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1.
Proc Natl Acad Sci U S A ; 118(6)2021 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-33536341

RESUMO

Tuberous sclerosis complex (TSC) is caused by mutations in either TSC1 or TSC2 genes and affects multiple organs, including kidney, lung, and brain. In the kidney, TSC presents with the enlargement of benign tumors (angiomyolipomata) and cysts, which eventually leads to kidney failure. The factors promoting cyst formation and tumor growth in TSC are incompletely understood. Here, we report that mice with principal cell-specific inactivation of Tsc1 develop numerous cortical cysts, which are overwhelmingly composed of hyperproliferating A-intercalated (A-IC) cells. RNA sequencing and confirmatory expression studies demonstrated robust expression of Forkhead Transcription Factor 1 (Foxi1) and its downstream targets, apical H+-ATPase and cytoplasmic carbonic anhydrase 2 (CAII), in cyst epithelia in Tsc1 knockout (KO) mice but not in Pkd1 mutant mice. In addition, the electrogenic 2Cl-/H+ exchanger (CLC-5) is significantly up-regulated and shows remarkable colocalization with H+-ATPase on the apical membrane of cyst epithelia in Tsc1 KO mice. Deletion of Foxi1, which is vital to intercalated cells viability and H+-ATPase expression, completely abrogated the cyst burden in Tsc1 KO mice, as indicated by MRI images and histological analysis in kidneys of Foxi1/Tsc1 double-knockout (dKO) mice. Deletion of CAII, which is critical to H+-ATPase activation, caused significant reduction in cyst burden and increased life expectancy in CAII/Tsc1 dKO mice vs. Tsc1 KO mice. We propose that intercalated cells and their acid/base/electrolyte transport machinery (H+-ATPase/CAII/CLC-5) are critical to cystogenesis, and their inhibition or inactivation is associated with significant protection against cyst generation and/or enlargement in TSC.

2.
J Physiol Sci ; 71(1): 5, 2021 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514305

RESUMO

The anion exchanger slc26a3 (DRA), which is mutated in congenital chloride-losing diarrhea, is expressed in the apical membrane of the cecum and middle-distal colon but not in the proximal colon of rodent large intestines. To elucidate the functional roles of DRA, we measured unidirectional 36Cl- and 22Na+ fluxes and HCO3- secretion in vitro in each of these segments using DRA-KO mice. Robust Cl- absorption, which was largely abolished after DRA deficiency, was present in the cecum and middle-distal colon but absent in the proximal colon. Na+ absorption was present in all three segments in both the control and DRA-KO mice. The luminal-Cl--dependent HCO3- secretions in the cecum and middle-distal colon were abolished in the DRA-KO mice. In conclusion, DRA mediates Cl- absorption and HCO3- secretion in the mouse cecum and middle-distal colon, and may have roles in H2O absorption and luminal acid/base regulation in these segments.

3.
J Neuroinflammation ; 17(1): 301, 2020 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-33054763

RESUMO

BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.

4.
Am J Physiol Renal Physiol ; 319(4): F712-F728, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32893663

RESUMO

Inhibitors of proximal tubular Na+-glucose cotransporter 2 (SGLT2) are natriuretic, and they lower blood pressure. There are reports that the activities of SGLT2 and Na+-H+ exchanger 3 (NHE3) are coordinated. If so, then part of the natriuretic response to an SGLT2 inhibitor is mediated by suppressing NHE3. To examine this further, we compared the effects of an SGLT2 inhibitor, empagliflozin, on urine composition and systolic blood pressure (SBP) in nondiabetic mice with tubule-specific NHE3 knockdown (NHE3-ko) and wild-type (WT) littermates. A single dose of empagliflozin, titrated to cause minimal glucosuria, increased urinary excretion of Na+ and bicarbonate and raised urine pH in WT mice but not in NHE3-ko mice. Chronic empagliflozin treatment tended to lower SBP despite higher renal renin mRNA expression and lowered the ratio of SBP to renin mRNA, indicating volume loss. This effect of empagliflozin depended on tubular NHE3. In diabetic Akita mice, chronic empagliflozin enhanced phosphorylation of NHE3 (S552/S605), changes previously linked to lesser NHE3-mediated reabsorption. Chronic empagliflozin also increased expression of genes involved with renal gluconeogenesis, bicarbonate regeneration, and ammonium formation. While this could reflect compensatory responses to acidification of proximal tubular cells resulting from reduced NHE3 activity, these effects were at least in part independent of tubular NHE3 and potentially indicated metabolic adaptations to urinary glucose loss. Moreover, empagliflozin increased luminal α-ketoglutarate, which may serve to stimulate compensatory distal NaCl reabsorption, while cogenerated and excreted ammonium balances urine losses of this "potential bicarbonate." The data implicate NHE3 as a determinant of the natriuretic effect of empagliflozin.


