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1.
J Am Soc Nephrol ; 31(7): 1479-1495, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32540856

RESUMO

BACKGROUND: Genetic mutations in α-actinin-4 (ACTN4)-an important actin crosslinking cytoskeletal protein that provides structural support for kidney podocytes-have been linked to proteinuric glomerulosclerosis in humans. However, the effect of post-translational modifications of ACTN4 on podocyte integrity and kidney function is not known. METHODS: Using mass spectrometry, we found that ACTN4 is phosphorylated at serine (S) 159 in human podocytes. We used phosphomimetic and nonphosphorylatable ACTN4 to comprehensively study the effects of this phosphorylation in vitro and in vivo. We conducted x-ray crystallography, F-actin binding and bundling assays, and immunofluorescence staining to evaluate F-actin alignment. Microfluidic organ-on-a-chip technology was used to assess for detachment of podocytes simultaneously exposed to fluid flow and cyclic strain. We then used CRISPR/Cas9 to generate mouse models and assessed for renal injury by measuring albuminuria and examining kidney histology. We also performed targeted mass spectrometry to determine whether high extracellular glucose or TGF-ß levels increase phosphorylation of ACTN4. RESULTS: Compared with the wild type ACTN4, phosphomimetic ACTN4 demonstrated increased binding and bundling activity with F-actin in vitro. Phosphomimetic Actn4 mouse podocytes exhibited more spatially correlated F-actin alignment and a higher rate of detachment under mechanical stress. Phosphomimetic Actn4 mice developed proteinuria and glomerulosclerosis after subtotal nephrectomy. Moreover, we found that exposure to high extracellular glucose or TGF-ß stimulates phosphorylation of ACTN4 at S159 in podocytes. CONCLUSIONS: These findings suggest that increased phosphorylation of ACTN4 at S159 leads to biochemical, cellular, and renal pathology that is similar to pathology resulting from human disease-causing mutations in ACTN4. ACTN4 may mediate podocyte injury as a consequence of both genetic mutations and signaling events that modulate phosphorylation.

2.
J Am Soc Nephrol ; 31(4): 783-798, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32086277

RESUMO

BACKGROUND: Increased nerve activity causes hypertension and kidney disease. Recent studies suggest that renal denervation reduces BP in patients with hypertension. Renal NE release is regulated by prejunctional α2A-adrenoceptors on sympathetic nerves, and α2A-adrenoceptors act as autoreceptors by binding endogenous NE to inhibit its own release. However, the role of α2A-adrenoceptors in the pathogenesis of hypertensive kidney disease is unknown. METHODS: We investigated effects of α2A-adrenoceptor-regulated renal NE release on the development of angiotensin II-dependent hypertension and kidney disease. In uninephrectomized wild-type and α2A-adrenoceptor-knockout mice, we induced hypertensive kidney disease by infusing AngII for 28 days. RESULTS: Urinary NE excretion and BP did not differ between normotensive α2A-adrenoceptor-knockout mice and wild-type mice at baseline. However, NE excretion increased during AngII treatment, with the knockout mice displaying NE levels that were significantly higher than those of wild-type mice. Accordingly, the α2A-adrenoceptor-knockout mice exhibited a systolic BP increase, which was about 40 mm Hg higher than that found in wild-type mice, and more extensive kidney damage. In isolated kidneys, AngII-enhanced renal nerve stimulation induced NE release and pressor responses to a greater extent in kidneys from α2A-adrenoceptor-knockout mice. Activation of specific sodium transporters accompanied the exaggerated hypertensive BP response in α2A-adrenoceptor-deficient kidneys. These effects depend on renal nerves, as demonstrated by reduced severity of AngII-mediated hypertension and improved kidney function observed in α2A-adrenoceptor-knockout mice after renal denervation. CONCLUSIONS: Our findings reveal a protective role of prejunctional inhibitory α2A-adrenoceptors in pathophysiologic conditions with an activated renin-angiotensin system, such as hypertensive kidney disease, and support the concept of sympatholytic therapy as a treatment.

