Mechanism of Hyperkalemia-Induced Metabolic Acidosis

Background Hyperkalemia in association with metabolic acidosis that are out of proportion to changes in glomerular filtration rate defines type 4 renal tubular acidosis (RTA), the most common RTA observed, but the molecular mechanisms underlying the associated metabolic acidosis are incompletely understood. We sought to determine whether hyperkalemia directly causes metabolic acidosis and, if so, the mechanisms through which this occurs.Methods We studied a genetic model of hyperkalemia that results from early distal convoluted tubule (DCT)–specific overexpression of constitutively active Ste20/SPS1-related proline-alanine–rich kinase (DCT-CA-SPAK).Results DCT-CA-SPAK mice developed hyperkalemia in association with metabolic acidosis and suppressed ammonia excretion; however, titratable acid excretion and urine pH were unchanged compared with those in wild-type mice. Abnormal ammonia excretion in DCT-CA-SPAK mice associated with decreased proximal tubule expression of the ammonia-generating enzymes phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and overexpression of the ammonia-recycling enzyme glutamine synthetase. These mice also had decreased expression of the ammonia transporter family member Rhcg and decreased apical polarization of H+-ATPase in the inner stripe of the outer medullary collecting duct. Correcting the hyperkalemia by treatment with hydrochlorothiazide corrected the metabolic acidosis, increased ammonia excretion, and normalized ammoniagenic enzyme and Rhcg expression in DCT-CA-SPAK mice. In wild-type mice, induction of hyperkalemia by administration of the epithelial sodium channel blocker benzamil caused hyperkalemia and suppressed ammonia excretion.Conclusions Hyperkalemia decreases proximal tubule ammonia generation and collecting duct ammonia transport, leading to impaired ammonia excretion that causes metabolic acidosis.

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NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis

Journal of the American Society of Nephrology ◽

10.1681/asn.2017080935 ◽

2018 ◽

Vol 29 (4) ◽

pp. 1182-1197 ◽

Cited By ~ 11

Author(s):

Hyun-Wook Lee ◽

Gunars Osis ◽

Autumn N. Harris ◽

Lijuan Fang ◽

Michael F. Romero ◽

...

Keyword(s):

Metabolic Acidosis ◽

Proximal Tubule ◽

Phosphoenolpyruvate Carboxykinase ◽

Molecular Mechanisms ◽

Collecting Duct ◽

Critical Role ◽

Ammonia Excretion ◽

Urine Ph ◽

Ammonia Metabolism ◽

Acid Loading

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

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Intercalated cell-specific Rh B glycoprotein deletion diminishes renal ammonia excretion response to hypokalemia

AJP Renal Physiology ◽

10.1152/ajprenal.00301.2012 ◽

2013 ◽

Vol 304 (4) ◽

pp. F422-F431 ◽

Cited By ~ 20

Author(s):

Jesse M. Bishop ◽

Hyun-Wook Lee ◽

Mary E. Handlogten ◽

Ki-Hwan Han ◽

Jill W. Verlander ◽

...

Keyword(s):

Metabolic Acidosis ◽

Collecting Duct ◽

Ammonia Excretion ◽

Ammonia Concentration ◽

Intercalated Cells ◽

Cortical Collecting Duct ◽

Intercalated Cell ◽

Transporter Family ◽

Total Ammonia ◽

Normal Increase

The ammonia transporter family member, Rh B Glycoprotein (Rhbg), is an ammonia-specific transporter heavily expressed in the kidney and is necessary for the normal increase in ammonia excretion in response to metabolic acidosis. Hypokalemia is a common clinical condition in which there is increased renal ammonia excretion despite the absence of metabolic acidosis. The purpose of this study was to examine Rhbg's role in this response through the use of mice with intercalated cell-specific Rhbg deletion (IC-Rhbg-KO). Hypokalemia induced by feeding a K+-free diet increased urinary ammonia excretion significantly. In mice with intact Rhbg expression, hypokalemia increased Rhbg protein expression in intercalated cells in the cortical collecting duct (CCD) and in the outer medullary collecting duct (OMCD). Deletion of Rhbg from intercalated cells inhibited hypokalemia-induced changes in urinary total ammonia excretion significantly and completely prevented hypokalemia-induced increases in urinary ammonia concentration, but did not alter urinary pH. We conclude that hypokalemia increases Rhbg expression in intercalated cells in the cortex and outer medulla and that intercalated cell Rhbg expression is necessary for the normal increase in renal ammonia excretion in response to hypokalemia.

