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1.
PLoS One ; 15(7): e0235756, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702059

RESUMO

Genome-wide association studies (GWAS) have routinely detected human quantitative trait loci (QTLs) for complex traits. Viewing that most GWAS single nucleotide polymorphisms (SNPs) are found in non-coding regions unrelated to the physiology of a polygenic trait of interest, a vital question to answer is whether or not any of these SNPs can functionally alter the phenotype with which it is associated. The study of blood pressure (BP) is a case in point. Conserved mechanisms in controlling BP by modularity is now unifying differing mammalian orders in that understanding mechanisms in rodents is tantamount to revealing the same in humans, while overcoming experimental limitations imposed by human studies. As a proof of principle, we used BP QTLs from Dahl salt-sensitive rats (DSS) as substitutes to capture distinct human functional orthologs. 3 DSS BP QTLs are located into distinct genome regions and correspond to several human GWAS genes. Each of the QTLs independently exerted a major impact on BP in vivo. BP was functionally changed by normotensive alleles from each of these QTLs, and yet, the human GWAS SNPs do not exist in the rat. They cannot be responsible for physiological alterations in BP caused by these QTLs. These SNPs are genome emblems for QTLs nearby, rather than being QTLs per se, since they only emerged during primate evolution after BP-regulating mechanisms have been established. We then identified specific mutated coding domains that are conserved between rodents and humans and that may implicate different steps of a common pathway or separate pathways.


Assuntos
Pressão Sanguínea/genética , Estudo de Associação Genômica Ampla , Hipertensão/genética , Hipertensão/prevenção & controle , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Animais , Determinação da Pressão Arterial , Mapeamento Cromossômico , Humanos , Masculino , Fenótipo , Ratos , Ratos Endogâmicos Dahl
2.
Am J Physiol Renal Physiol ; 319(1): F63-F75, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32463726

RESUMO

Diuretics and renin-angiotensin system blockers are often insufficient to control the blood pressure (BP) in salt-sensitive (SS) subjects. Abundant data support the proposal that the level of atrial natriuretic peptide may correlate with the pathogenesis of SS hypertension. We hypothesized here that increasing atrial natriuretic peptide levels with sacubitril, combined with renin-angiotensin system blockage by valsartan, can be beneficial for alleviation of renal damage in a model of SS hypertension, the Dahl SS rat. To induce a BP increase, rats were challenged with a high-salt 4% NaCl diet for 21 days, and chronic administration of vehicle or low-dose sacubitril and/or valsartan (75 µg/day each) was performed. Urine flow, Na+ excretion, and water consumption were increased on the high-salt diet compared with the starting point (0.4% NaCl) in all groups but remained similar among the groups at the end of the protocol. Upon salt challenge, we observed a mild decrease in systolic BP and urinary neutrophil gelatinase-associated lipocalin levels (indicative of alleviated tubular damage) in the valsartan-treated groups. Sacubitril, as well as sacubitril/valsartan, attenuated the glomerular filtration rate decline induced by salt. Alleviation of protein cast formation and lower renal medullary fibrosis were observed in the sacubitril/valsartan- and valsartan-treated groups, but not when sacubitril alone was administered. Interestingly, proteinuria was mildly mitigated only in rats that received sacubitril/valsartan. Further studies of the effects of sacubitril/valsartan in the setting of SS hypertension, perhaps involving a higher dose of the drug, are warranted to determine if it can interfere with the progression of the disease.


Assuntos
Aminobutiratos/administração & dosagem , Antagonistas de Receptores de Angiotensina/administração & dosagem , Pressão Sanguínea/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Nefropatias/tratamento farmacológico , Tetrazóis/administração & dosagem , Valsartana/administração & dosagem , Aminobutiratos/uso terapêutico , Antagonistas de Receptores de Angiotensina/uso terapêutico , Animais , Hipertensão/fisiopatologia , Nefropatias/fisiopatologia , Masculino , Ratos , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta , Tetrazóis/uso terapêutico , Valsartana/uso terapêutico
3.
Am J Physiol Renal Physiol ; 318(6): F1489-F1499, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32390513

RESUMO

Recently, we reported that obese Dahl salt-sensitive (SS) leptin receptor mutant (SSLepRmutant) rats display progressive renal injury. The present study demonstrated that the early development of renal injury in the SSLepRmutant strain is associated with an increase in the renal infiltration of macrophages compared with lean SS rats. We also examined whether depletion of macrophages with clodronate would reduce the early progression of renal injury in the SSLepRmutant strain. Four-week-old SS and SSLepRmutant rats were treated with either vehicle (PBS) or clodronate (50 mg/kg ip, 2 times/wk) for 4 wk. While the administration of clodronate did not reduce renal macrophage infiltration in SS rats, clodronate decreased macrophages in the kidneys of SSLepRmutant rats by >50%. Interestingly, clodronate significantly reduced plasma glucose, insulin, and triglyceride levels and markedly improved glucose tolerance in SSLepRmutant rats. Treatment with clodronate had no effect on the progression of proteinuria or renal histopathology in SS rats. In the SSLepRmutant strain, proteinuria was markedly reduced during the first 2 wk of treatment (159 ± 32 vs. 303 ± 52 mg/day, respectively). However, after 4 wk of treatment, the effect of clodronate was no longer observed in the SSLepRmutant strain (346 ± 195 vs. 399 ± 50 mg/day, respectively). The kidneys from SSLepRmutant rats displayed glomerular injury with increased mesangial expansion and renal fibrosis versus SS rats. Treatment with clodronate significantly decreased glomerular injury and renal fibrosis in the SSLepRmutant strain. Overall, these data indicate that the depletion of macrophages improves metabolic disease and slows the early progression of renal injury in SSLepRmutant rats.