Assuntos
Compostos Benzidrílicos/farmacologia , Diabetes Mellitus/tratamento farmacológico , Glucosídeos/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Natriurese/efeitos dos fármacos , Natriuréticos/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Transportador 2 de Glucose-Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio/metabolismo , Equilíbrio Ácido-Base/efeitos dos fármacos , Animais , Glicemia/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Diabetes Mellitus/metabolismo , Diabetes Mellitus/fisiopatologia , Modelos Animais de Doenças , Glicosúria/metabolismo , Glicosúria/fisiopatologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Trocador 3 de Sódio-Hidrogênio/deficiência , Trocador 3 de Sódio-Hidrogênio/genética
5.
Am J Physiol Cell Physiol ; 319(4): C641-C656, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32726161

RESUMO

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl- between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN-), a property that does not correspond with any of the Cl- channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN- permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl- channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN- conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN--selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.

6.
Int J Mol Sci ; 21(9)2020 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-32380787

RESUMO

Coronaviruses (CoVs), including Severe Acute Respiratory Syndrome (SARS), Middle East Respiratory Syndrome (MERS), and the novel coronavirus disease-2 (SARS-CoV-2) are a group of enveloped RNA viruses that cause a severe respiratory infection which is associated with a high mortality [...].


Assuntos
Lesão Renal Aguda/mortalidade , Lesão Renal Aguda/virologia , Betacoronavirus/patogenicidade , Infecções por Coronavirus/virologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/virologia , Pneumonia Viral/virologia , Lesão Renal Aguda/fisiopatologia , Lesão Renal Aguda/prevenção & controle , Angiotensina II/farmacologia , Catepsinas/metabolismo , Morte Celular/efeitos dos fármacos , Infecções por Coronavirus/mortalidade , Infecções por Coronavirus/patologia , Infecções por Coronavirus/fisiopatologia , Creatinina/sangue , Estado Terminal/mortalidade , Endossomos/efeitos dos fármacos , Endossomos/enzimologia , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Incidência , Túbulos Renais Proximais/fisiopatologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Lisossomos/metabolismo , Pandemias , Peptidil Dipeptidase A/metabolismo , Pneumonia Viral/mortalidade , Pneumonia Viral/patologia , Pneumonia Viral/fisiopatologia , Receptores Virais/metabolismo , Síndrome Respiratória Aguda Grave/sangue , Síndrome Respiratória Aguda Grave/mortalidade , Síndrome Respiratória Aguda Grave/fisiopatologia , Internalização do Vírus , Replicação Viral
7.
Br J Pharmacol ; 177(4): 898-911, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31658361

RESUMO

BACKGROUND AND PURPOSE: Although it has been reported that bovine carbonic anhydrase CAII is capable of generating NO from nitrite, the function and mechanism of CAII in nitrite-dependent NO formation and vascular responses remain controversial. We tested the hypothesis that CAII catalyses NO formation from nitrite and contributes to nitrite-dependent inhibition of platelet activation and vasodilation. EXPERIMENT APPROACH: The role of CAII in enzymatic NO generation was investigated by measuring NO formation from the reaction of isolated human and bovine CAII with nitrite using NO photolysis-chemiluminescence. A CAII-deficient mouse model was used to determine the role of CAII in red blood cell mediated nitrite reduction and vasodilation. KEY RESULTS: We found that the commercially available purified bovine CAII exhibited limited and non-enzymatic NO-generating reactivity in the presence of nitrite with or without addition of the CA inhibitor dorzolamide; the NO formation was eliminated with purification of the enzyme. There was no significant detectable NO production from the reaction of nitrite with recombinant human CAII. Using a CAII-deficient mouse model, there were no measurable changes in nitrite-dependent vasodilation in isolated aorta rings and in vivo in CAII-/- , CAII+/- , and wild-type mice. Moreover, deletion of the CAII gene in mice did not block nitrite reduction by red blood cells and the nitrite-NO-dependent inhibition of platelet activation. CONCLUSION AND IMPLICATIONS: These studies suggest that human, bovine and mouse CAII are not responsible for nitrite-dependent NO formation in red blood cells, aorta, or the systemic circulation.

