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1.
Artigo em Inglês | MEDLINE | ID: mdl-38961846

RESUMO

The prostaglandin E2 (PGE2) receptor EP3 has been detected in the thick ascending limb (TAL) and the collecting duct of the kidney, where its actions are proposed to inhibit water reabsorption. However, EP3 is also expressed in other cell types, including vascular endothelial cells. The aim here was to determine the contribution of EP3 in renal water handling in male and female adult mice by phenotyping a novel mouse model with doxycycline-dependent deletion of EP3 throughout the kidney tubule (EP3-/- mice). RNAscope demonstrated that EP3 was highly expressed in the cortical and medullary TAL of adult mice. Compared to controls EP3 mRNA expression was reduced by >80% in whole kidney (RT-qPCR) and non-detectable (RNAscope) in renal tubules of EP3-/- mice. Under basal conditions, there were no significant differences in control and EP3-/- mice of both genders in food and water intake, bodyweight, urinary output or clinical biochemistries. No differences were detectable between genotypes in handling of an acute water load, or in their response to the vasopressin analogue dDAVP. No differences in water handling were observed when PGE2 production was enhanced using a 1% NaCl load. Expression of proteins involved in kidney water handling were not different between genotypes. This study demonstrates that renal tubular EP3 is not essential for body fluid homeostasis in males or females, even when PGE2 levels are high. The mouse model is a novel tool for examining the role of EP3 in kidney function independently of potential developmental abnormalities or systemic effects.

2.
FASEB J ; 36(11): e22583, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36197017

RESUMO

Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5PC-KO ) had a very mild phenotype, with no clear differences in AQP2 abundance, and mild differences in renal water handling and maximal urinary concentrating capacity. In contrast, NFAT5 (NFAT5PC-KO ) mice had significantly higher water intake and their 24 h urine volume was almost 10-fold greater than controls. After challenging with dDAVP or 8 h water restriction, NFAT5PC-KO mice were unable to concentrate their urine, demonstrating that they suffer from NDI. The abundance of AQP2, other AQPs, and the urea transporter UT-A1 were greatly decreased in NFAT5PC-KO mice. In conclusion, NFAT5 is a major regulator of not only Aqp2 gene transcription, but also other genes important for water homeostasis and its absence leads to the development of NDI.


Assuntos
Diabetes Insípido Nefrogênico , Diabetes Mellitus , Túbulos Renais Coletores , Fatores de Transcrição/metabolismo , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Arginina Vasopressina/metabolismo , Desamino Arginina Vasopressina/metabolismo , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/metabolismo , Diabetes Mellitus/metabolismo , Fator V/metabolismo , Túbulos Renais Coletores/metabolismo , Camundongos , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Vasopressinas/metabolismo , Água/metabolismo
3.
J Biol Chem ; 297(2): 100915, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34174287

RESUMO

The thiazide-sensitive sodium-chloride cotransporter (NCC) in the renal distal convoluted tubule (DCT) plays a critical role in regulating blood pressure (BP) and K+ homeostasis. During hyperkalemia, reduced NCC phosphorylation and total NCC abundance facilitate downstream electrogenic K+ secretion and BP reduction. However, the mechanism for the K+-dependent reduction in total NCC levels is unknown. Here, we show that NCC levels were reduced in ex vivo renal tubules incubated in a high-K+ medium for 24-48 h. This reduction was independent of NCC transcription, but was prevented using inhibitors of the proteasome (MG132) or lysosome (chloroquine). Ex vivo, high K+ increased NCC ubiquitylation, but inhibition of the ubiquitin conjugation pathway prevented the high K+-mediated reduction in NCC protein. In tubules incubated in high K+ media ex vivo or in the renal cortex of mice fed a high K+ diet for 4 days, the abundance and phosphorylation of heat shock protein 70 (Hsp70), a key regulator of ubiquitin-dependent protein degradation and protein folding, were decreased. Conversely, in similar samples the expression of PP1α, known to dephosphorylate Hsp70, was also increased. NCC coimmunoprecipitated with Hsp70 and PP1α, and inhibiting their actions prevented the high K+-mediated reduction in total NCC levels. In conclusion, we show that hyperkalemia drives NCC ubiquitylation and degradation via a PP1α-dependent process facilitated by Hsp70. This mechanism facilitates K+-dependent reductions in NCC to protect plasma K+ homeostasis and potentially reduces BP.


Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Hipertensão/patologia , Túbulos Renais Distais/metabolismo , Potássio na Dieta/farmacologia , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Animais , Modelos Animais de Doenças , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteólise , Transdução de Sinais , Membro 3 da Família 12 de Carreador de Soluto/genética , Ubiquitinação
4.
Hepatology ; 74(4): 2102-2117, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-33982322

RESUMO

BACKGROUND AND AIMS: Induced pluripotent stem cells (iPSCs) provide an important tool for the generation of patient-derived cells, including hepatocyte-like cells, by developmental cues through an endoderm intermediate. However, most iPSC lines fail to differentiate into endoderm, with induction resulting in apoptosis. APPROACH AND RESULTS: To address this issue, we built upon published methods to develop an improved protocol. We discovered that doxycycline dramatically enhances the efficiency of iPSCs to endoderm differentiation by inhibiting apoptosis and promoting proliferation through the protein kinase B pathway. We tested this protocol in >70 iPSC lines, 90% of which consistently formed complete sheets of endoderm. Endoderm generated by our method achieves similar transcriptomic profiles, expression of endoderm protein markers, and the ability to be further differentiated to downstream lineages. CONCLUSIONS: Furthermore, this method achieves a 4-fold increase in endoderm cell number and will accelerate studies of human diseases in vitro and facilitate the expansion of iPSC-derived cells for transplantation studies.


Assuntos
Apoptose/efeitos dos fármacos , Doxiciclina/farmacologia , Endoderma , Células-Tronco Pluripotentes Induzidas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Antibacterianos/farmacologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Linhagem Celular , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/fisiologia , Endoderma/citologia , Endoderma/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
5.
J Am Soc Nephrol ; 30(8): 1454-1470, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31253651

RESUMO

BACKGROUND: The NaCl cotransporter NCC in the kidney distal convoluted tubule (DCT) regulates urinary NaCl excretion and BP. Aldosterone increases NaCl reabsorption via NCC over the long-term by altering gene expression. But the acute effects of aldosterone in the DCT are less well understood. METHODS: Proteomics, bioinformatics, and cell biology approaches were combined with animal models and gene-targeted mice. RESULTS: Aldosterone significantly increases NCC activity within minutes in vivo or ex vivo. These effects were independent of transcription and translation, but were absent in the presence of high potassium. In vitro, aldosterone rapidly increased intracellular cAMP and inositol phosphate accumulation, and altered phosphorylation of various kinases/kinase substrates within the MAPK/ERK, PI3K/AKT, and cAMP/PKA pathways. Inhibiting GPR30, a membrane-associated receptor, limited aldosterone's effects on NCC activity ex vivo, and NCC phosphorylation was reduced in GPR30 knockout mice. Phosphoproteomics, network analysis, and in vitro studies determined that aldosterone activates EGFR-dependent signaling. The EGFR immunolocalized to the DCT and EGFR tyrosine kinase inhibition decreased NCC activity ex vivo and in vivo. CONCLUSIONS: Aldosterone acutely activates NCC to modulate renal NaCl excretion.


Assuntos
Aldosterona/farmacologia , Túbulos Renais Distais/metabolismo , Transdução de Sinais , Tiazidas/farmacologia , Aldosterona/metabolismo , Animais , Pressão Sanguínea , Cálcio/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Biologia Computacional , AMP Cíclico/metabolismo , Receptores ErbB/metabolismo , Síndrome de Gitelman/metabolismo , Rim/metabolismo , Masculino , Camundongos , Mineralocorticoides/metabolismo , Fosforilação , Proteômica , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Cloreto de Sódio/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/metabolismo
6.
Mol Pharmacol ; 89(6): 686-93, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26993802

RESUMO

The aquaporin-1 (AQP1) water channel is a potentially important drug target, as AQP1 inhibition is predicted to have therapeutic action in edema, tumor growth, glaucoma, and other conditions. Here, we measured the AQP1 inhibition efficacy of 12 putative small-molecule AQP1 inhibitors reported in six recent studies, and one AQP1 activator. Osmotic water permeability was measured by stopped-flow light scattering in human and rat erythrocytes that natively express AQP1, in hemoglobin-free membrane vesicles from rat and human erythrocytes, and in plasma membrane vesicles isolated from AQP1-transfected Chinese hamster ovary cell cultures. As a positive control, 0.3 mM HgCl2 inhibited AQP1 water permeability by >95%. We found that none of the tested compounds at 50 µM significantly inhibited or increased AQP1 water permeability in these assays. Identification of AQP1 inhibitors remains an important priority.


