stages near term. This developmental shift in GABAergic transmission may contribute to fetal analgesia and neuroprotection at birth.

NKCC1 Activation Is Required for Myelinated Sensory Neurons Regeneration through JNK-Dependent Pathway.

After peripheral nerve injury, axons are able to regenerate, although specific sensory reinnervation and functional recovery are usually worse for large myelinated than for small sensory axons. The mechanisms that mediate the regeneration of different sensory neuron subpopulations are poorly known. The Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) is particularly relevant in setting the intracellular chloride concentration. After axotomy, increased NKCC1 phosphorylation has been reported to be important for neurite outgrowth of sensory neurons; however, the mechanisms underlying its effects are still unknown. In the present study we used in vitro and in vivo models to assess the differential effects of blocking NKCC1 activity on the regeneration of different types of dorsal root ganglia (DRGs) neurons after sciatic nerve injury in the rat. We observed that blocking NKCC1 activity by bumetanide administration induces a selective effect on neurite outgrowth and regeneration of myelinated fibers without affecting unmyelinated DRG neurons. To further study the mechanism underlying NKCC1 effects, we also assessed the changes in mitogen-activated protein kinase (MAPK) signaling under NKCC1 modulation. The inhibition of NKCC1 activity in vitro and in vivo modified pJNK1/2/3 expression in DRG neurons. Together, our study identifies a mechanism selectively contributing to myelinated axon regeneration, and point out the role of Cl(-) modulation in DRG neuron regeneration and in the activation of MAPKs, particularly those belonging to the JNK family.

Calcineurin and Sorting-Related Receptor with A-Type Repeats Interact to Regulate the Renal Na⁺-K⁺-2Cl⁻ Cotransporter.

The furosemide-sensitive Na(+)-K(+)-2Cl(-)-cotransporter (NKCC2) is crucial for NaCl reabsorption in kidney thick ascending limb (TAL) and drives the urine concentrating mechanism. NKCC2 activity is modulated by N-terminal phosphorylation and dephosphorylation. Serine-threonine kinases that activate NKCC2 have been identified, but less is known about phosphatases that deactivate NKCC2. Inhibition of calcineurin phosphatase has been shown to stimulate transport in the TAL and the distal convoluted tubule. Here, we identified NKCC2 as a target of the calcineurin Aβ isoform. Short-term cyclosporine administration in mice augmented the abundance of phospho-NKCC2, and treatment of isolated TAL with cyclosporine increased the chloride affinity and transport activity of NKCC2. Because sorting-related receptor with A-type repeats (SORLA) may affect NKCC2 phosphoregulation, we used SORLA-knockout mice to test whether SORLA is involved in calcineurin-dependent modulation of NKCC2. SORLA-deficient mice showed more calcineurin Aβ in the apical region of TAL cells and less NKCC2 phosphorylation and activity compared with littermate controls. In contrast, overexpression of SORLA in cultured cells reduced the abundance of endogenous calcineurin Aβ. Cyclosporine administration rapidly normalized the abundance of phospho-NKCC2 in SORLA-deficient mice, and a functional interaction between calcineurin Aβ and SORLA was further corroborated by binding assays in rat kidney extracts. In summary, we have shown that calcineurin Aβ and SORLA are key components in the phosphoregulation of NKCC2. These results may have clinical implications for immunosuppressive therapy using calcineurin inhibitors.

Calcineurin Inhibition Affects Na(K)Cl Cotransporter Activity in Kidney Distal Nephron

