Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 136
Filtrar
1.
Physiol Rev ; 103(4): 2827-2872, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37440209

RESUMO

The kidneys play a key role in maintaining total body homeostasis. The complexity of this task is reflected in the unique architecture of the organ. Ureteral obstruction greatly affects renal physiology by altering hemodynamics, changing glomerular filtration and renal metabolism, and inducing architectural malformations of the kidney parenchyma, most importantly renal fibrosis. Persisting pathological changes lead to chronic kidney disease, which currently affects ∼10% of the global population and is one of the major causes of death worldwide. Studies on the consequences of ureteral obstruction date back to the 1800s. Even today, experimental unilateral ureteral obstruction (UUO) remains the standard model for tubulointerstitial fibrosis. However, the model has certain limitations when it comes to studying tubular injury and repair, as well as a limited potential for human translation. Nevertheless, ureteral obstruction has provided the scientific community with a wealth of knowledge on renal (patho)physiology. With the introduction of advanced omics techniques, the classical UUO model has remained relevant to this day and has been instrumental in understanding renal fibrosis at the molecular, genomic, and cellular levels. This review details key concepts and recent advances in the understanding of obstructive nephropathy, highlighting the pathophysiological hallmarks responsible for the functional and architectural changes induced by ureteral obstruction, with a special emphasis on renal fibrosis.


Assuntos
Insuficiência Renal Crônica , Obstrução Ureteral , Humanos , Animais , Obstrução Ureteral/complicações , Obstrução Ureteral/patologia , Rim/metabolismo , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Hemodinâmica , Fibrose , Modelos Animais de Doenças
2.
Am J Physiol Renal Physiol ; 326(1): F69-F85, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-37855039

RESUMO

Poly(ADP-ribosyl)ation (PARylation), as a posttranslational modification mediated by poly(ADP-ribose) polymerases (PARPs) catalyzing the transfer of ADP-ribose from NAD+ molecules to acceptor proteins, involves a number of cellular processes. As mice lacking the PARP-1 gene (Parp1) produce more urine, we investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). In biotin-conjugated nicotinamide adenine dinucleotide (biotin-NAD+) pulldown and immunoprecipitation assays of poly(ADP)-ribose in mpkCCDc14 cells, immunoblots demonstrated that 1-deamino-8-D-arginine vasopressin (dDAVP) induced the PARylation of total proteins, associated with an increase in the cleavage of PARP-1 and cleaved caspase-3 expression. By inhibiting PARP-1 with siRNA, the abundance of dDAVP-induced AQP2 mRNA and protein was significantly diminished. In contrast, despite a substantial decrease in PARylation, the PARP-1 inhibitor (PJ34) had no effect on the dDAVP-induced regulation of AQP2 expression. The findings suggest that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. Bioinformatic analysis revealed that 408 proteins interact with PARP-1 in the collecting duct (CD) cells of the kidney. Among them, the signaling pathway of the vasopressin V2 receptor was identified for 49 proteins. In particular, ß-catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein. A significant decrease of ß-catenin phosphorylation (Ser552) in response to dDAVP was associated with siRNA-mediated PARP-1 knockdown. Taken together, PARP-1 is likely to play a role in vasopressin-induced AQP2 expression by interacting with ß-catenin in renal CD cells.NEW & NOTEWORTHY The poly(ADP-ribose) polymerase (PARP) family catalyzes poly(ADP-ribosylation) (PARylation), which is one of the posttranslational modifications of largely undetermined physiological significance. This study investigated the role of PARP-1, the most prevalent member of the PARP family, in the vasopressin-responsive expression of aquaporin-2 (AQP2). The results demonstrated that PARP-1 protein expression itself, and not PARP-1-mediated PARylation, is necessary for dDAVP-regulated AQP2 expression. ß-Catenin, which is phosphorylated at Ser552 by dDAVP, was identified as the PARP-1-interacting protein.


Assuntos
Aquaporina 2 , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Camundongos , Aquaporina 2/genética , beta Catenina/metabolismo , Biotina/metabolismo , Desamino Arginina Vasopressina/farmacologia , Rim/metabolismo , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Interferente Pequeno , Vasopressinas/farmacologia , Vasopressinas/metabolismo
3.
Int J Mol Sci ; 24(2)2023 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-36675199