Assuntos
Aquaporina 1/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Aquaporina 1/metabolismo , Células CHO , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Fluoresceínas/metabolismo , Hemoglobinas/metabolismo , Humanos , Osmose/efeitos dos fármacos , Ratos Wistar , Bibliotecas de Moléculas Pequenas/química , Água/metabolismo
7.
Medchemcomm ; 6: 1278-1284, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26191399

RESUMO

Kidney urea transporters are targets for development of small-molecule inhibitors with action as salt-sparing diuretics. A cell-based, functional high-throughput screen identified 2,7-bisacetamido fluorenone 3 as a novel inhibitor of urea transporters UT-A1 and UT-B. Here, we synthesized twenty-two 2,7-disubstituted fluorenone analogs by acylation. Structure-activity relationship analysis revealed: (a) the carbonyl moiety at C9 is required for UT inhibition; (b) steric limitation on C2, 7-substituents; and (c) the importance of a crescent-shape structure. The most potent fluorenones inhibited UT-A1 and UT-B urea transport with IC50 ~ 1 µM. Analysis of in vitro metabolic stability in hepatic microsomes indicated metabolism of 2,7-disubstituted fluorenones by reductase and subsequent elimination. Computational docking to a homology model of UT-A1 suggested UT inhibitor binding to the UT cytoplasmic domain at a site that does not overlap with the putative urea binding site.

8.
Lab Chip ; 15(16): 3380-90, 2015 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-26159099

RESUMO

Cell membrane water permeability is an important determinant of epithelial fluid secretion, tissue swelling, angiogenesis, tumor spread and other biological processes. Cellular water channels, aquaporins, are important drug targets. Water permeability is generally measured from the kinetics of cell volume change in response to an osmotic gradient. Here, we developed a microfluidic platform in which cells expressing a cytoplasmic, volume-sensing fluorescent dye are rapidly subjected to an osmotic gradient by solution mixing inside a ~0.1 nL droplet surrounded by oil. The solution mixing time was <10 ms. Osmotic water permeability was deduced from a single, time-integrated fluorescence image of an observation area in which the time after mixing was determined through spatial position. Water permeability was accurately measured in aquaporin-expressing erythrocytes with half-times for osmotic equilibration down to <50 ms. Compared with conventional water permeability measurements using costly stopped-flow instrumentation, the microfluidic platform here utilizes sub-microliter blood sample volume, does not suffer from mixing artifacts, and replaces challenging kinetic measurements by single image capture using a standard laboratory fluorescence microscope.


Assuntos
Eritrócitos/metabolismo , Técnicas Analíticas Microfluídicas/métodos , Água/metabolismo , Animais , Aquaporinas/deficiência , Aquaporinas/genética , Permeabilidade da Membrana Celular , Eritrócitos/química , Eritrócitos/citologia , Fluoresceínas/química , Proteínas de Membrana Transportadoras/deficiência , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência , Óleos/química , Água/química
9.
Kidney Int ; 88(2): 311-20, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25993324

RESUMO

Inhibitors of kidney urea transporter (UT) proteins have potential use as salt-sparing diuretics ('urearetics') with a different mechanism of action than diuretics that target salt transporters. To study UT inhibition in rats, we screened about 10,000 drugs, natural products and urea analogs for inhibition of rat UT-A1. Drug and natural product screening found nicotine, sanguinarine and an indolcarbonylchromenone with IC50 of 10-20 µM. Urea analog screening found methylacetamide and dimethylthiourea (DMTU). DMTU fully and reversibly inhibited rat UT-A1 and UT-B by a noncompetitive mechanism with IC50 of 2-3 mM. Homology modeling and docking computations suggested DMTU binding sites on rat UT-A1. Following a single intraperitoneal injection of 500 mg/kg DMTU, peak plasma concentration was 9 mM with t1/2 of about 10 h, and a urine concentration of 20-40 mM. Rats chronically treated with DMTU had a sustained, reversible reduction in urine osmolality from 1800 to 600 mOsm, a 3-fold increase in urine output, and mild hypokalemia. DMTU did not impair urinary concentrating function in rats on a low protein diet. Compared to furosemide-treated rats, the DMTU-treated rats had greater diuresis and reduced urinary salt loss. In a model of syndrome of inappropriate antidiuretic hormone secretion, DMTU treatment prevented hyponatremia and water retention produced by water-loading in dDAVP-treated rats. Thus, our results establish a rat model of UT inhibition and demonstrate the diuretic efficacy of UT inhibition.


