Cotransport Activity Research Articles

Glucocorticoid-induced leucine zipper protein regulates sodium and potassium balance in the distal nephron

Glucocorticoid induced leucine zipper protein (GILZ) is analdosterone-regulated protein that controls sodium transport in cultured kidney epithelial cells. Mice lacking GILZ have been reported previously to have electrolyte abnormalities. However, the mechanistic basis has not been explored. Here we provide evidence supporting a role for GILZ in modulating the balance of renal sodium and potassium excretion by regulating the sodium-chloride cotransporter (NCC) activity in the distal nephron. Gilz-/- mice have a higher plasma potassium concentration and lower fractional excretion of potassium than wild type mice. Furthermore, knockout mice are more sensitive to NCC inhibition by thiazides than are the wild type mice, and their phosphorylated NCC expression is higher. Despite increased NCC activity, knockout mice do not have higher blood pressure than wild type mice. However, during sodium deprivation, knockout mice come into sodium balance more quickly, than do the wild type, without a significant increase in plasma renin activity. Upon prolonged sodium restriction, knockout mice develop frank hyperkalemia. Finally, in HEK293T cells, exogenous GILZ inhibits NCC activity at least in part by inhibiting SPAK phosphorylation. Thus, GILZ promotes potassium secretion by inhibiting NCC and enhancing distal sodium delivery to the epithelial sodium channel. Additionally, Gilz-/- mice have features resembling familial hyperkalemic hypertension, a human disorder that manifests with hyperkalemia associated variably with hypertension.

Sodium-chloride cotransporter activity regulated by extracellular potassium

The thiazide-sensitive sodium-chloride cotransporter (NCC) is exclusively expressed in the apical membrane of the renal distal convoluted tubule (DCT), and it is important for maintaining fluid and salt balance. NCC is responsible for electroneutral NaCl reabsorption, and its activity is determined by phosphorylation, which was reported to be regulated by WNK-stimulated Ste20-type kinases, Ste20-related proline alanine-rich kinase, and oxidative stress response 1 kinase. WNK kinases have chloride-binding sites, and WNK4 has the highest sensitivity to intracellular chloride concentration ([Cl−]i) compared with WNK1 and WNK3. NCC dysfunction often comes together with abnormal urinary potassium excretion, which is not only highlighted by Mendelian disorders such as familial hyperkalemic hypertension (FHHt) and Gitelman syndrome but also presented more frequently by long-term usage of thiazides, specific inhibitors of NCC. Recent studies have shown that extracellular potassium (K+) can modulate DCT cell membrane voltage and in turn intracellular Cl−, which regulates phosphorylation of WNK kinases. Additional Cl− independent mechanisms were also reported by several groups. This paper is a brief review of the recent discoveries on mechanisms of NCC regulation by extracellular potassium.

Activation of Renal (Pro)Renin Receptor Contributes to High Fructose-Induced Salt Sensitivity.

A high-fructose diet is shown to induce salt-sensitive hypertension, but the underlying mechanism largely remains unknown. The major goal of the present study was to test the role of renal (pro)renin receptor (PRR) in this model. In Sprague-Dawley rats, high-fructose intake increased renal expression of full-length PRR, which were attenuated by allopurinol. High-fructose intake also upregulated renal mRNA and protein expression of sodium/hydrogen exchanger 3 and Na/K/2Cl cotransporter, as well as in vivo Na/K/2Cl cotransporter activity, all of which were nearly completely blocked by a PRR decoy inhibitor PRO20 or allopurinol treatment. Parallel changes were observed for indices of intrarenal renin-angiotensin-system including renal and urinary renin and angiotensin II levels. Radiotelemetry demonstrated that high-fructose or a high-salt diet alone did not affect mean arterial pressure, but the combination of the 2 maneuvers induced a ≈10-mm Hg increase of mean arterial pressure, which was blunted by PRO20 or allopurinol treatment. In cultured human kidney 2 cells, both fructose and uric acid increased protein expression of soluble PRR in a time- and dose-dependent manner; fructose-induced PRR upregulation was inhibited by allopurinol. Taken together, our data suggest that fructose via uric acid stimulates renal expression of PRR/soluble PRR that stimulate sodium/hydrogen exchanger 3 and Na/K/2Cl cotransporter expression and intrarenal renin-angiotensin system to induce salt-sensitive hypertension.

Reduced Efficacy of the KCC2 Cotransporter Promotes Epileptic Oscillations in a Subiculum Network Model.

