RESUMO
The renal response to acid-base disturbances involves phenotypic and remodeling changes in the collecting duct. This study examines whether the proximal tubule controls these responses. We examined mice with genetic deletion of proteins present only in the proximal tubule, either the A variant or both A and B variants of isoform 1 of the electrogenic Na+-bicarbonate cotransporter (NBCe1). Both knockout (KO) mice have spontaneous metabolic acidosis. We then determined the collecting duct phenotypic responses to this acidosis and the remodeling responses to exogenous acid loading. Despite the spontaneous acidosis in NBCe1-A KO mice, type A intercalated cells in the inner stripe of the outer medullary collecting duct (OMCDis) exhibited decreased height and reduced expression of H+-ATPase, anion exchanger 1, Rhesus B glycoprotein, and Rhesus C glycoprotein. Combined kidney-specific NBCe1-A/B deletion induced similar changes. Ultrastructural imaging showed decreased apical plasma membrane and increased vesicular H+-ATPase in OMCDis type A intercalated cell in NBCe1-A KO mice. Next, we examined the collecting duct remodeling response to acidosis. In wild-type mice, acid loading increased the proportion of type A intercalated cells in the connecting tubule (CNT) and OMCDis, and it decreased the proportion of non-A, non-B intercalated cells in the connecting tubule, and type B intercalated cells in the cortical collecting duct (CCD). These changes were absent in NBCe1-A KO mice. We conclude that the collecting duct phenotypic and remodeling responses depend on proximal tubule-dependent signaling mechanisms blocked by constitutive deletion of proximal tubule NBCe1 proteins.NEW & NOTEWORTHY This study shows that the proximal tubule regulates collecting duct phenotypic and remodeling responses to acidosis.
Assuntos
Acidose , Túbulos Renais Coletores , Simportadores de Sódio-Bicarbonato , Animais , Camundongos , Acidose/genética , Acidose/metabolismo , Glicoproteínas/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Proximais/metabolismo , Camundongos Knockout , ATPases Translocadoras de Prótons/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismoRESUMO
The molecular mechanisms regulating ammonia metabolism are fundamental to acid-base homeostasis. Deletion of the A splice variant of 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 purpose of the present study was to determine the effect of combined renal deletion of NBCe1-A and NBCe1-B on systemic and PT ammonia metabolism. We generated NBCe1-A/B deletion using Cre-loxP techniques and used Cre-negative mice as controls. As renal NBCe1-A and NBCe1-B expression is limited to the PT, Cre-positive mice had PT NBCe1-A/B deletion [PT-NBCe1-A/B knockout (KO)]. Although on a 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 the expression of phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and increased the expression of glutamine synthetase, an ammonia-recycling enzyme, in PTs 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 phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase expression in PTs in both the cortex and OM. We conclude that NBCe1-B, at least in the presence of NBCe1-A deletion, contributes to PT ammonia metabolism in the OM and thereby to systemic acid-base regulation.NEW & NOTEWORTHY The results of the present study show that combined deletion of both A and B splice variants of electrogenic Na+-bicarbonate cotransporter 1 from the proximal tubule impairs acid-base homeostasis and completely blocks changes in ammonia excretion in response to acidosis, indicating that both proteins are critical to acid-base homeostasis.
Assuntos
Equilíbrio Ácido-Base , Acidose/metabolismo , Amônia/metabolismo , Túbulos Renais Proximais/metabolismo , Simportadores de Sódio-Bicarbonato/deficiência , Acidose/genética , Acidose/fisiopatologia , Animais , Feminino , Deleção de Genes , Predisposição Genética para Doença , Glutamato-Amônia Ligase/metabolismo , Glutaminase/metabolismo , Concentração de Íons de Hidrogênio , Túbulos Renais Proximais/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Simportadores de Sódio-Bicarbonato/genéticaRESUMO
Sexual dimorphic variations are present in many aspects of biology and involve the structure and/or function of nearly every organ system. Acid-base homeostasis is critical for optimal health, and renal ammonia metabolism has a major role in the maintenance of acid-base homeostasis. Recent studies have shown sex-dependent differences in renal ammonia metabolism with regard to both basal ammonia excretion and the response to an exogenous acid load. These sexual dimorphisms are associated with structural changes in the proximal tubule and the collecting duct and variations in the expression of multiple proteins involved in ammonia metabolism and transport. Studies using orchiectomy-induced testosterone deficiency and physiological testosterone replacement have shown that testosterone underlies much of the sex-dependent differences in the proximal tubule. This parallels the finding that the canonical testosterone target receptor, androgen receptor (AR), is present exclusively in the proximal tubule. Thus testosterone, possibly acting through AR activation, regulates multiple components of renal structure and ammonia metabolism. The lack of detectable AR in the remainder of the nephron and collecting duct suggests that some dimorphisms in renal structure and ammonia transporter expression are mediated through mechanisms other than direct testosterone-dependent AR activation. A better understanding of the mechanism and biological implications of sex's effect on renal structure and ammonia metabolism is critical for optimizing our ability to care for both men and women with acid-base disturbances.