RESUMO

mpkCCDc14 cells, a polarized epithelial cell line derived from mouse kidney cortical collecting ducts, are known to express the vasopressin V2 receptor (V2R) and aquaporin-2 (AQP2) that are responsive to vasopressin. However, a low abundance of the endogenous AQP2 protein in the absence of vasopressin and heterogeneity of AQP2 protein abundance among the cultured cells may limit the further application of the cell line in AQP2 studies. To overcome the limitation, we aimed to establish mpkCCDc14 cells constitutively expressing V2R and AQP2 via CRISPR/Cas9-mediated genome engineering technology (i.e., V2R-AQP2 cells). 3'- and 5'-Junction PCR revealed that the V2R-AQP2 expression cassette with a long insert size (~2.2 kb) was correctly integrated. Immunoblotting revealed the expression of products of integrated Aqp2 genes. Cell proliferation rate and dDAVP-induced cAMP production were not affected by the knock-in of Avpr2 and Aqp2 genes. The AQP2 protein abundance was significantly higher in V2R-AQP2 cells compared with control mpkCCDc14 cells in the absence of dDAVP and the integrated AQP2 was detected. Immunocytochemistry demonstrated that V2R-AQP2 cells exhibited more homogenous and prominent AQP2 labeling intensity in the absence of dDAVP stimulation. Moreover, prominent AQP2 immunolabeling (both AQP2 and pS256-AQP2) in the apical domain of the genome-edited cells was observed in response to dDAVP stimulation, similar to that in the unedited control mpkCCDc14 cells. Taken together, mpkCCDc14 cells constitutively expressing V2R and AQP2 via genome engineering could be exploited for AQP2 studies.


Assuntos
Aquaporina 2 , Túbulos Renais Coletores , Camundongos , Animais , Aquaporina 2/metabolismo , Desamino Arginina Vasopressina/metabolismo , Túbulos Renais Coletores/metabolismo , Vasopressinas/metabolismo , Membrana Celular/metabolismo
4.
Am J Physiol Cell Physiol ; 320(5): C771-C777, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33625928

RESUMO

Aquaporin (AQP) water channels facilitate passive transport of water across cellular membranes following an osmotic gradient. AQPs are expressed in a multitude of epithelia, endothelia, and other cell types where they play important roles in physiology, especially in the regulation of body water homeostasis, skin hydration, and fat metabolism. AQP dysregulation is associated with many pathophysiological conditions, including nephrogenic diabetes insipidus, chronic kidney disease, and congestive heart failure. Moreover, AQPs have emerged as major players in a multitude of cancers where high expression correlates with metastasis and poor prognosis. Besides water transport, AQPs have been shown to be involved in cellular signaling, cell migration, cell proliferation, and regulation of junctional proteins involved in cell-cell adhesion; all cellular processes which are dysregulated in cancer. This review focuses on AQPs as regulators of junctional proteins involved in cell-cell adhesion.


Assuntos
Aquaporinas/metabolismo , Moléculas de Adesão Celular/metabolismo , Adesão Celular , Neoplasias/metabolismo , Água/metabolismo , Animais , Aquaporinas/química , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Humanos , Neoplasias/patologia , Estado de Hidratação do Organismo , Conformação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Equilíbrio Hidroeletrolítico
5.
Kidney Int ; 99(1): 117-133, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32853632

RESUMO

Cell therapy using genome-engineered stem cells has emerged as a novel strategy for the treatment of kidney diseases. By exploiting genome editing technology, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) secreting an angiogenic factors or an anti-inflammatory factor were generated for therapeutic application in acute kidney injury. Junction polymerase chain reaction analysis verified zinc finger nucleases-assisted integration of the desired gene into the hUC-MSCs. Flow cytometry and differentiation assays indicated that genome editing did not affect the differentiation potential of these mesenchymal stem cells. Protein measurement in conditioned media with the use of ELISA and immunoblotting revealed the production and secretion of each integrated gene product. For cell therapy in the bilateral ischemia-reperfusion mouse model of acute kidney injury, our innovative scaffold-free cell sheets were established using a non-biodegradable temperature-responsive polymer. One of each type of scaffold-free cell sheets of either the angiogenic factor vascular endothelial grown factor or angiopoietin-1, or the anti-inflammatory factor erythropoietin, or α-melanocyte-stimulating hormone-secreting hUC-MSCs was applied to the decapsulated kidney surface. This resulted in significant amelioration of kidney dysfunction in the mice with acute kidney injury, effects that were superior to intravenous administration of the same genome-engineered hUC-MSCs. Thus, our scaffold-free cell sheets of genome-engineered mesenchymal stem cells provides therapeutic effects by inhibiting acute kidney injury via angiogenesis or anti-inflammation.