3.
Acta Physiol (Oxf) ; 229(1): e13448, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31994810

RESUMO

AIM: Sexual dimorphisms are evident along the nephron: Females (F) exhibit higher ratios of renal distal to proximal Na+ transporters' abundance, greater lithium clearance (CLi ) more rapid natriuresis in response to saline infusion and lower plasma [K+ ] vs. males (M). During angiotensin II infusion hypertension (AngII-HTN) M exhibit distal Na+ transporter activation, lower proximal and medullary loop transporters, blunted natriuresis in response to saline load, and reduced plasma [K+ ]. This study aimed to determine whether responses of F to AngII-HTN mimicked those in M or were impacted by sexual dimorphisms evident at baseline. METHODS: Sprague Dawley rats and C57BL/6 mice were AngII infused via osmotic minipumps 2 and 3 weeks, respectively, and assessed by metabolic cage collections, tail-cuff sphygmomanometer, semi-quantitative immunoblotting of kidney and patch-clamp electrophysiology. RESULTS: In F rats, AngII-infusion increased BP to 190 mm Hg, increased phosphorylation of cortical NKCC2, NCC and cleavage of ENaC two to threefold, increased ENaC channel activity threefold and aldosterone 10-fold. K+ excretion increased and plasma [K+ ] decreased. Evidence of natriuresis in F included increased urine Na+ excretion and CLi , and decreased medullary NHE3, NKCC2 and Na,K-ATPase abundance. In C57BL/6 mice, AngII-HTN increased abundance of distal Na+ transporters, suppressed proximal-medullary transporters and reduced plasma [K+ ] in both F and M. CONCLUSION: Despite baseline sexual dimorphisms, AngII-HTN provokes similar increases in BP, aldosterone, distal transporters, ENaC channel activation and K+ loss accompanied by similar suppression of proximal and loop Na+ transporters, natriuresis and diuresis in females and males.

4.
Biol Sex Differ ; 10(1): 45, 2019 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-31484552

RESUMO

BACKGROUND: Obesity increases the risk for hypertension in both sexes, but the prevalence of hypertension is lower in females than in males until menopause, despite a higher prevalence of obesity in females. We previously demonstrated that angiotensin-converting enzyme 2 (ACE2), which cleaves the vasoconstrictor, angiotensin II (AngII), to generate the vasodilator, angiotensin-(1-7) (Ang-(1-7)), contributes to sex differences in obesity-hypertension. ACE2 expression in adipose tissue was influenced by obesity in a sex-specific manner, with elevated ACE2 expression in obese female mice. Moreover, estrogen stimulated adipose ACE2 expression and reduced obesity-hypertension in females. In this study, we hypothesized that deficiency of adipocyte ACE2 contributes to obesity-hypertension of females. METHODS: We generated a mouse model of adipocyte ACE2 deficiency. Male and female mice with adipocyte ACE2 deficiency or littermate controls were fed a low (LF) or a high fat (HF) diet for 16 weeks and blood pressure was quantified by radiotelemetry. HF-fed mice of each sex and genotype were challenged by an acute AngII injection, and blood pressure response was quantified. To translate these findings to humans, we performed a proof-of-principle study in obese transwomen in which systemic angiotensin peptides and blood pressure were quantified prior to and after 12 weeks of gender-affirming 17ß-estradiol hormone therapy. RESULTS: Adipocyte ACE2 deficiency had no effect on the development of obesity in either sex. HF feeding increased systolic blood pressures (SBP) of wild-type male and female mice compared to LF-fed controls. Adipocyte ACE2 deficiency augmented obesity-induced elevations in SBP in females, but not in males. Obese female, but not obese male mice with adipocyte ACE2 deficiency, had an augmented SBP response to acute AngII challenge. In humans, plasma 17ß-estradiol concentrations increased in obese transwomen administered 17ß-estradiol and correlated positively with plasma Ang-(1-7)/AngII balance, and negatively to SBP after 12 weeks of 17ß-estradiol administration. CONCLUSIONS: Adipocyte ACE2 protects female mice from obesity-hypertension, and reduces the blood pressure response to systemic AngII. In obese transwomen undergoing gender-affirming hormone therapy, 17ß-estradiol administration may regulate blood pressure via the Ang-(1-7)/AngII balance.


Assuntos
Adipócitos/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Obesidade/metabolismo , Peptidil Dipeptidase A/metabolismo , Angiotensina I/farmacologia , Animais , Dieta Hiperlipídica , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Fragmentos de Peptídeos/farmacologia , Peptidil Dipeptidase A/genética , Caracteres Sexuais
5.
Diabetes ; 67(10): 2096-2106, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30065034

RESUMO

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease worldwide, but its molecular pathogenesis is not well defined, and there are no specific treatments. In humans, there is a strong genetic component determining susceptibility to DN. However, specific genes controlling DN susceptibility in humans have not been identified. In this study, we describe a mouse model combining type 1 diabetes with activation of the renin-angiotensin system (RAS), which develops robust kidney disease with features resembling human DN: heavy albuminuria, hypertension, and glomerulosclerosis. Additionally, there is a powerful effect of genetic background regulating susceptibility to nephropathy; the 129 strain is susceptible to kidney disease, whereas the C57BL/6 strain is resistant. To examine the molecular basis of this differential susceptibility, we analyzed the glomerular transcriptome of young mice early in the course of their disease. We find dramatic differences in regulation of immune and inflammatory pathways, with upregulation of proinflammatory pathways in the susceptible (129) strain and coordinate downregulation in the resistant (C57BL/6) strain. Many of these pathways are also upregulated in rat models and in humans with DN. Our studies suggest that genes controlling inflammatory responses, triggered by hyperglycemia and RAS activation, may be critical early determinants of susceptibility to DN.