Ion regulation in the brain is a major determinant of neural excitability. Intracellular chloride in neurons, a partial determinant of the resting potential and the inhibitory reversal potentials, is regulated together with extracellular potassium via kation chloride cotransporters. During temporal lobe epilepsy, the homeostatic regulation of intracellular chloride is impaired in pyramidal cells, yet how this dysregulation may lead to seizures has not been explored. Using a realistic neural network model describing ion mechanisms, we show that chloride homeostasis pathology provokes seizure activity analogous to recordings from epileptogenic brain tissue. We show that there is a critical percentage of pathological cells required for seizure initiation. Our model predicts that restoration of the chloride homeostasis in pyramidal cells could be a viable antiepileptic strategy.

OP.7C.02] BLOOD PRESSURE-ASSOCIATED POLYMORPHISMS IN SLC4A7 (SODIUM/BICARBONATE CO-TRANSPORTER NBCN1) ARE LINKED WITH GENE EXPRESSION AND INTRACELLULAR PH REGULATION

Objective: Genomewide-association studies have identified a number of loci influencing blood pressure, one of which is located near SLC4A7 (lead SNP rs13082711). SLC4A7 encodes an electroneutral Na+, HCO3− co-transporter, NBCn1, which contributes to intracellular pH (pHi) regulation in a range of tissues, including vascular smooth muscle and endothelium. Additionally, the NBCn1 knockout mouse demonstrates an altered blood pressure phenotype. We investigated the potential influence of SLC4A7 genotype on DNA-protein interaction, gene expression, and pHi regulation. Design and method: Primary cultures of human umbilical vein endothelial cells and artery smooth muscle cells of different SLC4A7 genotypes were used. DNA-protein interactions were identified by formaldehyde-associated isolation of regulatory elements (FAIRE), electrophoretic mobility shift assays (EMSA), and DNA-pulldown assays. Gene expression was quantified by allelic imbalance analyses and protein immunoblots. Intracellular acidification was induced by the ammonium-prepulse method. NBCn1 protein function was evaluated as Na+, HCO3− co-transport activity, based on the rate of dimethyl-amiloride-insensitive, Na+- and HCO3−-dependent pHi recovery. A10 rat aortic smooth muscle cells transfected with either 326E or 326K SLC4A7 variants were used to verify primary cultures results. Results: FAIRE studies showed that the chromatin of the risk allele was preferentially open, allowing increased transcription. EMSAs demonstrated allele-dependent interactions with nuclear proteins. Protein sequencing from DNA-pulldown assays failed to identify the nuclear proteins involved. Consistent with allele-dependent DNA-protein interactions, the risk allele was associated with increased NBCn1 mRNA and protein expression. Furthermore, increased NBCn1 expression in risk allele carriers was associated with increased in vitro protein function, as demonstrated by greater Na+, HCO3− co-transport activity during intracellular acidosis and higher baseline steady-state pHi compared to protective allele homozygotes. A10 cells overexpressing either 326E or 326K NBCn1 variants demonstrated equivalent increases in Na+, HCO3− co-transport activity, suggesting the amino acid change is less important than expression levels. Conclusions: We have outlined a potential mechanism for the SLC4A7 locus influencing blood pressure via NBCn1 gene transcription, protein expression, and pHi regulation of human vascular smooth muscle and endothelial cells. This effect appears independent of the associated E326K amino acid change.

Abstract P189: Disruption of Cyp2c44 Increases Sodium Chloride Cotransporter Expression and Activity in the Distal Convoluted Tubule