8.
Int J Mol Sci ; 20(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561575

RESUMO

Acute kidney injury (AKI) refers to an abrupt decrease in kidney function. It affects approximately 7% of all hospitalized patients and almost 35% of intensive care patients. Mortality from acute kidney injury remains high, particularly in critically ill patients, where it can be more than 50%. The primary causes of AKI include ischemia/reperfusion (I/R), sepsis, or nephrotoxicity; however, AKI patients may present with a complicated etiology where many of the aforementioned conditions co-exist. Multiple bio-markers associated with renal damage, as well as metabolic and signal transduction pathways that are involved in the mediation of renal dysfunction have been identified as a result of the examination of models, patient samples, and clinical data of AKI of disparate etiologies. These discoveries have enhanced our ability to diagnose AKIs and to begin to elucidate the mechanisms involved in their pathogenesis. Studies in our laboratory revealed that the expression and activity of spermine/spermidine N1-acetyltransferase (SAT1), the rate-limiting enzyme in polyamine back conversion, were enhanced in kidneys of rats after I/R injury. Additional studies revealed that the expression of spermine oxidase (SMOX), another critical enzyme in polyamine catabolism, is also elevated in the kidney and other organs subjected to I/R, septic, toxic, and traumatic injuries. The maladaptive role of polyamine catabolism in the mediation of AKI and other injuries has been clearly demonstrated. This review will examine the biochemical and mechanistic basis of tissue damage brought about by enhanced polyamine degradation and discuss the potential of therapeutic interventions that target polyamine catabolic enzymes or their byproducts for the treatment of AKI.


Assuntos
Lesão Renal Aguda/etiologia , Lesão Renal Aguda/metabolismo , Poliaminas/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Biomarcadores , Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Humanos , Redes e Vias Metabólicas , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo
9.
Front Pharmacol ; 10: 815, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31543812

RESUMO

Thiazide diuretic (TZD)-mediated chronic reduction of arterial pressure is thought to occur through decreased total peripheral vascular resistance. Further, the decreased peripheral vascular resistance is accomplished through TZD activation of an extrarenal target, resulting in inhibition of vascular constriction. However, despite greater than five decades of investigation, little progress has been made into the identification of the TZD extrarenal target. Proposed mechanisms range from direct inhibition of constrictor and activation of relaxant signaling pathways in the vascular smooth muscle to indirect inhibition through decreased neurogenic and hormonal regulatory pathways. Surprisingly, particularly in view of this lack of progress, comprehensive reviews of the subject are absent. Moreover, even though it is well recognized that 1) several types of hypertension are insensitive to TZD reduction of arterial pressure and, further, TZD fail to reduce arterial pressure in normotensive subjects and animals, and 2) different mechanisms underlie acute and chronic TZD, findings derived from these models and parameters remain largely undifferentiated. This review 1) comprehensively describes findings associated with TZD reduction of arterial pressure; 2) differentiates between observations in TZD-sensitive and TZD-insensitive hypertension, normotensive subjects/animals, and acute and chronic effects of TZD; 3) critically evaluates proposed TZD extrarenal targets; 4) proposes guiding parameters for relevant investigations into extrarenal TZD target identification; and 5) proposes a working model for TZD chronic reduction of arterial pressure through vascular dilation.

