Regulation of renal NaDC1 expression and citrate excretion by NBCe1-A

Citrate is critical for acid-base homeostasis and to prevent calcium nephrolithiasis. Both metabolic acidosis and hypokalemia decrease citrate excretion and increase expression of Na+-dicarboxylate cotransporter 1 (NaDC1; SLC13A2), the primary protein involved in citrate reabsorption. However, the mechanisms transducing extracellular signals and mediating these responses are incompletely understood. The purpose of the present study was to determine the role of the Na+-coupled electrogenic bicarbonate cotransporter (NBCe1) A variant (NBCe1-A) in citrate metabolism under basal conditions and in response to acid loading and hypokalemia. NBCe1-A deletion increased citrate excretion and decreased NaDC1 expression in the proximal convoluted tubules (PCT) and proximal straight tubules (PST) in the medullary ray (PST-MR) but not in the PST in the outer medulla (PST-OM). Acid loading wild-type (WT) mice decreased citrate excretion. NaDC1 expression increased only in the PCT and PST-MR and not in the PST-MR. In NBCe1-A knockout (KO) mice, the acid loading change in citrate excretion was unaffected, changes in PCT NaDC1 expression were blocked, and there was an adaptive increase in PST-MR. Hypokalemia in WT mice decreased citrate excretion; NaDC1 expression increased only in the PCT and PST-MR. NBCe1-A KO blocked both the citrate and NaDC1 changes. We conclude that 1) adaptive changes in NaDC1 expression in response to metabolic acidosis and hypokalemia occur specifically in the PCT and PST-MR, i.e., in cortical proximal tubule segments; 2) NBCe1-A is necessary for normal basal, metabolic acidosis and hypokalemia-stimulated citrate metabolism and does so by regulating NaDC1 expression in cortical proximal tubule segments; and 3) adaptive increases in PST-OM NaDC1 expression occur in NBCe1-A KO mice in response to acid loading that do not occur in WT mice.

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NBCe1-A is required for the renal ammonia and K+ response to hypokalemia

AJP Renal Physiology ◽

10.1152/ajprenal.00481.2019 ◽

2020 ◽

Vol 318 (2) ◽

pp. F402-F421 ◽

Cited By ~ 1

Author(s):

Hyun-Wook Lee ◽

Autumn N. Harris ◽

Michael F. Romero ◽

Paul A. Welling ◽

Charles S. Wingo ◽

...

Keyword(s):

Metabolic Acidosis ◽

Proximal Tubule ◽

Splice Variant ◽

Ammonia Excretion ◽

Wild Type ◽

Outer Medulla ◽

Ammonia Metabolism ◽

Tissue Sections ◽

K Response

Hypokalemia increases ammonia excretion and decreases K+ excretion. The present study examined the role of the proximal tubule protein NBCe1-A in these responses. We studied mice with Na+-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion [knockout (KO) mice] and their wild-type (WT) littermates were provided either K+ control or K+-free diet. We also used tissue sections to determine the effect of extracellular ammonia on NaCl cotransporter (NCC) phosphorylation. The K+-free diet significantly increased proximal tubule NBCe1-A and ammonia excretion in WT mice, and NBCe1-A deletion blunted the ammonia excretion response. NBCe1-A deletion inhibited the ammoniagenic/ammonia recycling enzyme response in the cortical proximal tubule (PT), where NBCe1-A is present in WT mice. In the outer medulla, where NBCe1-A is not present, the PT ammonia metabolism response was accentuated by NBCe1-A deletion. KO mice developed more severe hypokalemia and had greater urinary K+ excretion during the K+-free diet than did WT mice. This was associated with blunting of the hypokalemia-induced change in NCC phosphorylation. NBCe1-A KO mice have systemic metabolic acidosis, but experimentally induced metabolic acidosis did not alter NCC phosphorylation. Although KO mice have impaired ammonia metabolism, experiments in tissue sections showed that lack of ammonia does impair NCC phosphorylation. Finally, urinary aldosterone was greater in KO mice than in WT mice, but neither expression of epithelial Na+ channel α-, β-, and γ-subunits nor of H+-K+-ATPase α1- or α2-subunits correlated with changes in urinary K+. We conclude that NBCe1-A is critical for the effect of diet-induced hypokalemia to increase cortical proximal tubule ammonia generation and for the expected decrease in urinary K+ excretion.