Assuntos
Diurese/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Cloreto de Sódio/urina , Tioureia/análogos & derivados , Animais , Sítios de Ligação , Modelos Animais de Doenças , Diuréticos/farmacologia , Cães , Avaliação Pré-Clínica de Medicamentos , Feminino , Furosemida/farmacologia , Hipopotassemia/induzido quimicamente , Hiponatremia/etiologia , Hiponatremia/prevenção & controle , Síndrome de Secreção Inadequada de HAD/complicações , Síndrome de Secreção Inadequada de HAD/tratamento farmacológico , Concentração Inibidora 50 , Células Madin Darby de Rim Canino , Proteínas de Membrana Transportadoras/química , Estrutura Molecular , Concentração Osmolar , Ratos , Ratos Wistar , Tioureia/sangue , Tioureia/química , Tioureia/farmacologia , Tioureia/uso terapêutico , Fatores de Tempo , Urina/química , Transportadores de Ureia
10.
Biochim Biophys Acta ; 1848(5): 1075-80, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25613743

RESUMO

Small-molecule inhibitors of urea transporter (UT) proteins in kidney have potential application as novel salt-sparing diuretics. The urea analog dimethylthiourea (DMTU) was recently found to inhibit the UT isoforms UT-A1 (expressed in kidney tubule epithelium) and UT-B (expressed in kidney vasa recta endothelium) with IC50 of 2-3 mM, and was shown to have diuretic action when administered to rats. Here, we measured UT-A1 and UT-B inhibition activity of 36 thiourea analogs, with the goal of identifying more potent and isoform-selective inhibitors, and establishing structure-activity relationships. The analog set systematically explored modifications of substituents on the thiourea including alkyl, heterocycles and phenyl rings, with different steric and electronic features. The analogs had a wide range of inhibition activities and selectivities. The most potent inhibitor, 3-nitrophenyl-thiourea, had an IC50 of ~0.2 mM for inhibition of both UT-A1 and UT-B. Some analogs such as 4-nitrophenyl-thiourea were relatively UT-A1 selective (IC50 1.3 vs. 10 mM), and others such as thioisonicotinamide were UT-B selective (IC50>15 vs. 2.8 mM).


Assuntos
Moduladores de Transporte de Membrana/farmacologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Tioureia/farmacologia , Ureia/metabolismo , Animais , Cães , Relação Dose-Resposta a Droga , Células Madin Darby de Rim Canino , Moduladores de Transporte de Membrana/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Tioureia/análogos & derivados , Tioureia/química , Transfecção , Transportadores de Ureia
11.
Nat Rev Nephrol ; 11(2): 113-23, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25488859

RESUMO

Conventional diuretics such as furosemide and thiazides target salt transporters in kidney tubules, but urea transporters (UTs) have emerged as alternative targets. UTs are a family of transmembrane channels expressed in a variety of mammalian tissues, in particular the kidney. UT knockout mice and humans with UT mutations exhibit reduced maximal urinary osmolality, demonstrating that UTs are necessary for the concentration of urine. Small-molecule screening has identified potent and selective inhibitors of UT-A, the UT protein expressed in renal tubule epithelial cells, and UT-B, the UT protein expressed in vasa recta endothelial cells. Data from UT knockout mice and from rodents administered UT inhibitors support the diuretic action of UT inhibition. The kidney-specific expression of UT-A1, together with high selectivity of the small-molecule inhibitors, means that off-target effects of such small-molecule drugs should be minimal. This Review summarizes the structure, expression and function of UTs, and looks at the evidence supporting the validity of UTs as targets for the development of salt-sparing diuretics with a unique mechanism of action. UT-targeted inhibitors may be useful alone or in combination with conventional diuretics for therapy of various oedemas and hyponatraemias, potentially including those refractory to treatment with current diuretics.