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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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Effects of ischemia-reperfusion injury on renal ammonia metabolism and the collecting duct

AJP Renal Physiology ◽

10.1152/ajprenal.00437.2006 ◽

2007 ◽

Vol 293 (4) ◽

pp. F1342-F1354 ◽

Cited By ~ 21

Author(s):

Ki-Hwan Han ◽

Hye-Young Kim ◽

Byron P. Croker ◽

Sirirat Reungjui ◽

Su-Youn Lee ◽

...

Keyword(s):

Metabolic Acidosis ◽

Proximal Tubule ◽

Renal Injury ◽

Collecting Duct ◽

Ischemia Reperfusion ◽

Ammonia Excretion ◽

Intercalated Cells ◽

Acute Renal Injury ◽

Intercalated Cell ◽

Medullary Collecting Duct

Acute renal injury induces metabolic acidosis, but its specific effects on the collecting duct, the primary site for urinary ammonia secretion, the primary component of net acid excretion, are incompletely understood. We induced ischemia-reperfusion (I/R) acute renal injury in Sprague-Dawley rats by clamping the renal pedicles bilaterally for 30 min followed by reperfusion for 6 h. Control rats underwent sham surgery without renal pedicle clamping. I/R injury decreased urinary ammonia excretion significantly but did not persistently alter urine volume, Na+, K+, or bicarbonate excretion. Histological examination demonstrated cellular damage in the outer and inner medullary collecting duct, as well as in the proximal tubule and the thick ascending limb of the loop of Henle. A subset of collecting duct cells were damaged and/or detached from the basement membrane; these cells were present predominantly in the outer medulla and were less frequent in the inner medulla. Immunohistochemistry identified that the damaged/detached cells were A-type intercalated cells, not principal cells. Both TdT-mediated dUTP nick-end labeling (TUNEL) staining and transmission electron microscopic examination demonstrated apoptosis but not necrosis. However, immunoreactivity for caspase-3 was observed in the proximal tubule, but not in collecting duct intercalated cells, suggesting that mechanism(s) of collecting duct intercalated cell apoptosis differ from those operative in the proximal tubule. We conclude that I/R injury decreases renal ammonia excretion and is associated with intercalated cell-specific detachment and apoptosis in the outer and inner medullary collecting duct. These effects likely contribute to the metabolic acidosis frequently observed in acute renal injury.

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Effect of collecting duct-specific deletion of both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on renal response to metabolic acidosis

AJP Renal Physiology ◽

10.1152/ajprenal.00176.2013 ◽

2014 ◽

Vol 306 (4) ◽

pp. F389-F400 ◽

Cited By ~ 25

Author(s):

Hyun-Wook Lee ◽

Jill W. Verlander ◽

Mary E. Handlogten ◽

Ki-Hwan Han ◽

I. David Weiner

Keyword(s):

Metabolic Acidosis ◽

Collecting Duct ◽

Ammonia Excretion ◽

Severe Metabolic Acidosis ◽

Urine Ph ◽

Renal Response ◽

Rh Glycoproteins ◽

Acid Loading ◽

Specific Deletion

The Rhesus (Rh) glycoproteins, Rh B and Rh C Glycoprotein (Rhbg and Rhcg, respectively), are ammonia-specific transporters expressed in renal distal nephron and collecting duct sites that are necessary for normal rates of ammonia excretion. The purpose of the current studies was to determine the effect of their combined deletion from the renal collecting duct (CD-Rhbg/Rhcg-KO) on basal and acidosis-stimulated acid-base homeostasis. Under basal conditions, urine pH and ammonia excretion and serum HCO3− were similar in control (C) and CD-Rhbg/Rhcg-KO mice. After acid-loading for 7 days, CD-Rhbg/Rhcg-KO mice developed significantly more severe metabolic acidosis than did C mice. Acid loading increased ammonia excretion, but ammonia excretion increased more slowly in CD-Rhbg/Rhcg-KO and it was significantly less than in C mice on days 1–5. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice on days 1, 3, and 5 of acid loading. Metabolic acidosis increased phosph enolpyruvate carboxykinase (PEPCK) and Na+/H+ exchanger NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that 1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis; 2) the significantly greater changes in PEPCK, NHE-3, and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared with acid-loaded C mice cause the role of Rhbg and Rhcg to be underestimated quantitatively; and 3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3, and GS expression despite the presence of persistent metabolic acidosis.

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Role of angiotensin II in the enhancement of ammonia production and secretion by the proximal tubule in metabolic acidosis

AJP Renal Physiology ◽

10.1152/ajprenal.00286.2007 ◽

2008 ◽

Vol 294 (4) ◽

pp. F874-F880 ◽

Cited By ~ 28

Author(s):

Glenn T. Nagami

Keyword(s):

Metabolic Acidosis ◽

Angiotensin Ii ◽

Proximal Tubule ◽

Phosphoenolpyruvate Carboxykinase ◽

Ammonia Excretion ◽

Angiotensin Ii Receptor Blocker ◽

Ammonia Production ◽

Angiotensin Ii Receptor ◽

Proximal Tubule Segments

Acidosis and angiotensin II stimulate ammonia production and transport by the proximal tubule. We examined the modulatory effect of the type 1 angiotensin II receptor blocker losartan on the ability of metabolic acidosis to stimulate ammonia production and secretion by mouse S2 proximal tubule segments. Mice given NH4Cl for 7 days developed metabolic acidosis (low serum bicarbonate concentration) and increased urinary excretion of ammonia. S2 tubule segments from acidotic mice displayed higher rates of ammonia production and secretion compared with those from control mice. However, when losartan was coadministered in vivo with NH4Cl, both the acidosis-induced increase in urinary ammonia excretion and the adaptive increase in ammonia production and secretion of microperfused S2 segments were largely blocked. In renal cortical tissue, losartan blocked the acid-induced increase in brush-border membrane NHE3 expression but had no effect on the acid-induced upregulation of phosphate-dependent glutaminase or phosphoenolpyruvate carboxykinase 1 in cortical homogenates. Addition of angiotensin II to the microperfusion solution enhanced ammonia secretion and production rates in tubules from NH4Cl-treated and control mice in a losartan-inhibitable manner. These results demonstrate that a 7-day acid challenge induces an adaptive increase in ammonia production and secretion by the proximal tubule and suggest that during metabolic acidosis, angiotensin II signaling is necessary for adaptive enhancements of ammonia excretion by the kidney and ammonia production and secretion by S2 proximal tubule segments, as mediated, in part, by angiotensin receptor-dependent enhancement of NHE3 expression.

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Role of the Rhesus glycoprotein, Rh B glycoprotein, in renal ammonia excretion

AJP Renal Physiology ◽

10.1152/ajprenal.00277.2010 ◽

2010 ◽

Vol 299 (5) ◽

pp. F1065-F1077 ◽

Cited By ~ 47

Author(s):

Jesse M. Bishop ◽

Jill W. Verlander ◽

Hyun-Wook Lee ◽

Raoul D. Nelson ◽

Arthur J. Weiner ◽

...

Keyword(s):