Assuntos
Ácido Clodrônico/farmacologia , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Obesidade/tratamento farmacológico , Receptores para Leptina/genética , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibrose , Insulina/sangue , Rim/metabolismo , Rim/patologia , Nefropatias/sangue , Nefropatias/etiologia , Nefropatias/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Mutação , Obesidade/sangue , Obesidade/complicações , Obesidade/genética , Ratos Endogâmicos Dahl , Fatores Sexuais , Fatores de Tempo , Triglicerídeos/sangue
4.
Am J Physiol Renal Physiol ; 318(5): F1237-F1245, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32223308

RESUMO

Changes in mitochondrial function are central to many forms of kidney disease, including acute injury, diabetic nephropathy, hypertension, and chronic kidney diseases. As such, there is an increasing need for reliable and fast methods for assessing mitochondrial respiratory function in renal cells. Despite being indispensable for many mechanistic studies, cultured cells or isolated mitochondria, however, often do not recapitulate in vivo or close to in vivo situations. Cultured and/or immortalized cells often change their bioenergetic profile and phenotype compared with in vivo or ex vivo situations, and isolated mitochondria are simply removed from their cellular milieu. This is especially important for extremely complex organs such as the kidney. Here, we report the development and validation of a new approach for the rapid assessment of mitochondrial oxygen consumption on freshly isolated glomeruli or proximal tubular fragments using Agilent SeaHorse XFe24 and XF96 Extracellular Flux Analyzers. We validated the technique in several healthy and diseased rodent models: the C57BL/6J mouse, the diabetic db/db mouse and matching db/+ control mouse, and the Dahl salt-sensitive rat. We compared the data to respiration from isolated mitochondria. The method can be adapted and used for the rapid assessment of mitochondrial oxygen consumption from any rodent model of the investigator's choice. The isolation methods presented here ensure viable and functional proximal tubular fragments and glomeruli, with a preserved cellular environment for studying mitochondrial function within the context of their surroundings and interactions.


Assuntos
Diabetes Mellitus/metabolismo , Metabolismo Energético , Hipertensão/metabolismo , Glomérulos Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Mitocôndrias/metabolismo , Animais , Respiração Celular , Diabetes Mellitus/patologia , Modelos Animais de Doenças , Feminino , Hipertensão/patologia , Glomérulos Renais/patologia , Túbulos Renais Proximais/patologia , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/patologia , Consumo de Oxigênio , Ratos Endogâmicos Dahl
5.
PLoS One ; 15(4): e0232067, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32324784

RESUMO

The heptapeptide angiotensin-(1-7) (Ang-(1-7)) is protective in the cardiovascular system through its induction of vasodilator production and angiogenesis. Despite acting antagonistically to the effects of elevated, pathophysiological levels of angiotensin II (AngII), recent evidence has identified convergent and beneficial effects of low levels of both Ang-(1-7) and AngII. Previous work identified the AngII receptor type I (AT1R) as a component of the protein complex formed when Ang-(1-7) binds its receptor, Mas1. Importantly, pharmacological blockade of AT1R did not alter the effects of Ang-(1-7). Here, we use a novel mutation of AT1RA in the Dahl salt-sensitive (SS) rat to test the hypothesis that interaction between Mas1 and AT1R contributes to proangiogenic Ang-(1-7) signaling. In a model of hind limb angiogenesis induced by electrical stimulation, we find that the restoration of skeletal muscle angiogenesis in SS rats by Ang-(1-7) infusion is impaired in AT1RA knockout rats. Enhancement of endothelial cell (EC) tube formation capacity by Ang-(1-7) is similarly blunted in AT1RA mutant ECs. Transcriptional changes elicited by Ang-(1-7) in SS rat ECs are altered in AT1RA mutant ECs, and tandem mass spectrometry-based proteomics demonstrate that the protein complex formed upon binding of Ang-(1-7) to Mas1 is altered in AT1RA mutant ECs. Together, these data support the hypothesis that interaction between AT1R and Mas1 contributes to proangiogenic Ang-(1-7) signaling.