Assuntos
Equilíbrio Ácido-Base , Amônia/metabolismo , Túbulos Renais Proximais/metabolismo , Testosterona/metabolismo , Animais , Feminino , Humanos , Concentração de Íons de Hidrogênio , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Receptores Androgênicos/metabolismo , Eliminação Renal , Reabsorção Renal , Caracteres Sexuais , Fatores SexuaisRESUMO
There are sex differences in renal ammonia metabolism and structure, many of which are mediated by testosterone. The goal of the present study was to determine the role of renal expression of testosterone's canonical receptor, androgen receptor (AR), in these sexual dimorphisms. We studied mice with kidney-specific AR deletion [KS-AR-knockout (KO)] generated using Cre/loxP techniques; control mice were Cre-negative littermates (wild type). 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 phosphoenolpyruvate carboxykinase (PEPCK) and Na+-K+-2Cl- cotransporter (NKCC2) expression and decreased Na+/H+ exchanger isoform 3 (NHE3) and electrogenic Na+-bicarbonate cotransporter 1 (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 [Formula: see text] significantly in either sex. In conclusion, AR-dependent signaling pathways in male, but not female, kidneys regulate PEPCK and NKCC2 expression and lead to the sexual differences in ammonia excretion. Opposing effects on NHE3 and NBCe1-A expression likely limit the magnitude of ammonia excretion changes. As AR is not present in the thick ascending limb, the effect of KS-AR-KO on NKCC2 expression is indirect. Finally, AR mediates the greater kidney size and proximal tubule volume density in male compared with female mice.NEW & NOTEWORTHY Sexual dimorphisms in ammonia metabolism involve androgen receptor (AR)-dependent signaling pathways in male, but not female, kidneys that lead to altered proximal tubule (PT), phosphoenolpyruvate carboxykinase, and thick ascending limb Na+-K+-2Cl- cotransporter expression. Adaptive responses in Na+/H+ exchanger 3 and electrogenic Na+-bicarbonate cotransporter 1-A expression limit the magnitude of the effect on ammonia excretion. Finally, the greater kidney size and PT volume density in male mice is the result of PT androgen signaling through AR.
Assuntos
Amônia/metabolismo , Rim/metabolismo , Receptores Androgênicos/metabolismo , Animais , Feminino , Rim/citologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Proximais/metabolismo , Masculino , Camundongos Knockout , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Receptores Androgênicos/genética , Eliminação Renal , Caracteres Sexuais , Fatores Sexuais , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismoRESUMO
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 phosphoenolpyruvate, 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.
Assuntos
Amônia/metabolismo , Terapia de Reposição Hormonal , Rim/efeitos dos fármacos , Eliminação Renal/efeitos dos fármacos , Testosterona/administração & dosagem , Animais , Feminino , Rim/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Orquiectomia , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Receptores Androgênicos/efeitos dos fármacos , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Fatores Sexuais , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Trocador 3 de Sódio-Hidrogênio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Testosterona/deficiênciaRESUMO
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-, ß-, 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.
Assuntos
Amônia/urina , Hipopotassemia/metabolismo , Túbulos Renais Proximais/metabolismo , Potássio na Dieta/sangue , Eliminação Renal , Simportadores de Sódio-Bicarbonato/metabolismo , Acidose/genética , Acidose/metabolismo , Acidose/fisiopatologia , Aldosterona/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Modelos Animais de Doenças , Canais Epiteliais de Sódio/metabolismo , Glutamato-Amônia Ligase/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Hipopotassemia/genética , Hipopotassemia/fisiopatologia , Túbulos Renais Proximais/fisiopatologia , Camundongos Knockout , Fosforilação , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genética , Membro 3 da Família 12 de Carreador de Soluto/metabolismoRESUMO
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.
