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

2016 ◽  
Vol 310 (11) ◽  
pp. F1229-F1242 ◽  
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.

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

2020 ◽  
Vol 318 (2) ◽  
pp. F402-F421 ◽  
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.

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

2012 ◽  
Vol 303 (3) ◽  
pp. F458-F466 ◽  
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.

scholarly journals Proximal tubule glutamine synthetase expression is necessary for the normal response to dietary protein restriction

2017 ◽  
Vol 313 (1) ◽  
pp. F116-F125 ◽  
Author(s):  
Hyun-Wook Lee ◽  
Gunars Osis ◽  
Mary E. Handlogten ◽  
Jill W. Verlander ◽  
I. David Weiner
Keyword(s):  
Glutamine Synthetase ◽  
Proximal Tubule ◽  
Dietary Protein ◽  
Acid Production ◽  
Protein Intake ◽  
Ammonia Excretion ◽  
Protein Restriction ◽  
Protein Diet ◽  
Acid Excretion ◽  
Dietary Protein Restriction

Dietary protein restriction has multiple benefits in kidney disease. Because protein intake is a major determinant of endogenous acid production, it is important that net acid excretion changes in parallel during changes in dietary protein intake. Dietary protein restriction decreases endogenous acid production and decreases urinary ammonia excretion, a major component of net acid excretion. Glutamine synthetase (GS) catalyzes the reaction of [Formula: see text] and glutamate, which regenerates the essential amino acid glutamine and decreases net ammonia generation. Because renal proximal tubule GS expression increases during dietary protein restriction, this could contribute to the decreased ammonia excretion. The purpose of the current study was to determine the role of proximal tubule GS in the renal response to protein restriction. We generated mice with proximal tubule-specific GS deletion (PT-GS-KO) using Cre-loxP techniques. Cre-negative (Control) and PT-GS-KO mice in metabolic cages were provided 20% protein diet for 2 days and were then changed to low-protein (6%) diet for the next 7 days. Additional PT-GS-KO mice were maintained on 20% protein diet. Dietary protein restriction caused a rapid decrease in urinary ammonia excretion in both genotypes, but PT-GS-KO blunted this adaptive response significantly. This occurred despite no significant genotype-dependent differences in urinary pH or in serum electrolytes. There were no significant differences between Control and PT-GS-KO mice in expression of multiple other proteins involved in renal ammonia handling. We conclude that proximal tubule GS expression is necessary for the appropriate decrease in ammonia excretion during dietary protein restriction.

scholarly journals Testosterone modulates renal ammonia metabolism

2020 ◽  
Vol 318 (4) ◽  
pp. F922-F935 ◽  
Author(s):  
Autumn N. Harris ◽  
Hyun-Wook Lee ◽  
Jill W. Verlander ◽  
I. David Weiner
Keyword(s):  
Proximal Tubule ◽  
Ammonia Excretion ◽  
Primary Component ◽  
Testosterone Replacement ◽  
Thick Ascending Limb ◽  
Ammonia Metabolism ◽  
Cellular Expression ◽  
Renal Structure ◽  
Bilateral Orchiectomy

There are substantial sex differences in renal structure and ammonia metabolism that correlate with differences in expression of proteins involved in ammonia generation and transport. This study determined the role of testis-derived testosterone in these differences. We studied 4-mo-old male C57BL/6 mice 4 and 8 wk after either bilateral orchiectomy (ORCH) or sham-operated control surgery and determined the effect of testosterone replacement to reverse the effects of ORCH. Finally, we determined the cellular expression of androgen receptor (AR), testosterone’s canonical target receptor. ORCH decreased kidney and proximal tubule size, and testosterone replacement reversed this effect. ORCH increased ammonia excretion in a testosterone-dependent fashion; this occurred despite similar food intake, which is the primary component of endogenous acid production. ORCH increased expression of both phospho enolpyruvate, a major ammonia-generating protein, and Na+-K+-2Cl− cotransporter, which mediates thick ascending limb ammonia reabsorption; these changes were reversed with testosterone replacement. Orchiectomy also decreased expression of Na+/H+ exchanger isoform 3, which mediates proximal tubule ammonia secretion, in a testosterone-dependent pattern. Finally, ARs are expressed throughout the proximal tubule in both the male and female kidney. Testosterone, possibly acting through ARs, has dramatic effects on kidney and proximal tubule size and decreases ammonia excretion through its effects on several key proteins involved in ammonia metabolism.