Assuntos
Injúria Renal Aguda , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Injúria Renal Aguda/genética , Injúria Renal Aguda/terapia , Animais , Diferenciação Celular , Camundongos , Cordão Umbilical
6.
FASEB J ; 34(2): 3379-3398, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31922312

RESUMO

Aquaporin-5 (AQP5) plays a role in breast cancer cell migration. This study aimed to identify AQP5-targeting miRNAs and examine their effects on breast cancer cell migration through exosome-mediated delivery. Bioinformatic analyses identified miR-1226-3p, miR-19a-3p, and miR-19b-3p as putative regulators of AQP5 mRNA. Immunoblotting revealed a decrease of AQP5 protein abundance when each of these miRNAs was transfected into human breast cancer MDA-MB-231 cells. Quantitative real-time PCR demonstrated the reduction of AQP5 mRNA expression by the transfection of miR-1226-3p and a luciferase reporter assay revealed the reduction of AQP5 translation after the transfection of miR-19b-3p in MDA-MB-231 cells. Consistently, the transfection of each miRNA impeded cell migration. Pathway enrichment analyses showed that these three miRNAs regulate target genes, which were predominantly enriched in the gap junction pathway. For the efficient delivery of AQP5-targeting miRNAs to breast cancer cells, exosomes expressing both miRNAs and a peptide targeting interleukin-4 receptor, which is highly expressed in breast cancer cells, were bioengineered and their inhibitory effects on AQP5 protein expression and cell migration were demonstrated in MDA-MB-231 cells. Taken together, AQP5-regulating miRNAs are identified, which could be exploited for the inhibition of breast cancer cell migration via the exosome-mediated delivery.


Assuntos
Neoplasias da Mama/metabolismo , Movimento Celular , Exossomos/metabolismo , MicroRNAs/metabolismo , Aquaporina 5/genética , Aquaporina 5/metabolismo , Feminino , Células HEK293 , Humanos , Subunidade alfa de Receptor de Interleucina-4/metabolismo , Células MCF-7 , MicroRNAs/genética , Oligopeptídeos/metabolismo
7.
FASEB J ; 33(6): 6980-6994, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30840830

RESUMO

Plasticity of epithelial cell-cell adhesion is vital in epithelial homeostasis and is regulated in multiple processes associated with cell migration, such as embryogenesis and wound healing. In cancer, cell-cell adhesion is compromised and is associated with increased cell migration and metastasis. Aquaporin (AQP) water channels facilitate water transport across cell membranes and are essential in the regulation of body water homeostasis. Increased expression of several AQPs, especially AQP5, is associated with increased cancer cell migration, metastasis, and poor prognosis. We found that AQP5 overexpression in normal epithelial cells induced cell detachment and dissemination from migrating cell sheets. AQP5 reduced both cell-cell coordination during collective migration and overall distance covered by the migrating cell sheets. AQP5 and the isoforms AQP1 and AQP4 decreased, whereas AQP3 increased, levels of plasma membrane-associated lateral junctional proteins. This regulation was mediated by the cytoplasmic domains of the AQPs. This shows that the AQPs have dual functions in epithelial physiology: as channel proteins and as differential regulators of cell-cell adhesiveness. This regulation may contribute to dynamic regulation of cell junctions in processes such as embryogenesis and wound healing and also explain the pivotal roles of AQPs in carcinogenesis and metastasis.-Login, F. H., Jensen, H. H., Pedersen, G. A., Koffman, J. S., Kwon, T.-H., Parsons, M., Nejsum, L. N. Aquaporins differentially regulate cell-cell adhesion in MDCK cells.


Assuntos
Aquaporinas/metabolismo , Adesão Celular/fisiologia , Animais , Aquaporinas/genética , Moléculas de Adesão Celular , Membrana Celular , Cães , Regulação da Expressão Gênica , Células Madin Darby de Rim Canino
8.
FASEB J ; 33(6): 7301-7314, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30860872

RESUMO

Hypercholesterolemia is reported to increase reactive oxygen species (ROS) and to promote breast cancer progression. ROS play an important role in tumor biology, and xanthine oxidase (XO) is an enzyme that generates ROS. The effects of febuxostat (FBX), an XO inhibitor, on breast cancer cell migration under LDL stimulation in vitro and metastasis of breast cancer associated with hypercholesterolemia in vivo were studied. In vitro, FBX significantly inhibited LDL-induced ROS production and cell migration. Treatment of small interfering RNA against XO was consistent with the findings of FBX treatment. In vivo, a significant increase of tumor growth and pulmonary metastasis was observed in a xenograft mouse model with 4T1 cells on a high cholesterol diet (HCD), both of which were markedly inhibited by FBX or allopurinol treatment. Moreover, ERK represented the main target-signaling pathway that was affected by FBX treatment in a xenograft mouse model on an HCD evaluated by NanoString nCounter analysis. Consistently, MEK/ERK inhibitors directly decreased the LDL-induced cell migration in vitro. In conclusion, FBX mitigates breast cancer cell migration and pulmonary metastasis in the hyperlipidemic condition, associated with decreased ROS generation and MAPK phosphorylation. The inhibition of ERK pathways is likely to underlie the XO inhibitor-mediated suppression of breast cancer cell migration.-Oh, S.-H., Choi, S.-Y., Choi, H.-J., Ryu, H.-M., Kim, Y.-J., Jung, H.-Y., Cho, J.-H., Kim, C.-D., Park, S.-H., Kwon, T.-H., Kim, Y.-L. The emerging role of xanthine oxidase inhibition for suppression of breast cancer cell migration and metastasis associated with hypercholesterolemia.