10.
Hypertension ; 74(3): 526-535, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31352824

RESUMO

The present study directly tested the hypothesis that the NHE3 (Na+/H+ exchanger 3) in the proximal tubules of the kidney is required for the development of Ang II (angiotensin II)-induced hypertension using PT-Nhe3-/- (proximal tubule-specific NHE3 knockout) mice. Specifically, PT-Nhe3-/- mice were generated using the SGLT2-Cre/Nhe3loxlox approach, whereas Ang II-induced hypertension was studied in 12 groups (n=5-12 per group) of adult male and female wild-type (WT) and PT-Nhe3-/- mice. Under basal conditions, systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure were significantly lower in male and female PT-Nhe3-/- than WT mice (P<0.01). A high pressor, 1.5 mg/kg per day, intraperitoneal or a slow pressor dose of Ang II, 0.5 mg/kg per day, intraperitoneal for 2 weeks significantly increased systolic blood pressure, diastolic blood pressure, and mean arterial blood pressure in male and female WT mice (P<0.01), but the hypertensive response to Ang II was markedly attenuated in male and female PT-Nhe3-/- mice (P<0.01). Ang II impaired the pressure-natriuresis response in WT mice, whereas proximal tubule-specific deletion of NHE3 improved the pressure-natriuresis response in Ang II-infused PT-Nhe3-/- mice (P<0.01). AT1 receptor blocker losartan completely blocked Ang II-induced hypertension in both WT and PT-Nhe3-/- mice (P<0.01). However, inhibition of nitric oxide synthase with L-NG-Nitroarginine methyl ester had no effect on Ang II-induced hypertension in WT or PT-Nhe3-/- mice (not significant). Furthermore, Ang II-induced hypertension was significantly attenuated by an orally absorbable NHE3 inhibitor AVE0657. In conclusion, NHE3 in the proximal tubules of the kidney may be a therapeutical target in hypertension induced by Ang II or with increased NHE3 expression in the proximal tubules.


Assuntos
Angiotensina II/farmacologia , Túbulos Renais Proximais/metabolismo , Losartan/administração & dosagem , Receptor Tipo 1 de Angiotensina/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética , Animais , Modelos Animais de Doenças , Feminino , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Injeções Intraperitoneais , Túbulos Renais Proximais/patologia , Masculino , Camundongos , Camundongos Knockout , Distribuição Aleatória , Valores de Referência , Trocadores de Sódio-Hidrogênio/metabolismo , Resultado do Tratamento
11.
Am J Physiol Renal Physiol ; 317(2): F419-F434, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31166707

RESUMO

Na+/H+ exchanger isoform 3 (NHE3) contributes to Na+/bicarbonate reabsorption and ammonium secretion in early proximal tubules. To determine its role in the diabetic kidney, type 1 diabetic Akita mice with tubular NHE3 knockdown [Pax8-Cre; NHE3-knockout (KO) mice] were generated. NHE3-KO mice had higher urine pH, more bicarbonaturia, and compensating increases in renal mRNA expression for genes associated with generation of ammonium, bicarbonate, and glucose (phosphoenolpyruvate carboxykinase) in proximal tubules and H+ and ammonia secretion and glycolysis in distal tubules. This left blood pH and bicarbonate unaffected in nondiabetic and diabetic NHE3-KO versus wild-type mice but was associated with renal upregulation of proinflammatory markers. Higher renal phosphoenolpyruvate carboxykinase expression in NHE3-KO mice was associated with lower Na+-glucose cotransporter (SGLT)2 and higher SGLT1 expression, indicating a downward tubular shift in Na+ and glucose reabsorption. NHE3-KO was associated with lesser kidney weight and glomerular filtration rate (GFR) independent of diabetes and prevented diabetes-associated albuminuria. NHE3-KO, however, did not attenuate hyperglycemia or prevent diabetes from increasing kidney weight and GFR. Higher renal gluconeogenesis may explain similar hyperglycemia despite lower SGLT2 expression and higher glucosuria in diabetic NHE3-KO versus wild-type mice; stronger SGLT1 engagement could have affected kidney weight and GFR responses. Chronic kidney disease in humans is associated with reduced urinary excretion of metabolites of branched-chain amino acids and the tricarboxylic acid cycle, a pattern mimicked in diabetic wild-type mice. This pattern was reversed in nondiabetic NHE3-KO mice, possibly reflecting branched-chain amino acids use for ammoniagenesis and tricarboxylic acid cycle upregulation to support formation of ammonia, bicarbonate, and glucose in proximal tubule. NHE3-KO, however, did not prevent the diabetes-induced urinary downregulation in these metabolites.