Assuntos
Angiotensina I/metabolismo , Músculo Esquelético/irrigação sanguínea , Fragmentos de Peptídeos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Animais , Estimulação Elétrica , Masculino , Espectrometria de Massas , Modelos Animais , Músculo Esquelético/metabolismo , Mutação , Neovascularização Fisiológica , Proteômica , Ratos , Ratos Endogâmicos Dahl , Transdução de Sinais
6.
Am J Physiol Renal Physiol ; 318(5): F1122-F1135, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174138

RESUMO

Emerging evidence has demonstrated that (pro)renin receptor (PRR)-mediated activation of intrarenal renin-angiotensin system (RAS) plays an essential role in renal handling of Na+ and water balance and blood pressure. The present study tested the possibility that the intrarenal RAS served as a molecular target for the protective action of ELABELA (ELA), a novel endogenous ligand of apelin receptor, in the distal nephron. By RNAscope and immunofluorescence, mRNA and protein expression of endogenous ELA was consistently localized to the collecting duct (CD). Apelin was also found in the medullary CDs as assessed by immunofluorescence. In cultured CD-derived M1 cells, exogenous ELA induced parallel decreases of full-length PRR (fPRR), soluble PRR (sPRR), and prorenin/renin protein expression as assessed by immunoblotting and medium sPRR and prorenin/renin levels by ELISA, all of which were reversed by 8-bromoadenosine 3',5'-cyclic monophosphate. Conversely, deletion of PRR in the CD or nephron in mice elevated Apela and Apln mRNA levels as well as urinary ELA and apelin excretion, supporting the antagonistic relationship between the two systems. Administration of exogenous ELA-32 infusion (1.5 mg·kg-1·day-1, minipump) to high salt (HS)-loaded Dahl salt-sensitive (SS) rats significantly lowered mean arterial pressure, systolic blood pressure, diastolic blood pressure, and albuminuria, accompanied with a reduction of urinary sPRR, angiotensin II, and prorenin/renin excretion. HS upregulated renal medullary protein expression of fPRR, sPRR, prorenin, and renin in Dahl SS rats, all of which were significantly blunted by exogenous ELA-32 infusion. Additionally, HS-induced upregulation of inflammatory cytokines (IL-1ß, IL-2, IL-6, IL-17A, IFN-γ, VCAM-1, ICAM-1, and MCP-1), fibrosis markers (TGF-ß1, FN, Col1A1, PAI-1, and TIMP-1), and kidney injury markers (NGAL, Kim-1, albuminuria, and urinary NGAL excretion) were markedly blocked by exogenous ELA infusion. Together, these results support the antagonistic interaction between ELA and intrarenal RAS in the distal nephron that appears to exert a major impact on blood pressure regulation.


Assuntos
Pressão Sanguínea , Hipertensão/metabolismo , Nefropatias/metabolismo , Rim/metabolismo , Hormônios Peptídicos/metabolismo , Sistema Renina-Angiotensina , Animais , Apelina/genética , Apelina/metabolismo , Receptores de Apelina/genética , Receptores de Apelina/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Hipertensão/tratamento farmacológico , Hipertensão/fisiopatologia , Rim/efeitos dos fármacos , Rim/patologia , Rim/fisiopatologia , Nefropatias/patologia , Nefropatias/fisiopatologia , Nefropatias/prevenção & controle , Masculino , Camundongos Knockout , Hormônios Peptídicos/administração & dosagem , Hormônios Peptídicos/genética , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Ratos Endogâmicos Dahl , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais
7.
Am J Physiol Renal Physiol ; 318(4): F982-F993, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32150444

RESUMO

Studies examining mechanisms of Dahl salt-sensitive (SS) hypertension have implicated the infiltration of leukocytes in the kidneys, which contribute to renal disease and elevated blood pressure. However, the signaling pathways by which leukocytes traffic to the kidneys remain poorly understood. The present study nominated a signaling pathway by analyzing a kidney RNA sequencing data set from SS rats fed either a low-salt (0.4% NaCl) diet or a high-salt (4.0% NaCl) diet. From this analysis, chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-C motif) receptor 2 (CCR2) were nominated as a potential pathway modifying renal leukocyte infiltration and contributing to SS hypertension. The functional role of the CCL2/CCR2 pathway was tested by daily administration of CCR2 antagonist (RS-102895 at 5 mg·kg-1·day-1 in DMSO) or DMSO vehicle for 3 or 21 days by intraperitoneal injections during the high salt challenge. Blood pressure, renal leukocyte infiltration, and renal damage were evaluated. The results demonstrated that RS-102895 treatment ameliorated renal damage (urinary albumin excretion; 43.4 ± 5.1 vs. 114.7 ± 15.2 mg/day in vehicle, P < 0.001) and hypertension (144.3 ± 2.2 vs. 158.9 ± 4.8 mmHg in vehicle, P < 0.001) after 21 days of high-salt diet. It was determined that renal leukocyte infiltration was blunted by day 3 of the high-salt diet (1.4 ± 0.1 vs. 1.9 ± 0.2 in vehicle × 106 CD45+ cells/kidney, P = 0.034). An in vitro chemotaxis assay validated the effect of RS-102895 on leukocyte chemotaxis toward CCL2. The results suggest that increased CCL2 in SS kidneys is important in the early recruitment of leukocytes, and blockade of this recruitment by administering RS-102895 subsequently blunted the renal damage and hypertension.