Assuntos
Acidose/urina , Amônia/urina , Rim/metabolismo , Acidose/patologia , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Feminino , Ácido Clorídrico/farmacologia , Concentração de Íons de Hidrogênio , Imuno-Histoquímica , Medula Renal/metabolismo , Medula Renal/patologia , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Caracteres SexuaisRESUMO
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.
Assuntos
Citratos/urina , Transportadores de Ácidos Dicarboxílicos/biossíntese , Transportadores de Ácidos Dicarboxílicos/genética , Transportadores de Ânions Orgânicos Dependentes de Sódio/biossíntese , Transportadores de Ânions Orgânicos Dependentes de Sódio/genética , Simportadores/biossíntese , Simportadores/genética , Acidose/metabolismo , Animais , Dieta , Feminino , Variação Genética , Hipopotassemia/metabolismo , Imuno-Histoquímica , Medula Renal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
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.
Assuntos
Acidose/metabolismo , Amônia/metabolismo , Túbulos Renais Proximais/metabolismo , Simportadores de Sódio-Bicarbonato/fisiologia , Equilíbrio Ácido-Base , Sequência de Aminoácidos , Amônia/urina , Animais , Sequência de Bases , Bicarbonatos/sangue , Transporte Biológico Ativo , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/genética , Membrana Celular/metabolismo , Indução Enzimática , Deleção de Genes , Glicoproteínas/biossíntese , Glicoproteínas/genética , Homeostase , Concentração de Íons de Hidrogênio , Túbulos Renais Coletores/metabolismo , Túbulos Renais Proximais/enzimologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alinhamento de Sequência , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição , Urina/químicaRESUMO
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.
Assuntos
Amônia/urina , Hiperpotassemia/genética , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Proteínas Serina-Treonina Quinases/genética , Acidose/etiologia , Aldosterona/urina , Amilorida/análogos & derivados , Animais , Proteínas de Transporte de Cátions/metabolismo , Diuréticos/uso terapêutico , Glutaminase/metabolismo , Hidroclorotiazida/uso terapêutico , Concentração de Íons de Hidrogênio , Hiperpotassemia/sangue , Hiperpotassemia/complicações , Hiperpotassemia/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , ATPases Translocadoras de Prótons/metabolismo , UrináliseRESUMO
Renal ammonia metabolism has a major role in the maintenance of acid-base homeostasis. Sex differences are well recognized as an important biological variable in many aspects of renal function, including fluid and electrolyte metabolism. However, sex differences in renal ammonia metabolism have not been previously reported. Therefore, the purpose of the current study was to investigate sex differences in renal ammonia metabolism. We studied 4-mo-old wild-type C57BL/6 mice fed a normal diet. Despite similar levels of food intake, and, thus, protein intake, which is the primary determinant of endogenous acid production, female mice excreted greater amounts of ammonia, but not titratable acids, than did male mice. This difference in ammonia metabolism was associated with fundamental structural differences between the female and male kidney. In the female mouse kidney, proximal tubules account for a lower percentage of the renal cortical parenchyma compared with the male kidney, whereas collecting ducts account for a greater percentage of the renal parenchyma than in male kidneys. To further investigate the mechanism(s) behind the greater ammonia excretion in female mice, we examined differences in the expression of proteins involved in renal ammonia metabolism and transport. Greater basal ammonia excretion in females was associated with greater expression of PEPCK, glutamine synthetase, NKCC2, Rhbg, and Rhcg than was observed in male mice. We conclude that there are sex differences in basal ammonia metabolism that involve both renal structural differences and differences in expression of proteins involved in ammonia metabolism.