Assuntos
Neoplasias da Mama/enzimologia , Hipercolesterolemia/complicações , Neoplasias Pulmonares/secundário , Proteínas de Neoplasias/antagonistas & inibidores , Xantina Oxidase/antagonistas & inibidores , Alopurinol/farmacologia , Alopurinol/uso terapêutico , Animais , Neoplasias da Mama/complicações , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular , Colesterol na Dieta/toxicidade , Progressão da Doença , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Febuxostat/farmacologia , Febuxostat/uso terapêutico , Feminino , Flavonoides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/prevenção & controle , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Proteínas de Neoplasias/genética , RNA Interferente Pequeno/farmacologia , Distribuição Aleatória , Espécies Reativas de Oxigênio , Imagem com Lapso de Tempo , Xantina Oxidase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Am Soc Nephrol ; 29(11): 2658-2670, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30305310

RESUMO

BACKGROUND: The bile acid-activated receptors, including the membrane G protein-coupled receptor TGR5 and nuclear farnesoid X receptor (FXR), have roles in kidney diseases. In this study, we investigated the role of TGR5 in renal water handling and the underlying molecular mechanisms. METHODS: We used tubule suspensions of inner medullary collecting duct (IMCD) cells from rat kidneys to investigate the effect of TGR5 signaling on aquaporin-2 (AQP2) expression, and examined the in vivo effects of TGR5 in mice with lithium-induced nephrogenic diabetes insipidus (NDI) and Tgr5 knockout (Tgr5-/-) mice. RESULTS: Activation of TGR5 by lithocholic acid (LCA), an endogenous TGR5 ligand, or INT-777, a synthetic TGR5-specific agonist, induced AQP2 expression and intracellular trafficking in rat IMCD cells via a cAMP-protein kinase A signaling pathway. In mice with NDI, dietary supplementation with LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly upregulated AQP2 expression in the kidney inner medulla. Supplementation with endogenous FXR agonist had no effect. In primary IMCD suspensions from lithium-treated rats, treatment with INT-767 (FXR and TGR5 dual agonist) or INT-777, but not INT-747 (FXR agonist), increased AQP2 expression. Tgr5-/- mice exhibited an attenuated ability to concentrate urine in response to dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium-treated Tgr5-/- mice, LCA treatment failed to prevent reduction of AQP2 expression. CONCLUSIONS: TGR5 stimulation increases renal AQP2 expression and improves impaired urinary concentration in lithium-induced NDI. TGR5 is thus involved in regulating water metabolism in the kidney.


Assuntos
Aquaporina 2/metabolismo , Túbulos Renais Coletores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Água/metabolismo , Animais , Aquaporina 2/genética , Ácidos e Sais Biliares/farmacologia , Células Cultivadas , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Ácidos Cólicos/farmacologia , Diabetes Insípido Nefrogênico/metabolismo , Homeostase , Túbulos Renais Coletores/efeitos dos fármacos , Ácido Litocólico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
10.
Am J Physiol Renal Physiol ; 314(5): F1020-F1025, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29357422

RESUMO

Lithium is widely used in treatment of bipolar affective disorders but often causes nephrogenic diabetes insipidus (NDI), a disorder characterized by severe urinary-concentrating defects. Lithium-induced NDI is caused by lithium uptake by collecting duct principal cells and altered expression of aquaporin-2 (AQP2), which are essential for water reabsorption of tubular fluid in the collecting duct. Sex hormones have previously been shown to affect the regulation of AQP2, so we tested whether tamoxifen (TAM), a selective estrogen receptor modulator, would attenuate lithium-induced alterations on renal water homeostasis. Rats were treated for 14 days with lithium, and TAM treatment was initiated 1 wk after onset of lithium administration. Lithium treatment resulted in severe polyuria and reduced AQP2 expression, which were ameliorated by TAM. Consistent with this, TAM attenuated downregulation of AQP2 and increased phosphorylation of the cAMP-responsive element-binding protein, which induced AQP2 expression in freshly isolated inner-medullary collecting duct suspension prepared from lithium-treated rats. In conclusion, TAM attenuated polyuria dose dependently and impaired urine concentration and downregulation of AQP2 protein expression in rats with lithium-induced NDI. These findings suggest that TAM is likely to be a novel therapeutic option for lithium-induced NDI.


