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1.
Physiol Rev ; 100(3): 1119-1147, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32347156

RESUMO

Intercalated cells (ICs) are found in the connecting tubule and the collecting duct. Of the three IC subtypes identified, type B intercalated cells are one of the best characterized and known to mediate Cl- absorption and HCO3- secretion, largely through the anion exchanger pendrin. This exchanger is thought to act in tandem with the Na+-dependent Cl-/HCO3- exchanger, NDCBE, to mediate net NaCl absorption. Pendrin is stimulated by angiotensin II and aldosterone administration via the angiotensin type 1a and the mineralocorticoid receptors, respectively. It is also stimulated in models of metabolic alkalosis, such as with NaHCO3 administration. In some rodent models, pendrin-mediated HCO3- secretion modulates acid-base balance. However, of probably more physiological or clinical significance is the role of these pendrin-positive ICs in blood pressure regulation, which occurs, at least in part, through pendrin-mediated renal Cl- absorption, as well as their effect on the epithelial Na+ channel, ENaC. Aldosterone stimulates ENaC directly through principal cell mineralocorticoid hormone receptor (ligand) binding and also indirectly through its effect on pendrin expression and function. In so doing, pendrin contributes to the aldosterone pressor response. Pendrin may also modulate blood pressure in part through its action in the adrenal medulla, where it modulates the release of catecholamines, or through an indirect effect on vascular contractile force. In addition to its role in Na+ and Cl- balance, pendrin affects the balance of other ions, such as K+ and I-. This review describes how aldosterone and angiotensin II-induced signaling regulate pendrin and the contribution of pendrin-positive ICs in the kidney to distal nephron function and blood pressure.


Assuntos
Rim/citologia , Rim/fisiologia , Transportadores de Sulfato/metabolismo , Equilíbrio Ácido-Base/efeitos dos fármacos , Equilíbrio Ácido-Base/fisiologia , Aldosterona/farmacologia , Angiotensina II/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos
2.
Physiol Rev ; 97(2): 465-494, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28151423

RESUMO

Acid-base homeostasis is critical to maintenance of normal health. Renal ammonia excretion is the quantitatively predominant component of renal net acid excretion, both under basal conditions and in response to acid-base disturbances. Although titratable acid excretion also contributes to renal net acid excretion, the quantitative contribution of titratable acid excretion is less than that of ammonia under basal conditions and is only a minor component of the adaptive response to acid-base disturbances. In contrast to other urinary solutes, ammonia is produced in the kidney and then is selectively transported either into the urine or the renal vein. The proportion of ammonia that the kidney produces that is excreted in the urine varies dramatically in response to physiological stimuli, and only urinary ammonia excretion contributes to acid-base homeostasis. As a result, selective and regulated renal ammonia transport by renal epithelial cells is central to acid-base homeostasis. Both molecular forms of ammonia, NH3 and NH4+, are transported by specific proteins, and regulation of these transport processes determines the eventual fate of the ammonia produced. In this review, we discuss these issues, and then discuss in detail the specific proteins involved in renal epithelial cell ammonia transport.


Assuntos
Equilíbrio Ácido-Base/fisiologia , Amônia/metabolismo , Transporte Biológico/fisiologia , Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Homeostase/fisiologia , Humanos
3.
Am J Physiol Renal Physiol ; 326(2): F202-F218, 2024 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-38059296

RESUMO

Angiotensin II increases apical plasma membrane pendrin abundance and function. This study explored the role of the small GTPase Rac1 in the regulation of pendrin by angiotensin II. To do this, we generated intercalated cell (IC) Rac1 knockout mice and observed that IC Rac1 gene ablation reduced the relative abundance of pendrin in the apical region of intercalated cells in angiotensin II-treated mice but not vehicle-treated mice. Similarly, the Rac1 inhibitor EHT 1864 reduced apical pendrin abundance in angiotensin II-treated mice, through a mechanism that does not require aldosterone. This IC angiotensin II-Rac1 signaling cascade modulates pendrin subcellular distribution without significantly changing actin organization. However, NADPH oxidase inhibition with APX 115 reduced apical pendrin abundance in vivo in angiotensin II-treated mice. Moreover, superoxide dismutase mimetics reduced Cl- absorption in angiotensin II-treated cortical collecting ducts perfused in vitro. Since Rac1 is an NADPH subunit, Rac1 may modulate pendrin through NADPH oxidase-mediated reactive oxygen species production. Because pendrin gene ablation blunts the pressor response to angiotensin II, we asked if pendrin blunts the angiotensin II-induced increase in kidney superoxide. Although kidney superoxide was similar in vehicle-treated wild-type and pendrin knockout mice, it was lower in angiotensin II-treated pendrin-null kidneys than in wild-type kidneys. We conclude that angiotensin II acts through Rac1, independently of aldosterone, to increase apical pendrin abundance. Rac1 may stimulate pendrin, at least partly, through NADPH oxidase. This increase in pendrin abundance contributes to the increment in blood pressure and kidney superoxide content seen in angiotensin II-treated mice.NEW & NOTEWORTHY This study defines a new signaling mechanism by which angiotensin II modulates oxidative stress and blood pressure.


Assuntos
Angiotensina II , Transportadores de Sulfato , Proteínas rac1 de Ligação ao GTP , Animais , Camundongos , Aldosterona/farmacologia , Aldosterona/metabolismo , Angiotensina II/farmacologia , Angiotensina II/metabolismo , Camundongos Knockout , NADPH Oxidases/metabolismo , Transportadores de Sulfato/genética , Superóxidos/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo
4.
Pflugers Arch ; 475(5): 607-620, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-36977894

RESUMO

The intercalated cell Cl-/HCO3- exchanger, pendrin, modulates ENaC subunit abundance and function. Whether ENaC modulates pendrin abundance and function is however unknown. Because αENaC mRNA has been detected in pendrin-positive intercalated cells, we hypothesized that ENaC, or more specifically the αENaC subunit, modulates intercalated cell function. The purpose of this study was therefore to determine if αENaC is expressed at the protein level in pendrin-positive intercalated cells and to determine if αENaC gene ablation or constitutively upregulating ENaC activity changes pendrin abundance, subcellular distribution, and/or function. We observed diffuse, cytoplasmic αENaC label in pendrin-positive intercalated cells from both mice and rats, with much lower label intensity in pendrin-negative, type A intercalated cells. However, while αENaC gene ablation within principal and intercalated cells of the CCD reduced Cl- absorption, it did not change pendrin abundance or subcellular distribution in aldosterone-treated mice. Further experiments used a mouse model of Liddle's syndrome to explore the effect of increasing ENaC channel activity on pendrin abundance and function. The Liddle's variant did not increase either total or apical plasma membrane pendrin abundance in aldosterone-treated or in NaCl-restricted mice. Similarly, while the Liddle's mutation increased total Cl- absorption in CCDs from aldosterone-treated mice, it did not significantly affect the change in Cl- absorption seen with pendrin gene ablation. We conclude that in rats and mice, αENaC localizes to pendrin-positive ICs where its physiological role remains to be determined. While pendrin modulates ENaC abundance, subcellular distribution, and function, ENaC does not have a similar effect on pendrin.


Assuntos
Aldosterona , Proteínas de Transporte de Ânions , Camundongos , Ratos , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Aldosterona/metabolismo , Canais Epiteliais de Sódio/genética , Canais Epiteliais de Sódio/metabolismo , Pressão Sanguínea/fisiologia , Transportadores de Sulfato/genética
5.
Am J Physiol Renal Physiol ; 324(1): F12-F29, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36264886

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/metabolismo
6.
Am J Physiol Renal Physiol ; 322(2): F208-F224, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35001662

RESUMO

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/genética
7.
Am J Physiol Renal Physiol ; 322(5): F486-F497, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35224991

RESUMO

Pendrin is an intercalated cell Cl-/[Formula: see text] exchanger thought to participate in K+-sparing NaCl absorption. However, its role in K+ homeostasis has not been clearly defined. We hypothesized that pendrin-null mice will develop hypokalemia with dietary K+ restriction. We further hypothesized that pendrin knockout (KO) mice mitigate urinary K+ loss by downregulating the epithelial Na+ channel (ENaC). Thus, we examined the role of ENaC in Na+ and K+ balance in pendrin KO and wild-type mice following dietary K+ restriction. To do so, we examined the relationship between Na+ and K+ balance and ENaC subunit abundance in K+-restricted pendrin-null and wild-type mice that were NaCl restricted or replete. Following a NaCl-replete, K+-restricted diet, K+ balance and serum K+ were similar in both groups. However, following a Na+, K+, and Cl--deficient diet, pendrin KO mice developed hypokalemia from increased K+ excretion. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. The fall in serum K+ observed in K+-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. However, reducing ENaC activity also reduced blood pressure and increased apparent intravascular volume contraction, since KO mice had lower serum Na+, higher blood urea nitrogen and hemoglobin, greater weight loss, greater metabolic alkalosis, and greater NaCl excretion. We conclude that dietary Na+ and K+ restriction induces hypokalemia in pendrin KO mice. Pendrin-null mice limit renal K+ loss by downregulating ENaC. However, this ENaC downregulation occurs at the expense of intravascular volume.NEW & NOTEWORTHY Pendrin is an apical Cl-/[Formula: see text] exchanger that provides renal K+-sparing NaCl absorption. The pendrin-null kidney has an inability to fully conserve K+ and limits renal K+ loss by downregulating the epithelial Na+ channel (ENaC). However, with Na+ restriction, the need to reduce ENaC for K+ balance conflicts with the need to stimulate ENaC for intravascular volume. Therefore, NaCl restriction stimulates ENaC less in pendrin-null mice than in wild-type mice, which mitigates their kaliuresis and hypokalemia but exacerbates volume contraction.


Assuntos
Hipopotassemia , Animais , Proteínas de Transporte de Ânions/metabolismo , Dieta , Canais Epiteliais de Sódio/metabolismo , Camundongos , Camundongos Knockout
8.
Am J Physiol Renal Physiol ; 321(5): F629-F644, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34605272

RESUMO

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/metabolismo
9.
Am J Physiol Renal Physiol ; 320(5): F706-F718, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33719570

RESUMO

Cellular metabolic rates in the kidney are critical for maintaining normal renal function. In a hypoxic milieu, cells rely on glycolysis to meet energy needs, resulting in the generation of pyruvate and NADH. In the absence of oxidative phosphorylation, the continuation of glycolysis is dependent on the regeneration of NAD+ from NADH accompanied by the fermentation of pyruvate to lactate. This reaction is catalyzed by lactate dehydrogenase (LDH) isoform A (LDHA), whereas LDH isoform B (LDHB) catalyzes the opposite reaction. LDH is widely used as a potential injury marker as it is released from damaged cells into the urine and serum; however, the precise isoform-specific cellular localization of the enzyme along the nephron has not been characterized. By combining immunohistochemistry results and single-cell RNA-sequencing data on healthy mouse kidneys, we identified that LDHA is primarily expressed in proximal segments, whereas LDHB is expressed in the distal parts of the nephron. In vitro experiments in mouse and human renal proximal tubule cells showed an increase in LDHA following hypoxia with no change in LDHB. Using immunofluorescence, we observed that the overall expression of both LDHA and LDHB proteins decreased following renal ischemia-reperfusion injury as well as in the adenine-diet-induced model of chronic kidney disease. Single-nucleus RNA-sequencing analyses of kidneys following ischemia-reperfusion injury revealed a significant decline in the number of cells expressing detectable levels of Ldha and Ldhb; however, cells that were positive showed increased average expression postinjury, which subsided during the recovery phase. These data provide information on the cell-specific expression of LDHA and LDHB in the normal kidney as well as following acute and chronic kidney disease.NEW & NOTEWORTHY Cellular release of lactate dehydrogenase (LDH) is being used as an injury marker; however, the exact localization of LDH within the nephron remains unclear. We show that LDH isoform A is expressed proximally, whereas isoform B is expressed distally. Both subunit expressions were significantly altered in models of acute kidney injury and chronic kidney disease. Our study provides new insights into basal and postinjury renal lactate metabolism.


Assuntos
Injúria Renal Aguda/enzimologia , Rim/enzimologia , L-Lactato Desidrogenase/metabolismo , Insuficiência Renal Crônica/enzimologia , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Biomarcadores/metabolismo , Hipóxia Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação Enzimológica da Expressão Gênica , Humanos , Isoenzimas , Rim/patologia , L-Lactato Desidrogenase/genética , Masculino , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Fatores de Tempo
10.
J Am Soc Nephrol ; 31(3): 483-499, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32054691

RESUMO

BACKGROUND: Aldosterone activates the intercalated cell mineralocorticoid receptor, which is enhanced with hypokalemia. Whether this receptor directly regulates the intercalated cell chloride/bicarbonate exchanger pendrin is unclear, as are potassium's role in this response and the receptor's effect on intercalated and principal cell function in the cortical collecting duct (CCD). METHODS: We measured CCD chloride absorption, transepithelial voltage, epithelial sodium channel activity, and pendrin abundance and subcellular distribution in wild-type and intercalated cell-specific mineralocorticoid receptor knockout mice. To determine if the receptor directly regulates pendrin, as well as the effect of serum aldosterone and potassium on this response, we measured pendrin label intensity and subcellular distribution in wild-type mice, knockout mice, and receptor-positive and receptor-negative intercalated cells from the same knockout mice. RESULTS: Ablation of the intercalated cell mineralocorticoid receptor in CCDs from aldosterone-treated mice reduced chloride absorption and epithelial sodium channel activity, despite principal cell mineralocorticoid receptor expression in the knockout mice. With high circulating aldosterone, intercalated cell mineralocorticoid receptor gene ablation directly reduced pendrin's relative abundance in the apical membrane region and pendrin abundance per cell whether serum potassium was high or low. Intercalated cell mineralocorticoid receptor ablation blunted, but did not eliminate, aldosterone's effect on pendrin total and apical abundance and subcellular distribution. CONCLUSIONS: With high circulating aldosterone, intercalated cell mineralocorticoid receptor ablation reduces chloride absorption in the CCD and indirectly reduces principal cell epithelial sodium channel abundance and function. This receptor directly regulates pendrin's total abundance and its relative abundance in the apical membrane region over a wide range in serum potassium concentration. Aldosterone regulates pendrin through mechanisms both dependent and independent of the IC MR receptor.


Assuntos
Aldosterona/metabolismo , Proteínas de Transporte de Ânions/fisiologia , Túbulos Renais Coletores/metabolismo , Potássio/sangue , Receptores de Mineralocorticoides/metabolismo , Transportadores de Sulfato/genética , Angiotensina II/farmacologia , Animais , Células Cultivadas , Antiportadores de Cloreto-Bicarbonato/metabolismo , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Técnicas In Vitro , Transporte de Íons/fisiologia , Túbulos Renais Coletores/citologia , Camundongos , Camundongos Knockout , Sensibilidade e Especificidade , Transdução de Sinais/efeitos dos fármacos , Canais de Sódio/genética
11.
Am J Physiol Renal Physiol ; 318(4): F922-F935, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32116019

RESUMO

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/deficiência
12.
Am J Physiol Renal Physiol ; 318(2): F402-F421, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31841393

RESUMO

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/metabolismo
13.
Am J Physiol Renal Physiol ; 318(6): F1463-F1477, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32338037

RESUMO

The renal circadian clock has a major influence on the function of the kidney. Aryl hydrocarbon receptor nuclear translocator-like protein 1 [ARNTL; also known as brain and muscle ARNT-like 1 (BMAL1)] is a core clock protein and transcription factor that regulates the expression of nearly half of all genes. Using male and female kidney-specific cadherin BMAL1 knockout (KS-BMAL1 KO) mice, we examined the role of renal distal segment BMAL1 in blood pressure control and solute handling. We confirmed that this mouse model does not express BMAL1 in thick ascending limb, distal convoluted tubule, and collecting duct cells, which are the final locations for solute and fluid regulation. Male KS-BMAL1 KO mice displayed a substantially lower basal systolic blood pressure compared with littermate control mice, yet their circadian rhythm in pressure remained unchanged [male control mice: 127 ± 0.7 mmHg (n = 4) vs. male KS-BMAL KO mice: 119 ± 2.3 mmHg (n = 5), P < 0.05]. Female mice, however, did not display a genotype difference in basal systolic blood pressure [female control mice: 120 ± 1.6 mmHg (n = 5) vs. female KS-BMAL1 KO mice: 119 ± 1.5 mmHg (n = 7), P = 0.4]. In addition, male KS-BMAL1 KO mice had less Na+ retention compared with control mice in response to a K+-restricted diet (15% less following 5 days of treatment). However, there was no genotype difference in Na+ handling after a K+-restricted diet in female mice. Furthermore, there was evidence indicating a sex-specific response to K+ restriction where female mice reabsorbed less Na+ in response to this dietary challenge compared with male mice. We propose that BMAL1 in the distal nephron and collecting duct contributes to blood pressure regulation and Na+ handling in a sex-specific manner.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Pressão Sanguínea , Ritmo Circadiano , Néfrons/metabolismo , Reabsorção Renal , Sódio/metabolismo , Fatores de Transcrição ARNTL/deficiência , Fatores de Transcrição ARNTL/genética , Animais , Feminino , Genótipo , Homeostase , Túbulos Renais Coletores/metabolismo , Masculino , Camundongos Knockout , Fenótipo , Potássio na Dieta/metabolismo , Fatores Sexuais , Fatores de Tempo
14.
Proc Natl Acad Sci U S A ; 114(46): E9989-E9998, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29089413

RESUMO

Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.