Assuntos
Amônia/metabolismo , Rim/metabolismo , Eliminação Renal , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Feminino , Regulação da Expressão Gênica , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Rim/anatomia & histologia , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxiquinase (ATP)/genética , Fosfoenolpiruvato Carboxiquinase (ATP)/metabolismo , Fatores Sexuais , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismoRESUMO
Sodium-coupled bicarbonate transporters are critical for renal electrolyte transport. The electrogenic, sodium-coupled bicarbonate cotransporter, isoform 1 (NBCe1), encoded by the SLC4A4 geneencoded by the SLC4A4 gene has five multiple splice variants; the A splice variant, NBCe1-A, is the primary basolateral bicarbonate transporter in the proximal convoluted tubule. This study's purpose was to determine if there is expression of additional NBCe1 splice variants in the mouse kidney, their cellular distribution, and their regulation by metabolic acidosis. In wild-type mice, an antibody reactive only to NBCe1-A showed basolateral immunolabel only in cortical proximal tubule (PT) segments, whereas an antibody reactive to all NBCe1 splice variants (pan-NBCe1) showed basolateral immunolabel in PT segments in both the cortex and outer medulla. In mice with NBCe1-A deletion, the pan-NBCe1 antibody showed basolateral PT immunolabel in both the renal cortex and outer stripe of the outer medulla, and immunoblot analysis showed expression of a ~121-kDa protein. RT-PCR of mRNA from NBCe1-A knockout mice directed at splice variant-specific regions showed expression of only NBCe1-B mRNA. In wild-type kidney, RT-PCR confirmed expression of mRNA for the NBCe1-B splice variant and absence of mRNA for the C, D, and E splice variants. Finally, exogenous acid loading increased expression in the proximal straight tubule in the outer stripe of the outer medulla. These studies demonstrate that the NBCe1-B splice variant is present in the PT, and its expression increases in response to exogenous acid loading, suggesting it participates in the PT contribution to acid-base homeostasis.
Assuntos
Equilíbrio Ácido-Base , Acidose/metabolismo , Túbulos Renais Proximais/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Acidose/genética , Acidose/fisiopatologia , Animais , Western Blotting , Modelos Animais de Doenças , Regulação da Expressão Gênica , Imuno-Histoquímica , Camundongos Knockout , Isoformas de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genéticaRESUMO
Mammalian kidneys are specialized to maintain fluid and electrolyte homeostasis. The epithelial transport processes along the renal tubule that match output to input have long been the subject of experimental and theoretical study. However, emerging data have identified a new dimension of investigation: sex. Like most tissues, the structure and function of the kidney is regulated by sex hormones and chromosomes. Available data demonstrate sex differences in the abundance of kidney solute and electrolyte transporters, establishing that renal tubular organization and operation are distinctly different in females and males. Newer studies have provided insights into the physiological consequences of these sex differences. Computational simulations predict that sex differences in transporter abundance are likely driven to optimize reproduction, enabling adaptive responses to the nutritional requirements of serial pregnancies and lactation - normal life-cycle changes that challenge the ability of renal transporters to maintain fluid and electrolyte homeostasis. Later in life, females may also undergo menopause, which is associated with changes in disease risk. Although numerous knowledge gaps remain, ongoing studies will provide further insights into the sex-specific mechanisms of sodium, potassium, acid-base and volume physiology throughout the life cycle, which may lead to therapeutic opportunities.
Assuntos
Rim , Caracteres Sexuais , Animais , Feminino , Humanos , Masculino , Rim/fisiologia , Túbulos Renais , Eletrólitos , Sódio , MamíferosRESUMO
BACKGROUND: Lymphoma has been implicated as a possible cause of proteinuria in dogs. However, information about the potential importance of proteinuria in dogs with lymphoma is limited. HYPOTHESIS: To determine if the presence of proteinuria at diagnosis was associated with median survival times in dogs with lymphoma and if lymphoma stage (I-V) or type (B vs T) were associated with the presence of proteinuria. ANIMALS: Eighty-six client-owned dogs with a new diagnosis of lymphoma between 2008 and 2020. METHODS: This was a retrospective cross-sectional study with dogs divided into proteinuric or nonproteinuric groups based on dipstick urine protein (protein ≥30 mg/dL classified as proteinuric) or a ratio of dipstick protein to urine specific gravity (ratio ≥1.5 classified as proteinuric). Dogs were excluded for: (1) treatment within 2 months with glucocorticoid, anti-neoplastic, or anti-proteinuric therapies, (2) diagnosed hypercortisolism or renal lymphoma, (3) active urine sediment, or (4) urine pH >8. Survival analysis utilized a Kaplan-Meier estimator and log-rank testing. RESULTS: There was a significant difference in median survival between proteinuric and nonproteinuric dogs classified by urine dipstick (245 days [91, 399] vs 335 days [214, 456]; P = .03) or UP : USG (237 days [158, 306] vs 304 days [173, 434]; P = .03). No difference in prevalence of proteinuria was identified between stages (I-V) or types (B and T). CONCLUSIONS AND CLINICAL IMPORTANCE: Proteinuria appears to be negatively associated with survival time in dogs newly diagnosed with lymphoma.