Assuntos
Diabetes Insípido Nefrogênico/prevenção & controle , Hipoglicemiantes/farmacologia , Capacidade de Concentração Renal/efeitos dos fármacos , Túbulos Renais Coletores/efeitos dos fármacos , Cloreto de Lítio , Tamoxifeno/farmacologia , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Proteína de Ligação a CREB/metabolismo , Diabetes Insípido Nefrogênico/induzido quimicamente , Diabetes Insípido Nefrogênico/metabolismo , Diabetes Insípido Nefrogênico/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/efeitos dos fármacos , Receptor beta de Estrogênio/genética , Receptor beta de Estrogênio/metabolismo , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/fisiopatologia , Masculino , Fosforilação , Poliúria/induzido quimicamente , Poliúria/fisiopatologia , Poliúria/prevenção & controle , Ratos Sprague-Dawley , Fatores de Tempo
11.
Am J Physiol Renal Physiol ; 314(3): F329-F342, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29070573

RESUMO

Mineralocorticoids trigger a profibrotic process in the kidney. In mouse cortical collecting duct cells, the present study addressed two main questions: 1) what are microRNAs (miRNAs) and their target genes that are changed by aldosterone? and 2) what do miRNAs, in response to aldosterone, regulate regarding signaling pathways related to fibrosis? A microarray chip assay was done in cells in the absence or presence of aldosterone treatment (10-6 M; 3 days). The candidate miRNAs were identified by the criteria of >30% of fold change among the significantly changed miRNAs ( P < 0.05). Twenty-nine miRNAs were upregulated (>1.3-fold), and 27 miRNAs were downregulated (<0.7-fold). Putative target genes of identified miRNAs were associated with 74 Kyoto Encyclopedia of Genes and Genomes pathways. Among them, the wingless-related integration site (Wnt) signaling pathway was highly ranked, where 15 mature miRNAs were observed. These miRNAs were further analyzed by real-time quantitative PCR, and among them, miR-130b-3p, miR-34c-5p, and miR-146a-5p were selected. Through the identification of putative target genes of these three miRNAs, mRNA and protein expression of the Ca2+/calmodulin-dependent protein kinase type II ß-chain ( Camk2b) gene (a target gene of miR-34c-5p) were found to be increased significantly in aldosterone-treated cells, where fibronectin (FN) and α-smooth muscle actin were induced. When CaMKIIß small interfering RNA or the miR-34c-5p mimic was transfected, aldosterone-induced FN expression was significantly attenuated, along with reduced CaMKIIß protein expression. A luciferase reporter assay revealed a decrease of CaMKIIß translation in cells transfected with miRNA mimics of miR-34c-5p. In conclusion, aldosterone-induced downregulation of miR-34c-5p in the Wnt signaling pathway and a consequent increase of CaMKIIß expression are likely to be involved in aldosterone-induced fibrosis.


Assuntos
Aldosterona , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Nefropatias/enzimologia , Túbulos Renais Coletores/enzimologia , MicroRNAs/metabolismo , Actinas/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Linhagem Celular , Modelos Animais de Doenças , Fibronectinas/metabolismo , Fibrose , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Nefropatias/induzido quimicamente , Nefropatias/genética , Nefropatias/patologia , Túbulos Renais Coletores/patologia , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Transcriptoma , Via de Sinalização Wnt
12.
Biochem Biophys Res Commun ; 493(3): 1210-1216, 2017 11 25.
Artigo em Inglês | MEDLINE | ID: mdl-28958942

RESUMO

Aberrant levels of aquaporin-5 (AQP5) expression have been observed in several types of cancer, including breast cancer, where AQP5 overexpression is associated with metastasis and poor prognosis. In cultured cancer cells, AQP5 facilitates cell migration and activates Ras signaling. Both increased cell migration and Ras activation are associated with cancer metastasis, but so far it is unknown if AQP5 also affects these processes in vivo. Therefore, we investigated if high AQP5 expression in breast cancer tissue correlated with increased activation of Ras and of Rac1, which is a GTPase also involved in cell migration. This was accomplished by immunohistochemical analysis of invasive ductal carcinoma of breast tissue sections from human patients, followed by qualitative and quantitative correlation analysis between AQP5 and activated Ras and Rac1. Immunohistochemistry revealed that activation of Ras and Rac1 was positively correlated. There was, however, no correlation between high AQP5 expression and activation of Ras, whereas a nonsignificant, but positive, tendency between the levels of AQP5 and activated Rac1 levels was observed. In summary, this is the first report that correlates AQP5 expression levels to downstream signaling partners in breast cancer tissue sections. The results suggest Rac1 as a potential downstream signaling partner of AQP5 in vivo.