Assuntos
Diuréticos/farmacologia , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Animais , Humanos , Transportadores de Ureia
12.
Subcell Biochem ; 73: 165-77, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25298345

RESUMO

Urea transporter (UT) proteins, which include isoforms of UT-A in kidney tubule epithelia and UT-B in vasa recta endothelia and erythrocytes, facilitate urinary concentrating function. Inhibitors of urea transporter function have potential clinical applications as sodium-sparing diuretics, or 'urearetics,' in edema from different etiologies, such as congestive heart failure and cirrhosis, as well as in syndrome of inappropriate antidiuretic hormone (SIADH). High-throughput screening of drug-like small molecules has identified UT-A and UT-B inhibitors with nanomolar potency. Inhibitors have been identified with different UT-A versus UT-B selectivity profiles and putative binding sites on UT proteins. Studies in rodent models support the utility of UT inhibitors in reducing urinary concentration, though testing in clinically relevant animal models of edema has not yet been done.


Assuntos
Proteínas de Membrana Transportadoras/química , Estrutura Terciária de Proteína , Bibliotecas de Moléculas Pequenas/química , Ureia/química , Animais , Transporte Biológico/efeitos dos fármacos , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Ureia/metabolismo , Transportadores de Ureia
13.
Am J Nephrol ; 39(6): 528-35, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24942911

RESUMO

BACKGROUND/AIMS: Cyclosporine (CsA) is a calcineurin inhibitor widely used as an immunosuppressant in organ transplantation. Previous studies demonstrated the relationship between CsA and renal sodium transporters such as the Na-K-2Cl cotransporter in the loop of Henle (NKCC2). Experimental models of CsA-induced hypertension have shown an increase in renal NKCC2. METHODS: Using immunoblotting of urinary exosomes, we investigated in CsA-treated kidney transplant patients (n = 39) the excretion of NKCC2 and Na-Cl cotransporter (NCC) and its association with blood pressure (BP) level. We included 8 non-CsA-treated kidney transplant patients as a control group. Clinical data, immunosuppression and hypertension treatments, blood and 24-hour urine tests, and 24-hour ambulatory BP monitoring were recorded. RESULTS: CsA-treated patients tended to excrete a higher amount of NKCC2 than non-CsA-treated patients (mean ± SD, 175 ± 98 DU and 90 ± 70.3 DU, respectively; p = 0.05) and showed higher BP values (24-hour systolic BP 138 ± 17 mm Hg and 112 ± 12 mm Hg, p = 0.003; 24-hour diastolic BP, 83.8 ± 9.8 mm Hg and 72.4 ± 5.2 mm Hg, p = 0.015, respectively). Within the CsA-treated group, there was no correlation between either NKCC2 or NCC excretion and BP levels. This was confirmed by a further analysis including potential confounding factors. On the other hand, a significant positive correlation was observed between CsA blood levels and the excretion of NKCC2 and NCC. CONCLUSION: Overall, these results support the hypothesis that CsA induces an increase in NKCC2 and NCC in urinary exosomes of renal transplant patients. The fact that the increase in sodium transporters in urine did not correlate with the BP level suggests that in kidney transplant patients, other mechanisms could be implicated in CsA-induced hypertension.


Assuntos
Ciclosporina/uso terapêutico , Exossomos/metabolismo , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Rim , Rim/metabolismo , Sódio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Adulto , Idoso , Pressão Sanguínea , Estudos de Casos e Controles , Ciclosporina/farmacologia , Exossomos/efeitos dos fármacos , Feminino , Humanos , Imunossupressores/farmacologia , Rim/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Membro 1 da Família 12 de Carreador de Soluto/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/efeitos dos fármacos , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Urina , Adulto Jovem
14.
FASEB J ; 28(9): 3878-90, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24843071

RESUMO

Urea transport (UT) proteins of the UT-A class are expressed in epithelial cells in kidney tubules, where they are required for the formation of a concentrated urine by countercurrent multiplication. Here, using a recently developed high-throughput assay to identify UT-A inhibitors, a screen of 50,000 synthetic small molecules identified UT-A inhibitors of aryl-thiazole, γ-sultambenzosulfonamide, aminocarbonitrile butene, and 4-isoxazolamide chemical classes. Structure-activity analysis identified compounds that inhibited UT-A selectively by a noncompetitive mechanism with IC50 down to ∼1 µM. Molecular modeling identified putative inhibitor binding sites on rat UT-A. To test compound efficacy in rats, formulations and administration procedures were established to give therapeutic inhibitor concentrations in blood and urine. We found that intravenous administration of an indole thiazole or a γ-sultambenzosulfonamide at 20 mg/kg increased urine output by 3-5-fold and reduced urine osmolality by ∼2-fold compared to vehicle control rats, even under conditions of maximum antidiuresis produced by 1-deamino-8-D-arginine vasopressin (DDAVP). The diuresis was reversible and showed urea > salt excretion. The results provide proof of concept for the diuretic action of UT-A-selective inhibitors. UT-A inhibitors are first in their class salt-sparing diuretics with potential clinical indications in volume-overload edemas and high-vasopressin-associated hyponatremias.


Assuntos
Transporte Biológico/efeitos dos fármacos , Diurese/efeitos dos fármacos , Capacidade de Concentração Renal/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Bibliotecas de Moléculas Pequenas/farmacologia , Urina/química , Animais , Cromatografia Líquida , Diurese/fisiologia , Cães , Ensaios de Triagem em Larga Escala , Células Madin Darby de Rim Canino , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Concentração Osmolar , Ratos , Ratos Wistar , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/farmacocinética , Cloreto de Sódio , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Distribuição Tecidual , Sistema Urinário/efeitos dos fármacos , Sistema Urinário/metabolismo , Transportadores de Ureia
15.
FASEB J ; 28(3): 1446-53, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24334548

RESUMO

Aquaporin 1 (AQP1) is a plasma membrane water-transporting protein expressed strongly in tumor microvascular endothelia. We previously reported impaired angiogenesis in implanted tumors in AQP1-deficient mice and reduced migration of AQP1-deficient endothelial cells in vitro. Here, we investigated the consequences of AQP1 deficiency in mice that spontaneously develop well-differentiated, luminal-type breast adenomas with lung metastases [mouse mammary tumor virus-driven polyoma virus middle T oncogene (MMTV-PyVT)]. AQP1(+/+) MMTV-PyVT mice developed large breast tumors with total tumor mass 3.5 ± 0.5 g and volume 265 ± 36 mm(3) (SE, 11 mice) at age 98 d. Tumor mass (1.6±0.2 g) and volume (131±15 mm(3), 12 mice) were greatly reduced in AQP1(-/-) MMTV-PyVT mice (P<0.005). CD31 immunofluorescence showed abnormal microvascular anatomy in tumors of AQP1(-/-) MMTV-PyVT mice, with reduced vessel density. HIF-1α expression was increased in tumors in AQP1(-/-) MMTV-PyVT mice. The number of lung metastases (5±1/mouse) was much lower than in AQP1(+/+) MMTV-PyVT mice (31±8/mouse, P<0.005). These results implicate AQP1 as an important determinant of tumor angiogenesis and, hence, as a potential drug target for adjuvant therapy of solid tumors.


Assuntos
Aquaporina 1/genética , Neoplasias da Mama/patologia , Deleção de Genes , Neoplasias Pulmonares/secundário , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/genética , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética
16.
Chem Biol ; 20(10): 1235-44, 2013 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-24055006

RESUMO

Urea transporter (UT) proteins, including UT-A in kidney tubule epithelia and UT-B in vasa recta microvessels, facilitate urinary concentrating function. A screen for UT-A inhibitors was developed in MDCK cells expressing UT-A1, water channel aquaporin-1, and YFP-H148Q/V163S. An inwardly directed urea gradient produces cell shrinking followed by UT-A1-dependent swelling, which was monitored by YFP-H148Q/V163S fluorescence. Screening of ~90,000 synthetic small molecules yielded four classes of UT-A1 inhibitors with low micromolar half-maximal inhibitory concentration that fully and reversibly inhibited urea transport by a noncompetitive mechanism. Structure-activity analysis of >400 analogs revealed UT-A1-selective and UT-A1/UT-B nonselective inhibitors. Docking computations based on homology models of UT-A1 suggested inhibitor binding sites. UT-A inhibitors may be useful as diuretics ("urearetics") with a mechanism of action that may be effective in fluid-retaining conditions in which conventional salt transport-blocking diuretics have limited efficacy.


Assuntos
Avaliação Pré-Clínica de Medicamentos , Proteínas de Membrana Transportadoras/metabolismo , Bibliotecas de Moléculas Pequenas/análise , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sítios de Ligação , Cães , Ensaios de Triagem em Larga Escala , Células Madin Darby de Rim Canino , Proteínas de Membrana Transportadoras/química , Simulação de Acoplamento Molecular , Conformação Proteica , Ratos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Transportadores de Ureia
17.
Nephron Physiol ; 114(3): p25-34, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20068364

RESUMO

BACKGROUND: Altered renal sodium handling has a major pathogenic role in salt-sensitive hypertension. Renal sodium transporters are present in urinary exosomes. We hypothesized that sodium transporters would be excreted into the urine in different amounts in response to sodium intake in salt-sensitive versus salt-resistant patients. METHODS: Urinary exosomes were isolated by ultracentrifugation, and their content of Na-K-2Cl cotransporter (NKCC2) and Na-Cl cotransporter (NCC) was analyzed by immunoblotting. Animal studies: NKCC2 and NCC excretion was measured in 2 rat models to test whether changes in sodium transporter excretion are indicative of regulated changes in the kidney tissue. Human studies: in hypertensive patients (n = 41), we investigated: (1) a possible correlation between sodium reabsorption and urinary exosomal excretion of sodium transporters, and (2) the profile of sodium transporter excretion related to blood pressure (BP) changes with salt intake. A 24-hour ambulatory BP monitoring and a 24-hour urine collection were performed after 1 week on a low- and 1 week on a high-salt diet. RESULTS: Animal studies: urinary NKCC2 and NCC excretion rates correlated well with their abundance in the kidney. Human studies: 6 patients (15%) were classified as salt sensitive. The NKCC2 and NCC abundance did not decrease after the high-salt period, when the urinary sodium reabsorption decreased from 99.7 to 99.0%. In addition, the changes in BP with salt intake were not associated with a specific profile of exosomal excretion. CONCLUSIONS: Our results do not support the idea that excretion levels of NKCC2 and NCC via urinary exosomes are markers of tubular sodium reabsorption in hypertensive patients.


Assuntos
Exossomos , Hipertensão/metabolismo , Túbulos Renais/metabolismo , Simportadores de Cloreto de Sódio/urina , Sódio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/urina , Animais , Pressão Sanguínea , Feminino , Humanos , Hipertensão/urina , Rim/metabolismo , Masculino , Ratos , Sódio/urina , Simportadores de Cloreto de Sódio/metabolismo , Sódio na Dieta/administração & dosagem , Membro 1 da Família 12 de Carreador de Soluto/metabolismo
18.
Med Clin (Barc) ; 129(11): 433-7, 2007 Sep 29.
Artigo em Espanhol | MEDLINE | ID: mdl-17927939

RESUMO

Renal sodium and water reabsorption is mediated by renal sodium transporters and water channels or aquaporins which are localized in the apical and basolateral membranes of tubular epithelial cells. The main apical sodium transporters and water channels located along the nephron are: sodium-proton exchanger subtype 3 (NHE-3) which reabsorbs most of the sodium coming from the glomerular filtrate, sodium-phosphate type II cotransporter (NaPiII) and aquaporin-1, all of which are located in the proximal tubule; sodium-potassium-2 chloride cotransporter (NKCC2) which plays a key role in sodium reabsorption in the thick ascending limb; the sodium-chloride cotransporter (NCC) in the distal tubule; and the epithelial sodium channel (ENaC) and aquaporin-2 located in the collecting tubule. There are some experimental studies in which the role of these proteins has been associated with the pathophysiology of several sodium and water balance disorders. In humans, urine is the perfect source to obtain biomarkers useful for the diagnosis of kidney diseases and the assessment of disease progression without the use of invasive procedures. Thus, some of the renal sodium transporters or the aquaporins located in the apical membrane which are excreted in the tubular lumen and detected in urine could become biomarkers of some sodium and water balance disorders. Nowadays there are many studies investigating the role of these proteins in humans in clinical settings.


Assuntos
Aquaporinas/metabolismo , Rim/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sódio/metabolismo , Biomarcadores/metabolismo , Humanos
19.
Med. clín (Ed. impr.) ; 129(11): 433-437, sept. 2007. ilus, tab
Artigo em Es | IBECS | ID: ibc-057968