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BACKGROUND: Ammonium excretion decreases as kidney function decreases in several species, including cats, and may have predictive or prognostic value in patients with chronic kidney disease (CKD). Urine ammonia measurement is not readily available in clinical practice, and urine anion gap (UAG) has been proposed as a surrogate test. OBJECTIVES: Evaluate the correlation between urine ammonia-to-creatinine ratio (UACR) and UAG in healthy cats and those with CKD and determine if a significant difference exists between UAG of healthy cats and cats with CKD. ANIMALS: Urine samples collected from healthy client-owned cats (n = 59) and those with stable CKD (n = 17). METHODS: Urine electrolyte concentrations were measured using a commercial chemistry analyzer and UAG was calculated as ([sodium] + [potassium]) - [chloride]. Urine ammonia and creatinine concentrations had been measured previously using commercially available enzymatic assays and used to calculate UACR. Spearman's rank correlation coefficient between UAG and UACR was calculated for both groups. The UAG values of healthy cats and cats with CKD were assessed using the Mann-Whitney test (P < .05). RESULTS: The UAG was inversely correlated with UACR in healthy cats (P < .002, r0 = -0.40) but not in cats with CKD (P = .55; r0 = -0.15). A significant difference was found between UAG in healthy cats and those with CKD (P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: The UAG calculation cannot be used as a substitute for UACR in cats. The clinical relevance of UAG differences between healthy cats and those with CKD remains unknown.
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Doenças do Gato , Insuficiência Renal Crônica , Humanos , Gatos , Animais , Equilíbrio Ácido-Base , Creatinina/urina , Amônia , Insuficiência Renal Crônica/urina , Insuficiência Renal Crônica/veterinária , PrognósticoRESUMO
BACKGROUND: Disruption of acid-base homeostasis can lead to many clinical problems. Ammonia excretion by the kidneys is critical to maintaining acid-base homeostasis through bicarbonate production. Measurement of ammonia excretion may help determine if the kidneys are properly functioning in maintaining acid-base balance. Reference intervals are essential tools for clinical decision-making but do not currently exist for urinary ammonia-to-creatinine ratio (UACR) in feline patients. OBJECTIVE: This study aimed to generate a reference interval (RI) for UACR in healthy adult cats. METHODS: The study used samples from client-owned adult healthy cats that presented to the University of Florida Primary Care and Dentistry service (n = 92). Physical examination, serum biochemistry, urinalysis, urine ammonia, and creatinine concentrations were measured. Cats were excluded if there were significant abnormalities in their urinalysis or biochemistry panel. The RI for UACR was calculated according to the recommendation of the American Society for Veterinary Clinical Pathology. The UACR was evaluated for correlation with serum bicarbonate, weight, age, and sex. RESULTS: The RI for UACR was 3.4-20.7 with 90% confidence intervals for the lower and upper limits of (3.0-3.7) and (16.0-23.7), respectively. No significant correlation with age, sex, or weight was found. There was no discernable relationship between serum bicarbonate and UACR. CONCLUSIONS: Establishing an RI for UACR in healthy adult cats will allow further studies to determine if changes in UACR are observed during specific disease states.
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Amônia , Doenças do Gato , Gatos , Animais , Creatinina/urina , Bicarbonatos , Urinálise/veterinária , Rim , Albuminúria/urina , Albuminúria/veterináriaRESUMO
Acid-base homeostasis is critical to the maintenance of normal health. The kidneys have a central role in bicarbonate generation, which occurs through the process of net acid excretion. Renal ammonia excretion is the predominant component of renal net acid excretion under basal conditions and in response to acid-base disturbances. Ammonia produced in the kidney is selectively transported into the urine or the renal vein. The amount of ammonia produced by the kidney that is excreted in the urine varies dramatically in response to physiological stimuli. Recent studies have advanced our understanding of ammonia metabolism's molecular mechanisms and regulation. Ammonia transport has been advanced by recognizing that the specific transport of NH3 and NH4+ by specific membrane proteins is critical to ammonia transport. Other studies show that proximal tubule protein, NBCe1, specifically the A variant, significantly regulates renal ammonia metabolism. This review discusses these critical aspects of the emerging features of ammonia metabolism and transport.