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Acid-Base Effects of Combined Renal Deletion of NBCe1-A and NBCe1-B

AJP Renal Physiology ◽

10.1152/ajprenal.00358.2021 ◽

2022 ◽

Author(s):

Hyun-Wook Lee ◽

Jill W. Verlander ◽

Gary E Shull ◽

Autumn N. Harris ◽

I. David Weiner

Keyword(s):

Proximal Tubule ◽

Splice Variant ◽

Molecular Mechanisms ◽

Ammonia Excretion ◽

Basal Diet ◽

Acid Base ◽

Outer Medulla ◽

Ammonia Metabolism ◽

Induced Changes ◽

Acid Loading

The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deleting the A splice variant of the Na⁺-bicarbonate cotransporter, electrogenic, isoform 1 (NBCe1-A) partially blocks the effect of acidosis to increase urinary ammonia excretion, and this appears to involve the dysregulated expression of ammoniagenic enzymes in the proximal tubule (PT) in the cortex, but not in the outer medulla (OM). A second NBCe1 splice variant, NBCe1-B, is present throughout the PT, including the OM, where NBCe1-A is not present. The current studies determined the effects of combined renal deletion of NBCe1-A and NBCe1-B on systemic and proximal tubule ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. Since renal NBCe1-A and NBCe1-B expression is limited to the proximal tubule, Cre-positive mice had proximal tubule NBCe1-A/B deletion (PT-NBCe1-A/B KO). While on basal diet, PT-NBCe1-A/B KO mice had severe metabolic acidosis, yet urinary ammonia excretion was not changed significantly. PT-NBCe1-A/B KO decreased expression of phosphate-dependent glutaminase (PDG) and phosphoenolpyruvate carboxykinase (PEPCK) and increased expression of glutamine synthetase (GS), an ammonia recycling enzyme, in PT in both the cortex and OM. Exogenous acid-loading increased ammonia excretion in control mice, but PT-NBCe1-A/B KO prevented any increase. PT-NBCe1-A/B KO significantly blunted acid loading-induced changes in PDG, PEPCK, and GS expression in the proximal tubule in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to proximal tubule ammonia metabolism in the OM and thereby to systemic acid-base regulation.

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Role of KCNE1-Dependent K+ Fluxes in Mouse Proximal Tubule

Journal of the American Society of Nephrology ◽

10.1681/asn.v12102003 ◽

2001 ◽

Vol 12 (10) ◽

pp. 2003-2011

Author(s):

VOLKER VALLON ◽

FLORIAN GRAHAMMER ◽

KERSTIN RICHTER ◽

MARKUS BLEICH ◽

FLORIAN LANG ◽

...

Keyword(s):

Proximal Tubule ◽

Coupled Transport ◽

Wild Type ◽

Electrochemical Gradient ◽

Volume Depletion ◽

Luminal Membrane ◽

Clearance Studies ◽

Electrophysiological Studies ◽

Kidney Micropuncture

Abstract. The electrochemical gradient for K+ across the luminal membrane of the proximal tubule favors K+ fluxes to the lumen. Here it was demonstrated by immunohistochemistry that KCNE1 and KCNQ1, which form together the slowly activated component of the delayed rectifying K+ current in the heart, also colocalize in the luminal membrane of proximal tubule in mouse kidney. Micropuncture experiments revealed a reduced K+ concentration in late proximal and early distal tubular fluid as well as a reduced K+ delivery to these sites in KCNE1 knockout (-/-), compared with wild-type (+/+) mice. These observations would be consistent with KCNE1-dependent K+ fluxes to the lumen in proximal tubule. Electrophysiological studies in isolated perfused proximal tubules indicated that this K+ flux is essential to counteract membrane depolarization due to electrogenic Na+-coupled transport of glucose or amino acids. Clearance studies revealed an enhanced fractional urinary excretion of fluid, Na+, Cl-, and glucose in KCNE1 -/- compared with KCNE1 +/+ mice that may relate to an attenuated transport in proximal tubule and contribute to volume depletion in these mice, as indicated by higher hematocrit values.