RESUMO

En la reabsorción tubular de sodio y agua están implicados los transportadores renales de sodio y los canales de agua o aquaporinas, que se localizan en las membranas apicales y basolaterales de las células epiteliales a lo largo de los túbulos renales. Los principales transportadores de sodio y canales de agua apicales situados a lo largo de la nefrona son: el intercambiador sodio-protones tipo 3 (NHE-3), que reabsorbe la mayoría del sodio procedente del filtrado glomerular, el cotransportador sodio-fosfato tipo II (NaPiII) y la aquaporina-1, todos ellos localizados en el túbulo proximal; el cotransportador de sodio-potasio-2 cloros (NKCC2), clave en la reabsorción de sodio en la rama gruesa del asa de Henle; el cotransportador sodio-cloro (NCC) en el túbulo distal y el canal epitelial de sodio (ENaC) y la aquaporina-2, ubicados en el túbulo colector. Hay varios estudios experimentales en los que se ha puesto de manifiesto el papel de estas proteínas en determinadas enfermedades asociadas a trastornos en el balance hidrosalino. En humanos, la orina es la fuente ideal para la obtención no invasiva de biomarcadores útiles en el diagnóstico y el seguimiento de enfermedades renales. Así, proteínas de localización apical, como algunos transportadores renales de sodio o aquaporinas que se excretan a la luz tubular, pueden detectarse en orina y podrían resultar biomarcadores de determinados trastornos en el balance hidrosalino. En la actualidad se está realizando numerosos trabajos en patología humana para definir mejor el papel de estas proteínas en la clínica


Renal sodium and water reabsorption is mediated by renal sodium transporters and water channels or aquaporins which are localized in the apical and basolateral membranes of tubular epithelial cells. The main apical sodium transporters and water channels located along the nephron are: sodium-proton exchanger subtype 3 (NHE-3) which reabsorbs most of the sodium coming from the glomerular filtrate, sodium-phosphate type II cotransporter (NaPiII) and aquaporin-1, all of which are located in the proximal tubule; sodium-potassium-2 chloride cotransporter (NKCC2) which plays a key role in sodium reabsorption in the thick ascending limb; the sodium-chloride cotransporter (NCC) in the distal tubule; and the epithelial sodium channel (ENaC) and aquaporin-2 located in the collecting tubule. There are some experimental studies in which the role of these proteins has been associated with the pathophysiology of several sodium and water balance disorders. In humans, urine is the perfect source to obtain biomarkers useful for the diagnosis of kidney diseases and the assessment of disease progression without the use of invasive procedures. Thus, some of the renal sodium transporters or the aquaporins located in the apical membrane which are excreted in the tubular lumen and detected in urine could become biomarkers of some sodium and water balance disorders. Nowadays there are many studies investigating the role of these proteins in humans in clinical settings


Assuntos
Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio , Túbulos Renais/fisiologia , Aquaporinas/fisiologia , Nefropatias/fisiopatologia , Biomarcadores/análise , Desequilíbrio Hidroeletrolítico/fisiopatologia
20.
Nephrol Dial Transplant ; 22(10): 2810-6, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17595192

RESUMO

BACKGROUND: Hypertension induced by cyclosporine is associated with renal sodium and water retention. Using immunoblotting of kidney homogenates, we investigated the regulation of sodium and water transport proteins in a rat model of cyclosporine-induced hypertension. METHODS: Rats were treated with cyclosporine (25 mg/kg/day intraperitoneally) during 7 days. Control rats received vehicle. RESULTS: Cyclosporine-treated rats had an increase in blood pressure with a decrease in renal sodium excretion compared with control rats. There were no differences either in sodium intake or in plasma creatinine levels between the two groups of rats. These data suggest that the decrease in sodium excretion in the cyclosporine-treated rats was due to an increase in renal sodium absorption. The densitometric analysis of the renal immunoblot showed an increase in the Na-K-2Cl cotransporter of the loop of Henle (NKCC2) in cyclosporine-treated rats (178% +/- 36) compared with control rats (100% +/- 18; P < 0.05*). This protein rise was associated with an increase in the NKCC2 mRNA pointing to a transcriptional regulation of this sodium transporter. There were no statistically significant changes in the sodium proton exchange (NHE-3) of the proximal tubule although in this renal segment, aquaporin-1 was increased in cyclosporine-treated rats compared with control rats (control 100% +/- 6 vs cyclosporine 119% +/- 6; P < 0.05*). CONCLUSIONS: Our results pointed to the thick ascending limb of the loop of Henle as an important site of sodium retention in cyclosporine-induced hypertension. This data may have potential clinical implications for the treatment of hypertension induced by cyclosporine.


Assuntos
Ciclosporina/farmacologia , Hipertensão/tratamento farmacológico , Simportadores de Cloreto de Sódio-Potássio/fisiologia , Sódio/metabolismo , Animais , Transporte Biológico , Modelos Animais de Doenças , Imunossupressores/farmacologia , Rim/metabolismo , Alça do Néfron/metabolismo , Modelos Biológicos , Ratos , Ratos Wistar , Trocador 3 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 1 da Família 12 de Carreador de Soluto , Água/metabolismo
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