Assuntos
Equilíbrio Ácido-Base , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/metabolismo , Túbulos Renais/metabolismo , Reabsorção Renal , Trocador 3 de Sódio-Hidrogênio/deficiência , Sódio/urina , Equilíbrio Ácido-Base/genética , Aminoácidos de Cadeia Ramificada/urina , Amônia/urina , Animais , Bicarbonatos/urina , Biomarcadores/urina , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 1/urina , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/urina , Modelos Animais de Doenças , Metabolismo Energético/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Concentração de Íons de Hidrogênio , Túbulos Renais/fisiopatologia , Masculino , Metabolômica/métodos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transportador 1 de Glucose-Sódio/genética , Transportador 1 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/metabolismo , Trocador 3 de Sódio-Hidrogênio/genética
12.
Physiol Rep ; 7(2): e13983, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30675765

RESUMO

Tuberous sclerosis complex (TSC) is a tumor predisposition syndrome with significant renal cystic and solid tumor disease. While the most common renal tumor in TSC, the angiomyolipoma, exhibits a loss of heterozygosity associated with disease, we have discovered that the renal cystic epithelium is composed of type A intercalated cells that have an intact Tsc gene that have been induced to exhibit Tsc-mutant disease phenotype. This mechanism appears to be different than that for ADPKD. The murine models described here closely resemble the human disease and both appear to be mTORC1 inhibitor responsive. The induction signaling driving cystogenesis may be mediated by extracellular vesicle trafficking.


Assuntos
Doenças Renais Císticas/patologia , Esclerose Tuberosa/patologia , Animais , Modelos Animais de Doenças , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Feminino , Doenças Renais Císticas/genética , Doenças Renais Císticas/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Knockout , Esclerose Tuberosa/genética , Esclerose Tuberosa/metabolismo , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 1 do Complexo Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo
13.
Naunyn Schmiedebergs Arch Pharmacol ; 392(1): 117-121, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30470918

RESUMO

The chronic reduction of arterial blood pressure by thiazide diuretics (TZD) in hypertensive patients is mediated through an extra-renal mechanism. It is widely held that this extra-renal mechanism is a direct TZD inhibition of vasoconstriction. This study tested whether the TZD, hydrochlorothiazide (HCTZ), inhibited agonist constriction of mesenteric arterioles ex vivo. Mice deficient in the kidney distal convoluted tubule Na+/Cl- cotransporter (NCC), i.e., the target of thiazide inhibition-mediated diuresis, and wild type (WT), were subjected to Na+-restricted diet. Mesenteric arterioles from NCC knockout and WT mice were then isolated, placed under constant pressure, and the inhibitory effects of HCTZ (100 µM) on phenylephrine constriction determined. HCTZ did not inhibit phenylephrine constriction of arterioles from NCC knockout and wild type (WT) mice subjected to Na+-restricted diet. This study suggests that future investigations to identify the extra-renal site of chronic TZD treatment should (1) focus on indirect inhibition of vascular constriction and (2) be determined under clinically relevant conditions. These conditions include chronic TZD at relevant concentrations in hypertensive animals.


Assuntos
Arteríolas/efeitos dos fármacos , Diuréticos/farmacologia , Hidroclorotiazida/farmacologia , Vasoconstrição/efeitos dos fármacos , Animais , Arteríolas/fisiologia , Feminino , Masculino , Mesentério/irrigação sanguínea , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenilefrina/farmacologia , Membro 3 da Família 12 de Carreador de Soluto/genética , Vasoconstritores/farmacologia
14.
Hypertension ; 72(6): 1328-1336, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30571224

RESUMO

The present study directly tested the hypothesis that deletion of the NHE3 (Na+/H+ exchanger 3) selectively in the proximal tubules of the kidney lowers basal blood pressure by increasing the pressure-natriuresis response in mice. Adult male and female, age-matched wild-type (WT) littermates and proximal tubule-specific NHE3 knockout mice (PT- Nhe3-/-; n=6-16 per group) were studied for (1) basal phenotypes of electrolytes and pH, blood pressure, and kidney function; (2) the pressure-natriuresis response using the mesenteric, celiac, and abdominal arterial occlusion technique; and (3) the natriuretic responses to acute saline expansion (0.9% NaCl, 10% body weight, intraperitoneal) or 2-week of 2% NaCl diet. Under basal conditions, PT- Nhe3-/- mice showed significantly lower systolic, diastolic, and mean arterial blood pressure ( P<0.01) than WT mice ( P<0.01). PT- Nhe3-/- mice also exhibited significantly greater diuretic ( P<0.01) and natriuretic responses than WT mice ( P<0.01), without altering 24-hour fecal Na+ excretion, plasma pH, Na+, and bicarbonate levels. In response to increased renal perfusion pressure by 30 mm Hg, the pressure-natriuresis response increased 5-fold in WT mice ( P<0.01), but it increased 8-fold in PT- Nhe3-/- mice ( P<0.01). In response to 10% acute saline expansion or 2-week 2% NaCl diet, more pronounced natriuretic responses were demonstrated in PT- Nhe3-/- than WT mice ( P<0.01). Our results support the scientific premise and physiological relevance that NHE3 in the proximal tubules plays an essential role in maintaining basal blood pressure homeostasis, and genetic deletion of NHE3 selectively in the proximal tubules of the kidney lowers blood pressure by increasing the pressure natriuretic response.


Assuntos
Pressão Sanguínea/fisiologia , Túbulos Renais Proximais/metabolismo , Natriurese/fisiologia , Trocador 3 de Sódio-Hidrogênio/metabolismo , Animais , Rim/metabolismo , Camundongos , Camundongos Knockout , Cloreto de Sódio na Dieta , Trocador 3 de Sódio-Hidrogênio/genética
15.
JCI Insight ; 3(20)2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30333321

RESUMO

Defects in genes mediating thyroid hormone biosynthesis result in dyshormonogenic congenital hypothyroidism (CH). Here, we report homozygous truncating mutations in SLC26A7 in 6 unrelated families with goitrous CH and show that goitrous hypothyroidism also occurs in Slc26a7-null mice. In both species, the gene is expressed predominantly in the thyroid gland, and loss of function is associated with impaired availability of iodine for thyroid hormone synthesis, partially corrected in mice by iodine supplementation. SLC26A7 is a member of the same transporter family as SLC26A4 (pendrin), an anion exchanger with affinity for iodide and chloride (among others), whose gene mutations cause congenital deafness and dyshormonogenic goiter. However, in contrast to pendrin, SLC26A7 does not mediate cellular iodide efflux and hearing in affected individuals is normal. We delineate a hitherto unrecognized role for SLC26A7 in thyroid hormone biosynthesis, for which the mechanism remains unclear.


Assuntos
Antiporters/genética , Hipotireoidismo Congênito/genética , Bócio/genética , Transportadores de Sulfato/genética , Adulto , Animais , Criança , Pré-Escolar , Códon sem Sentido , Hipotireoidismo Congênito/diagnóstico , Análise Mutacional de DNA , Feminino , Bócio/congênito , Bócio/diagnóstico , Células HEK293 , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Linhagem , Glândula Tireoide/patologia , Sequenciamento Completo do Exoma
16.
Cell Physiol Biochem ; 50(4): 1361-1375, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30355950

RESUMO

BACKGROUND/AIMS: The sodium-dependent bicarbonate transporter Slc4a8 (a.k.a NDCBE) mediates the co-transport of sodium and bicarbonate in exchange for chloride. It is abundantly detected in the brain, with low expression levels in the kidney. The cell distribution and subcellular localization of Slc4a8 in the kidney and its role in acid/base and electrolyte homeostasis has been the subject of conflicting reports. There are no conclusive localization or functional studies to pinpoint the location and demonstrate the function of Slc4a8 in the kidney. METHODS: Molecular techniques, including RT-PCR and in situ hybridization, were performed on kidney sections and tagged epitopes were used to examine the membrane targeting of Slc4a8 in polarized kidney cells. Crispr/Cas9 was used to generate and examine Slc4a8 KO mice. RESULTS: Zonal distribution and in situ hybridization studies showed very little expression for Slc4a8 (NDCBE) in the cortex or in cortical collecting ducts (CCD). Slc4a8 was predominantly detected in the outer and inner medullary collecting ducts (OMCD and IMCD), and was targeted to the basolateral membrane of osmotically tolerant MDCK cells. Slc4a8 KO mice did not show any abnormal salt or bicarbonate wasting under baseline conditions or in response to bicarbonate loading, salt restriction or furosemide-induced diuresis. CONCLUSION: Slc4a8 (NDCBE) is absent in the CCD and is predominantly localized on the basolateral membrane of medullary collecting duct cells. Further, Slc4a8 deletion does not cause significant acid base or electrolyte abnormalities in pathophysiologic states. Additional studies are needed to examine the role of Slc4a8 (NDCBE) in intracellular pH and volume regulation in medullary collecting duct cells.