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Basolateral membrane H-OH-HCO3 transport in the proximal tubule

AJP Renal Physiology ◽

10.1152/ajprenal.1989.256.5.f751 ◽

1989 ◽

Vol 256 (5) ◽

pp. F751-F765

Author(s):

P. A. Preisig ◽

R. J. Alpern

Keyword(s):

Metabolic Acidosis ◽

Proximal Tubule ◽

Basolateral Membrane ◽

Respiratory Acidosis ◽

Cell Voltage ◽

Transport Mechanisms ◽

Transport Capacity ◽

Chronic Metabolic Acidosis

This review focuses on the basolateral membrane mechanisms of H-OH-HCO3 transport in the proximal tubule. The mechanism that has the greatest transport capacity and mediates most of transepithelial H-HCO3 transport is the electrogenic, Na-3HCO3 cotransporter. This transporter has been extensively characterized in the salamander, rat, and rabbit proximal tubule, and has now been found in a number of other epithelia that effect transepithelial NaHCO3 transport. Transporter rate is sensitive to intra- and extracellular [Na], intra- and extracellular [HCO3]/pH, and cell voltage. Adaptations in transporter activity have been demonstrated in chronic metabolic acidosis and alkalosis, chronic respiratory acidosis and alkalosis, and chronic hyperfiltration. In addition to the Na-3HCO3 cotransporter, the basolateral membrane possesses both Na-dependent and -independent Cl-HCO3 exchangers, a H leak, and in the S3 proximal tubule an Na-H antiporter. The role of these H-OH-HCO3 transport mechanisms in transcellular HCO3 and Cl absorption and pHi defense is discussed.

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Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.2.f298 ◽

1999 ◽

Vol 277 (2) ◽

pp. F298-F302 ◽

Cited By ~ 68

Author(s):

Tong Wang ◽

Chao-Ling Yang ◽

Thecla Abbiati ◽

Patrick J. Schultheis ◽

Gary E. Shull ◽

...

Keyword(s):

Atpase Activity ◽

Proximal Tubule ◽

Apical Membrane ◽

Significant Inhibition ◽

Fluid Absorption ◽

Wild Type ◽

Significant Component ◽

Nhe Isoforms

NHE3 is the predominant isoform responsible for apical membrane Na+/H+exchange in the proximal tubule. Deletion of NHE3 by gene targeting results in an NHE3−/−mouse with greatly reduced proximal tubule[Formula: see text] absorption compared with NHE3+/+ animals (P. J. Schultheis, L. L. Clarke, P. Meneton, M. L. Miller, M. Soleimani, L. R. Gawenis, T. M. Riddle, J. J. Duffy, T. Doetschman, T. Wang, G. Giebisch, P. S. Aronson, J. N. Lorenz, and G. E. Shull. Nature Genet. 19: 282–285, 1998). The purpose of the present study was to evaluate the role of other acidification mechanisms in mediating the remaining component of proximal tubule [Formula: see text] reabsorption in NHE3−/− mice. Proximal tubule transport was studied by in situ microperfusion. Net rates of[Formula: see text] ( J HCO3) and fluid absorption ( J v) were reduced by 54 and 63%, respectively, in NHE3 null mice compared with controls. Addition of 100 μM ethylisopropylamiloride (EIPA) to the luminal perfusate caused significant inhibition of J HCO3 and J v in NHE3+/+ mice but failed to inhibit J HCO3 or J v in NHE3−/− mice, indicating lack of activity of NHE2 or other EIPA-sensitive NHE isoforms in the null mice. Addition of 1 μM bafilomycin caused a similar absolute decrement in J HCO3 in wild-type and NHE3 null mice, indicating equivalent rates of[Formula: see text] absorption mediated by H+-ATPase. Addition of 10 μM Sch-28080 did not reduce J HCO3 in either wild-type or NHE3 null mice, indicating lack of detectable H+-K+-ATPase activity in the proximal tubule. We conclude that, in the absence of NHE3, neither NHE2 nor any other EIPA-sensitive NHE isoform contributes to mediating [Formula: see text] reabsorption in the proximal tubule. A significant component of[Formula: see text] reabsorption in the proximal tubule is mediated by bafilomycin-sensitive H+-ATPase, but its activity is not significantly upregulated in NHE3 null mice.

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Role of TASK2 Potassium Channels Regarding Volume Regulation in Primary Cultures of Mouse Proximal Tubules

The Journal of General Physiology ◽

10.1085/jgp.200308820 ◽

2003 ◽

Vol 122 (2) ◽

pp. 177-190 ◽

Cited By ~ 71

Author(s):

Herve Barriere ◽

Radia Belfodil ◽

Isabelle Rubera ◽

Michel Tauc ◽

Florian Lesage ◽

...