Assuntos
Quimiocina CCL2/metabolismo , Quimiotaxia de Leucócito , Hipertensão/metabolismo , Rim/metabolismo , Leucócitos/metabolismo , Cloreto de Sódio na Dieta , Animais , Anti-Hipertensivos/farmacologia , Pressão Arterial , Benzoxazinas/farmacologia , Células Cultivadas , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Quimiotaxia de Leucócito/efeitos dos fármacos , Modelos Animais de Doenças , Hipertensão/patologia , Hipertensão/fisiopatologia , Hipertensão/prevenção & controle , Rim/efeitos dos fármacos , Rim/patologia , Leucócitos/efeitos dos fármacos , Leucócitos/patologia , Masculino , Piperidinas/farmacologia , Ratos Endogâmicos Dahl , Receptores CCR2/antagonistas & inibidores , Receptores CCR2/metabolismo , Transdução de Sinais , Regulação para Cima
8.
Curr Hypertens Rep ; 22(2): 13, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32016562

RESUMO

PURPOSE OF REVIEW: This review will provide an in-depth coverage of the epidemiological and pre-clinical literature surrounding the role of dietary protein in hypertension, with a special emphasis on the history of our work on the Dahl salt-sensitive rat. RECENT FINDINGS: Our studies have dedicated much effort into understanding the relationship between dietary protein and its effect on the development of salt-sensitive hypertension and renal injury. Our evidence over the last 15 years have demonstrated that both the source and amount of dietary protein can influence the severity of disease, where we have determined mechanisms related to immunity, the maternal environment during pregnancy, and more recently the gut microbiota, which significantly contribute to these diet-induced effects. Deeper understanding of these dietary protein-related mechanisms may provide insight on the plausibility of dietary modifications as future therapeutic avenues for hypertension and renal disease.


Assuntos
Proteínas na Dieta , Hipertensão , Nefropatias , Animais , Pressão Sanguínea , Feminino , Humanos , Rim , Gravidez , Ratos , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta
9.
Am J Physiol Renal Physiol ; 318(4): F911-F921, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32068459

RESUMO

The present study examined whether development of renal injury in the nondiabetic obese Dahl salt-sensitive leptin receptor mutant (SSLepRmutant) strain is associated with elevations in glomerular filtration rate and renal lipid accumulation. Baseline mean arterial pressure at 6 wk of age was similar between Dahl salt-sensitive wild-type (SSWT) and SSLepRmutant rats. However, by 18 wk of age, the SSLepRmutant strain developed hypertension, while the elevation in mean arterial pressure was not as severe in SSWT rats (192 ± 4 and 149 ± 6 mmHg, respectively). At baseline, proteinuria was fourfold higher in SSLepRmutant than SSWT rats and remained elevated throughout the study. The early development of progressive proteinuria was associated with renal hyperfiltration followed by a decline in renal function over the course of study in the SSLepRmutant compared with SSWT rats. Kidneys from the SSLepRmutant strain displayed more glomerulosclerosis and glomerular lipid accumulation than SSWT rats. Glomeruli were isolated from the renal cortex of both strains at 6 and 18 wk of age, and RNA sequencing was performed to identify genes and pathways driving glomerular injury. We observed significant increases in expression of the influx lipid transporters, chemokine (C-X-C motif) ligand 16 (Cxcl16) and scavenger receptor and fatty acid translocase (Cd36), respectively, and a significant decrease in expression of the efflux lipid transporter, ATP-binding cassette subfamily A member 2 (Abca2; cholesterol efflux regulatory protein 2), in SSLepRmutant compared with SSWT rats at 6 and 18 wk of age, which were validated by RT-PCR analysis. These data suggest an association between glomerular hyperfiltration and glomerular lipid accumulation during the early development of proteinuria associated with obesity.


Assuntos
Tecido Adiposo/metabolismo , Hemodinâmica , Hipertensão/metabolismo , Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Metabolismo dos Lipídeos , Mutação , Obesidade/metabolismo , Receptores para Leptina/genética , Circulação Renal , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Tecido Adiposo/patologia , Tecido Adiposo/fisiopatologia , Adiposidade , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Quimiocina CXCL16/genética , Quimiocina CXCL16/metabolismo , Modelos Animais de Doenças , Predisposição Genética para Doença , Taxa de Filtração Glomerular , Hipertensão/genética , Hipertensão/patologia , Hipertensão/fisiopatologia , Nefropatias/genética , Nefropatias/patologia , Nefropatias/fisiopatologia , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Metabolismo dos Lipídeos/genética , Obesidade/genética , Obesidade/patologia , Obesidade/fisiopatologia , Fenótipo , Proteinúria/metabolismo , Proteinúria/patologia , Proteinúria/fisiopatologia , Ratos Endogâmicos Dahl , Cloreto de Sódio na Dieta
10.
Biochem Biophys Res Commun ; 524(1): 184-189, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-31982132

RESUMO

Plasma aldosterone concentration increases in proportion to the severity of heart failure, even during treatment with renin-angiotensin system inhibitors. This study investigated alternative regulatory mechanisms of aldosterone production that are significant in heart failure. Dahl salt-sensitive rats on a high-salt diet, a rat model of heart failure with cardio-renal syndrome, had high plasma aldosterone levels and elevated ß3-adrenergic receptor expression in hypoxic zona glomerulosa cells. In H295R cells (a human adrenocortical cell line), hypoxia-induced ß3-adrenergic receptor expression. Hypoxia-mediated ß3-adrenergic receptor expression augmented aldosterone production by facilitating hydrolysis of lipid droplets though ERK-mediated phosphorylation of hormone-sensitive lipase, also known as cholesteryl ester hydrolase. Hypoxia also accelerated the synthesis of cholesterol esters by acyl-CoA:cholesterol acyltransferase, thereby increasing the cholesterol ester content in lipid droplets. Thus, hypoxia enhanced aldosterone production by zona glomerulosa cells via promotion of the accumulation and hydrolysis of cholesterol ester in lipid droplets. In conclusion, hypoxic zona glomerulosa cells with heart failure show enhanced aldosterone production via increased catecholamine responsiveness and activation of cholesterol trafficking, irrespective of the renin-angiotensin system.