Assuntos
Nefropatias Diabéticas/genética , Nefropatias Diabéticas/imunologia , Inflamação/genética , Inflamação/imunologia , Nefropatias/genética , Nefropatias/imunologia , Animais , Glicemia/genética , Glicemia/imunologia , Western Blotting , Predisposição Genética para Doença/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real
6.
Cell Chem Biol ; 25(10): 1195-1207.e32, 2018 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-30033129

RESUMO

Sustained vascular smooth muscle hypercontractility promotes hypertension and cardiovascular disease. The etiology of hypercontractility is not completely understood. New therapeutic targets remain vitally important for drug discovery. Here we report that Pim kinases, in combination with DAPK3, regulate contractility and control hypertension. Using a co-crystal structure of lead molecule (HS38) in complex with DAPK3, a dual Pim/DAPK3 inhibitor (HS56) and selective DAPK3 inhibitors (HS94 and HS148) were developed to provide mechanistic insight into the polypharmacology of hypertension. In vitro and ex vivo studies indicated that Pim kinases directly phosphorylate smooth muscle targets and that Pim/DAPK3 inhibition, unlike selective DAPK3 inhibition, significantly reduces contractility. In vivo, HS56 decreased blood pressure in spontaneously hypertensive mice in a dose-dependent manner without affecting heart rate. These findings suggest including Pim kinase inhibition within a multi-target engagement strategy for hypertension management. HS56 represents a significant step in the development of molecularly targeted antihypertensive medications.


Assuntos
Proteínas Quinases Associadas com Morte Celular/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Pressão Sanguínea/efeitos dos fármacos , Cristalografia por Raios X , Proteínas Quinases Associadas com Morte Celular/química , Proteínas Quinases Associadas com Morte Celular/metabolismo , Humanos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Camundongos , Modelos Moleculares , Terapia de Alvo Molecular , Contração Muscular/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-pim-1/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Ratos Sprague-Dawley , Alinhamento de Sequência
7.
Am J Physiol Renal Physiol ; 314(4): F531-F542, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29187372

RESUMO

Diabetic nephropathy is a major cause of end-stage renal disease in developed countries. While angiotensin-converting enzyme (ACE) inhibitors are used to treat diabetic nephropathy, how intrarenal ACE contributes to diabetic renal injury is uncertain. Here, two mouse models with different patterns of renal ACE expression were studied to determine the specific contribution of tubular vs. glomerular ACE to early diabetic nephropathy: it-ACE mice, which make endothelial ACE but lack ACE expression by renal tubular epithelium, and ACE 3/9 mice, which lack endothelial ACE and only express renal ACE in tubular epithelial cells. The absence of endothelial ACE normalized the glomerular filtration rate and endothelial injury in diabetic ACE 3/9 mice. However, these mice developed tubular injury and albuminuria and displayed low renal levels of megalin that were similar to those observed in diabetic wild-type mice. In diabetic it-ACE mice, despite hyperfiltration, the absence of renal tubular ACE greatly reduced tubulointerstitial injury and albuminuria and increased renal megalin expression compared with diabetic wild-type and diabetic ACE 3/9 mice. These findings demonstrate that endothelial ACE is a central regulator of the glomerular filtration rate while tubular ACE is a key player in the development of tubular injury and albuminuria. These data suggest that tubular injury, rather than hyperfiltration, is the main cause of microalbuminuria in early diabetic nephropathy.


Assuntos
Albuminúria/enzimologia , Diabetes Mellitus Experimental/enzimologia , Nefropatias Diabéticas/enzimologia , Túbulos Renais/enzimologia , Peptidil Dipeptidase A/metabolismo , Albuminúria/genética , Albuminúria/patologia , Albuminúria/fisiopatologia , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/genética , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/fisiopatologia , Células Endoteliais/enzimologia , Taxa de Filtração Glomerular , Glomérulos Renais/enzimologia , Glomérulos Renais/fisiopatologia , Túbulos Renais/patologia , Túbulos Renais/fisiopatologia , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos Knockout , Peptidil Dipeptidase A/deficiência , Peptidil Dipeptidase A/genética , RNA Interferente Pequeno/genética , Estreptozocina
8.
Curr Opin Nephrol Hypertens ; 27(1): 1-7, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29045337