Assuntos
Aquaporina 5/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas ras/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Feminino , Humanos , Imuno-Histoquímica , Transdução de Sinais
13.
Am J Physiol Renal Physiol ; 311(6): F1318-F1328, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27760771

RESUMO

The kidney collecting duct is an important renal tubular segment for regulation of body water homeostasis and urine concentration. Water reabsorption in the collecting duct principal cells is controlled by vasopressin, a peptide hormone that induces the osmotic water transport across the collecting duct epithelia through regulation of water channel proteins aquaporin-2 (AQP2) and aquaporin-3 (AQP3). In particular, vasopressin induces both intracellular translocation of AQP2-bearing vesicles to the apical plasma membrane and transcription of the Aqp2 gene to increase AQP2 protein abundance. The signaling pathways, including AQP2 phosphorylation, RhoA phosphorylation, intracellular calcium mobilization, and actin depolymerization, play a key role in the translocation of AQP2. This review summarizes recent data demonstrating the regulation of AQP2 as the underlying molecular mechanism for the homeostasis of water balance in the body.


Assuntos
Aquaporina 2/metabolismo , Homeostase/fisiologia , Túbulos Renais Coletores/metabolismo , Transdução de Sinais/fisiologia , Equilíbrio Hidroeletrolítico/fisiologia , Animais , Aquaporina 3/metabolismo , Água Corporal/metabolismo , Humanos , Fosforilação
14.
Am J Physiol Renal Physiol ; 311(6): F1294-F1307, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27733367

RESUMO

The carboxyl terminus of aquaporin-2 (AQP2c) undergoes posttranslational modifications, including phosphorylation and ubiquitination, in the process of regulating aquaporin-2 (AQP2) translocation and protein abundance. We aimed to identify novel proteins interacting with AQP2c. Recombinant AQP2c protein was made in Escherichia coli BL21 (DE3) cells by exploiting the pET32 TrxA fusion system. Lysates of rat kidney inner medullary collecting duct (IMCD) tubule suspensions interacted with rat AQP2c bound to Ni2+-resin were subjected to LC-MS/MS proteomic analysis. Potential interacting proteins were identified, including vacuolar protein sorting-associated protein 35 (Vps35). Coimmunoprecipitation assay demonstrated that Vps35 interacted with AQP2c. Immunohistochemistry of rat kidney revealed that AQP2 and Vps35 were partly colocalized at the intracellular vesicles in collecting duct cells. The role of Vps35 in AQP2 regulation induced by 1-deamino-8D-arginine vasopressin (dDAVP) was examined in mpkCCDc14 cells. Cell surface biotinylation assay demonstrated that dDAVP-induced apical translocation of AQP2 was significantly decreased under siRNA-mediated Vps35 knockdown. dDAVP-induced AQP2 upregulation was less prominent in the cells with Vps35 knockdown. Moreover, AQP2 protein abundance was decreased to a greater extent during the withdrawal period after dDAVP stimulation under Vps35 knockdown, which was significantly inhibited by chloroquine (a blocker of the lysosomal pathway) but not by MG132 (a proteasome inhibitor). Immunocytochemistry demonstrated that internalized AQP2 was more associated with lysosomal-associated membrane protein 1 (LAMP-1) in primary cultured IMCD cells under a Vps35 knockdown situation. Taken together, our results show that Vps35 interacts with AQP2c, and depletion of Vps35 is likely to be associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2 protein.


Assuntos
Aquaporina 2/metabolismo , Rim/metabolismo , Lisossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Técnicas de Silenciamento de Genes , Túbulos Renais Coletores/metabolismo , Fosforilação , RNA Interferente Pequeno , Ratos , Espectrometria de Massas em Tandem , Proteínas de Transporte Vesicular/genética
15.
Am J Physiol Renal Physiol ; 310(11): F1317-27, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-26962105

RESUMO

In the kidney, metabolic processes are different among the cortex (COR), outer medulla (OM), and inner medulla (IM). Using matrix-assisted laser desorption/ionization (MALDI) and imaging mass spectrometry (IMS), we examined the change of metabolites in the COR, OM, and IM of the rat kidney after furosemide treatment compared with vehicle-treated controls. Osmotic minipumps were implanted in male Sprague-Dawley rats to deliver 12 mg·day(-1)·rat(-1) of furosemide. Vehicle-treated (n = 14) and furosemide-treated (furosemide rats, n = 15) rats in metabolic cages received a fixed amount of rat chow (15 g·220 g body wt(-1)·day(-1) for each rat) with free access to water intake for 6 days. At day 6, higher urine output (32 ± 4 vs. 9 ± 1 ml/day) and lower urine osmolality (546 ± 44 vs. 1,677 ± 104 mosmol/kgH2O) were observed in furosemide rats. Extracts of COR, OM, and IM were analyzed by ultraperformance liquid chromatography coupled with quadrupole time-of-flight (TOF) mass spectrometry, where multivariate analysis revealed significant differences between the two groups. Several metabolites, including acetylcarnitine, betaine, carnitine, choline, and glycerophosphorylcholine (GPC), were significantly changed. The changes of metabolites were further identified by MALDI-TOF/TOF and IMS. Their spatial distribution and relative quantitation in the kidneys were analyzed by IMS. Carnitine compounds were increased in COR and IM, whereas carnitine and acetylcarnitine were decreased in OM. Choline compounds were increased in COR and OM but decreased in IM from furosemide rats. Betaine and GPC were decreased in OM and IM. Taken together, MALDI-TOF/TOF and IMS successfully provide the spatial distribution and relative quantitation of metabolites in the kidney.