Assuntos
Córtex Suprarrenal/patologia , Aldosterona/biossíntese , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/patologia , Hipóxia/metabolismo , Hipóxia/patologia , Córtex Suprarrenal/efeitos dos fármacos , Animais , Síndrome Cardiorrenal/complicações , Catecolaminas/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Colesterol/metabolismo , Modelos Animais de Doenças , Humanos , Hipóxia/complicações , Masculino , Fosforilação/efeitos dos fármacos , Ratos Endogâmicos Dahl , Receptores Adrenérgicos beta 3/metabolismo , Esterol Esterase/metabolismo , Zona Glomerulosa/metabolismo , Zona Glomerulosa/patologia
11.
Circulation ; 141(9): 751-767, 2020 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-31948273

RESUMO

BACKGROUND: Myocardial fibrosis is a hallmark of cardiac remodeling and functionally involved in heart failure development, a leading cause of deaths worldwide. Clinically, no therapeutic strategy is available that specifically attenuates maladaptive responses of cardiac fibroblasts, the effector cells of fibrosis in the heart. Therefore, our aim was to develop novel antifibrotic therapeutics based on naturally derived substance library screens for the treatment of cardiac fibrosis. METHODS: Antifibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts, subsequent validation, and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of human cardiac fibroblasts, modulation of apoptosis, and extracellular matrix expression. In vitro findings were confirmed in vivo with an angiotensin II-mediated murine model of cardiac fibrosis in both preventive and therapeutic settings, as well as in the Dahl salt-sensitive rat model. To investigate the mechanism underlying the antifibrotic potential of the lead compounds, treatment-dependent changes in the noncoding RNAome in primary human cardiac fibroblasts were analyzed by RNA deep sequencing. RESULTS: High-throughput natural compound library screening identified 15 substances with antiproliferative effects in human cardiac fibroblasts. Using multiple in vitro fibrosis assays and stringent selection algorithms, we identified the steroid bufalin (from Chinese toad venom) and the alkaloid lycorine (from Amaryllidaceae species) to be effective antifibrotic molecules both in vitro and in vivo, leading to improvement in diastolic function in 2 hypertension-dependent rodent models of cardiac fibrosis. Administration at effective doses did not change plasma damage markers or the morphology of kidney and liver, providing the first toxicological safety data. Using next-generation sequencing, we identified the conserved microRNA 671-5p and downstream the antifibrotic selenoprotein P1 as common effectors of the antifibrotic compounds. CONCLUSIONS: We identified the molecules bufalin and lycorine as drug candidates for therapeutic applications in cardiac fibrosis and diastolic dysfunction.


Assuntos
Alcaloides de Amaryllidaceae/farmacologia , Bufanolídeos/farmacologia , Cardiomiopatias/prevenção & controle , Fármacos Cardiovasculares/farmacologia , Fibroblastos/efeitos dos fármacos , Fenantridinas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cardiomiopatias/etiologia , Cardiomiopatias/metabolismo , Cardiomiopatias/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Diástole , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Ensaios de Triagem em Larga Escala , Humanos , Hipertensão/complicações , Hipertensão/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Miocárdio/metabolismo , Miocárdio/patologia , Ratos Endogâmicos Dahl , Selenoproteína P/genética , Selenoproteína P/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos
12.
Am J Physiol Renal Physiol ; 318(1): F148-F159, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31608671

RESUMO

Reduced nitric oxide (NO) and a decrease in cGMP signaling mediated by soluble guanylate cyclase (sGC) has been linked to the development of several cardiorenal diseases. Stimulation of sGC is a potential means for enhancing cGMP production in conditions of reduced NO bioavailability. The purpose of our studies was to determine the effects of praliciguat, a clinical-stage sGC stimulator, in a model of cardiorenal failure. Dahl salt-sensitive rats fed a high-salt diet to induce hypertension and organ damage were treated with the sGC stimulator praliciguat to determine its effects on hemodynamics, biomarkers of inflammation, fibrosis, tissue function, and organ damage. Praliciguat treatment reduced blood pressure, improved cardiorenal damage, and attenuated the increase in circulating markers of inflammation and fibrosis. Notably, praliciguat affected markers of renal damage at a dose that had minimal effect on blood pressure. In addition, liver fibrosis and circulating markers of tissue damage were attenuated in praliciguat-treated rats. Stimulation of the NO-sGC-cGMP pathway by praliciguat attenuated or normalized indicators of chronic inflammation, fibrosis, and tissue dysfunction in the Dahl salt-sensitive rat model. Stimulation of sGC by praliciguat may present an effective mechanism for treating diseases linked to NO deficiency, particularly those associated with cardiac and renal failure. Praliciguat is currently being evaluated in patients with diabetic nephropathy and heart failure with preserved ejection fraction.


Assuntos
Fibrose/tratamento farmacológico , Agonistas da Guanilil Ciclase C/farmacologia , Inflamação/tratamento farmacológico , Rim/efeitos dos fármacos , Pirazóis/farmacologia , Pirimidinas/farmacologia , Insuficiência Renal/tratamento farmacológico , Animais , Biomarcadores/sangue , Pressão Sanguínea/efeitos dos fármacos , Quimiocina CCL2/sangue , GMP Cíclico/metabolismo , Fibrose/patologia , Agonistas da Guanilil Ciclase C/uso terapêutico , Inflamação/patologia , Rim/patologia , Masculino , Peptídeo Natriurético Encefálico/sangue , Óxido Nítrico/metabolismo , Osteopontina/sangue , Fragmentos de Peptídeos/sangue , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Ratos , Ratos Endogâmicos Dahl , Insuficiência Renal/patologia , Transdução de Sinais/efeitos dos fármacos , Guanilil Ciclase Solúvel/metabolismo , Inibidor Tecidual de Metaloproteinase-1/sangue
13.
Hypertension ; 75(2): 492-499, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31865789

RESUMO

The endoplasmic reticulum (ER) plays a pivotal role in syntheses of proteins and steroid hormones and regulation of intracellular Ca2+ level. We aimed to investigate ER-associated genes in aldosterone-producing adenomas (APAs) and clarify their effect on aldosterone production. Microarray analysis targeting 288 ER-associated genes was conducted using nonfunctioning adrenocortical adenomas (n=5) and APAs (n=19). Immunohistochemistry and quantitative polymerase chain reaction analyses were performed with 13 nonfunctioning adrenocortical adenoma and 48 APA samples. Functional studies were performed with human adrenocortical carcinoma (HAC15) cells, some of which were genetically modified using lentiviruses. The ER chaperone calmegin (CLGN) was the most highly expressed ER-associated gene in APAs relative to nonfunctioning adrenocortical adenomas. Analysis with quantitative polymerase chain reaction revealed CLGN to be 9.5-fold upregulated in APAs relative to nonfunctioning adrenocortical adenomas. There were no differences among different APA genotypes affecting aldosterone production. Immunohistochemistry analysis revealed that CLGN was strongly expressed in APAs and aldosterone-producing cell clusters. Angiotensin II stimulation or KCNJ5 T158A overexpression in HAC15 cells did not affect CLGN mRNA levels. CLGN overexpression in HAC15 cells increased aldosterone levels but did not stimulate CYP11B2 mRNA levels. Pathway and gene ontology analyses using RNA sequencing results showed that tRNA aminoacyl metabolism was the most enriched pathway in CLGN-overexpressing cells. CYP11B2 (aldosterone synthase) and HSD3B2 (3 beta-hydroxysteroid dehydrogenase/delta 5->4-isomerase type 2) protein expression were more abundant in CLGN-overexpressing cells. CLGN knockdown using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) method in HAC15 cells that carry the KCNJ5 mutation did not affect aldosterone production. To summarize, CLGN was upregulated and associated with aldosterone production via translational regulation of CYP11B2 in APAs.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/metabolismo , Proteínas de Ligação ao Cálcio/genética , Retículo Endoplasmático/metabolismo , Regulação Neoplásica da Expressão Gênica , Chaperonas Moleculares/genética , Neoplasias Experimentais , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/patologia , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Animais , Proteínas de Ligação ao Cálcio/biossíntese , Masculino , Chaperonas Moleculares/biossíntese , RNA Neoplásico/genética , Ratos , Ratos Endogâmicos Dahl , Testículo , Regulação para Cima
14.
Hypertension ; 75(2): 431-438, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31865796

RESUMO

Mechanical stretch raises intracellular Ca (Cai) in many cell types. Luminal flow-derived stretch stimulates O2- production by thick ascending limbs (THALs). Renal O2- is greater in Dahl salt-sensitive (SS) than salt-resistant (SR) rats. We hypothesized that mechanical stretch stimulates Ca influx via TRPV4 (transient receptor potential vanilloid type 4) which in turn raises Cai in THALs; these increases in Cai are necessary for stretch to augment O2- production; and stretch-stimulated, and therefore flow-induced, O2- production is enhanced in SS compared with SR THALs due to elevated Ca influx and increased Cai. Cai and O2- were measured in SS and SR THALs from rats on normal salt using Fura2-acetoxymethyl ester and dihydroethidium, respectively. Stretch raised Cai in SS by 270.4±48.9 nmol/L and by 123.6±27.0 nmol/L in SR THALs (P<0.02). Removing extracellular Ca eliminated the increases and differences in Cai between strains. Knocking down TRPV4 in SS THALs reduced stretch-induced Cai to SR levels (SS: 92.0±15.9 nmol/L; SR: 123.6±27.0 nmol/L). RN1734, a TRPV4 inhibitor, blunted stretch-elevated Cai by ≈75% and ≈66% in SS (P<0.03) and SR (P<0.04), respectively. Stretch augmented O2- production by 58.6±10.2 arbitrary fluorescent units/min in SS and by 24.4±2.6 arbitrary fluorescent units/min in SR THALs (P<0.05). Removal of extracellular Ca blunted stretch-induced increases in O2- and eliminated differences between strains. RN1734 reduced stretch-induced O2- by ≈70% in SS (P<0.005) and ≈60% in SR (P<0.01). Conclusions are as follows: (1) stretch activates TRPV4, which raises Cai in THALs; (2) the increase in Cai stimulates O2- production; and (3) stretch-induced O2- production is enhanced in SS THALs due to greater increases in Cai.