RESUMO

PURPOSE OF REVIEW: The renin-angiotensin system (RAS) plays a critical role in the pathogenesis of hypertension. Homeostatic actions of the RAS, such as increasing blood pressure (BP) and vasoconstriction, are mediated via type 1 (AT1) receptors for angiotensin II. All components of the RAS are present in the renal proximal tubule, which reabsorbs the bulk of the glomerular filtrate, making this segment of the nephron a location of great interest for solute handling under RAS influence. This review highlights recent studies that illustrate the key role of renal proximal tubule AT1 receptors in BP regulation. RECENT FINDINGS: A variety of investigative approaches have demonstrated that angiotensin II signaling via AT1a receptors, specifically in the renal proximal tubule, is a major regulator of BP and sodium homeostasis. Reduction of proximal tubule AT1a receptors led to lower BPs, whereas overexpression generally caused increased BPs. SUMMARY: AT1a receptors in the proximal tubule are critical to the regulation of BP by the kidney and the RAS. The pattern of BP modulation is associated with alterations in sodium transporters. As a key site for sodium homeostasis, the renal proximal tubule could hence be a potential target in the treatment of hypertension.


Assuntos
Pressão Sanguínea , Túbulos Renais Proximais/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiologia , Animais , Homeostase , Humanos , Receptor Tipo 1 de Angiotensina/fisiologia , Transdução de Sinais , Sódio/metabolismo
9.
Nat Rev Nephrol ; 14(1): 48-56, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29062142

RESUMO

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease in the developed world. Accordingly, an urgent need exists for new, curative treatments as well as for biomarkers to stratify risk of DN among individuals with diabetes mellitus. A barrier to progress in these areas has been a lack of animal models that faithfully replicate the main features of human DN. Such models could be used to define the pathogenesis, identify drug targets and test new therapies. Owing to their tractability for genetic manipulation, mice are widely used to model human diseases, including DN. Questions have been raised, however, about the general utility of mouse models in human drug discovery. Standard mouse models of diabetes typically manifest only modest kidney abnormalities, whereas accelerated models, induced by superimposing genetic stressors, recapitulate key features of human DN. Incorporation of systems biology approaches and emerging data from genomics and metabolomics studies should enable further model refinement. Here, we discuss the current status of mouse models for DN, their limitations and opportunities for improvement. We emphasize that future efforts should focus on generating robust models that reproduce the major clinical and molecular phenotypes of human DN.


Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 2 , Nefropatias Diabéticas , Modelos Animais de Doenças , Falência Renal Crônica , Camundongos , Animais , Análise de Sistemas , Biologia de Sistemas
10.
J Exp Ther Oncol ; 11(2): 107-115, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28976133

RESUMO

OBJECTIVE: The renin-angiotensin system, through its type 1 and type 2 angiotensin receptors (AT1R and AT2R, respectively) may have a role in prostate cancer. The objective of this pilot study was to explore that potential role by determining whether the AT1R blocker, losartan, would reduce the growth of LAPC-4 prostate cancer xenografts in nude mice. We also evaluated the tumor growth effects of using angiotensin II to activate both AT1R and AT2R simultaneously. Our data showed that losartan decreased tumor volumes by 56% versus control. This decrease reached statistical significance at day 54 (p = 0.0014). By day 54, Ki67 was also reduced in the losartan group, though not significantly so (p = 0.077). Losartan had no significant effect on AT1R or AT2R expression. Despite significant increases in both AT1R and AT2R at day 29 (p = 0.043 and 0.038, respectively), the administration of angiotensin II did not result in any significant differences in tumor volumes or ki67 at any time point. These data suggest that selective activation and induction of AT2R coupled with blockade of AT1R may slow prostate cancer growth. Future larger studies are needed to confirm these results.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Losartan/farmacologia , Neoplasias da Próstata/patologia , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/genética , Carga Tumoral/efeitos dos fármacos , Angiotensina II/farmacologia , Animais , Linhagem Celular Tumoral , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias da Próstata/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/efeitos dos fármacos , Transdução de Sinais , Transcriptoma/efeitos dos fármacos , Vasoconstritores/farmacologia
11.
Cardiovasc Res ; 113(13): 1551-1559, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-29048463

RESUMO

Aims: Chronic kidney disease (CKD) is a powerful independent risk factor for cardiovascular events, including vein graft failure. Because CKD impairs the clearance of small proteins, we tested the hypothesis that CKD exacerbates vein graft disease by elevating serum levels of critical cytokines that promote vein graft neointimal hyperplasia. Methods and results: We modelled CKD in C57BL/6 mice with 5/6ths nephrectomy, which reduced glomerular filtration rate by 60%, and we modelled vein grafting with inferior-vena-cava-to-carotid interposition grafting. CKD increased vein graft neointimal hyperplasia four-fold, decreased vein graft re-endothelialization two-fold, and increased serum levels of interleukin-9 (IL-9) five-fold. By quantitative immunofluorescence and histochemical staining, vein grafts from CKD mice demonstrated a ∼two-fold higher prevalence of mast cells, and a six-fold higher prevalence of activated mast cells. Concordantly, vein grafts from CKD mice showed higher levels of TNF and NFκB activation, as judged by phosphorylation of NFκB p65 on Ser536 and by expression of VCAM-1. Arteriovenous fistula veins from humans with CKD also showed up-regulation of mast cells and IL-9. Treating CKD mice with IL-9-neutralizing IgG reduced vein graft neointimal area four-fold, increased vein graft re-endothelialization ∼two-fold, and reduced vein graft total and activated mast cell levels two- and four-fold, respectively. Treating CKD mice with the mast cell stabilizer cromolyn reduced neointimal hyperplasia and increased re-endothelialization in vein grafts. In vitro, IL-9 promoted endothelial cell apoptosis but had no effect on smooth muscle cell proliferation. Conclusion: CKD aggravates vein graft disease through mechanisms involving IL-9 and mast cell activation.