The calcineurin inhibitors (CNI) tacrolimus and cyclosporine A (CsA) are widely used for immunosuppression. However, side effects such as hypertension and electrolyte disorders commonly occur. Our previous data suggest that tacrolimus exerts local effects in kidney distal convoluted tubule (DCT), leading to activation of the Na+, Cl- cotransporter (NCC), salt retention and hypertension. This study aims to provide the molecular basis for the renal action of CsA. Calcineurin Aa and Ab isoforms were ubiquitously detected along mouse isolated nephron segments by immunoblotting and qPCR. Calcineurin Aa was preferentially colocalized with NCC in DCT subapical region, whereas calcineurin Ab was enriched together with Na+, K+, Cl- cotransporter (NKCC2) in the thick ascending limb (TAL). Immunophilins, mediating the inhibitory effects of CsA, were detected in both TAL and DCT of mice and rats using immunofluorescence and in situ hybridization. Acute as well as chronic administration of CsA in rats induced strong increases of NKCC2 and NCC phosphorylation without changes in their abundance, suggesting that activation of both cotransporters occurred at the posttranslational level. Analysis of the WNK-SPAK/OSR1 kinase cascade, controlling phosphorylation of the transporters, suggested enhanced activity in both distal segments. In sum, our data identify local components of calcineurin signalling in TAL and DCT which are likely involved in the activation of NKCC2 and NCC upon CNI. These results may have implications for antihypertensive therapy during immune suppression.

A Fructose‐Enriched Diet Induces Salt‐Sensitive Hypertension and Enhances NKCC2 and SPAK/OSR1 Phosphorylation in Thick Ascending Limbs (TALs).

Fructose consumption increased in the past three decades and has been implicated in the epidemic of hypertension, diabetes and obesity. 10% of the US population consumes up to 20% of their calories from fructose. The effect of fructose intake in sodium handling by the kidney and blood pressure regulation is not understood. In models of salt-sensitive hypertension, the Na-K-2Cl cotransporter (NKCC2) presents higher activity. We hypothesized that 20% fructose (but not glucose) induces salt sensitive hypertension in part by enhancing NKCC2 activity. Sprague-Dawley rats were given 20% fructose or 20% glucose in drinking water for 1 week after which high salt diet (4% Na in chow) was started. Systolic blood pressure (SBP) was measured by tail cuff. In fructose-fed rats, the addition of 4% NaCl diet increased SBP from 120±10 to 128±6 mmHg on day 2 (p<0.01) and to 144±18 mmHg after 2 weeks (p<0.01). In rats fed glucose or high salt alone SBP was not changed. NKCC2 phosphorylation at Thr96,101 was enhanced by 161±51% (p<0.02) in rats fed fructose plus salt compared to salt alone and phospho-Ser126 was also enhanced by 151±40%. The phosphorylation of SPAK/OSR1 kinases was enhanced by 100±30% (p<0.04). Furosemide sensitive NaCl transport in TALs was enhanced by 36±11% in rats fed fructose plus high salt compared to salt alone (p<0.02). We concluded that a high fructose but not high glucose diet induces salt-sensitive hypertension in normal rats. The salt sensitivity may be in part due to higher NKCC2 phosphorylation and activity which was maintained during high salt intake.

Functional Cross‐talk in the Kidney between ß 3 Adrenergic Receptors and NKCC2 in the Thick Ascending Limb of the Loop of Henle

The thick ascending limb of the loop of Henle (TAL) reabsorbs ∼30% of the filtered NaCl in the kidney, in a process mediated by the apical Na-K-2Cl cotransporter NKCC2, which thus plays a crucial role in blood pressure regulation. It is known that catecholamine enhances NaCl reabsorption in the TAL acting through either ß1 or ß2 adrenergic receptors (BAR1 and BAR2). The presence and the possible functional role of ß3 adrenergic receptors (BAR3) in the TAL are still debated. We found that BAR3 are expressed at the basolateral membrane of TAL cells in mouse kidney. Tracking the expression of BAR3 along the nephron, we found that they are expressed also in other vasopressin-sensitive segments, suggesting their possible role in salt and water homeostasis. Interestingly, we found that NKCC2 phosphorylation, but not NKCC2 protein expression, significantly increased in mouse kidney slices stimulated with the BAR3 selective agonist BRL37344, demonstrating for the first time that the selective activation of BAR3 stimulates NKCC2 activity in the kidney. Of note, mice knock-out for both BAR1 and BAR2 showed a remarkable increase in NKCC2 phosphorylation compared to the control strain even in resting conditions demonstrating that BAR3, even when expressed without the other subtypes of the beta adrenergic receptors in the TAL, are able to regulate the activity of NKCC2. These data show the physiological relevance of BAR3 in the regulation of NKCC2 activity in the kidney and suggest BAR3 antagonists as possible and novel pharmacological tools for the treatment of hypertension.