Assuntos
Cálcio/metabolismo , Hipertensão/genética , Líquido Intracelular/metabolismo , Alça do Néfron/metabolismo , Oxigênio/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Modelos Animais de Doenças , Hipertensão/metabolismo , Masculino , Ratos , Ratos Endogâmicos Dahl , Cloreto de Sódio/metabolismo
15.
Biomed Res Int ; 2019: 2816959, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31886193

RESUMO

Backgrounds: Long noncoding RNAs (lncRNAs) play an important role in various biological processes. However, their functions in salt-sensitive hypertension are largely unknown. In this study, the lncRNA-seq technique was employed to compare the expression profiles of lncRNAs and mRNAs in salt-sensitive hypertensive rats. Methods: Blood pressure, serum sodium, and urinary creatinine were texted in salt-sensitive and salt-insensitive rats fed with different salt concentrations. High-throughput sequencing was used to detect the expression of lncRNAs and mRNA in the renal medulla of the two groups. Results: Blood pressure and urinary sodium/creatinine of high-salt diets of the sensitive group were significantly higher than that in the control group. Serum sodium has no significant difference between the two groups in high-salt diets. NONRATG007131.2 and NONRATG012674.2 were the most different lncRNAs in the high salt-sensitive group. Correlation analysis reveals that Matn1, Serpinb12, Anxa8, and Hspa5 may play an important role in salt-sensitive hypertension. Conclusion: This study analyzed the difference in lncRNA and mRNA between salt-sensitive and salt-insensitive rats with different salt diets by high-throughput sequencing. Salt sensitivity and salt concentration were two key factors for the induction of hypertension. We found some potential genes that play an important role in salt-sensitive hypertension.


Assuntos
Hipertensão/genética , RNA Longo não Codificante/genética , Transcriptoma/efeitos dos fármacos , Animais , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hipertensão/sangue , Hipertensão/patologia , Hipertensão/urina , RNA Longo não Codificante/isolamento & purificação , Ratos , Ratos Endogâmicos Dahl/sangue , Ratos Endogâmicos Dahl/genética , Cloreto de Sódio/sangue , Cloreto de Sódio/urina , Cloreto de Sódio na Dieta/farmacologia , Sequenciamento Completo do Exoma
16.
Nutrients ; 11(11)2019 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-31684148

RESUMO

Diets rich in (poly)phenols are associated with a reduced reduction in the incidence of cardiovascular disorders. While the absorption and metabolism of (poly)phenols has been described, it is not clear how their metabolic fate is affected under pathological conditions. This study evaluated the metabolic fate of berry (poly)phenols in an in vivo model of hypertension as well as the associated microbiota response. Dahl salt-sensitive rats were fed either a low-salt diet (0.26% NaCl) or a high-salt diet (8% NaCl), with or without a berry mixture (blueberries, blackberries, raspberries, Portuguese crowberry and strawberry tree fruit) for 9 weeks. The salt-enriched diet promoted an increase in the urinary excretion of berry (poly)phenol metabolites, while the abundance of these metabolites decreased in faeces, as revealed by UPLC-MS/MS. Moreover, salt and berries modulated gut microbiota composition as demonstrated by 16S rRNA analysis. Some changes in the microbiota composition were associated with the high-salt diet and revealed an expansion of the families Proteobacteria and Erysipelotrichaceae. However, this effect was mitigated by the dietary supplementation with berries. Alterations in the metabolic fate of (poly)phenols occur in parallel with the modulation of gut microbiota in hypertensive rats. Thus, beneficial effects of (poly)phenols could be related with these interlinked modifications, between metabolites and microbiota environments.


Assuntos
Frutas , Microbioma Gastrointestinal/fisiologia , Fenóis/metabolismo , Animais , Dieta , Disbiose/metabolismo , Fezes/microbiologia , Microbioma Gastrointestinal/genética , Glicosídeos/metabolismo , Masculino , Fenóis/análise , Compostos Fitoquímicos/análise , Compostos Fitoquímicos/metabolismo , Ratos , Ratos Endogâmicos Dahl , Sódio na Dieta
17.
Am J Physiol Renal Physiol ; 317(5): F1398-F1403, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31588797

RESUMO

Our current knowledge of the properties of renal ion channels responsible for electrolytes and cell energy homeostasis mainly relies on rodent studies. However, it has not been established yet to what extent their characteristics can be generalized to those of humans. The present study was designed to develop a standardized protocol for the isolation of well-preserved glomeruli and renal tubules from rodent and human kidneys and to assess the functional suitability of the obtained materials for physiological studies. Separation of nephron segments from human and rodent kidneys was achieved using a novel vibrodissociation technique. The integrity of isolated renal tubules and glomeruli was probed via electrophysiological analysis and fluorescence microscopy, and the purity of the collected fractions was confirmed using quantitative RT-PCR with gene markers for specific cell types. The developed approach allows rapid isolation of well-preserved renal tubules and glomeruli from human and rodent kidneys amenable for electrophysiological, Ca2+ imaging, and omics studies. Analysis of the basic electrophysiological parameters of major K+ and Na+ channels expressed in human cortical collecting ducts revealed that they exhibited similar biophysical properties as previously reported in rodent studies. Using vibrodissociation for nephron segment isolation has several advantages over existing techniques: it is less labor intensive, requires little to no enzymatic treatment, and produces large quantities of well-preserved experimental material in pure fractions. Applying this method for the separation of nephron segments from human and rodent kidneys may be a powerful tool for the indepth assessment of kidney function in health and disease.