Assuntos
Derivação Arteriovenosa Cirúrgica , Artéria Carótida Primitiva/cirurgia , Interleucina-9/metabolismo , Mastócitos/metabolismo , Insuficiência Renal Crônica/complicações , Doenças Vasculares/complicações , Veia Cava Inferior/transplante , Animais , Apoptose , Artéria Carótida Primitiva/imunologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Hiperplasia , Interleucina-9/imunologia , Mastócitos/imunologia , Camundongos Endogâmicos C57BL , Neointima , Fosforilação , Insuficiência Renal Crônica/imunologia , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , Fatores de Tempo , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Doenças Vasculares/imunologia , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia , Veia Cava Inferior/imunologia , Veia Cava Inferior/metabolismo , Veia Cava Inferior/patologia
12.
FASEB J ; 31(12): 5520-5529, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28842425

RESUMO

Accurate analysis of placental and fetal oxygenation is critical during pregnancy. Photoacoustic imaging (PAI) combines laser technology with ultrasound in real time. We tested the sensitivity and accuracy of PAI for analysis of placental and fetal oxygen saturation (sO2) in mice. The placental labyrinth (L) had a higher sO2 than the junctional zone plus decidua region (JZ+D) in C57Bl/6 mice. Changing maternal O2 from 100 to 20% in C57Bl/6 mice lowered sO2 in these regions. C57Bl/6 mice were treated with the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME) from gestational day (GD) 11 to GD18 to induce hypertension. L-NAME decreased sO2 in L and JZ+D at GD14 and GD18 in association with fetal growth restriction and higher blood pressure. Hypoxia-inducible factor 1α immunostaining was higher in L-NAME vs control mice at GD14. Fetal sO2 levels were similar between l-NAME and control mice at GD14 and GD18. In contrast to untreated C57Bl/6, L-NAME decreased placental sO2 at GD14 and GD18 vs GD10 or GD12. Placental sO2 was lower in fetal growth restriction in an angiotensin-converting enzyme 2 knockout mouse model characterized by placental hypoxia. On phantom studies, patterns of sO2 measured directly correlated with those measured by PAI. In summary, PAI enables the detection of placental and fetal oxygenation during normal and pathologic pregnancies in mice.-Yamaleyeva, L. M., Sun, Y., Bledsoe, T., Hoke, A., Gurley, S. B., Brosnihan, K. B. Photoacoustic imaging for in vivo quantification of placental oxygenation in mice.


Assuntos
Oxigênio/metabolismo , Técnicas Fotoacústicas/métodos , Placenta/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/metabolismo , Hipertensão/induzido quimicamente , Hipertensão/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Placenta/efeitos dos fármacos , Gravidez
13.
JCI Insight ; 2(7): e92720, 2017 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-28405625

RESUMO

The renal collecting duct (CD), as the terminal segment of the nephron, is responsible for the final adjustments to the amount of sodium excreted in urine. While angiotensin II modulates reabsorptive functions of the CD, the contribution of these actions to physiological homeostasis is not clear. To examine this question, we generated mice with cell-specific deletion of AT1A receptors from the CD. Elimination of AT1A receptors from both principal and intercalated cells (CDKO mice) had no effect on blood pressures at baseline or during successive feeding of low- or high-salt diets. In contrast, the severity of hypertension caused by chronic infusion of angiotensin II was paradoxically exaggerated in CDKO mice compared with controls. In wild-type mice, angiotensin II induced robust expression of cyclooxygenase-2 (COX-2) in renal medulla, primarily localized to intercalated cells. Upregulation of COX-2 was diminished in CDKO mice, resulting in reduced generation of vasodilator prostanoids. This impaired expression of COX-2 has physiological consequences, since administration of a specific COX-2 inhibitor to CDKO and control mice during angiotensin II infusion equalized their blood pressures. Stimulation of COX-2 was also triggered by exposure of isolated preparations of medullary CDs to angiotensin II. Deletion of AT1A receptors from principal cells alone did not affect angiotensin II-dependent COX2 stimulation, implicating intercalated cells as the main source of COX2 in this setting. These findings suggest a novel paracrine role for the intercalated cell to attenuate the severity of hypertension. Strategies for preserving or augmenting this pathway may have value for improving the management of hypertension.


Assuntos
Angiotensina II/farmacologia , Ciclo-Oxigenase 2/metabolismo , Hipertensão/fisiopatologia , Túbulos Renais Coletores/citologia , Animais , Pressão Sanguínea , Inibidores de Ciclo-Oxigenase 2/farmacologia , Modelos Animais de Doenças , Hipertensão/metabolismo , Camundongos , Camundongos Knockout , Receptor Tipo 1 de Angiotensina/genética
14.
Lab Anim ; 51(2): 138-146, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27166392

RESUMO

Genotyping of genetically-engineered mice is necessary for the effective design of breeding strategies and identification of mutant mice. This process relies on the identification of DNA markers introduced into genomic sequences of mice, a task usually performed using the polymerase chain reaction (PCR). Clearly, the limiting step in genotyping is isolating pure genomic DNA. Isolation of mouse DNA for genotyping typically involves painful procedures such as tail snip, digit removal, or ear punch. Although the harvesting of hair has previously been proposed as a source of genomic DNA, there has been a perceived complication and reluctance to use this non-painful technique because of low DNA yields and fear of contamination. In this study we developed a simple, economic, and efficient strategy using Chelex® resins to purify genomic DNA from hair roots of mice which are suitable for genotyping. Upon comparison with standard DNA purification methods using a commercially available kit, we demonstrate that Chelex® efficiently and consistently purifies high-quality DNA from hair roots, minimizing pain, shortening time and reducing costs associated with the determination of accurate genotypes. Therefore, the use of hair roots combined with Chelex® is a reliable and more humane alternative for DNA genotyping.


Assuntos
Quelantes/química , DNA/isolamento & purificação , Técnicas de Genotipagem/métodos , Folículo Piloso/química , Camundongos/genética , Poliestirenos/química , Polivinil/química , Animais , Animais Geneticamente Modificados/genética , Feminino , Masculino
15.
Sci Rep ; 6: 33678, 2016 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-27649628

RESUMO

Cardiovascular and renal pathologies are frequently associated with an activated renin-angiotensin-system (RAS) and increased levels of its main effector and vasoconstrictor hormone angiotensin II (Ang II). Angiotensin-converting-enzyme-2 (ACE2) has been described as a crucial enzymatic player in shifting the RAS towards its so-called alternative vasodilative and reno-protective axis by enzymatically converting Ang II to angiotensin-(1-7) (Ang-(1-7)). Yet, the relative contribution of ACE2 to Ang-(1-7) formation in vivo has not been elucidated. Mass spectrometry based quantification of angiotensin metabolites in the kidney and plasma of ACE2 KO mice surprisingly revealed an increase in Ang-(1-7), suggesting additional pathways to be responsible for alternative RAS activation in vivo. Following assessment of angiotensin metabolism in kidney homogenates, we identified neprilysin (NEP) to be a major source of renal Ang-(1-7) in mice and humans. These findings were supported by MALDI imaging, showing NEP mediated Ang-(1-7) formation in whole kidney cryo-sections in mice. Finally, pharmacologic inhibition of NEP resulted in strongly decreased Ang-(1-7) levels in murine kidneys. This unexpected new role of NEP may have implications for the combination therapy with NEP-inhibitors and angiotensin-receptor-blockade, which has been shown being a promising therapeutic approach for heart failure therapy.


Assuntos
Rim/fisiologia , Neprilisina/metabolismo , Sistema Renina-Angiotensina/fisiologia , Aminobutiratos/farmacologia , Angiotensina I/metabolismo , Angiotensina II/genética , Angiotensina II/metabolismo , Animais , Biomarcadores , Biópsia , Compostos de Bifenilo/farmacologia , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Córtex Renal/fisiologia , Camundongos , Camundongos Knockout , Neprilisina/antagonistas & inibidores , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Renina/genética , Renina/metabolismo
16.
J Am Geriatr Soc ; 64(10): 2154-2158, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27377350

RESUMO

Older adults with advanced chronic kidney disease (CKD) experience functional impairment that can complicate CKD management. Failure to recognize functional impairment may put these individuals at risk of further functional decline, nursing home placement, and missed opportunities for timely goals-of-care conversations. Routine geriatric assessment could be a useful tool for identifying older adults with CKD who are at risk of functional decline and provide contextual information to guide clinical decision-making. Two innovative programs were implemented in the Veterans Health Administration that incorporate geriatric assessment into a nephrology visit. In one program, a geriatrician embedded in a nephrology clinic used standardized geriatric assessment tools with individuals with CKD aged 70 and older (Comprehensive Geriatric Assessment for CKD) (CGA-4-CKD). In the second program, a nephrology clinic used comprehensive appointments for individuals aged 75 and older to conduct geriatric assessments and CKD care (Renal Silver). Data on 68 veterans who had geriatric assessments through these programs between November 2013 and May 2015 are reported. In CGA-4-CKD, difficulty with one or more activities of daily living (ADLs), history of falls, and cognitive impairment were each found in 27.3% of participants. ADL difficulty was found in 65.7%, falls in 28.6%, and cognitive impairment in 51.6% of participants in Renal Silver. Geriatric assessment guided care processes in 45.4% (n = 15) of veterans in the CGA-4-CKD program and 37.1% (n = 13) of those in Renal Silver. Findings suggest there is a significant burden of functional impairment in older adults with CKD. Knowledge of this impairment is applicable to CKD management.


Assuntos
Atividades Cotidianas , Avaliação Geriátrica/métodos , Serviços de Saúde para Idosos/organização & administração , Competência Mental , Insuficiência Renal Crônica , Acidentes por Quedas/estatística & dados numéricos , Idoso , Tomada de Decisão Clínica , Feminino , Idoso Fragilizado , Humanos , Masculino , Modelos Organizacionais , Nefrologia/métodos , Insuficiência Renal Crônica/diagnóstico , Insuficiência Renal Crônica/fisiopatologia , Insuficiência Renal Crônica/psicologia , Medição de Risco/métodos , Fatores de Risco , Estados Unidos , Saúde dos Veteranos
17.
J Hypertens ; 34(9): 1752-65, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27379538

RESUMO

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) deletion worsens kidney injury, and its amplification ameliorates diabetic nephropathy. Male sex increases the incidence, prevalence, and progression of chronic kidney disease in our environment. METHOD: Here, we studied the effect of ACE2 deficiency and gonadectomy (GDX) on diabetic nephropathy and its relationship with fibrosis, protein kinase B (Akt) activation, and the expression of several components of the renin-angiotensin system (RAS).Mice were injected with streptozotocin to induce diabetes and followed for 19 weeks. Physiological and renal parameters were studied in wild-type and ACE2 knockout (ACE2KO) male mice with and without GDX. RESULTS: Diabetic ACE2KO showed increased blood pressure (BP), glomerular injury, and renal fibrosis compared with diabetic wild-type. Gonadectomized diabetic ACE2KO presented a decrease in BP. In the absence of ACE2, GDX attenuated albuminuria and renal lesions, such as mesangial matrix expansion and podocyte loss. Both, α-smooth muscle actin accumulation and collagen deposition were significantly decreased in renal cortex of gonadectomized diabetic ACE2KO but not diabetic wild-type mice. GDX also reduced circulating ACE activity in ACE2KO mice. Loss of ACE2 modified the effect of GDX on cortical gene expression of RAS in diabetic mice. Akt phosphorylation in renal cortex was increased by diabetes and loss of ACE2 and decreased by GDX in control and diabetic ACE2KO but not in wild-type mice. CONCLUSIONS: Our results suggest that GDX may exert a protective effect within the kidney under pathological conditions of diabetes and ACE2 deficiency. This renoprotection may be ascribed to different mechanisms such as decrease in BP, modulation of RAS, and downregulation of Akt-related pathways.


Assuntos
Pressão Sanguínea/fisiologia , Diabetes Mellitus Experimental/metabolismo , Glomérulos Renais/fisiopatologia , Orquiectomia , Peptidil Dipeptidase A/metabolismo , Sistema Renina-Angiotensina/fisiologia , Animais , Masculino , Camundongos , Camundongos Knockout
18.
Hypertension ; 67(6): 1291-7, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27141055

RESUMO

The main actions of the renin-angiotensin system to control blood pressure (BP) are mediated by the angiotensin type 1 receptors (AT1Rs). The major murine AT1R isoform, AT1AR, is expressed throughout the nephron, including the collecting duct in both principal and intercalated cells. Principal cells play the major role in sodium and water reabsorption. Although aldosterone is considered to be the dominant regulator of sodium reabsorption by principal cells, recent studies suggest a role for direct actions of AT1R. To specifically examine the contributions of AT1AR in principal cells to BP regulation and the development of hypertension in vivo, we generated inbred 129/SvEv mice with deletion of AT1AR from principal cells (PCKO). At baseline, we found that BPs measured by radiotelemetry were similar between PCKOs and controls. During 1-week of low-salt diet (<0.02% NaCl), BPs fell significantly (P<0.05) and to a similar extent in both groups. On a high-salt (6% NaCl) diet, BP increased but was not different between groups. During the initial phase of angiotensin II-dependent hypertension, there was a modest but significant attenuation of hypertension in PCKOs (163±6 mm Hg) compared with controls (178±2 mm Hg; P<0.05) that was associated with enhanced natriuresis and decreased alpha epithelial sodium channel activation in the medulla of PCKOs. However, from day 9 onward, BPs were indistinguishable between groups. Although effects of AT1AR on baseline BP and adaptation to changes in dietary salt are negligible, our studies suggest that direct actions of AT1AR contribute to the initiation of hypertension and epithelial sodium channel activation.