Cytochrome-P450 (CYP) epoxygenase plays an important role in the regulation of renal sodium transport. Previous studies have demonstrated that global disruption of Cyp2c44 , a major epoxygenase in the renal tubules, increased ENaC activity in the collecting duct. In addition, we have demonstrated that an increase in dietary Na intake significantly increased the mRNA expression of CYP2c44 in the DCT. DCT is responsible for the reabsorption of 5-9% of filter Na load and is the target for thiazide diuretic. Na absorption in the DCT is mediated by NaCl cotransporter (NCC) and also by ENaC in the late part of DCT. However, the role of epoxyeicosatrienoic acid (EET) in the regulation Na transport in the DCT is not clear. We now examine the role of CYP2c44 and EET in the regulation of NCC expression. The disruption of Cyp2c44 increases the expression of NCC and ENaC activity in the DCT. The disruption of CYP2c44 also suppressed the renal expression of GPR75, a receptor which is activated by 20-hydroxyeicosatetraenoic acid (20-HETE). Renal Na clearance study also demonstrates that the effect of hydrochlorothiazide on Na excretion in Cyp2c44 knockout (KO) mice was significantly higher than those of WT mice. In addition, the disruption of Cyp2c44 significantly increased the expression of ste20-proline and alanine-rich kinase (SPAK) which is known to stimulate NCC activity by phosphorylation. Increased dietary Na intake decreases NCC expression and this effect is blunted in Cyp2c44 KO mice. The role of EET in the regulation of NCC expression is further suggested by the finding that high fat-diet induced increase in NCC expression is inhibited by application of EET. We conclude that CYP2c44-derived EET plays an important role in inhibiting NCC and ENaC in the DCT.

The regulation of Na+Cl- cotransporter by with-no-lysine kinase 4.

Abundant evidence supports that the NaCl cotransporter (NCC) activity is tightly regulated by the with-no-lysine (WNK) kinases. Here, we summarize the data regarding NCC regulation by WNKs, with a particular emphasis on WNK4. Several studies involving in-vivo and in-vitro models have provided paradoxical data regarding WNK4 regulation of the NCC. Although some studies show that WNK4 can activate the NCC, other equally compelling studies show that WNK4 inhibits the NCC. Recent studies have shown that WNK4 is regulated by the intracellular chloride concentration ([Cl]i), which could account for these paradoxical results. In conditions of high [Cl]i, WNK4 could act as an inhibitor via heterodimer formation with other WNKs. In contrast, when [Cl]i is low, WNK4 can activate Ste20-related, proline-alanine-rich kinase (SPAK)/oxidative stress responsive kinase 1 (OSR1) and thus the NCC. Modulation of WNK4 by [Cl]i has been shown to account for the potassium-sensing properties of the distal convoluted tubule. Other regulators of WNK4 include hormones and ubiquitination. Modulation of WNK4 activity by [Cl]i can account for its dual role on the NCC, and this has important physiological implications regarding the regulation of extracellular potassium concentration. Defective regulation of WNKs by ubiquitination explains most cases of familial hyperkalemic hypertension.

Abstract 125: Interleukin 6 Activates the Mineralocorticoid Receptor via Rac1, Increasing Sodium Chloride Cotransporter Expression and Activity

Hypertension is characterized by increased sodium (Na + ) reabsorption along the aldosterone-sensitive distal nephron (ASDN), as well as a chronic systemic inflammation. Interleukin-6 (IL-6) is thought to be a mediator of this inflammatory process. Interestingly, increased Na + reabsorption within the ASDN does not always correlate with increases in serum aldosterone (Aldo), the hormone that modulates Na + reabsorption via the mineralocorticoid receptor (MR). Thus, understanding how increased MR-mediated Na + reabsorption may occur independent of Aldo stimulation is critical. We hypothesize that IL-6 transactivates the MR via Rac1, increasing sodium chloride cotransporter (NCC) expression and activity. To demonstrate IL-6-mediated MR activation via Rac1, mDCT15 cells were transfected with mineralocorticoid response element (MRE)-luciferase reporter constructs, treated with vehicle, Aldo (100nM), IL-6 (100ng/mL) and/or Rac1 inhibitor (EHT1864, 20uMol), and luciferase assays performed (48hrs). To determine if IL-6 increases NCC expression, in vivo , we perfused kidneys with IL-6 (intra-renal, 16ng/hr, 1-3d) and the cortex was isolated for WB analysis of total NCC expression. To determine direct IL-6 mediated effects on NCC activity, we performed thiazide-sensitive 22 Na + -uptake studies in cell monolayers in presence of: vehicle, IL-6 (100ng/mL for 45min or 6hrs) or IL-6+MR antagonist (spironolactone, 20uMol). IL-6 treatment significantly increased luciferase activity (2.6±0.9 fold/MRE-only), which was reduced with Rac1 inhibition (0.2±0.08, p<0.01, n=8-10) demonstrating a Rac1-dependent activation of MR via IL-6. In the kidney cortex, IL-6 infusion increased total NCC expression (>2.5 fold) after day 3, as compared to vehicle. In addition, 22 Na + -uptake studies (n=6) revealed an IL-6-mediated increase in Na + transport (1927±40 nmol/mg/20min vs. 1503±7 nmol/mg/20min, p<0.0001), which was reduced with the MR antagonist (1721±25nmol/mg, p<0.001). These data suggest that IL-6 activates the MR via Rac1, leading to increased NCC expression and activity. These data provide evidence for alternate mechanisms of increased ASDN Na + uptake during inflammatory and hypertensive conditions, independently of Aldo-mediated signaling.