Assuntos
Rim/metabolismo , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Bicarbonatos/metabolismo , Sistemas CRISPR-Cas/genética , Diurese/efeitos dos fármacos , Cães , Furosemida/farmacologia , Hibridização In Situ , Túbulos Renais Coletores/metabolismo , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Oligorribonucleotídeos Antissenso/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Sódio/urina , Cloreto de Sódio/metabolismo , Simportadores de Sódio-Bicarbonato/deficiência , Simportadores de Sódio-Bicarbonato/genética
17.
Curr Drug Metab ; 19(12): 1012-1020, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29962339

RESUMO

BACKGROUND: Thiazides are the most commonly used medications for the treatment of mild and moderate hypertension. Despite their recognized effect, the mechanism by which thiazides reduce systemic blood pressure remains uncertain. The prevailing belief is that thiazides reduce blood pressure primarily via enhancement of salt excretion consequent to the inhibition of the Na-Cl Cotransporter (NCC) in the Distal Convoluted Tubules (DCT). However, recent reports point to a reduction in peripheral vascular resistance as a major mechanism of antihypertensive effect of thiazides. It is plausible that both mechanisms, renal and extra-renal, may be operating simultaneously. Recent studies point to compensatory mechanisms in the kidney distal nephron that may play a role in blunting the diuretic effect of thiazides. Not much information is available about the efficacy of thiazides in controlling blood pressure in individuals with Chronic Kidney Disease (CKD). OBJECTIVE: This review will discuss the latest updates on the use and efficacy of thiazides derivatives as diuretics and antihypertensive medications in CKD patients. CONCLUSION: Thiazides remain effective as diuretics and antihypertensive agents in individuals with low GFR.


Assuntos
Anti-Hipertensivos/uso terapêutico , Diuréticos/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , Tiazidas/uso terapêutico , Animais , Anti-Hipertensivos/farmacologia , Diuréticos/farmacologia , Humanos , Rim/efeitos dos fármacos , Tiazidas/farmacologia
18.
Front Physiol ; 9: 849, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30050451

RESUMO

Background: Probenecid is a uricosuric agent that in addition to exerting a positive ionotropic effect in the heart, blocks the ATP transporter Pannexin 1 and inhibits the Cl-/HCO3- exchanger, pendrin. In the kidney, pendrin blunts the loss of salt wasting secondary to the inhibition of the thiazide-sensitive Na+-Cl- co-transporter (NCC/SLC12A3). Hypothesis: Pre-treatment with probenecid down-regulates pendrin; therefore, leaving NCC as the main salt absorbing transporter in the distal nephron, and hence enhances the hydrochlorothiazide (HCTZ)-induced diuresis. Methods: Daily balance studies, blood and urine chemical analysis, immunofluorescence, as well as western and northern blot analyses were utilized to examine the effects of probenecid alone (at 250 mg/kg/day) or in combination with HCTZ (at 40 mg/kg/day) on kidney function and on salt and water transporters in the collecting duct. Results: Male Sprague Dawley rats were subjected to three different protocols: (1) HCTZ for 4 days, (2) probenecid for 10 days, and (3) primed with probenecid for 6 days followed by probenecid and HCTZ for 4 additional days. Treatment protocol 1 (HCTZ for 4 days) only mildly increased the urine volume (U Vol) from a baseline of 9.8-13.4 ml/day. In response to treatment protocol 2 (probenecid for 10 days), U Vol increased to 15.9 ml/24 h. Treatment protocol 3 (probenecid for 6 days followed by probenecid and HCTZ for 4 additional days) increased the U Vol to 42.9 ml/day on day 4 of co-treatment with HCTZ and probenecid (compared to probenecid p = 0.003, n = 5 or HCTZ alone p = 0.001, n = 5). Probenecid treatment at 250 mg/kg/day downregulated the expression of pendrin and led to a decrease in AQP2 expression. Enhanced diuresis by probenecid plus HCTZ was not associated with volume depletion. Conclusion: Probenecid pre-treatment downregulates pendrin and robustly enhances diuresis by HCTZ-mediated NCC inhibition in kidney.