Assuntos
Diuréticos/farmacologia , Furosemida/farmacologia , Rim/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Ingestão de Líquidos/efeitos dos fármacos , Rim/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley , Micção/efeitos dos fármacos
16.
Nanotechnology ; 27(17): 175303, 2016 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-26984937

RESUMO

Mimicking the nanoscale surface texture of the extracellular matrix can affect the regulation of cellular behavior, including adhesion, differentiation, and neurite outgrowth. In this study, SU-8-based polymer surfaces with well-ordered nanowell arrays were fabricated using nanosphere lithography with polystyrene nanoparticles. We show that the SU-8 surface with nanowells resulted in similar neuronal development of rat pheochromocytoma (PC12) cells compared with an unpatterned poly-L-lysine (PLL)-coated SU-8 surface. Additionally, even after soaking the substrate in cell culture medium for two weeks, cells on the nanowell SU-8 surface showed long-term neurite outgrowth compared to cells on the PLL-coated SU-8 surface. The topographical surface modification of the nanowell array demonstrates potential as a replacement for cell adhesive material coatings such as PLL, for applications requiring long-term use of polymer-based implantable devices.


Assuntos
Compostos de Epóxi/química , Nanosferas/química , Polímeros/química , Animais , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Neurônios/citologia , Neurônios/metabolismo , Células PC12 , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Polilisina/química , Polímeros/farmacologia , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Receptor trkA/genética , Receptor trkA/metabolismo , Propriedades de Superfície
17.
Int J Mol Sci ; 17(12)2016 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-27941618

RESUMO

Aquaporins (AQPs) are water channel proteins robustly expressed in the central nervous system (CNS). A number of previous studies described the cellular expression sites and investigated their major roles and function in the brain and spinal cord. Among thirteen different mammalian AQPs, AQP1 and AQP4 have been mainly studied in the CNS and evidence has been presented that they play important roles in the pathogenesis of CNS injury, edema and multiple diseases such as multiple sclerosis, neuromyelitis optica spectrum disorders, amyotrophic lateral sclerosis, glioblastoma multiforme, Alzheimer's disease and Parkinson's disease. The objective of this review is to highlight the current knowledge about AQPs in the spinal cord and their proposed roles in pathophysiology and pathogenesis related to spinal cord lesions and injury.


Assuntos
Aquaporinas/metabolismo , Medula Espinal/metabolismo , Animais , Aquaporina 4/metabolismo , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Humanos
18.
Am J Physiol Renal Physiol ; 308(7): F737-48, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25651562

RESUMO

Kidney collecting duct cells are continuously exposed to the changes of extracellular pH (pHe). We aimed to study the effects of altered pHe on desmopressin (dDAVP)-induced phosphorylation (Ser(256), Ser(261), Ser(264), and Ser(269)) and apical targeting of aquaporin-2 (AQP2) in rat kidney inner medullary collecting duct (IMCD) cells. When freshly prepared IMCD tubule suspensions exposed to HEPES buffer with pH 5.4, 6.4, 7.4, or 8.4 for 1 h were stimulated with dDAVP (10(-10) M, 3 min), AQP2 phosphorylation at Ser(256), Ser(264), and Ser(269) was significantly attenuated under acidic conditions. Next, IMCD cells primary cultured in transwell chambers were exposed to a transepithelial pH gradient for 1 h (apical pH 6.4, 7.4, or 8.4 vs. basolateral pH 7.4 and vice versa). Immunocytochemistry and cell surface biotinylation assay revealed that exposure to either apical pH 6.4 or basolateral pH 6.4 for 1 h was associated with decreased dDAVP (10(-9) M, 15 min, basolateral)-induced apical targeting of AQP2 and surface expression of AQP2. Fluorescence resonance energy transfer analysis revealed that the dDAVP (10(-9) M)-induced increase of PKA activity was significantly attenuated when LLC-PK1 cells were exposed to pHe 6.4 compared with pHe 7.4 and 8.4. In contrast, forskolin (10(-7) M)-induced PKA activation and dDAVP (10(-9) M)-induced increases of intracellular Ca(2+) were not affected. Taken together, dDAVP-induced phosphorylation and apical targeting of AQP2 are attenuated in IMCD cells under acidic pHe, likely via an inhibition of vasopressin V2 receptor-G protein-cAMP-PKA actions.