Assuntos
Aquaporina 2/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Coletores/metabolismo , Rim/metabolismo , Análise de Sequência de RNA/métodos , Transcriptoma , Animais , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Proteínas de Transporte de Ânions/metabolismo , Sequência de Bases , Biomarcadores/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Proteína Jagged-1/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA/metabolismo , Receptor Notch2/metabolismo , Transdução de Sinais , Transportadores de Sulfato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcriptoma/genética
15.
Am J Physiol Renal Physiol ; 317(4): F890-F905, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390234

RESUMO

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 Sexuais
16.
Am J Physiol Renal Physiol ; 316(5): F1026-F1040, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30810063

RESUMO

Conditional gene targeting using Cre recombinase has offered a powerful tool to modify gene function precisely in defined cells/tissues and at specific times. However, in mammalian cells, Cre recombinase can be genotoxic. The importance of including Cre-expressing control mice to avoid misinterpretation and to maximize the validity of the experimental results has been increasingly recognized. While studying the role of podocytes in the pathogenesis of glomerular basement membrane (GBM) thickening, we used Cre recombinase driven by the podocyte-specific podocin promoter (NPHS2-Cre) to generate a conditional knockout. By conventional structural and functional measures (histology by periodic acid-Schiff staining, albuminuria, and plasma creatinine), we did not detect significant differences between NPHS2-Cre transgenic and wild-type control mice. However, surprisingly, the group that expressed Cre transgene alone developed signs of podocyte toxicity, including marked GBM thickening, loss of normal foot process morphology, and reduced Wilms tumor 1 expression. GBM thickening was characterized by altered expression of core structural protein laminin isoform α5ß2γ1. RNA sequencing analysis of extracted glomeruli identified 230 genes that were significant and differentially expressed (applying a q < 0.05-fold change ≥ ±2 cutoff) in NPHS2-Cre mice compared with wild-type control mice. Many biological processes were reflected in the RNA sequencing data, including regulation of the extracellular matrix and pathways related to apoptosis and cell death. This study highlights the importance of including the appropriate controls for potential Cre-mediated toxicity in conditional gene-targeting experiments. Indeed, omitting the Cre transgene control can result in critical errors during interpretation of experimental data.


Assuntos
Marcação de Genes/efeitos adversos , Membrana Basal Glomerular/enzimologia , Integrases/metabolismo , Podócitos/enzimologia , Animais , Regulação da Expressão Gênica , Membrana Basal Glomerular/ultraestrutura , Integrases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Laminina/genética , Laminina/metabolismo , Proteínas de Membrana/genética , Camundongos Transgênicos , Podócitos/ultraestrutura , Regiões Promotoras Genéticas , Fatores de Tempo , Proteínas WT1/genética , Proteínas WT1/metabolismo
17.
Am J Physiol Renal Physiol ; 317(2): F489-F501, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31188034

RESUMO

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 Knockout
18.
J Am Soc Nephrol ; 29(4): 1182-1197, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29483156

RESUMO

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/química
19.
J Am Soc Nephrol ; 29(5): 1411-1425, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29483157

RESUMO

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 , Urinálise
20.
J Am Soc Nephrol ; 29(6): 1706-1719, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29773687

RESUMO

BackgroundNedd4-2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a correlation between Nedd4-2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4-2 gene ablation on IC transporter abundance and function and on BP.Methods We generated IC Nedd4-2 knockout mice using Cre-lox technology and produced global pendrin/Nedd4-2 null mice by breeding global Nedd4-2 null (Nedd4-2-/- ) mice with global pendrin null (Slc26a4-/- ) mice. Mice ate a diet with 1%-4% NaCl; BP was measured by tail cuff and radiotelemetry. We measured transepithelial transport of Cl- and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry.Results IC Nedd4-2 gene ablation markedly increased electroneutral Cl-/HCO3- exchange in the cortical collecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4-2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, or the Na+-dependent Cl-/HCO3- exchanger (Slc4a8). However, IC Nedd4-2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4-2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4-2 knockout mice.Conclusions IC Nedd4-2 regulates Cl-/HCO3- exchange in ICs., Nedd4-2 gene ablation increases BP in part through its action in these cells.


Assuntos
Pressão Sanguínea/genética , Canais Epiteliais de Sódio/metabolismo , Transporte de Íons/genética , Ubiquitina-Proteína Ligases Nedd4/genética , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Animais , Bicarbonatos/metabolismo , Membrana Celular/metabolismo , Canais de Cloreto/metabolismo , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Troca Iônica , Túbulos Renais Coletores/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , ATPases Translocadoras de Prótons/metabolismo , Prótons , Reabsorção Renal/efeitos dos fármacos , Simportadores de Sódio-Bicarbonato/metabolismo , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo , Tiazidas/farmacologia
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