19.
Metabolism ; 85: 298-304, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29715464

RESUMO

OBJECTIVE: Cold and ß3-adrenergic receptor (AR) agonists activate beige adipocyte biogenesis in white adipose tissue (WAT). The two stimuli also induce expression of inflammatory cytokines in WAT. The low-grade inflammation may further promote WAT browning. However, the mechanisms to reconcile these two biological processes remain to be elucidated. In this study, we aim to investigate the roles of the rate-limiting polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SAT1) in regulating beige adipocyte biogenesis and inflammation. METHODS: Adipose-specific SAT1 knockout mice (SAT1-aKO) were generated by crossing adiponectin-cre to SAT1-lox/lox mice. Metabolic phenotype was investigated. Primary pre-adipocytes were isolated from inguinal WAT (iWAT) and differentiated to adipocytes for studying beige adipocyte biogenesis. RESULT: The expression and enzymatic activity of SAT1 were up-regulated in iWAT upon cold and ß3-AR stimulation. SAT1-aKO mice developed late-onset obesity on a high-fat diet with impaired cold-induced beige adipocyte biogenesis and energy expenditure. RNA-seq analysis of iWAT from cold-challenged SAT1-aKO mice revealed that, in addition to beige adipocyte biogenesis signatures, the immune response markers were highly enriched among reduced genes. In cultured adipocytes, SAT1 overexpression or pharmacological activation with N1, N11-diethylnorspermine (DENSpm) elevated oxygen consumption and increased the expression of beige adipocyte marker UCP1 and PGC-1α. DENSpm treatment of adipocytes also increased the expression of inflammatory genes. SAT1 activation enhanced hydrogen peroxide production in adipocytes. Antioxidant N-acetylcysteine abrogated the elevated UCP1 expression and reversed some inflammatory genes induced by SAT1 activation. CONCLUSIONS: SAT1 activation plays a key role in cold and ß3-AR agonist-induced beige adipocyte biogenesis and low-grade inflammation.


Assuntos
Acetiltransferases/metabolismo , Adipócitos Bege/metabolismo , Metabolismo dos Lipídeos/fisiologia , Termogênese/fisiologia , Acetiltransferases/genética , Animais , Metabolismo Energético/fisiologia , Peróxido de Hidrogênio/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos , Camundongos Knockout , Consumo de Oxigênio/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo
20.
J Biol Chem ; 293(17): 6259-6268, 2018 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-29530983

RESUMO

The solute carrier family 26 (SLC26) gene family encodes at least 10 different anion exchangers. SLC26 member 6 (SLC26A6 or CFEX/PAT-1) and the cystic fibrosis transmembrane conductance regulator (CFTR) co-localize to the apical membrane of pancreatic duct cells, where they act in concert to drive HCO3- and fluid secretion. In contrast, in the small intestine, SLC26A6 serves as the major pathway for oxalate secretion. However, little is known about the function of Slc26a6 in murine salivary glands. Here, RNA sequencing-based transcriptional profiling and Western blots revealed that Slc26a6 is highly expressed in mouse submandibular and sublingual salivary glands. Slc26a6 localized to the apical membrane of salivary gland acinar cells with no detectable immunostaining in the ducts. CHO-K1 cells transfected with mouse Slc26a6 exchanged Cl- for oxalate and HCO3-, whereas two other anion exchangers known to be expressed in salivary gland acinar cells, Slc4a4 and Slc4a9, mediated little, if any, Cl-/oxalate exchange. Of note, both Cl-/oxalate exchange and Cl-/HCO3- exchange were significantly reduced in acinar cells isolated from the submandibular glands of Slc26a6-/- mice. Oxalate secretion in submandibular saliva also decreased significantly in Slc26a6-/- mice, but HCO3- secretion was unaffected. Taken together, our findings indicate that Slc26a6 is located at the apical membrane of salivary gland acinar cells, where it mediates Cl-/oxalate exchange and plays a critical role in the secretion of oxalate into saliva.


Assuntos
Células Acinares/metabolismo , Antiporters/metabolismo , Membrana Celular/metabolismo , Ácido Oxálico/metabolismo , Glândula Submandibular/metabolismo , Transportadores de Sulfato/metabolismo , Células Acinares/citologia , Animais , Antiporters/genética , Bicarbonatos/metabolismo , Células CHO , Membrana Celular/genética , Antiportadores de Cloreto-Bicarbonato/genética , Antiportadores de Cloreto-Bicarbonato/metabolismo , Cloretos/metabolismo , Cricetulus , Camundongos , Camundongos Knockout , Saliva/metabolismo , Simportadores de Sódio-Bicarbonato/genética , Simportadores de Sódio-Bicarbonato/metabolismo , Glândula Submandibular/citologia , Transportadores de Sulfato/genética
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