Assuntos
Angiotensina II/farmacologia , Pressão Sanguínea/fisiologia , Hipertensão/fisiopatologia , Túbulos Renais Coletores/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Sistema Renina-Angiotensina/fisiologia , Análise de Variância , Animais , Células Cultivadas , Modelos Animais de Doenças , Hipertensão/metabolismo , Túbulos Renais Coletores/citologia , Camundongos , Distribuição Aleatória , Valores de Referência
19.
J Am Soc Nephrol ; 27(8): 2257-64, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26744488

RESUMO

Inappropriate activation of the renin-angiotensin system (RAS) contributes to many CKDs. However, the role of the RAS in modulating AKI requires elucidation, particularly because stimulating type 1 angiotensin II (AT1) receptors in the kidney or circulating inflammatory cells can have opposing effects on the generation of inflammatory mediators that underpin the pathogenesis of AKI. For example, TNF-α is a fundamental driver of cisplatin nephrotoxicity, and generation of TNF-α is suppressed or enhanced by AT1 receptor signaling in T lymphocytes or the distal nephron, respectively. In this study, cell tracking experiments with CD4-Cre mT/mG reporter mice revealed robust infiltration of T lymphocytes into the kidney after cisplatin injection. Notably, knockout of AT1 receptors on T lymphocytes exacerbated the severity of cisplatin-induced AKI and enhanced the cisplatin-induced increase in TNF-α levels locally within the kidney and in the systemic circulation. In contrast, knockout of AT1 receptors on kidney epithelial cells ameliorated the severity of AKI and suppressed local and systemic TNF-α production induced by cisplatin. Finally, disrupting TNF-α production specifically within the renal tubular epithelium attenuated the AKI and the increase in circulating TNF-α levels induced by cisplatin. These results illustrate discrepant tissue-specific effects of RAS stimulation on cisplatin nephrotoxicity and raise the concern that inflammatory mediators produced by renal parenchymal cells may influence the function of remote organs by altering systemic cytokine levels. Our findings suggest selective inhibition of AT1 receptors within the nephron as a promising intervention for protecting patients from cisplatin-induced nephrotoxicity.


Assuntos
Lesão Renal Aguda/fisiopatologia , Rim/metabolismo , Receptor Tipo 1 de Angiotensina/fisiologia , Linfócitos T , Lesão Renal Aguda/induzido quimicamente , Animais , Cisplatino/administração & dosagem , Epitélio/metabolismo , Feminino , Camundongos , Receptor Tipo 1 de Angiotensina/biossíntese , Linfócitos T/metabolismo
20.
Curr Opin Nephrol Hypertens ; 25(1): 59-66, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26575394

RESUMO

PURPOSE OF REVIEW: Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that metabolizes angiotensin II (AngII). AngII levels can be modulated by ACE2 in tissues where the enzyme is highly expressed, such as the kidney. In the kidney, ACE2 has the potential to regulate the intrarenal renin-angiotensin system (RAS), which can impact blood pressure and renal injury. Thus, in disease states where the RAS is often upregulated, the function of ACE2 plays a particularly important role. This review highlights the results of recent studies that demonstrate the interplay between ACE2 and the kidney under normal and pathological conditions. RECENT FINDINGS: ACE2 has been reported to play a key role in renal and cardiovascular function. Recent studies have implicated shedding of the membrane-bound ectodomain of ACE2 as one way in which the enzyme can be regulated and enzymatic activity altered. This posttranslational modification releases a fragment which retains enzymatic activity, and thus provides a novel mechanism by which the RAS can be altered in response to physiological stimuli. Decreased ACE2 activity is associated with increased blood pressure, diabetes, and oxidative stress, whereas, increased levels of ACE2 appear to be renoprotective. SUMMARY: A growing body of evidence, involving both experimental and human studies, points out the crucial role that ACE2 plays on the modulation of renal injury. Thus, therapeutic targets aiming to increase ACE2 activity and the ACE2-Ang(1-7)-MasR axis could potentially become clinically relevant, especially in disease states where the renal RAS is upregulated.


Assuntos
Nefropatias/etiologia , Peptidil Dipeptidase A/fisiologia , Animais , Pressão Sanguínea , Nefropatias Diabéticas/metabolismo , Humanos , Rim/enzimologia , Rim/metabolismo , Nefropatias/enzimologia , Estresse Oxidativo , Peptidil Dipeptidase A/química , Peptidil Dipeptidase A/deficiência
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