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Amônia , Compostos de Amônio , Rim , Néfrons , Veias RenaisRESUMO
BACKGROUND: Heart failure-associated hypochloremia can be depletional from diuretics or dilutional from water retention. Serum osmolality reflects water balance but has not been evaluated in dogs with heart disease. HYPOTHESIS: To determine if serum osmolality is related to heart disease stage and amount of mathematical correction of serum chloride (Cl- ) concentrations in healthy dogs and dogs with myxomatous mitral valve degeneration (MMVD). ANIMALS: Seventy-seven dogs (20 healthy, 25 Stage B MMVD, 32 Stage C/D MMVD). METHODS: Serum Cl- concentrations were mathematically corrected. Osmolality was calculated (calOsm) and directly measured by freezing point depression (dmOsm) and compared by Bland-Altman analysis. Biochemical variables and osmolality were compared among healthy, Stage B, and Stage C/D dogs. Correlations were explored between osmolality and biochemical variables. Median and range are presented. P < .05 was considered significant. RESULTS: The calOsm was different among groups (P = .003), with Stage B (310 mOsm/kg; 306, 316) and C/D dogs (312 mOsm/kg; 308, 319) having higher calOsm than healthy dogs (305 mOsm/kg; 302, 308). Osmolality methods were moderately correlated (P < .0001, rs = .46) but with proportional bias and poor agreement. The amount of Cl- correction was negatively correlated with calOsm (P < .0001, rs = -.78) and dmOsm (P = .004, rs = -.33). Serum bicarbonate concentration was negatively correlated with Cl- (P < .0001, rs = -.67). CONCLUSIONS AND CLINICAL IMPORTANCE: Dogs with Stage B and Stage C/D heart disease had higher calOsm than healthy dogs. Osmolality was inversely related to the amount of Cl- correction, which supports its use in assessing relative body water content. Poor agreement between calOsm and dmOsm prevents methodological interchange.
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Doenças do Cão , Cardiopatias , Insuficiência Cardíaca , Prolapso da Valva Mitral , Cães , Animais , Cloretos , Cardiopatias/veterinária , Prolapso da Valva Mitral/veterinária , Insuficiência Cardíaca/veterinária , Concentração Osmolar , ÁguaRESUMO
A 5-year-old female spayed French Bulldog presented for anorexia and increased respiratory rate. On presentation, she was dyspneic with stridor and increased bronchovesicular sounds. Point-of-care ultrasound identified pericardial effusion. Thoracic radiographs identified pleural effusion, a wide cranial mediastinum, and multifocal unstructured interstitial pulmonary opacities. Bloodwork revealed a moderate leukocytosis characterized by a mature neutrophilia with a left shift, hypoalbuminemia, mildly increased alkaline phosphatase activity, and moderate hypokalemia. Thoracic CT findings revealed moderate pericardial and bilateral pleural effusion, mediastinal effusion, and moderate cranial mediastinal lymphadenopathy. Diagnostic thoracocentesis and pericardiocentesis revealed septic exudates with bacilli. Two days later, a median sternotomy and pericardiectomy were performed. Aerobic cultures of the effusions grew Achromobacter xylosoxidans ss deitrificans. The patient was treated with Amoxicillin-clavulanate and enrofloxacin for 12 weeks and clinically fully recovered. Achromobacter xylosoxidans has not been reported as a cause of purulent pericarditis and pyothorax in a dog. Uniquely, this patient is suspected of developing this infection secondary to immunosuppression.
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OBJECTIVE: To characterize the signalment, clinical signs, clinical pathological and histologic findings, and outcome in 8 related Bracchi Italiani with proteinuric kidney disease. ANIMALS: 8 client-owned Bracchi Italiani. PROCEDURES: Health records submitted to the Bracco Italiano Health Foundation and the Bracco Italiano Club of America between 2012 and 2019 were reviewed for dogs with evidence of nephropathy for which histologic diagnoses were obtained. Pedigree, signalment, clinical signs, diagnostic test results (including microscopic examination of kidney tissue samples collected ante- or postmortem), and outcome were acquired. Results were presented as descriptive statistics. RESULTS: The most common clinical sign in affected dogs was inappetence. All dogs were proteinuric, and 4 dogs were azotemic. Seven dogs developed clinical signs of kidney disease and were euthanized a median of 75 days postdiagnosis. Six dogs had glomerular amyloidosis, and 1 dog each had nephrosclerosis and nonamyloidotic fibrillar glomerulopathy. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that the clinical presentation may vary in affected dogs, and proteinuria in young or middle-aged Bracchi Italiani should raise the concern for hereditary nephropathy. Prognosis is likely poor once clinical signs are noted.