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1.
Biochim Biophys Acta Mol Cell Res ; 1865(7): 995-1001, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29694915

RESUMO

In susceptible tumor cells, DNA-damaging antineoplastic agents induce an increase in intracellular pH during the premitochondrial stage of apoptosis. The rate of nonenzymatic deamidation of two asparagines in the anti-apoptotic protein Bcl-xL is accelerated by this increase in pH. Deamidation of these asparagines is a signal for the degradation of Bcl-xL, which is a component of the apoptotic response to DNA damage. It has previously been shown that the increase in pH is mediated by the ion transporter Na+/H+ exchanger 1 in some cells. Here we demonstrate that one or more additional ion transporters also have a role in the regulation of Bcl-xL deamidation in at least some tumor cell lines and fibroblasts. As a second, independent finding, we report that there are histidines in close proximity to the Bcl-xL deamidation sites that are highly conserved in land-dwelling species and we present evidence that deamidation of human Bcl-xL is intramolecularly catalyzed in a manner that is dependent upon these histidines. Further, we present evidence that these histidines act as a pH-sensitive switch that enhances the effect of the increase in pH on the rate of Bcl-xL deamidation. The conservation of such histidines implies that human Bcl-xL is in essence "designed" to be deamidated, which provides further evidence that deamidation serves as a bona fide regulatory post-translational modification of Bcl-xL.


Assuntos
Histidina/química , Bombas de Íon/metabolismo , Proteína bcl-X/química , Proteína bcl-X/metabolismo , Células 3T3 , Animais , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , Desaminação , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Proteína bcl-X/genética
2.
J Am Soc Nephrol ; 27(10): 2974-2982, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26940092

RESUMO

Endoplasmic reticulum (ER) stress and disrupted proteostasis contribute to the pathogenesis of a variety of glomerular and tubular diseases. Thus, it is imperative to develop noninvasive biomarkers for detecting ER stress in podocytes or tubular cells in the incipient stage of disease, when a kidney biopsy is not yet clinically indicated. Mesencephalic astrocyte-derived neurotrophic factor (MANF) localizes to the ER lumen and is secreted in response to ER stress in several cell types. Here, using mouse models of human nephrotic syndrome caused by mutant laminin ß2 protein-induced podocyte ER stress and AKI triggered by tunicamycin- or ischemia-reperfusion-induced tubular ER stress, we examined MANF as a potential urine biomarker for detecting ER stress in podocytes or renal tubular cells. ER stress upregulated MANF expression in podocytes and tubular cells. Notably, urinary MANF excretion concurrent with podocyte or tubular cell ER stress preceded clinical or histologic manifestations of the corresponding disease. Thus, MANF can potentially serve as a urine diagnostic or prognostic biomarker in ER stress-related kidney diseases to help stratify disease risk, predict disease progression, monitor treatment response, and identify subgroups of patients who can be treated with ER stress modulators in a highly targeted manner.


Assuntos
Estresse do Retículo Endoplasmático , Nefropatias/urina , Fatores de Crescimento Neural/urina , Animais , Biomarcadores/urina , Nefropatias/etiologia , Camundongos
3.
PLoS Biol ; 11(6): e1001588, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23823868

RESUMO

The cellular concentration of Bcl-xL is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-xL undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-xL is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-xL for degradation. Additionally, we show that degradation of deamidated Bcl-xL is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-xL, underscoring its importance in Bcl-xL regulation. Our findings strongly suggest that deamidation-regulated Bcl-xL degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli.


Assuntos
Amidas/metabolismo , Proteólise , Proteína bcl-X/metabolismo , Sequência de Aminoácidos , Animais , Calpaína/metabolismo , Linhagem Celular , Sequência Conservada , Dano ao DNA , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteína bcl-X/química
4.
J Urol ; 193(5 Suppl): 1784-90, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25301096

RESUMO

PURPOSE: The calcineurin-NFAT signaling pathway regulates the transcription of genes important for development. It is impacted by various genetic and environmental factors. We investigated the potential role of NFAT induced transcriptional dysregulation in the pathogenesis of congenital abnormalities of the kidneys and urinary tract. MATERIALS AND METHODS: A murine model of conditional NFATc1 activation in the ureteric bud was generated and examined for histopathological changes. Metanephroi were also cultured in vitro to analyze branching morphogenesis in real time. RESULTS: NFATc1 activation led to defects resembling multicystic dysplastic kidney. These mutants showed severe disorganization of branching morphogenesis characterized by decreased ureteric bud branching and the disconnection of ureteric bud derivatives from the main collecting system. The orphan ureteric bud derivatives may have continued to induce nephrogenesis and likely contributed to the subsequent formation of blunt ended filtration units and cysts. The ureter also showed irregularities consistent with impaired epithelial-mesenchymal interaction. CONCLUSIONS: This study reveals the profound effects of NFAT signaling dysregulation on the ureteric bud and provides insight into the pathogenesis of multicystic dysplastic kidney. Our results suggest that the obstruction hypothesis and the bud theory may not be mutually exclusive to explain the pathogenesis of multicystic dysplastic kidney. Ureteric bud dysfunction such as that induced by NFAT activation can disrupt ureteric bud-metanephric mesenchyma interaction, causing primary defects in branching morphogenesis, subsequent dysplasia and cyst formation. Obstruction of the main collecting system can further enhance these defects, producing the pathological changes associated with multicystic dysplastic kidney.


Assuntos
Rim Displásico Multicístico/embriologia , Fatores de Transcrição NFATC/fisiologia , Organogênese/fisiologia , Ureter/embriologia , Animais , Calcineurina/fisiologia , Camundongos , Transdução de Sinais/fisiologia , Transcrição Gênica
5.
J Urol ; 193(5 Suppl): 1860-1869, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25813565

RESUMO

PURPOSE: Congenital urinary tract obstruction is a leading cause of renal maldevelopment and pediatric kidney disease. Nonetheless, few groups have examined its molecular pathogenesis in humans. We evaluated the role of BMP-7, a protein required for renal injury repair and nephrogenesis, in disease progression in patients with obstructive uropathy. MATERIALS AND METHODS: Whole kidney and cell specific BMP-7 expression was examined in a murine model of unilateral ureteral obstruction and in patients with congenital ureteropelvic junction obstruction. Findings were correlated with molecular markers of renal injury and clinical parameters. RESULTS: Unilateral ureteral obstruction led to a dramatic decrease in BMP-7 expression in the proximal and distal tubules before the onset of significant loss of renal architecture and fibrosis, suggesting that this is a critical molecular event that drives early stage disease progression. Loss of BMP-7 expression then extended to the collecting ducts and glomeruli in end stage kidney disease. When translating these findings to patients with ureteropelvic junction obstruction, global loss of BMP-7 expression correlated with a decreased number of nephrons, loss of renal architecture, severe renal fibrosis and loss of kidney function. CONCLUSIONS: Given that BMP-7 has a critical role in renal injury repair and nephrogenesis, these findings show that cell specific changes in BMP-7 expression contribute to the onset of irreversible renal injury and impaired kidney development secondary to congenital urinary tract obstruction. Accordingly therapies that target these cell populations to restore BMP-7 activity may limit disease progression in patients with obstructive uropathy.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Nefropatias/metabolismo , Túbulos Renais Distais/metabolismo , Túbulos Renais Proximais/metabolismo , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Caspase 3/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Humanos , Túbulos Renais Distais/citologia , Túbulos Renais Proximais/citologia , Camundongos Endogâmicos C57BL
6.
J Urol ; 191(1): 242-52, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23820056

RESUMO

PURPOSE: Recombinant BMP-7 inhibits the pathogenesis of renal injury in response to various stimuli. However, little is known about the molecular regulation of endogenous BMP-7 and its renal protective functions. We examined transcriptional regulation of Bmp-7 and its role in the pathogenesis of renal injury resulting from urinary tract dysfunction. MATERIALS AND METHODS: Obstruction induced renal injury was modeled in vivo in mice by unilateral ureteral obstruction and in vitro in primary kidney cells by treatment with transforming growth factor-ß, a profibrotic cytokine that is increased in the obstructed kidney. RESULTS: Unilateral ureteral obstruction resulted in the loss of BMP-7 expression in conjunction with histone deacetylation and transcriptional repression of the Bmp-7 promoter. The histone deacetylase inhibitor trichostatin A stimulated Bmp-7 expression in primary kidney cells. Trichostatin A also inhibited the expression of transforming growth factor-ß dependent profibrotic genes in a manner that depended on BMP receptor signaling. These findings extended to the obstructed kidney in vivo, in which trichostatin A treatment restored the expression of Bmp-7 along with BMP-7 mediated suppression of transforming growth factor-ß dependent signaling pathways. Finally, trichostatin A stimulated activation of the BMP-7 pathway the ameliorated obstruction induced renal injury by preventing disruption of the renal architecture and the development of renal fibrosis. CONCLUSIONS: These findings show that histone deacetylase dependent repression of Bmp-7 transcription is a critical event during the pathogenesis of renal injury in obstructive uropathy. Accordingly, treatment with histone deacetylase inhibitors represents a potentially effective strategy to restore BMP-7 expression and its renal protective functions during treatment of obstructive uropathy.


Assuntos
Proteína Morfogenética Óssea 7/biossíntese , Histona Desacetilases/metabolismo , Nefropatias/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Obstrução Ureteral/metabolismo , Animais , Proteína Morfogenética Óssea 7/antagonistas & inibidores , Células Cultivadas , Modelos Animais de Doenças , Fibrose , Nefropatias/etiologia , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/biossíntese , Transcrição Gênica , Obstrução Ureteral/complicações
7.
Cancer Cell ; 5(1): 3-4, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14749119

RESUMO

The discovery two decades ago that the Philadelphia chromosome encodes an oncogenic fusion of Bcr and Abl remains among the most important contributions to our understanding of the process of malignant transformation. We now know that Bcr-Abl is one of more than 30 aberrantly activated tyrosine kinases that are expressed in a variety of tumors. Conventional treatment of the tumors in which these proteins are expressed is usually doomed to failure because the activated tyrosine kinases render the tumor cells stubbornly resistant to apoptosis. In this context, it is notable that Zhao and coworkers have uncovered a novel weapon in the resistance armamentarium of these rogue kinases, the suppression of the inactivating deamidation of Bcl-xL (this issue of Cancer Cell).


Assuntos
Apoptose/fisiologia , Proteínas de Fusão bcr-abl/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Transformação Celular Neoplásica/metabolismo , Dimerização , Feminino , Genes erbB-2/fisiologia , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Camundongos , Cromossomo Filadélfia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Estrogênio/metabolismo , Regulação para Cima , Proteína bcl-X
8.
Front Med (Lausanne) ; 9: 892746, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35646977

RESUMO

Bladder outlet obstruction (BOO) induces bladder dysfunction and altered bladder architecture. Irrespective of the release of the obstruction, persistent bladder dysfunction severely affects the quality of life. A better understanding of the repair process offers an opportunity to enhance postintervention management. We subsequently evaluated the postobstructive repair process in mice subjected to 24 h BOO followed by release. Male and female mice bladders were obstructed for 24 h by placing a clip around the bladder neck. After the release of obstruction, the mice were studied for 3, 7, and 14 days to observe the bladder repair process over time. Voiding frequency and volume were recorded using the voiding spot assay, and the transcutaneous glomerular filtration rate (tGFR) was measured. Fibrogenesis and associated gene expressions and altered protein levels were evaluated in the bladder using histology, quantatative polymerase chain reaction (qPCR), and Western blot analyses. Bladder wall thickness was increased in both genders over time but occurred later in female mice. Moreover, collagen deposition in the smooth muscle layer increased over time in both genders. Male mice showed a decreased average voided volume at 3 days post release, while female mice showed no significant change during the time course. Fibrosis-related molecular events, including upregulation of fibronectin (FN) protein and Collagen-3 (Col-3) mRNA expression, were transient and normalized again at 14 days in both genders. Transforming growth factor-ß (TGF-ß) and bone morphogenic protein (BMP)-7 mRNA expressions were upregulated at 14 days post release in both genders. Transcutaneous GFR remained normal during the time course. Release of 24 h BOO initiated a bladder remodeling process. The animal model enables a wide range of experiments to study bladder remodeling, and gender differences offer potential targets for understanding bladder fibrosis and adaptation with BOO.

9.
Am J Physiol Renal Physiol ; 301(6): F1293-302, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21880836

RESUMO

Although obstructive uropathies are frequently correctable through surgery, the potential for permanent renal injury remains even following the successful correction of obstructions. Little is known about the intrinsic mechanisms that determine the reversibility of renal injuries. We and others found that exogenous bone morphogenic protein 7 (BMP-7) inhibits the pathogenesis of renal injury. Here, we examine the role of endogenous BMP-7 in the outcome of renal recovery following the correction of obstructive uropathies using a reversible murine model of ureteral obstruction. The role of BMP-7 was determined by examining the regulation of BMP-7 during renal recovery and by treating with either BMP-7-neutralizing antibodies or exogenous BMP-7. While BMP-7 is upregulated following the correction of obstructions that lead to reversible renal injury, the upregulation of BMP-7 is diminished following the correction of prolonged obstructions that lead to irreversible renal injury. The activation of the BMP-7 pathway is required for several processes that contribute to renal recovery including the suppression of transforming growth factor-ß-dependent profibrotic pathways, the restoration of renal architecture, and the resolution of fibrotic changes in the kidney. Importantly, the therapeutic restoration of BMP-7 enhances renal recovery following the correction of prolonged obstructions that typically lead to irreversible renal injury. Together, these findings show that, while BMP-7 plays a critical role in the repair of obstruction-induced renal injuries, the potential for renal recovery from prolonged obstruction is diminished, in part, due to the dysregulation of BMP-7. Accordingly, renal recovery from obstructive uropathies may be optimized through timely intervention and adjuvant approaches to restore BMP-7 activity.


Assuntos
Proteína Morfogenética Óssea 7/fisiologia , Nefropatias/fisiopatologia , Rim/lesões , Obstrução Ureteral/complicações , Animais , Proteína Morfogenética Óssea 7/antagonistas & inibidores , Proteína Morfogenética Óssea 7/farmacologia , Nefropatias/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima
10.
J Urol ; 185(6 Suppl): 2523-30, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21527199

RESUMO

PURPOSE: Urinary tract obstruction causes hydroureteronephrosis and requires surgical intervention to prevent permanent renal injury. While many studies have focused on the development of renal injury, we examined the molecular mechanisms that promote renal recovery after correcting obstruction. MATERIALS AND METHODS: A reversible murine model of ureteral obstruction was used to examine the bone morphogenic protein-7 and transforming growth factor-ß signaling pathways during renal recovery after obstruction induced injury. Analysis was done using standard molecular techniques, including reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunoblotting and co-immunoprecipitation. RESULTS: After correcting obstruction the up-regulation of bone morphogenic protein-7 inhibited the transforming growth factor-ß dependent profibrotic pathways that are central to renal injury pathogenesis. The inhibitory effects of bone morphogenic protein-7 were mediated in part by the activation of its downstream target proteins, SMA and MAD related proteins 1, 5 and 8, which suppress the activity of transforming growth factor-ß dependent Smad proteins and in turn inhibit the expression of transforming growth factor-ß dependent genes. Activation of the bone morphogenic protein-7-Smad related protein 1/5/8 pathway during renal recovery promoted renal architecture restoration and fibrosis resolution in the kidney after correcting obstruction. CONCLUSIONS: Together these findings show that the bone morphogenic protein-7-Smad1/5/8 pathway promotes kidney repair after obstruction induced injury. Accordingly the pathway represents an important therapeutic target to stimulate this innate repair mechanisms of the kidney during treatment for obstruction induced renal injury.


Assuntos
Proteína Morfogenética Óssea 7/fisiologia , Nefropatias/prevenção & controle , Proteína Smad1/fisiologia , Animais , Nefropatias/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Obstrução Ureteral/complicações
11.
J Urol ; 184(4 Suppl): 1686-91, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20728136

RESUMO

PURPOSE: Bladder outlet obstruction results in smooth muscle cell hyperplasia, decreased bladder wall compliance, and lower and upper urinary tract pathology. The cyclin-dependent kinase inhibitor p27(KIP1) regulates bladder smooth muscle cell proliferation in response to bladder outlet obstruction but little is known about its physiological role in the bladder. We investigated the role of p27(KIP1) in the structure and function of the detrusor layer of the bladder wall. MATERIALS AND METHODS: We used immunoblotting and reverse transcriptase-polymerase chain reaction to examine cell cycle regulation in response to increased mechanical tension in an in vitro model of tension induced smooth muscle cell proliferation and an in vivo model of bladder outlet obstruction. We compared unobstructed bladders of p27(+/+) and p27(-/-) mice (Jackson Laboratory, Bar Harbor, Maine) structurally by histological staining and functionally by in vivo cystometric measurements of bladder capacity, detrusor compliance and detrusor leak point pressure. RESULTS: Increased tension decreased p27(KIP1) at the protein level in human bladder smooth muscle cells and in intact murine bladder smooth muscle. p27(-/-) mice had bladder smooth muscle cell hyperplasia even in the absence of bladder outlet obstruction. While p27 loss had little effect on detrusor leak point pressure, p27(-/-) mice had significantly decreased bladder capacity and detrusor compliance. CONCLUSIONS: To our knowledge we provide the first report of the in vivo significance of p27(KIP1) in the regulation of detrusor function using a cystometric approach. We identified a role for p27(KIP1) in protecting against dysregulated smooth muscle cell proliferation, bladder capacity and detrusor compliance under normotensive conditions.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Músculo Liso/fisiologia , Bexiga Urinária/fisiologia , Animais , Fenômenos Biomecânicos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27 , Humanos , Camundongos
12.
JCI Insight ; 5(12)2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32396531

RESUMO

Renal fibrosis features exaggerated inflammation, extracellular matrix (ECM) deposition, and peritubular capillary loss. We previously showed that IL-10 stimulates high-molecular weight hyaluronan (HMW-HA) expression by fibroblasts, and we hypothesize that HMW-HA attenuates renal fibrosis by reducing inflammation and ECM remodeling. We studied the effects of IL-10 overexpression on HA production and scarring in mouse models of unilateral ureteral obstruction (UUO) and ischemia/reperfusion (I/R) to investigate whether IL-10 antifibrotic effects are HA dependent. C57BL/6J mice were fed with the HA synthesis inhibitor, 4-methylumbelliferone (4-MU), before UUO. We observed that in vivo injury increased intratubular spaces, ECM deposition, and HA expression at day 7 and onward. IL-10 overexpression reduced renal fibrosis in both models, promoted HMW-HA synthesis and stability in UUO, and regulated cell proliferation in I/R. 4-MU inhibited IL-10-driven antifibrotic effects, indicating that HMW-HA is necessary for cytokine-mediated reduction of fibrosis. We also found that IL-10 induces in vitro HMW-HA production by renal fibroblasts via STAT3-dependent upregulation of HA synthase 2. We propose that IL-10-induced HMW-HA synthesis plays cytoprotective and antifibrotic roles in kidney injury, thereby revealing an effective strategy to attenuate renal fibrosis in obstructive and ischemic pathologies.


Assuntos
Fibroblastos/metabolismo , Rim/lesões , Rim/metabolismo , Peso Molecular , Animais , Proliferação de Células/efeitos dos fármacos , Citocinas/metabolismo , Matriz Extracelular/metabolismo , Inflamação/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL
14.
JCI Insight ; 2(23)2017 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-29212948

RESUMO

ER stress has emerged as a signaling platform underlying the pathogenesis of various kidney diseases. Thus, there is an urgent need to develop ER stress biomarkers in the incipient stages of ER stress-mediated kidney disease, when a kidney biopsy is not yet clinically indicated, for early therapeutic intervention. Cysteine-rich with EGF-like domains 2 (CRELD2) is a newly identified protein that is induced and secreted under ER stress. For the first time to our knowledge, we demonstrate that CRELD2 can serve as a sensitive urinary biomarker for detecting ER stress in podocytes or renal tubular cells in murine models of podocyte ER stress-induced nephrotic syndrome and tunicamycin- or ischemia-reperfusion-induced acute kidney injury (AKI), respectively. Most importantly, urinary CRELD2 elevation occurs in patients with autosomal dominant tubulointerstitial kidney disease caused by UMOD mutations, a prototypical tubular ER stress disease. In addition, in pediatric patients undergoing cardiac surgery, detectable urine levels of CRELD2 within postoperative 6 hours strongly associate with severe AKI after surgery. In conclusion, our study has identified CRELD2 as a potentially novel urinary ER stress biomarker with potential utility in early diagnosis, risk stratification, treatment response monitoring, and directing of ER-targeted therapies in selected patient subgroups in the emerging era of precision nephrology.


Assuntos
Injúria Renal Aguda/urina , Moléculas de Adesão Celular/urina , Estresse do Retículo Endoplasmático/fisiologia , Proteínas da Matriz Extracelular/urina , Síndrome Nefrótica/urina , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/fisiopatologia , Animais , Biomarcadores/urina , Procedimentos Cirúrgicos Cardíacos , Moléculas de Adesão Celular/fisiologia , Criança , Proteínas da Matriz Extracelular/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mutação , Nefrite Intersticial/genética , Nefrite Intersticial/fisiopatologia , Nefrite Intersticial/urina , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/fisiopatologia , Podócitos/metabolismo , Complicações Pós-Operatórias/urina , Uromodulina/genética
15.
PLoS One ; 11(7): e0158908, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27454431

RESUMO

Emerging evidence has highlighted the pivotal role of microvasculature injury in the development and progression of renal fibrosis. Angiopoietin-1 (Ang-1) is a secreted vascular growth factor that binds to the endothelial-specific Tie2 receptor. Ang-1/Tie2 signaling is critical for regulating blood vessel development and modulating vascular response after injury, but is dispensable in mature, quiescent vessels. Although dysregulation of vascular endothelial growth factor (VEGF) signaling has been well studied in renal pathologies, much less is known about the role of the Ang-1/Tie2 pathway in renal interstitial fibrosis. Previous studies have shown contradicting effects of overexpressing Ang-1 systemically on renal tubulointerstitial fibrosis when different engineered forms of Ang-1 are used. Here, we investigated the impact of site-directed expression of native Ang-1 on the renal fibrogenic process and peritubular capillary network by exploiting a conditional transgenic mouse system [Pax8-rtTA/(TetO)7 Ang-1] that allows increased tubular Ang-1 production in adult mice. Using a murine unilateral ureteral obstruction (UUO) fibrosis model, we demonstrate that targeted Ang-1 overexpression attenuates myofibroblast activation and interstitial collagen I accumulation, inhibits the upregulation of transforming growth factor ß1 and subsequent phosphorylation of Smad 2/3, dampens renal inflammation, and stimulates the growth of peritubular capillaries in the obstructed kidney. Our results suggest that Ang-1 is a potential therapeutic agent for targeting microvasculature injury in renal fibrosis without compromising the physiologically normal vasculature in humans.


Assuntos
Angiopoietina-1/genética , Expressão Gênica , Nefropatias/genética , Nefropatias/patologia , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica , Nefropatias/metabolismo , Camundongos , Camundongos Transgênicos , Microcirculação , Neovascularização Patológica/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
16.
Vitam Horm ; 99: 91-144, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26279374

RESUMO

Chronic kidney disease (CKD) is a significant health problem that most commonly results from congenital abnormalities in children and chronic renal injury in adults. The therapeutic potential of BMP-7 was first recognized nearly two decades ago with studies demonstrating its requirement for kidney development and ability to inhibit the pathogenesis of renal injury in models of CKD. Since this time, our understanding of CKD has advanced considerably and treatment strategies have evolved with the identification of many additional signaling pathways, cell types, and pathologic processes that contribute to disease progression. The purpose of this review is to revisit the seminal studies that initially established the importance of BMP-7, highlight recent advances in BMP-7 research, and then integrate this knowledge with current research paradigms. We will provide an overview of the evolutionarily conserved roles of BMP proteins and the features that allow BMP signaling pathways to function as critical signaling nodes for controlling biological processes, including those related to CKD. We will discuss the multifaceted functions of BMP-7 during kidney development and the potential for alterations in BMP-7 signaling to result in congenital abnormalities and pediatric kidney disease. We will summarize the renal protective effects of recombinant BMP-7 in experimental models of CKD and then propose a model to describe the potential physiological role of endogenous BMP-7 in the innate repair mechanisms of the kidneys that respond to renal injury. Finally, we will highlight emerging clinical approaches for applying our knowledge of BMP-7 toward improving the treatment of patients with CKD.


Assuntos
Proteína Morfogenética Óssea 7/metabolismo , Rim/crescimento & desenvolvimento , Rim/lesões , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais/fisiologia , Adulto , Animais , Proteína Morfogenética Óssea 7/administração & dosagem , Proteína Morfogenética Óssea 7/farmacologia , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos
18.
Mol Cell Biol ; 29(18): 5104-14, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19596792

RESUMO

The proliferation of all nontransformed adherent cells is dependent upon the development of mechanical tension within the cell; however, little is known about the mechanisms by which signals regulated by mechanical tension are integrated with those regulated by growth factors. We show here that Skp2, a component of a ubiquitin ligase complex that mediates the degradation of several proteins that inhibit proliferation, is upregulated when increased mechanical tension develops in intact smooth muscle and that its upregulation is critical for the smooth muscle proliferative response to increased mechanical tension. Notably, whereas growth factors regulate Skp2 at the level of protein stability, we found that mechanical tension regulates Skp2 at the transcriptional level. Importantly, we demonstrate that the calcium-regulated transcription factor NFATc1 is a critical mediator of the effect of increased mechanical tension on Skp2 transcription. These findings identify Skp2 as a node at which signals from mechanical tension and growth factors are integrated to regulate proliferation, and they define calcium-NFAT-Skp2 signaling as a critical pathway in the mechanoregulation of proliferation.


Assuntos
Mecanotransdução Celular , Animais , Sequência de Bases , Sítios de Ligação , Núcleo Celular/metabolismo , Proliferação de Células , Separação Celular , Sequência Consenso , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Dados de Sequência Molecular , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Fatores de Transcrição NFATC/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Quinases Associadas a Fase S/genética , Proteínas Quinases Associadas a Fase S/metabolismo , Solubilidade , Estresse Mecânico , Técnicas de Cultura de Tecidos , Transcrição Gênica
19.
J Biol Chem ; 277(4): 2554-61, 2002 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-11707430

RESUMO

The cytotoxic side effects of anti-neoplastic drugs are increased in patients with either type 1 or type 2 diabetes mellitus by a mechanism that is not clearly defined. We report that the circulating glucose metabolite, methylglyoxal (MGO), enhances cisplatin-induced apoptosis by activating protein kinase Cdelta (PKCdelta). We found that treatment of myeloma cells with the antioxidant N-acetylcysteine completely blocked cisplatin-dependent intracellular GSH oxidation, reactive oxygen species (ROS) generation, poly(ADP-ribose) polymerase cleavage, and apoptosis. Importantly, co-treatment of cells with the reactive carbonyl MGO and cisplatin increased apoptosis by 90% over the expected additive effect of combined MGO and cisplatin treatment. This same synergism was also observed when ROS generation was examined. MGO and cisplatin increased PKCdelta activity by 4-fold, and this effect was blocked by the PKCdelta inhibitor rottlerin but not by NAC. Furthermore, rottlerin blocked combined MGO and cisplatin-induced ROS generation and apoptosis. Finally, MGO and cisplatin induced c-Abl activation and c-Abl:PKCdelta association. Rottlerin blocked c-Abl activation, but the c-Abl inhibitor STI-571 increased MGO and cisplatin-induced apoptosis by 50%. Taken together these data indicate that MGO synergistically enhances cisplatin-induced apoptosis through activation of PKCdelta and that PKCdelta is critical to both cell death and cell survival pathways. These findings suggest that in the patient with diabetes mellitus heightened oxidative stress can enhance the cytotoxicity of agents that induce DNA damage.


Assuntos
Cisplatino/toxicidade , Isoenzimas/metabolismo , Proteína Quinase C/metabolismo , Aldeído Pirúvico/metabolismo , Acetilcisteína/farmacologia , Anexina A5/farmacologia , Antioxidantes/farmacologia , Apoptose , Western Blotting , Caspases/metabolismo , Morte Celular , Sobrevivência Celular , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Glutationa/metabolismo , Humanos , Peróxidos/metabolismo , Ligação Proteica , Proteína Quinase C-delta , Proteínas Proto-Oncogênicas c-abl/metabolismo , Aldeído Pirúvico/farmacologia , Espécies Reativas de Oxigênio , Fatores de Tempo , Células Tumorais Cultivadas , Regulação para Cima
20.
J Urol ; 172(4 Pt 2): 1662-5; discussion 1666, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15371785

RESUMO

PURPOSE: The alpha1 antagonist doxazosin is used to treat lower urinary tract symptoms and is believed to function primarily as a smooth muscle relaxant. However, doxazosin has been shown to inhibit proliferation and induce apoptosis in nonbladder smooth muscle. Consequently, we examined the effects of doxazosin on human bladder smooth muscle cell (SMC) proliferation and apoptosis. MATERIALS AND METHODS: Primary human bladder SMCs were cultured in M199 with 10% fetal bovine serum (FBS) until they reached 65% confluency and then they were made quiescent by serum starvation in M199 with 0.4% FBS for 24 hours. The quiescent bladder SMCs were pretreated for 30 minutes with doxazosin or vehicle (dimethyl sulfoxide) and then stimulated with 10% FBS for 24 hours. Measurement of 5'-bromo-2'-deoxyuridine (BrdU) uptake by flow cytometry was used to determine the effect of doxazosin on cell cycle progression. Western immunoblot was used to examine cell cycle protein expression and phosphorylation of the retinoblastoma protein (Rb) and cyclin A, both of which regulate cell cycle progression. RESULTS: Cellular proliferation was inhibited in a dose dependent manner by doxazosin. There was nearly a 50% decrease in BrdU uptake at 10 microM doxazosin and an approximately 90% decrease in BrdU at 25 microM doxazosin. Notably, doxazosin inhibited phosphorylation of Rb and expression of cyclin A, both of which are necessary for cell cycle progression. At concentrations of 25 microM doxazosin or greater apoptosis was induced in the bladder SMCs, as indicated by an increase in subG1 DNA content. CONCLUSIONS: Our study demonstrates that doxazosin inhibits mitogen induced proliferation of human bladder SMC by blocking cell cycle progression at the of G1/S border. Doxazosin induced cell cycle inhibition appears to be at least in part due to an inhibition of mitogen induced Rb phosphorylation and cyclin A expression. At higher concentrations doxazosin induces apoptosis in human bladder SMCs.


Assuntos
Antagonistas Adrenérgicos alfa/farmacologia , Apoptose/efeitos dos fármacos , Doxazossina/farmacologia , Substâncias de Crescimento/fisiologia , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Humanos
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