Basolateral Kir4.1 activity in the distal convoluted tubule regulates K secretion by determining NaCl cotransporter activity.

Renal potassium (K) secretion plays a key role in maintaining K homeostasis. The classic mechanism of renal K secretion is focused on the connecting tubule and cortical collecting duct, in which K is uptaken by basolateral Na-K-ATPase and is secreted into the lumen by apical ROMK (Kir1.1) and Ca-activated big conductance K channel. Recently, genetic studies and animal models have indicated that inwardly rectifying K channel 4.1 (Kir4.1 or Kcnj10) in the distal convoluted tubule (DCT) may play a role in the regulation of K secretion in the aldosterone-sensitive distal nephron by targeting the NaCl cotransporter (NCC). This review summarizes recent progresses regarding the role of Kir4.1 in the regulation of NCC and K secretion. Kir4.1 is expressed in the basolateral membrane of the DCT, and plays a predominant role in contributing to the basolateral K conductance and in participating in the generation of negative membrane potential. Kir4.1 is also the substrate of src-family tyrosine kinase and the stimulation of src-family tyrosine kinase activates Kir4.1 activity in the DCT. The genetic deletion or functional inhibition of Kir4.1 depolarizes the membrane of the DCT, inhibits ste20-proline-alanine rich kinase, and suppresses NCC activity. Moreover, the downregulation of Kir4.1 increases epithelial Na channel expression in the collecting duct and urinary K excretion. Finally, mice with low Kir4.1 activity in the DCT are hypomagnesemia and hypokalemia. Recent progress in exploring the regulation and the function of Kir4.1 in the DCT strongly indicates that Kir4.1plays an important role in initiating the regulation of renal K secretion by targeting NCC and it may serves as a K sensor in the kidney.

KCC3 deficiency-induced disruption of paranodal loops and impairment of axonal excitability in the peripheral nervous system

The autosomal recessive Hereditary Motor and Sensory Neuropathy with Agenesis of the Corpus Callosum (HMSN/ACC) is associated with the dysfunction of the K+–Cl− cotransporter type 3 (KCC3), which is an electroneutral cotransporter. We previously found that the inhibition of KCC3 cotransporter activity reduces the propagation of action potentials in the peripheral nervous system (PNS). However, the pathogenesis by which KCC3 deficiency impairs peripheral nerve function remains to be examined. Thus, we conducted imaging and electrophysiological studies in the peripheral nerves of KCC3−/− mice at various ages. Analysis using transmission electron microscopy (TEM) revealed an age-dependent progressive swelling of microvilli and disorganization of paranodal loops in KCC3−/− nerves. Yet, no mislocated voltage-dependent channels were observed between the nodes and juxtaparanodes of KCC3−/− nerves. However, electrophysiological studies using the threshold tracking technique indicated a reduced stimulus–response curve slope with an elevated rheobase, a decreased strength–duration time constant, diminished persistent Na+ currents, and an outward deviation of threshold electrotonus in KCC3−/− nerves compared to wild-type nerves. These functional changes indicate an overall reduction in axonal excitability and suggest an increase in paranodal conductance, which was relevant to the pathology at the paranode. Altogether, our findings highlight the importance of KCC3 in maintaining paranodal integrity and in optimizing the propagation of action potentials along peripheral nerves.

A novel mutant Na+/HCO3− cotransporter NBCe1 in a case of compound‐heterozygous inheritance of proximal renal tubular acidosis