Keyword(s):

Cell Volume ◽

Volume Regulation ◽

Primary Cultures ◽

Regulatory Volume Decrease ◽

Cell Volume Regulation ◽

Proximal Tubules ◽

Wild Type ◽

Convoluted Tubules ◽

K Currents

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using β-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 μM 293B, but blocked by 500 μM quinidine and 10 μM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.

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Upregulation of apical sodium-chloride cotransporter and basolateral chloride channels is responsible for the maintenance of salt-sensitive hypertension

AJP Renal Physiology ◽

10.1152/ajprenal.00340.2007 ◽

2008 ◽

Vol 295 (2) ◽

pp. F556-F567 ◽

Cited By ~ 35

Author(s):

Giovambattista Capasso ◽

Maria Rizzo ◽

Maria Lisa Garavaglia ◽

Francesco Trepiccione ◽

Miriam Zacchia ◽

...

Keyword(s):

Chloride Channels ◽

Renal Cortex ◽

Outer Medulla ◽

Western Blots ◽

Sodium Chloride Cotransporter ◽

Normotensive Strain ◽

Active Channels ◽

Salt Sensitive Hypertension ◽

Convoluted Tubules

We investigated which of the NaCl transporters are involved in the maintenance of salt-sensitive hypertension. Milan hypertensive (MHS) rats were studied 3 mo after birth. In MHS, compared with normotensive strain (MNS), mRNA abundance, quantified by competitive PCR on isolated tubules, was unchanged, both for Na+/H+ isoform 3 (NHE3) and Na+-K+-2Cl− (NKCC2), but higher (119%, n = 5, P < 0.005) for Na+-Cl− (NCC) in distal convoluted tubules (DCT). These results were confirmed by Western blots, which revealed: 1) unchanged NHE3 in the cortex and NKCC2 in the outer medulla; 2) a significant increase (52%, n = 6, P < 0.001) of NCC in the cortex; 3) α- and β-sodium channels [epithelial Na+ channel (ENaC)] unaffected in renal cortex and slightly reduced in the outer medulla, while γ-ENaC remained unchanged. Pendrin protein expression was unaffected. The role of NCC was reinforced by immunocytochemical studies showing increased NCC on the apical membrane of DCT cells of MHS animals, and by clearance experiments demonstrating a larger sensitivity ( P < 0.001) to bendroflumethiazide in MHS rats. Kidney-specific chloride channels (ClC-K) were studied by Western blot experiments on renal cortex and by patch-clamp studies on primary culture of DCT dissected from MNS and MHS animals. Electrophysiological characteristics of ClC-K channels were unchanged in MHS rats, but the number of active channels in a patch was 0.60 ± 0.21 ( n = 35) in MNS rats and 2.17 ± 0.59 ( n = 23) in MHS rats ( P < 0.05). The data indicate that, in salt-sensitive hypertension, there is a strong upregulation, both of NCC and ClC-K along the DCT, which explains the persistence of hypertension.

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Ammonia production and secretion by S3 proximal tubule segments from acidotic mice: role of ANG II

AJP Renal Physiology ◽

10.1152/ajprenal.00189.2003 ◽

2004 ◽

Vol 287 (4) ◽

pp. F707-F712 ◽

Cited By ~ 21

Author(s):

Glenn T. Nagami

Keyword(s):

Proximal Tubule ◽

Ang Ii ◽

Ammonia Production ◽

Low Sodium ◽

Luminal Perfusion ◽

And Control ◽

Acid Loading ◽

Proximal Tubule Segments ◽

Secretion Rates

ANG II has potent effects on ammonia production and secretion rates by the proximal tubule and is found in substantial concentrations in the lumen of the proximal tubule in vivo. Because our previous studies demonstrated that acid loading enhanced the stimulatory effects of ANG II on ammonia production and secretion by S2 proximal tubule segments, we examined the effect of ANG II on ammonia production and secretion by isolated, perfused S3 segments from nonacidotic control mice and acidotic mice given NH4Cl for 7 days. In the absence of ANG II, ammonia production and secretion rates were no different in S3 segments from acidotic and control mice. By contrast, when ANG II was present in the luminal perfusion solution, ammonia production and secretion rates were stimulated, in a losartan-inhibitable manner, to a greater extent in S3 segments from acidotic mice. Ammonia secretion rates in S3 segments were largely inhibited by perfusion with a low-sodium solution containing amiloride in the presence or absence of ANG II. These results demonstrated that isolated, perfused mouse S3 proximal tubule segments produce and secrete ammonia, that NH4Cl-induced acidosis does not affect the basal rates of ammonia production and secretion, and that ANG II, added to the luminal fluid, stimulates ammonia production and secretion to a greater extent in S3 segments from acidotic mice. These findings suggest that S3 segments, in the presence of ANG II, can contribute to the enhanced renal excretion that occurs with acid loading.