scholarly journals NBCe1 expression is required for normal renal ammonia metabolism

2015 ◽  
Vol 309 (7) ◽  
pp. F658-F666 ◽  
Author(s):  
Mary E. Handlogten ◽  
Gunars Osis ◽  
Hyun-Wook Lee ◽  
Michael F. Romero ◽  
Jill W. Verlander ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Gene Deletion ◽  
Ammonia Excretion ◽  
Ammonia Metabolism ◽  
Gene Dose ◽  
Ammonia Transport ◽  
Microscopy Examination ◽  
Lower Urinary

The mechanisms regulating proximal tubule ammonia metabolism are incompletely understood. The present study addressed the role of the proximal tubule basolateral electrogenic Na+-coupled bicarbonate cotransporter (NBCe1; Slc4a4) in renal ammonia metabolism. We used mice with heterozygous and homozygous NBCe1 gene deletion and compared these mice with their wild-type littermates. Because homozygous NBCe1 gene deletion causes 100% mortality before day 25, we studied mice at day 8 (±1 day). Both heterozygous and homozygous gene deletion caused a gene dose-related decrease in serum bicarbonate. The ability to lower urinary pH was intact, and even accentuated, with NBCe1 deletion. However, in contrast to the well-known effect of metabolic acidosis to increase urinary ammonia excretion, NBCe1 deletion caused a gene dose-related decrease in ammonia excretion. There was no identifiable change in proximal tubule structure by light microscopy. Examination of proteins involved in renal ammonia metabolism showed decreased expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase, key enzymes in proximal tubule ammonia generation, and increased expression of glutamine synthetase, which recycles intrarenal ammonia and regenerates glutamine. Expression of key proteins involved in ammonia transport outside of the proximal tubule (rhesus B glycoprotein and rhesus C glycoprotein) was not significantly changed by NBCe1 deletion. We conclude from these findings that NBCe1 expression is necessary for normal proximal tubule ammonia metabolism.

scholarly journals Role of the Renal Androgen Receptor in Ammonia Metabolism

2021 ◽  
Author(s):  
Autumn N. Harris ◽  
Rebeca A Castro ◽  
Hyun-Wook Lee ◽  
Jill W. Verlander ◽  
I. David Weiner
Keyword(s):  
Sex Differences ◽  
Androgen Receptor ◽  
Proximal Tubule ◽  
Ammonia Excretion ◽  
Volume Density ◽  
Plasma Testosterone ◽  
Kidney Size ◽  
Ammonia Metabolism ◽  
Female Mice

Background: There are sex differences in renal ammonia metabolism and structure, many of which are mediated by testosterone. This study's goal was to determine the role of renal expression of testosterone's canonical receptor, androgen receptor (AR), in these sexual dimorphisms. Methods: We studied mice with kidney-specific AR deletion (KS-AR-KO) generated using Cre/loxP techniques; control mice were Cre-negative littermates (WT). Results: In male, but not female, mice, KS-AR-KO increased ammonia excretion, which eliminated sex differences. Although renal structural size typically parallel ammonia excretion, KS-AR-KO decreased kidney size, cortical proximal tubule volume density and cortical proximal tubule cell height in males; neither were altered in females and collecting duct volume density was unaltered in both sexes. Analysis of key protein involved in ammonia handling showed in male mice that KS-AR-KO increased both PEPCK and NKCC2 expression, and decreased NHE3 and NBCe1-A expression. In female mice, KS-AR-KO did not alter these parameters. These effects occurred even though KS-AR-KO did not alter plasma testosterone, food intake or serum Na+, K+, or HCO3- significantly in either sex. Conclusions: AR-dependent signaling pathways in male, but not female, kidney regulate PEPCK and NKCC2 expression and lead to the sexual differences in ammonia excretion. Opposing effects on NHE-3 and NBCe1-A expression likely limit the magnitude of ammonia excretion changes. Since AR is not present in the TAL, the effect of KS-AR-KO on NKCC2 expression is indirect. Finally, AR mediates the greater kidney size and PT volume density in male than in female mice.

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

2010 ◽  
Vol 299 (5) ◽  
pp. F1065-F1077 ◽  
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.

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

2018 ◽  
Vol 29 (4) ◽  
pp. 1182-1197 ◽  
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.

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

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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