The inheritance of two defective alleles of SLC4A4, the gene that encodes the widely-expressed electrogenic sodium bicarbonate cotransporter NBCe1, results in the bicarbonate-wasting disease proximal renal tubular acidosis (pRTA). In the present study, we report the first case of compound-heterozygous inheritance of pRTA (p.Arg510His/p.Gln913Arg) in an individual with low blood pH, blindness and neurological signs that resemble transient ischaemic attacks. We employ fluorescence microscopy on non-polarized (human embryonic kidney) and polarized (Madin-Darby canine kidney) renal cell lines and electrophysiology on Xenopus oocytes to characterize the mutant transporters (R510H and Q913R). Both mutant transporters exhibit enhanced intracellular retention in renal cells, an observation that probably explains the HCO3- transport deficit in the individual. Both mutants retain a close-to-normal per molecule Na+ /HCO3- cotransport activity in Xenopus oocytes, suggesting that they are suitable candidates for folding-correction therapy. However, Q913R expression is uniquely associated with a depolarizing, HCO3- independent, Cl- -conductance in oocytes that could have pathological consequences if expressed in the cells of patients. Proximal renal tubular acidosis (pRTA) is a rare, recessively-inherited disease characterized by abnormally acidic blood, blindness, as well as below average height and weight. pRTA is typically associated with homozygous mutation of the solute carrier 4 family gene SLC4A4. SLC4A4 encodes the electrogenic sodium bicarbonate cotransporter NBCe1, a membrane protein that acts to maintain intracellular and plasma pH. We present the first description of a case of compound-heterozygous inheritance of pRTA. The individual has inherited two mutations in NBCe1: p.Arg510His (R510H) and p.Gln913Arg (Q913R), one from each parent. In addition to the usual features of pRTA, the patient exhibits unusual signs, such as muscle spasms and fever. We have recreated these mutant transporters for expression in model systems. We find that both of the mutant proteins exhibit substantial intracellular retention when expressed in mammalian renal cell lines. When expressed in Xenopus oocytes, we find that the R510H and Q913R-mutant NBCe1 molecules exhibit apparently normal Na+ /HCO3- cotransport activity but that Q913R is associated with an unusual HCO3- independent anion-leak. We conclude that a reduced accumulation of NBCe1 protein in the basolateral membrane of proximal-tubule epithelia is the most probable cause of pRTA in this case. We further note that the Q913R-associated anion-leak could itself be pathogenic if expressed in the plasma membrane of mammalian cells, compromising the benefit of strategies aiming to enhance mutant NBCe1 accumulation in the plasma membrane.

The EP3 receptor regulates water excretion in response to high salt intake

The mechanisms by which prostanoids contribute to the maintenance of whole body water homeostasis are complex and not fully understood. The present study demonstrates that an EP3-dependent feedback mechanism contributes to the regulation of water homeostasis under high-salt conditions. Rats on a normal diet and tap water were placed in metabolic cages and given either sulprostone (20 μg·kg-1·day-1) or vehicle for 3 days to activate EP3 receptors in the thick ascending limb (TAL). Treatment was continued for another 3 days in rats given either 1% NaCl in the drinking water or tap water. Sulprostone decreased expression of cyclooxygenase 2 (COX-2) expression by ∼75% in TAL tubules from rats given 1% NaCl concomitant with a ∼60% inhibition of COX-2-dependent PGE2 levels in the kidney. Urine volume increased after ingestion of 1% NaCl but was reduced ∼40% by sulprostone. In contrast, the highly selective EP3 receptor antagonist L-798106 (100 μg·kg-1·day-1), which increased COX-2 expression and renal PGE2 production, increased urine volume in rats given 1% NaCl. Sulprostone increased expression of aquaporin-2 (AQP2) in the inner medullary collecting duct plasma membrane in association with an increase in phosphorylation at Ser269 and decrease in Ser261 phosphorylation; antagonism of EP3 with L-798106 reduced AQP2 expression. Thus, although acute activation of EP3 by PGE2 in the TAL and collecting duct inhibits the Na-K-2Cl cotransporter and AQP2 activity, respectively, chronic activation of EP3 in vivo limits the extent of COX-2-derived PGE2 synthesis, thereby mitigating the inhibitory effects of PGE2 on these transporters and decreasing urine volume.

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.

Interleukin-17A Regulates Renal Sodium Transporters and Renal Injury in Angiotensin II-Induced Hypertension.