Assuntos
Técnicas de Preparação Histocitológica/métodos , Néfrons , Animais , Cálcio/metabolismo , Humanos , Camundongos , Ratos , Ratos Endogâmicos Dahl , Vibração
18.
Hypertension ; 74(4): 854-863, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31476910

RESUMO

The Dahl salt-sensitive (SS) rat is an established model of SS hypertension and renal damage. In addition to salt, other dietary components were shown to be important determinants of hypertension in SS rats. With previous work eliminating the involvement of genetic differences, grain-fed SS rats from Charles River Laboratories (SS/CRL; 5L2F/5L79) were less susceptible to salt-induced hypertension and renal damage compared with purified diet-fed SS rats bred at the Medical College of Wisconsin (SS/MCW; 0.4% NaCl, AIN-76A). With the known role of immunity in hypertension, the present study characterized the immune cells infiltrating SS/MCW and SS/CRL kidneys via flow cytometry and RNA sequencing in T-cells isolated from the blood and kidneys of rats maintained on their respective parental diet or on 3 weeks of high salt (4.0% NaCl, AIN-76A). SS/CRL rats were protected from salt-induced hypertension (116.5±1.2 versus 141.9±14.4 mm Hg), albuminuria (21.7±3.5 versus 162.9±22.2 mg/d), and renal immune cell infiltration compared with SS/MCW. RNA-seq revealed >50% of all annotated genes in the entire transcriptome to be significantly differentially expressed in T-cells isolated from blood versus kidney, regardless of colony or chow. Pathway analysis of significantly differentially expressed genes between low and high salt conditions demonstrated changes related to inflammation in SS/MCW renal T-cells compared with metabolism-related pathways in SS/CRL renal T-cells. These functional and transcriptomic T-cell differences between SS/MCW and SS/CRL show that dietary components in addition to salt may influence immunity and the infiltration of immune cells into the kidney, ultimately impacting susceptibility to salt-induced hypertension and renal damage.


Assuntos
Pressão Sanguínea/fisiologia , Hipertensão/patologia , Rim/patologia , Cloreto de Sódio na Dieta/farmacologia , Linfócitos T/metabolismo , Transcriptoma , Animais , Pressão Sanguínea/efeitos dos fármacos , Citometria de Fluxo , Hipertensão/metabolismo , Rim/metabolismo , Masculino , Ratos , Ratos Endogâmicos Dahl
20.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514290

RESUMO

In salt-sensitive hypertension, reactive oxygen species (ROS) play a major role in the progression of renal disease partly through the activation of the mineralocorticoid receptor (MR). We have previously demonstrated that urinary vanin-1 is an early biomarker of oxidative renal tubular injury. However, it remains unknown whether urinary vanin-1 might reflect the treatment effect. The objective of this study was to clarify the treatment effect for renal tubular damage in Dahl salt-sensitive (DS) rats. DS rats (six weeks old) were given one of the following for four weeks: high-salt diet (8% NaCl), high-salt diet plus a superoxide dismutase mimetic, tempol (3 mmol/L in drinking water), high-salt diet plus eplerenone (100 mg/kg/day), and normal-salt diet (0.3% NaCl). After four-week treatment, blood pressure was measured and kidney tissues were evaluated. ROS were assessed by measurements of malondialdehyde and by immunostaining for 4-hydroxy-2-nonenal. A high-salt intake for four weeks caused ROS and histological renal tubular damages in DS rats, both of which were suppressed by tempol and eplerenone. Proteinuria and urinary N-acetyl-ß-D-glucosaminidase exhibited a significant decrease in DS rats receiving a high-salt diet plus eplerenone, but not tempol. In contrast, urinary vanin-1 significantly decreased in DS rats receiving a high-salt diet plus eplerenone as well as tempol. Consistent with these findings, immunohistochemical analysis revealed that vanin-1 was localized in the renal proximal tubules but not the glomeruli in DS rats receiving a high-salt diet, with the strength attenuated by tempol or eplerenone treatment. In conclusion, these results suggest that urinary vanin-1 is a potentially sensitive biomarker for ameliorating renal tubular damage in salt-sensitive hypertension.


Assuntos
Amidoidrolases/metabolismo , Túbulos Renais/patologia , Estresse Oxidativo , Amidoidrolases/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Pressão Sanguínea/efeitos dos fármacos , Óxidos N-Cíclicos/farmacologia , Eplerenona/farmacologia , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/fisiopatologia , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos Endogâmicos Dahl , Marcadores de Spin , Sístole/efeitos dos fármacos
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