Metabolic Acidosis ◽

Glutamine Synthetase ◽

Collecting Duct ◽

Ammonia Excretion ◽

Acid Excretion ◽

Urine Ph ◽

Outer Medulla ◽

Intercalated Cell ◽

Titratable Acid ◽

Acid Loading

Rh B glycoprotein (Rhbg) is a member of the Rh glycoprotein family of ammonia transporters. In the current study, we examine Rhbg's role in basal and acidosis-stimulated acid-base homeostasis. Metabolic acidosis induced by HCl administration increased Rhbg expression in both the cortex and outer medulla. To test the functional significance of increased Rhbg expression, we used a Cre-loxP approach to generate mice with intercalated cell-specific Rhbg knockout (IC-Rhbg-KO). On normal diet, intercalated cell-specific Rhbg deletion did not alter urine ammonia excretion, pH, or titratable acid excretion significantly, but it did decrease glutamine synthetase expression in the outer medulla significantly. After metabolic acidosis was induced, urinary ammonia excretion was significantly less in IC-Rhbg-KO than in control (C) mice on days 2–4 of acid loading, but not on day 5. Urine pH and titratable acid excretion and dietary acid intake did not differ significantly between acid-loaded IC-Rhcg-KO and C mice. In IC-Rhbg-KO mice, acid loading increased connecting segment (CNT) cell and outer medullary collecting duct principal cell Rhbg expression. In both C and IC-Rhbg-KO mice, acid loading decreased glutamine synthetase in both the cortex and outer medulla; the decrease on day 3 was similar in IC-Rhbg-KO and C mice, but on day 5 it was significantly greater in IC-Rhbg-KO than in C mice. We conclude 1) intercalated cell Rhbg contributes to acidosis-stimulated renal ammonia excretion, 2) Rhbg in CNT and principal cells may contribute to renal ammonia excretion, and 3) decreased glutamine synthetase expression may enable normal rates of ammonia excretion under both basal conditions and on day 5 of acid loading in IC-Rhbg-KO mice.

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Differences in acidosis-stimulated renal ammonia metabolism in the male and female kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00244.2019 ◽

2019 ◽

Vol 317 (4) ◽

pp. F890-F905 ◽

Cited By ~ 4

Author(s):

Autumn N. Harris ◽

Hyun-Wook Lee ◽

Lijuan Fang ◽

Jill W. Verlander ◽

I. David Weiner

Keyword(s):

Phosphoenolpyruvate Carboxykinase ◽

Collecting Duct ◽

Ammonia Excretion ◽

Volume Density ◽

Male Mice ◽

Ammonia Metabolism ◽

Female Mice ◽

Predominant Component ◽

Acid Load ◽

Acid Loading

Renal ammonia excretion is a critical component of acid-base homeostasis, and changes in ammonia excretion are the predominant component of increased net acid excretion in response to metabolic acidosis. We recently reported substantial sex-dependent differences in basal ammonia metabolism that correlate with sex-dependent differences in renal structure and expression of key proteins involved in ammonia metabolism. The purpose of the present study was to investigate the effect of sex on the renal ammonia response to an exogenous acid load. We studied 4-mo-old C57BL/6 mice. Ammonia excretion, which was less in male mice under basal conditions, increased in response to acid loading to a greater extent in male mice, such that maximal ammonia excretion did not differ between the sexes. Fundamental structural sex differences in the nonacid-loaded kidney persisted after acid loading, with less cortical proximal tubule volume density in the female kidney than in the male kidney, whereas collecting duct volume density was greater in the female kidney. To further investigate sex-dependent differences in the response to acid loading, we examined the expression of proteins involved in ammonia metabolism. The change in expression of phosphoenolpyruvate carboxykinase and Rh family B glycoprotein with acid loading was greater in male mice than in female mice, whereas Na+-K+-2Cl– cotransporter and inner stripe of the outer medulla intercalated cell Rh family C glycoprotein expression were significantly greater in female mice than in male mice. There was no significant sex difference in glutamine synthetase, Na+/H+ exchanger isoform 3, or electrogenic Na+-bicarbonate cotransporter 1 variant A protein expression in response to acid loading. We conclude that substantial sex-dependent differences in the renal ammonia response to acid loading enable a similar maximum ammonia excretion response.

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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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Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis

AJP Renal Physiology ◽

10.1152/ajprenal.00226.2012 ◽

2012 ◽

Vol 303 (3) ◽

pp. F458-F466 ◽

Cited By ~ 29

Author(s):

Sara M. Molinas ◽

Laura Trumper ◽

Raúl A. Marinelli

Keyword(s):

Metabolic Acidosis ◽

Protein Expression ◽

Proximal Tubule ◽

Ammonia Excretion ◽

Renal Proximal Tubule ◽

In Vivo Studies ◽

Proximal Tubule Cell ◽

Inner Mitochondrial Membrane ◽

Ammonia Transport ◽

In Cells

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% ( P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (−31%, P < 0.05) as well as in cells exposed to acid (−90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH4Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.

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