Angiotensin II-induced hypertension is associated with an increase in T-cell production of interleukin-17A (IL-17A). Recently, we reported that IL-17A(-/-) mice exhibit blunted hypertension, preserved natriuresis in response to a saline challenge, and decreased renal sodium hydrogen exchanger 3 expression after 2 weeks of angiotensin II infusion compared with wild-type mice. In the current study, we performed renal transporter profiling in mice deficient in IL-17A or the related isoform, IL-17F, after 4 weeks of Ang II infusion, the time when the blood pressure reduction in IL-17A(-/-) mice is most prominent. Deficiency of IL-17A abolished the activation of distal tubule transporters, specifically the sodium-chloride cotransporter and the epithelial sodium channel and protected mice from glomerular and tubular injury. In human proximal tubule (HK-2) cells, IL-17A increased sodium hydrogen exchanger 3 expression through a serum and glucocorticoid-regulated kinase 1-dependent pathway. In mouse distal convoluted tubule cells, IL-17A increased sodium-chloride cotransporter activity in a serum and glucocorticoid-regulated kinase 1/Nedd4-2-dependent pathway. In both cell types, acute treatment with IL-17A induced phosphorylation of serum and glucocorticoid-regulated kinase 1 at serine 78, and treatment with a serum and glucocorticoid-regulated kinase 1 inhibitor blocked the effects of IL-17A on sodium hydrogen exchanger 3 and sodium-chloride cotransporter. Interestingly, both HK-2 and mouse distal convoluted tubule 15 cells produce endogenous IL-17A. IL17F had little or no effect on blood pressure or renal sodium transporter abundance. These studies provide a mechanistic link by which IL-17A modulates renal sodium transport and suggest that IL-17A inhibition may improve renal function in hypertension and other autoimmune disorders.

The expression, regulation, and function of Kir4.1 (Kcnj10) in the mammalian kidney.

Kir4.1 is an inwardly rectifying potassium (K(+)) channel and is expressed in the brain, inner ear, and kidney. In the kidney, Kir4.1 is expressed in the basolateral membrane of the late thick ascending limb (TAL), the distal convoluted tubule (DCT), and the connecting tubule (CNT)/cortical collecting duct (CCD). It plays a role in K(+) recycling across the basolateral membrane in corresponding nephron segments and in generating negative membrane potential. The renal phenotypes of the loss-function mutations of Kir4.1 include mild salt wasting, hypomagnesemia, hypokalemia, and metabolic alkalosis, suggesting that the disruption of Kir4.1 mainly impairs the transport in the DCT. Patch-clamp experiments and immunostaining demonstrate that Kir4.1 plays a predominant role in determining the basolateral K(+) conductance in the DCT. However, the function of Kir4.1 in the TAL and CNT/CCD is not essential, because K(+) channels other than Kir4.1 are also expressed. The downregulation of Kir4.1 in the DCT reduced basolateral chloride (Cl(-)) conductance, suppressed the expression of ste20 proline-alanine-rich kinase (SPAK), and decreased Na-Cl cotransporter (NCC) expression and activity. This suggests that Kir4.1 regulates NCC expression by the modulation of the Cl(-)-sensitive with-no-lysine kinase-SPAK pathway.

Ae4 (Slc4a9) is an electroneutral monovalent cation-dependent Cl-/HCO3- exchanger.

Ae4 (Slc4a9) belongs to the Slc4a family of Cl(-)/HCO3 (-) exchangers and Na(+)-HCO3 (-) cotransporters, but its ion transport cycle is poorly understood. In this study, we find that native Ae4 activity in mouse salivary gland acinar cells supports Na(+)-dependent Cl(-)/HCO3 (-) exchange that is comparable with that obtained upon heterologous expression of mouse Ae4 and human AE4 in CHO-K1 cells. Additionally, whole cell recordings and ion concentration measurements demonstrate that Na(+) is transported by Ae4 in the same direction as HCO3 (-) (and opposite to that of Cl(-)) and that ion transport is not associated with changes in membrane potential. We also find that Ae4 can mediate Na(+)-HCO3 (-) cotransport-like activity under Cl(-)-free conditions. However, whole cell recordings show that this apparent Na(+)-HCO3 (-) cotransport activity is in fact electroneutral HCO3 (-)/Na(+)-HCO3 (-) exchange. Although the Ae4 anion exchanger is thought to regulate intracellular Cl(-) concentration in exocrine gland acinar cells, our thermodynamic calculations predict that the intracellular Na(+), Cl(-), and HCO3 (-) concentrations required for Ae4-mediated Cl(-) influx differ markedly from those reported for acinar secretory cells at rest or under sustained stimulation. Given that K(+) ions share many properties with Na(+) ions and reach intracellular concentrations of 140-150 mM (essentially the same as extracellular [Na(+)]), we hypothesize that Ae4 could mediate K(+)-dependent Cl(-)/HCO3 (-) exchange. Indeed, we find that Ae4 mediates Cl(-)/HCO3 (-) exchange activity in the presence of K(+) as well as Cs(+), Li(+), and Rb(+) In summary, our results strongly suggest that Ae4 is an electroneutral Cl(-)/nonselective cation-HCO3 (-) exchanger. We postulate that the physiological role of Ae4 in secretory cells is to promote Cl(-) influx in exchange for K(+)(Na(+)) and HCO3 (-) ions.