Assuntos
Aquaporina 2/metabolismo , Membrana Celular/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Animais , Aquaporina 2/efeitos dos fármacos , Linhagem Celular , Membrana Celular/metabolismo , Desamino Arginina Vasopressina/farmacologia , Espaço Extracelular/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Células LLC-PK1 , Masculino , Fosforilação/fisiologia , Transporte Proteico/efeitos dos fármacos , Ratos Sprague-Dawley , Fármacos Renais/farmacologia , Suínos
19.
Am J Physiol Renal Physiol ; 308(7): F749-64, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25651560

RESUMO

Mature microRNA (miRNA) acts as an important posttranscriptional regulator. We aimed to profile vasopressin-responsive miRNAs in kidney inner medullary collecting duct cells and to identify aquaporin-2 (AQP2)-targeting miRNAs. Microarray chip assay was carried out in inner medullary collecting duct tubule suspensions from rat kidneys in the absence or presence of desmopressin (dDAVP) stimulation (10(-9) M, 2 h). The results demonstrated 19 miRNAs, including both precursor and mature miRNAs, as potential candidates that showed significant changes in expression after dDAVP stimulation (P < 0.05). Nine mature miRNAs exhibiting >1.3-fold changes in expression on the microarray (miR-127, miR-1, miR-873, miR-16, miR-206, miR-678, miR-496, miR-298, and miR-463) were further examined by quantitative real-time PCR, and target genes of the selected miRNAs were predicted. Next, to identify AQP2-targeting miRNAs, in silico analysis was performed. Four miRNAs (miR-32, miR-137, miR-216a, and miR-216b) target the 3'-untranslated region of rat AQP2 mRNA. Target seed regions of miR-32 and miR-137 were also conserved in the 3'-untranslated region of mouse AQP2 mRNA. Quantitative real-time PCR and immunoblot analysis demonstrated that dDAVP-induced AQP2 expression was significantly attenuated in mpkCCDc14 cells when cells were transfected with miRNA mimics of miR-32 or miR-137. Moreover, luciferase reporter assay demonstrated a significant decrease of AQP2 translation in mpkCCDc14 cells transfected with miRNA mimics of miR-32 or miR-137. The present study provides novel insights into the regulation of AQP2 by RNA interference; however, vasopressin-regulated miRNAs did not include miR-32 or miR-137, indicating that the interaction of miRNAs with the AQP2 regulatory pathway requires further analysis.


Assuntos
Aquaporina 2/metabolismo , Túbulos Renais Coletores/metabolismo , MicroRNAs/metabolismo , Vasopressinas/farmacologia , Animais , Linhagem Celular , Desamino Arginina Vasopressina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Medula Renal/metabolismo , Masculino , Transporte Proteico/genética , Ratos Sprague-Dawley
20.
Am J Physiol Renal Physiol ; 309(5): F474-83, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26109089

RESUMO

Because cyclophosphamide-induced hyponatremia was reported to occur without changes in plasma vasopressin in a patient with central diabetes insipidus, we hypothesized that cyclophosphamide or its active metabolite, 4-hydroperoxycyclophosphamide (4-HC), may directly dysregulate the expression of water channels or sodium transporters in the kidney. To investigate whether intrarenal mechanisms for urinary concentration are activated in vivo and in vitro by treatment with cyclophosphamide and 4-HC, respectively, we used water-loaded male Sprague-Dawley rats, primary cultured inner medullary collecting duct (IMCD) cells, and IMCD suspensions prepared from male Sprague-Dawley rats. In cyclophosphamide-treated rats, significant increases in renal expression of aquaporin-2 (AQP2) and Na-K-2Cl cotransporter type 2 (NKCC2) were shown by immunoblot analysis and immunohistochemistry. Apical translocation of AQP2 was also demonstrated by quantitative immunocytochemistry. In both rat kidney and primary cultured IMCD cells, significant increases in AQP2 and vasopressin receptor type 2 (V2R) mRNA expression were demonstrated by real-time quantitative PCR analysis. Confocal laser-scanning microscopy revealed that apical translocation of AQP2 was remarkably increased when primary cultured IMCD cells were treated with 4-HC in the absence of vasopressin stimulation. Moreover, AQP2 upregulation and cAMP accumulation in response to 4-HC were significantly reduced by tolvaptan cotreatment in primary cultured IMCD cells and IMCD suspensions, respectively. We demonstrated that, in the rat kidney, cyclophosphamide may activate V2R and induce upregulation of AQP2 in the absence of vasopressin stimulation, suggesting the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis (NSIAD).


Assuntos
Aquaporina 2/metabolismo , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacologia , Túbulos Renais Coletores/efeitos dos fármacos , Rim/efeitos dos fármacos , Vasopressinas/metabolismo , Animais , Células Cultivadas , Rim/metabolismo , Túbulos Renais Coletores/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Membro 1 da Família 12 de Carreador de Soluto/metabolismo
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa