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2.
Res Sq ; 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38659788

RESUMO

Background: The evaluation of volume status is essential to clinical decision-making, yet multiple studies have shown that physical exam does not reliably estimate a patient's intravascular volume. Venous excess ultrasound score (VExUS) is an emerging volume assessment tool that utilizes inferior vena cava (IVC) diameter and pulse-wave Doppler waveforms of the portal, hepatic and renal veins to evaluate venous congestion. A point-of-care ultrasound exam initially developed by Beaubein-Souligny et al., VExUS represents a reproducible, non-invasive and accurate means of assessing intravascular congestion. VExUS has recently been validated against RHC-the gold-standard of hemodynamic evaluation for volume assessment. While VExUS scores were shown to correlate with elevated cardiac filling pressures (i.e., right atrial pressure (RAP) and pulmonary capillary wedge pressure (PCWP)) at a static point in time, the ability of VExUS to capture dynamic changes in volume status has yet to be elucidated. We hypothesized that paired VExUS examinations performed before and after hemodialysis (HD) would reflect changes in venous congestion in a diverse patient population. Methods: Inpatients with end-stage renal disease undergoing intermittent HD were evaluated with transabdominal VExUS and lung ultrasonography before and following HD. Paired t-tests were conducted to assess differences between pre-HD and post-HD VExUS scores, B-line scores and dyspnea scores. Results: Fifty-six patients were screened for inclusion in this study. Ten were excluded due to insufficient image quality or incomplete exams, and forty-six patients (ninety-two paired ultrasound exams) were included in the final analysis. Paired t-test analysis of pre-HD and post-HD VExUS scores revealed a mean VExUS grade change of 0.82 (p<0.001) on a VExUS scale ranging from 0 to 4. The mean difference in B-line score following HD was 0.8 (p=0.001). There was no statistically significant difference in subjective dyspnea score (p=0.41). Conclusions: Large-volume fluid removal with HD was represented by changes in VExUS score, highlighting the utility of the VExUS exam to capture dynamic shifts in intravascular volume status. Future studies should evaluate change in VExUS grade with intravenous fluid or diuretic administration, with the ultimate goal of evaluating the capacity of a standardized bedside ultrasound protocol to guide inpatient volume optimization.

4.
JCI Insight ; 8(12)2023 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-37345660

RESUMO

Innate and adaptive immune cells modulate the severity of autosomal dominant polycystic kidney disease (ADPKD), a common kidney disease with inadequate treatment options. ADPKD has parallels with cancer, in which immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have previously shown that in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of programmed cell death protein 1 (PD-1)/cytotoxic T lymphocyte associated protein 4 (CTLA-4) on T cells and programmed cell death ligand 1 (PD-L1)/CD80 on macrophages and epithelial cells in Pkd1RC/RC mice versus WT, paralleling disease severity. PD-L1/CD80 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti-PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti-PD-1 plus anti-CTLA-4, blocking 2 immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD severity. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers correlative with PKD severity. Together, our data suggest that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.


Assuntos
Doenças Renais Policísticas , Rim Policístico Autossômico Dominante , Adulto , Humanos , Animais , Camundongos , Antígeno B7-H1 , Rim , Terapia Combinada , Antígeno B7-1
7.
Am J Physiol Renal Physiol ; 322(1): F105-F119, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34866403

RESUMO

15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.


Assuntos
Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/metabolismo , Citocinas/metabolismo , Metabolismo Energético , Mediadores da Inflamação/metabolismo , Rim/enzimologia , Metaboloma , Nefrite/etiologia , Obstrução Ureteral/complicações , Animais , Araquidonato 12-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/genética , Microambiente Celular , Citocinas/genética , Modelos Animais de Doenças , Metabolismo Energético/efeitos dos fármacos , Fibrose , Rim/efeitos dos fármacos , Rim/patologia , Leucócitos/enzimologia , Inibidores de Lipoxigenase/farmacologia , Macrófagos/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nefrite/enzimologia , Nefrite/patologia , Nefrite/prevenção & controle , Fenótipo , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/enzimologia , Obstrução Ureteral/patologia
8.
Kidney360 ; 1(10): 1052-1059, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-35368786

RESUMO

Background: Interest in nephrology as a career has declined dramatically over the past several years. Only 62% of nephrology fellowship positions are filled for the upcoming 2020 appointment year. The purpose of this study was to identify perceptions, attitudes, motivators, and barriers to a career in nephrology among internal medicine residents. Methods: We recruited focus groups of internal medicine residents (N=25) from the University of Colorado, and asked questions aimed at exploring perceptions, attitudes, and barriers to a career in nephrology, and ways to increase interest in nephrology. All focus groups were conducted on the University of Colorado Denver Anschutz Medical Campus. Focus group sessions were recorded and transcribed. Thematic analysis was used to identify key concepts and themes. Results: Residents described many barriers to a career in nephrology, including lack of exposure, lack of advances in the field, low monetary compensation, high complexity, lack of role models/mentors, and low-prestige/noncompetitive nature of the field. Most residents had no exposure to outpatient nephrology. Lack of new therapeutics was a significant deterrent to nephrology. Nephrology teaching in medical school was described as not clinically relevant and too complicated. Several residents felt they were not smart enough for nephrology. Only three residents had a role model within nephrology. Residents used the word "stigmatized" to describe nephrology, and discussed how low prestige decreased their interest in a field. Participants expressed suggestions to increase interest in nephrology through earlier and more outpatient nephrology exposure, enhanced interactions with nephrologists, and research and advancements in the field. Conclusions: Residents identified several modifiable barriers to a career in nephrology. Changing how nephrology is taught in medical school, enhancing interactions with nephrologists through increased exposure, and highlighting research and advancements in nephrology may change the perception of nephrology and increase the number of residents entering the field.


Assuntos
Internato e Residência , Nefrologia , Escolha da Profissão , Bolsas de Estudo , Grupos Focais , Humanos , Nefrologia/educação
9.
Arterioscler Thromb Vasc Biol ; 40(2): 394-403, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31852223

RESUMO

OBJECTIVE: Pathological vascular remodeling and excessive perivascular fibrosis are major contributors to reduced vessel compliance that exacerbates cardiovascular diseases, for instance, promoting clinically relevant myocardial remodeling. Inflammation plays a significant role in both pathological vascular remodeling and fibrosis. We previously demonstrated that smooth muscle cell-specific PTEN depletion promotes significant vascular fibrosis and accumulation of inflammatory cells. In the current study, we aimed to determine the beneficial role of systemic PTEN elevation on Ang II (angiotensin II)-induced vascular fibrosis and remodeling. Approach and Results: Transgenic mice carrying additional copies of the wild-type Pten gene (super PTEN [sPTEN]) and WT littermates were subjected to Ang II or saline infusion for 14 or 28 days. Compared with WT, Ang II-induced vascular fibrosis was significantly blunted in sPTEN mice, as shown by histochemical stainings and label-free second harmonic generation imaging. The protection against Ang II was recapitulated in sPTEN mice bearing WT bone marrow but not in WT mice reconstituted with sPTEN bone marrow. Ang II-induced elevation of profibrotic and proinflammatory gene expression observed in WT mice was blocked in aortic tissue of sPTEN mice. Immunofluorescent staining and flow cytometry both indicated that perivascular infiltration of T cells and macrophages was significantly inhibited in sPTEN mice. In vitro induction of PTEN expression suppressed Ang II-induced Ccl2 expression in vascular smooth muscle cells. CONCLUSIONS: Systemic PTEN elevation mediates protection against Ang II-induced vascular inflammation and fibrosis predominantly through effects in resident vascular cells. Our data highly support that pharmacological upregulation of PTEN could be a novel and viable approach for the treatment of pathological vascular fibrosis.


Assuntos
Regulação da Expressão Gênica , Músculo Liso Vascular/metabolismo , PTEN Fosfo-Hidrolase/genética , Doenças Vasculares/genética , Remodelação Vascular/genética , Angiotensina II/toxicidade , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Fibrose/genética , Fibrose/metabolismo , Fibrose/patologia , Citometria de Fluxo , Masculino , Camundongos , Camundongos Transgênicos , Músculo Liso Vascular/patologia , PTEN Fosfo-Hidrolase/biossíntese , RNA/genética , Ratos , Doenças Vasculares/metabolismo , Doenças Vasculares/patologia
10.
Am J Physiol Renal Physiol ; 316(4): F732-F742, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649890

RESUMO

In inflammatory diseases, the 5-lipoxygenase (5-LO) pathway contributes to epithelial damage and fibrosis by catalyzing the production of leukotrienes (LTs). Antagonists of the 5-LO pathway are currently approved for use in patients and are well tolerated. We found that expression of 5-LO is strongly induced in three models of chronic kidney disease: unilateral ureteral obstruction (UUO), folate nephropathy, and an orthologous mouse model of polycystic kidney disease. Immunohistochemistry showed that macrophages are the dominant source of 5-LO. Zileuton, a US Food and Drug Administration-approved antagonist of 5-LO, significantly reduced fibrosis at 7 and 14 days after UUO; these findings were confirmed using a genetically modified [5-LO-associated protein-knockout ( Alox5ap-/-)] mouse strain. Inhibition of 5-LO did not appear to change infiltration of leukocytes after UUO as measured by flow cytometry. However, fluorescence-lifetime imaging microscopy showed that 5-LO inhibitors reversed the glycolytic switch in renal tubular epithelial cells after UUO. Two downstream enzymes of 5-LO, LTA4 hydrolase (LTA4H) and LTC4 synthase (LTC4S), are responsible for the synthesis of LTB4 and cysteinyl LTs, respectively. Fibrosis was reduced after UUO in Ltc4s-/-, but not Lta4h-/-, mice. In contrast, using the folate nephropathy model, we found reduced fibrosis and improved renal function in both Ltc4s-/- and Lta4h-/- mice. In summary, our studies suggest that manipulation of the 5-LO pathway may represent a novel treatment approach for chronic kidney disease.


Assuntos
Rim/patologia , Inibidores de Lipoxigenase/uso terapêutico , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/patologia , Animais , Araquidonato 5-Lipoxigenase/genética , Fibrose , Túbulos Renais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Renais Policísticas/tratamento farmacológico , Receptores de Leucotrienos/genética , Receptores de Leucotrienos/metabolismo , Insuficiência Renal Crônica/induzido quimicamente , Transdução de Sinais/efeitos dos fármacos , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/etiologia
11.
Kidney Int ; 94(6): 1127-1140, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30249452

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited nephropathy. To date, therapies alleviating the disease have largely focused on targeting abnormalities in renal epithelial cell signaling. ADPKD has many hallmarks of cancer, where targeting T cells has brought novel therapeutic interventions. However, little is known about the role and therapeutic potential of T cells in ADPKD. Here, we used an orthologous ADPKD model, Pkd1 p.R3277C (RC), to begin to define the role of T cells in disease progression. Using flow cytometry, we found progressive increases in renal CD8+ and CD4+ T cells, correlative with disease severity, but with selective activation of CD8+ T cells. By immunofluorescence, T cells specifically localized to cystic lesions and increased levels of T-cell recruiting chemokines (CXCL9/CXCL10) were detected by qPCR/in situ hybridization in the kidneys of mice, patients, and ADPKD epithelial cell lines. Importantly, immunodepletion of CD8+ T cells from one to three months in C57Bl/6 Pkd1RC/RC mice resulted in worsening of ADPKD pathology, decreased apoptosis, and increased proliferation compared to IgG-control, consistent with a reno-protective role of CD8+ T cells. Thus, our studies suggest a functional role for T cells, specifically CD8+ T cells, in ADPKD progression. Hence, targeting this pathway using immune-oncology agents may represent a novel therapeutic approach for ADPKD.


Assuntos
Imunidade Adaptativa , Linfócitos T CD8-Positivos/microbiologia , Rim Policístico Autossômico Dominante/imunologia , Animais , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular , Modelos Animais de Doenças , Progressão da Doença , Células Epiteliais , Feminino , Humanos , Imunoterapia/métodos , Rim/citologia , Rim/imunologia , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Rim Policístico Autossômico Dominante/terapia , Transdução de Sinais/imunologia , Canais de Cátion TRPP/genética
12.
JCI Insight ; 3(4)2018 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-29467331

RESUMO

Phosphatase and tensin homolog (PTEN) is an essential regulator of the differentiated vascular smooth muscle cell (SMC) phenotype. Our goal was to establish that PTEN loss promotes SMC dedifferentiation and pathological vascular remodeling in human atherosclerotic coronary arteries and nonatherosclerotic coronary arteries exposed to continuous-flow left ventricular assist devices (CF-LVADs). Arteries were categorized as nonatherosclerotic hyperplasia (NAH), atherosclerotic hyperplasia (AH), or complex plaque (CP). NAH coronary arteries from CF-LVAD patients were compared to NAH coronaries from non-LVAD patients. Intimal PTEN and SMC contractile protein expression was reduced compared with the media in arteries with NAH, AH, or CP. Compared with NAH, PTEN and SMC contractile protein expression was reduced in the media and intima of arteries with AH and CP. NAH arteries from CF-LVAD patients showed marked vascular remodeling and reduced PTEN and α-smooth muscle actin (αSMA) in medial SMCs compared with arteries from non-LVAD patients; this correlated with increased medial collagen deposition. Mechanistically, compared with ApoE-/- mice, SMC-specific PTEN-null/ApoE-/- double-knockout mice exhibited accelerated atherosclerosis progression and increased vascular fibrosis. By microarray and validated quantitative RT-PCR analysis, SMC PTEN deficiency promotes a global upregulation of proinflammatory and profibrotic genes. We propose that PTEN is an antiinflammatory, antifibrotic target that functions to maintain SMC differentiation. SMC loss of PTEN results in pathological vascular remodeling of human arteries.


Assuntos
Aterosclerose/patologia , Vasos Coronários/patologia , Coração Auxiliar/efeitos adversos , Miócitos de Músculo Liso/patologia , PTEN Fosfo-Hidrolase/deficiência , Remodelação Vascular , Actinas/metabolismo , Adulto , Idoso , Animais , Aterosclerose/genética , Diferenciação Celular , Vasos Coronários/citologia , Modelos Animais de Doenças , Endotélio Vascular , Feminino , Fibrose , Insuficiência Cardíaca/cirurgia , Humanos , Hiperplasia/patologia , Masculino , Camundongos , Camundongos Knockout para ApoE , Pessoa de Meia-Idade , Músculo Liso Vascular/citologia , Músculo Liso Vascular/patologia , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo
13.
J Lipid Res ; 59(2): 380-390, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29229740

RESUMO

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme directs a complex "eicosanoid storm" that accompanies the tissue response to injury. cPLA2α and its downstream eicosanoid mediators are also implicated in the pathogenesis of fibrosis in many organs, including the kidney. We aimed to determine the role of cPLA2α in bone marrow-derived cells in a murine model of renal fibrosis, unilateral ureteral obstruction (UUO). WT C57BL/6J mice were irradiated and engrafted with donor bone marrow from either WT mice [WT-bone marrow transplant (BMT)] or mice deficient in cPLA2α (KO-BMT). After full engraftment, mice underwent UUO and kidneys were collected 3, 7, and 14 days after injury. Using picrosirius red, collagen-3, and smooth muscle α actin staining, we determined that renal fibrosis was significantly attenuated in KO-BMT animals as compared with WT-BMT animals. Lipidomic analysis of homogenized kidneys demonstrated a time-dependent upregulation of pro-inflammatory eicosanoids after UUO; KO-BMT animals had lower levels of many of these eicosanoids. KO-BMT animals also had fewer infiltrating pro-inflammatory CD45+CD11b+Ly6Chi macrophages and reduced message levels of pro-inflammatory cytokines. Our results indicate that cPLA2α and/or its downstream mediators, produced by bone marrow-derived cells, play a major role in eicosanoid production after renal injury and in renal fibrinogenesis.


Assuntos
Medula Óssea/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Nefropatias/metabolismo , Obstrução Ureteral/metabolismo , Animais , Fibrose/metabolismo , Fibrose/patologia , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/genética , Nefropatias/patologia , Camundongos , Camundongos Endogâmicos C57BL , Obstrução Ureteral/patologia
14.
Kidney Int ; 92(4): 784-786, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28938948

RESUMO

Lymphangiogenesis appears to accompany renal fibrosis, but signals that regulate the lymphangiogenic growth factor vascular endothelial growth factor C are not well understood. Kinashi et al. have shown that conditionally deleting connective tissue growth factor reduces renal fibrosis, vascular endothelial growth factor C, and lymphangiogenesis. Connective tissue growth factor has pleiotropic effects in the setting of renal fibrosis; this study adds a potentially new mechanism for the profibrotic effects of connective tissue growth factor.


Assuntos
Fator de Crescimento do Tecido Conjuntivo , Fator C de Crescimento do Endotélio Vascular , Fibrose , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Nefropatias , Linfangiogênese
15.
Biomed Opt Express ; 7(9): 3519-3530, 2016 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-27699117

RESUMO

Phasor approach to fluorescence lifetime microscopy is used to study development of fibrosis in the unilateral ureteral obstruction model (UUO) of kidney in mice. Traditional phasor analysis has been modified to create a multiparametric analysis scheme that splits the phasor points in four equidistance segments based on the height of peak of the phasor distribution and calculates six parameters including average phasor positions, the shape of each segment, the angle of the distribution and the number of points in each segment. These parameters are used to create a spectrum of twenty four points specific to the phasor distribution of each sample. Comparisons of spectra from diseased and healthy tissues result in quantitative separation and calculation of statistical parameters including AUC values, positive prediction values and sensitivity. This is a new method in the evolving field of analyzing phasor distribution of FLIM data and provides further insights. Additionally, the progression of fibrosis with time is detected using this multiparametric approach to phasor analysis.

16.
Artigo em Inglês | MEDLINE | ID: mdl-27554058

RESUMO

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme controls the release of arachidonic acid from membrane bound phospholipids and is the rate-limiting step in production of eicosanoids. A variety of different kidney injuries activate cPLA2α, therefore we hypothesized that cPLA2α activity would regulate pathologic processes in HK-2 cells, a human renal tubular epithelial cell line, by regulating cell phenotype and proliferation. In two lentiviral cPLA2α-silenced knockdowns, we observed decreased proliferation and increased apoptosis compared to control HK-2 cells. cPLA2α-silenced cells also demonstrated an altered morphology, had increased expression E-cadherin, and decreased expression of Ncadherin. Increased levels of E-cadherin were associated with increased promoter activity and decreased levels of SNAIL1, SNAIL2, and ZEB1, transcriptional repressors of E-cadherin expression. Addition of exogenous arachidonic acid, but not PGE2, reversed the phenotypic changes in cPLA2α-silenced cells. These data suggest that cPLA2α may play a key role in renal repair after injury through a PGE2-independent mechanism.


Assuntos
Desdiferenciação Celular , Células Epiteliais/citologia , Fosfolipases A2 do Grupo IV/metabolismo , Túbulos Renais/citologia , Ácido Araquidônico/farmacologia , Caderinas/genética , Desdiferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dinoprostona/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Inativação Gênica , Fosfolipases A2 do Grupo IV/deficiência , Fosfolipases A2 do Grupo IV/genética , Humanos , Fenótipo , Regiões Promotoras Genéticas/genética
17.
Kidney Int ; 90(5): 1123-1128, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27555119

RESUMO

All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust assessment procedure independent of observer interpretation and let us create criteria to quantify the extent of fibrosis directly from the image. Thus, the FLIM-SHG technique shows remarkable improvement in quantification of renal fibrosis compared to standard histological techniques.


Assuntos
Rim/patologia , Microscopia de Fluorescência , Nefroesclerose/diagnóstico , Imagem Óptica , Animais , Modelos Animais de Doenças , Fibrose , Camundongos
18.
Mol Pharmacol ; 86(5): 570-9, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25169989

RESUMO

The retinoid X receptor (RXR) partners with numerous nuclear receptors, such as the peroxisome proliferator activated receptor (PPAR) family, liver X receptors (LXRs), and farnesoid X receptor (FXR). Although each heterodimer can be activated by specific ligands, a subset of these receptors, defined as permissive nuclear receptors, can also be activated by RXR agonists known as rexinoids. Many individual RXR heterodimers have beneficial effects in vascular smooth muscle cells (SMCs). Because rexinoids can potently activate multiple RXR pathways, we hypothesized that treating SMCs with rexinoids would more effectively reverse the pathophysiologic effects of angiotensin II than an individual heterodimer agonist. Cultured rat aortic SMCs were pretreated with either an RXR agonist (bexarotene or 9-cis retinoic acid) or vehicle (dimethylsulfoxide) for 24 hours before stimulation with angiotensin II. Compared with dimethylsulfoxide, bexarotene blocked angiotensin II-induced SM contractile gene induction (calponin and smooth muscle-α-actin) and protein synthesis ([(3)H]leucine incorporation). Bexarotene also decreased angiotensin II-mediated inflammation, as measured by decreased expression of monocyte chemoattractant protein-1 (MCP-1). Activation of p38 mitogen-activated protein (MAP) kinase but not extracellular signal-related kinase (ERK) or protein kinase B (Akt) was also blunted by bexarotene. We compared bexarotene to five agonists of nuclear receptors (PPARα, PPARγ, PPARδ, LXR, and FXR). Bexarotene had a greater effect on calponin reduction, MCP-1 inhibition, and p38 MAP kinase inhibition than any individual agonist. PPARγ knockout cells demonstrated blunted responses to bexarotene, indicating that PPARγ is necessary for the effects of bexarotene. These data demonstrate that RXR is a potent modulator of angiotensin II-mediated responses in the vasculature, partially through inhibition of p38.


Assuntos
Angiotensina II/metabolismo , Expressão Gênica/genética , Inflamação/genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Actinas/genética , Actinas/metabolismo , Angiotensina II/genética , Animais , Bexaroteno , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Tetra-Hidronaftalenos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Calponinas
19.
J Am Heart Assoc ; 2(3): e000188, 2013 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-23727701

RESUMO

BACKGROUND: Pulmonary vascular remodeling in pulmonary hypertension (PH) is characterized by increased vascular smooth muscle cell (SMC) and adventitial fibroblast proliferation, small vessel occlusion, and inflammatory cell accumulation. The underlying molecular mechanisms driving progression remain poorly defined. We have focused on loss of the phosphatase PTEN in SMCs as a major driver of pathological vascular remodeling. Our goal was to define the role of PTEN in human PH and in hypoxia-induced PH using a mouse model with inducible deletion of PTEN in SMCs. METHODS AND RESULTS: Staining of human biopsies demonstrated enhanced inactive PTEN selectively in the media from hypertensive patients compared to controls. Mice with induced deletion of PTEN in SMCs were exposed to normoxia or hypoxia for up to 4 weeks. Under normoxia, SMC PTEN depletion was sufficient to induce features of PH similar to those observed in wild-type mice exposed to chronic hypoxia. Under hypoxia, PTEN depletion promoted an irreversible progression of PH characterized by increased pressure, extensive pulmonary vascular remodeling, formation of complex vascular lesions, and increased macrophage accumulation associated with synergistic increases in proinflammatory cytokines and proliferation of both SMCs and nonSMCs. CONCLUSIONS: Chronic inactivation of PTEN selectively in SMC represents a critical mediator of PH progression, leading to cell autonomous events and increased production of factors correlated to proliferation and recruitment of adventitial and inflammatory cells, resulting in irreversible progression of the disease.


Assuntos
Hipertensão Pulmonar/etiologia , Hipóxia/complicações , PTEN Fosfo-Hidrolase/fisiologia , Animais , Progressão da Doença , Masculino , Camundongos , Miócitos de Músculo Liso , Ratos , Índice de Gravidade de Doença
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