Extreme blood pressure polymorphisms influence the bicarbonate‐independent conductance of NBCn1 but not its Na/HCO3 cotransport activity

Genome‐wide association studies identified SLC4A7 as a locus associated with extreme blood pressure levels (Ehret G.B. et al. Nature. 2011 vol. 478 p.103). The product of this gene, the electroneutral Na+‐coupled HCO3− cotransporter NBCn1 is an important mediator of acid–base movement wherever it is expressed, which includes epithelial tissues, neurons and vascular smooth muscle. In addition to Na/HCO3 cotransport, NBCn1 mediates a steady‐state conductance (Gn1) that is independent of HCO3− transport. Exome sequencing of SLC4A7 was conducted among African and European American individuals from the Atherosclerosis Risk in Communities (ARIC) study. Individuals with whole exome sequence data were selected from the extremes (above upper quartile and below lower quartile) of the systolic and diastolic blood pressure distributions. In total, 42 single‐nucleotide polymorphisms (SNPs) associated with extremes of blood pressure were found within SLC4A7. Six SNPs located in the NBCn1 coding sequence (these result in the E252D, L261M, E326K, T563A, N756S, and P896S missense mutations) were selected for insertion into the human NBCn1‐B variant to test their function when expressed in Xenopus oocytes. All six NBCn1‐B mutants exhibited a near‐normal Na/HCO3 cotransport activity as determined by the rate of intracellular pH (pHi) recovery from a CO2‐induced acidification. However, when the Gn1 is normalized to cotransport activity to control for surface presentation, five of the mutants displayed a 12–35% reduction in the measured Gn1. The sixth mutation, P896S, which resides in the fourth extracellular loop (EL4) of NBCn1, caused a reduction in Gn1 of 84% on the wild‐type E326 background and 76% on the extreme blood‐pressure–associated K326 background. We conclude from this work that the polymorphisms we tested have little, if any effect on the Na/HCO3 cotransport rate, but can have substantial effects on the Gn1. Thus, these polymorphisms would not be expected to have a major, direct effect on pHi, but would be expected to affect cell membrane voltage (Vm). In particular, reduced Gn1 would be expected to hyperpolarize cells (i.e., shift Vm toward more negative voltages). Such shifts in Vm would be expected to secondarily affect a wide range of ion channels and electrogenic solute carriers in the SLC superfamily.Support or Funding InformationR01‐HL090969, R01‐DK30344, R01‐DK81567

Expression of apical Na(+)-L-glutamine co-transport activity, B(0)-system neutral amino acid co-transporter (B(0)AT1) and angiotensin-converting enzyme 2 along the jejunal crypt-villus axis in young pigs fed a liquid formula.

Gut apical amino acid (AA) transport activity is high at birth and during suckling, thus being essential to maintain luminal nutrient-dependent mucosal growth through providing AA as essential metabolic fuel, substrates and nutrient stimuli for cellular growth. Because system-B(0) Na(+)-neutral AA co-transporter (B(0)AT1, encoded by the SLC6A19 gene) plays a dominant role for apical uptake of large neutral AA including L-Gln, we hypothesized that high apical Na(+)-Gln co-transport activity, and B(0)AT1 (SLC6A19) in co-expression with angiotensin-converting enzyme 2 (ACE2) were expressed along the entire small intestinal crypt-villus axis in young animals via unique control mechanisms. Kinetics of Na(+)-Gln co-transport activity in the apical membrane vesicles, prepared from epithelial cells sequentially isolated along the jejunal crypt-villus axis from liquid formula-fed young pigs, were measured with the membrane potential being clamped to zero using thiocyanate. Apical maximal Na(+)-Gln co-transport activity was much higher (p<0.05) in the upper villus cells than in the middle villus (by 29%) and the crypt (by 30%) cells, whereas Na(+)-Gln co-transport affinity was lower (p<0.05) in the upper villus cells than in the middle villus and the crypt cells. The B(0)AT1 (SLC6A19) mRNA abundance was lower (p<0.05) in the crypt (by 40-47%) than in the villus cells. There were no significant differences in B(0)AT1 and ACE2 protein abundances on the apical membrane among the upper villus, the middle villus and the crypt cells. Our study suggests that piglet fast growth is associated with very high intestinal apical Na(+)-neutral AA uptake activities via abundantly co-expressing B(0)AT1 and ACE2 proteins in the apical membrane and by transcribing the B(0)AT1 (SLC6A19) gene in the epithelia along the entire crypt-villus axis.

The influence of erythrocyte maturity on ion transport and membrane lipid composition in the rat.

Significant relationships between ion transport and membrane lipid composition (cholesterol, total phospholipids and sphingomyelins) were found in erythrocytes of salt hypertensive Dahl rats. In these animals mean cellular hemoglobin content correlated negatively with Na(+)-K(+) pump activity and Na(+) leak but positively with Na(+)-K(+) cotransport activity. Immature erythrocytes exhibit lower mean cellular hemoglobin content (MCHC) than mature ones. The aim of the present study was to find a relationship between erythrocyte maturity, membrane lipid composition and ion transport activity in Wistar rats aged three months which were subjected to repeated hemorrhage (blood loss 2 ml/day for 6 days) to enrich circulating erythrocytes with immature forms. Immature and mature erythrocyte fractions in control and hemorrhaged rats were separated by repeated centrifugation. Hemorrhaged rats had increased number of reticulocytes but reduced hematocrit and MCHC compared to control rats. Immature erythrocytes of hemorrhaged rats differed from mature ones of control animals by elevated Na(+)-K(+) pump activity, reduced Na(+)-K(+) cotransport activity and increased Rb(+) leak. These ion transport changes in immature erythrocytes were accompanied by higher concentration of total phospholipids in their cell membranes. Membrane phospholipid content correlated positively with Na(+)-K(+) pump activity and cation leaks but negatively with Na(+)-K(+) cotransport activity. Moreover, they were also negatively related with MCHC which correlated negatively with Na(+)-K(+) pump activity and Rb(+) leak but positively with Na(+)-K(+) cotransport activity. Thus certain abnormalities of erythrocyte ion transport and membrane lipid composition detected in hypertensive animals might be caused by higher incidence of immature cells.

Ventricular Na+-HCO3- Cotransporter Activity and Intracellular H+ Mobility are Remodeled in Cardiac Hypertrophy and Heart Failure

Intracellular pH (pHi) in ventricular myocytes (VMs) is tightly regulated by the action of sarcolemmal transporters (Na+/H+ exchanger, NHE1 and Na+-HCO3- cotransporters, NBCs). Upregulation of NHE1 activity may act causally in the development of maladaptive hypertrophy and heart failure (HF). We have investigated whether remodeling of other aspects of pHi regulation is a common feature in animal models of hypertrophy and HF.Compensated cardiac hypertrophy was induced in adult C57BL6 mice by delivery of subcutaneous isoprenaline (10mg/kg/day) for 2 weeks. Decompensated HF was induced in adult female sheep by transvenous right ventricular tachypacing for 5-8 weeks. VMs were isolated by enzymatic digestion and H+-equivalent fluxes on NHE1 and NBCs were measured in cells loaded with SNARF1-AM from pHi-recovery rates following a 20mM ammonium prepulse in HEPES- or HCO3--buffered superfusates.In VMs from the mouse hypertrophy model, NHE1 flux was unchanged, while flux on NBCs significantly increased (by 3.1±1.7mM/min at pHi 6.9; 73% increase vs. control). Immunoblotting demonstrated an increase in NBCe1 and NBCn1 protein expression in hypertrophied mouse VMs compared with control. In VMs from HF sheep, NBC-dependent flux also increased significantly (by 6.9±3.6mM/min at pHi 6.8; 510% increase vs. control), while NHE1-dependent flux was unchanged. This consequent increase in total Na+-dependent acid efflux was accompanied by a 3-fold decrease in intrinsic cytoplasmic H+ mobility (control 125 μm2/s vs. HF 40μm2/s).In conclusion, in VMs from a mouse model of cardiac hypertrophy and a sheep model of HF, NBC activity was significantly increased, while H+ mobility significantly decreased in HF, suggesting a profound remodeling of the pHi regulatory system that occurs early in the development of hypertrophy and is sustained in HF.Funded by BHF (RDVJ, AT, ML) & The Physiological Society (RVDJ).