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Proximal tubule-specific glutamine synthetase deletion alters basal and acidosis-stimulated ammonia metabolism

AJP Renal Physiology ◽

10.1152/ajprenal.00547.2015 ◽

2016 ◽

Vol 310 (11) ◽

pp. F1229-F1242 ◽

Cited By ~ 18

Author(s):

Hyun-Wook Lee ◽

Gunars Osis ◽

Mary E. Handlogten ◽

Wouter H. Lamers ◽

Farrukh A. Chaudhry ◽

...

Keyword(s):

Metabolic Acidosis ◽

Glutamine Synthetase ◽

Proximal Tubule ◽

Ammonia Excretion ◽

Renal Proximal Tubule ◽

Acid Excretion ◽

Ammonia Metabolism ◽

Adaptive Changes ◽

Multiple Components

Glutamine synthetase (GS) catalyzes the recycling of NH4+ with glutamate to form glutamine. GS is highly expressed in the renal proximal tubule (PT), suggesting ammonia recycling via GS could decrease net ammoniagenesis and thereby limit ammonia available for net acid excretion. The purpose of the present study was to determine the role of PT GS in ammonia metabolism under basal conditions and during metabolic acidosis. We generated mice with PT-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Under basal conditions, PT-GS-KO increased urinary ammonia excretion significantly. Increased ammonia excretion occurred despite decreased expression of key proteins involved in renal ammonia generation. After the induction of metabolic acidosis, the ability to increase ammonia excretion was impaired significantly by PT-GS-KO. The blunted increase in ammonia excretion occurred despite greater expression of multiple components of ammonia generation, including SN1 (Slc38a3), phosphate-dependent glutaminase, phospho enolpyruvate carboxykinase, and Na+-coupled electrogenic bicarbonate cotransporter. We conclude that 1) GS-mediated ammonia recycling in the PT contributes to both basal and acidosis-stimulated ammonia metabolism and 2) adaptive changes in other proteins involved in ammonia metabolism occur in response to PT-GS-KO and cause an underestimation of the role of PT GS expression.

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Regulation of sodium transporters in the thick ascending limb of rat kidney: response to angiotensin II

AJP Renal Physiology ◽

10.1152/ajprenal.00307.2002 ◽

2003 ◽

Vol 285 (1) ◽

pp. F152-F165 ◽

Cited By ~ 87

Author(s):

Tae-Hwan Kwon ◽

Jakob Nielsen ◽

Young-Hee Kim ◽

Mark A. Knepper ◽

Jørgen Frøkiær ◽

...

Keyword(s):

Proximal Tubule ◽

Brush Border ◽

Rat Kidney ◽

Thick Ascending Limb ◽

Ang Ii ◽

Outer Medulla ◽

Protein Protein Interaction ◽

Sodium Transporters ◽

Outer Strip

The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) Na+ transporters. Rats were on a fixed intake of Na+ and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng·min-1·kg-1 ANG II (sc). Semiquantitative immunoblotting revealed that Na+/H+ exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 ± 28 (ANG II-50, n = 5), 166 ± 23 (ANG II-100, n = 7), and 167 ± 19% (ANG II-200, n = 4) of control levels ( n = 6, P < 0.05), whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive Na+-K+-2Cl- cotransporter (BSC-1) in the ISOM was also increased to 187 ± 28 (ANG II-50), 162 ± 23 (ANG II-100), and 166 ± 19% (ANG II-200) of control levels ( P < 0.05), but there were no changes in the abundance of Na+-K+-ATPase and the electroneutral Na+-HCO3 cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng·min-1·kg-1 for 5 days, n = 6) had a higher urinary pH compared with controls. NH4Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal Na+ and HCO3 reabsorption in response to ANG II.

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