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1.
J Biol Chem ; 289(25): 17454-67, 2014 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-24817115

RESUMO

Podocytes are highly differentiated cells and critical elements for the filtration barrier of the kidney. Loss of their foot process (FP) architecture (FP effacement) results in urinary protein loss. Here we show a novel role for the neutral amino acid glutamine in structural and functional regulation of the kidney filtration barrier. Metabolic flux analysis of cultured podocytes using genetic, toxic, and immunologic injury models identified increased glutamine utilization pathways. We show that glutamine uptake is increased in diseased podocytes to couple nutrient support to increased demand during the disease state of FP effacement. This feature can be utilized to transport increased amounts of glutamine into damaged podocytes. The availability of glutamine determines the regulation of podocyte intracellular pH (pHi). Podocyte alkalinization reduces cytosolic cathepsin L protease activity and protects the podocyte cytoskeleton. Podocyte glutamine supplementation reduces proteinuria in LPS-treated mice, whereas acidification increases glomerular injury. In summary, our data provide a metabolic opportunity to combat urinary protein loss through modulation of podocyte amino acid utilization and pHi.


Assuntos
Podócitos/metabolismo , Proteinúria/metabolismo , Animais , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/imunologia , Células Cultivadas , Citoesqueleto/genética , Citoesqueleto/imunologia , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Concentração de Íons de Hidrogênio , Camundongos , Camundongos Knockout , Podócitos/imunologia , Podócitos/patologia , Proteinúria/genética , Proteinúria/imunologia , Proteinúria/patologia
2.
Gut ; 61(10): 1454-64, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22234980

RESUMO

BACKGROUND AND AIMS: Pigment epithelium-derived factor (PEDF), a non-inhibitory SERPIN with potent antiangiogenic activity, has been recently implicated in metabolism and adipogenesis, both of which are known to influence pancreatic cancer progression. Increased pancreatic fat in human pancreatic tumour correlates with greater tumour dissemination while PEDF deficiency in mice promotes pancreatic hyperplasia and visceral obesity. Oncogenic Ras, the most common mutation in pancreatic ductal adenocarcinoma (PDAC), has similarly been shown to promote adipogenesis and premalignant lesions. METHODS: In order to determine whether concurrent loss of PEDF is sufficient to promote adipogenesis and tumorigenesis in the pancreas, the authors ablated PEDF in an EL-Kras(G12D) mouse model of non-invasive cystic papillary neoplasms. RESULTS: EL-Kras(G12D)/PEDF deficient mice developed invasive PDAC associated with enhanced matrix metalloproteinase (MMP)-2 and MMP-9 expression and increased peripancreatic fat with adipocyte hypertrophy and intrapancreatic adipocyte infiltration (pancreatic steatosis). In support of increased adipogenesis, the stroma of the pancreas of EL-Kras(G12D)/PEDF deficient mice demonstrated higher tissue levels of two lipid droplet associated proteins, tail-interacting protein 47 (TIP47, perilipin 3) and adipose differentiation-related protein (ADRP, Pperilipin 2), while adipose triglyceride lipase, a key factor in lipolysis, was decreased. In patients with PDAC, both tissue and serum levels of PEDF were decreased, stromal TIP47 expression was higher and the tissue VEGF to PEDF ratio was increased (p<0.05). CONCLUSIONS: These data highlight the importance of lipid metabolism in the tumour microenvironment and identify PEDF as a critical negative regulator of both adiposity and tumour invasion in the pancreas.


Assuntos
Adipócitos Brancos/patologia , Biomarcadores Tumorais/deficiência , Carcinoma Ductal Pancreático/metabolismo , Fatores de Crescimento Neural/deficiência , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Serpinas/deficiência , Adipócitos Brancos/metabolismo , Adiposidade , Animais , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proteínas do Olho , Marcadores Genéticos , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Mutação , Invasividade Neoplásica , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Reação em Cadeia da Polimerase em Tempo Real , Células Estromais/metabolismo , Células Estromais/patologia
3.
Am J Pathol ; 179(4): 1719-32, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21839714

RESUMO

The transient receptor potential channel C6 (TRPC6) is a slit diaphragm-associated protein in podocytes involved in regulating glomerular filter function. Gain-of-function mutations in TRPC6 cause hereditary focal segmental glomerulosclerosis (FSGS), and several human acquired proteinuric diseases show increased glomerular TRPC6 expression. Angiotensin II (AngII) is a key contributor to glomerular disease and may regulate TRPC6 expression in nonrenal cells. We demonstrate that AngII regulates TRPC6 mRNA and protein levels in cultured podocytes and that AngII infusion enhances glomerular TRPC6 expression in vivo. In animal models for human FSGS (doxorubicin nephropathy) and increased renin-angiotensin system activity (Ren2 transgenic rats), glomerular TRPC6 expression was increased in an AngII-dependent manner. TRPC6 expression correlated with glomerular damage markers and glomerulosclerosis. We show that the regulation of TRPC6 expression by AngII and doxorubicin requires TRPC6-mediated Ca(2+) influx and the activation of the Ca(2+)-dependent protein phosphatase calcineurin and its substrate nuclear factor of activated T cells (NFAT). Accordingly, calcineurin inhibition by cyclosporine decreased TRPC6 expression and reduced proteinuria in doxorubicin nephropathy, whereas podocyte-specific inducible expression of a constitutively active NFAT mutant increased TRPC6 expression and induced severe proteinuria. Our findings demonstrate that the deleterious effects of AngII on podocytes and its pathogenic role in glomerular disease involve enhanced TRPC6 expression via a calcineurin/NFAT positive feedback signaling pathway.


Assuntos
Angiotensina II/farmacologia , Retroalimentação Fisiológica/efeitos dos fármacos , Fatores de Transcrição NFATC/metabolismo , Podócitos/patologia , Transdução de Sinais/efeitos dos fármacos , Canais de Cátion TRPC/metabolismo , Antagonistas de Receptores de Angiotensina/farmacologia , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Calcineurina/metabolismo , Cálcio/metabolismo , Doxorrubicina , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Nefropatias/induzido quimicamente , Nefropatias/complicações , Nefropatias/metabolismo , Nefropatias/patologia , Camundongos , Modelos Biológicos , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Proteinúria/complicações , Proteinúria/metabolismo , Proteinúria/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Renina/farmacologia , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6
4.
J Am Soc Nephrol ; 20(5): 950-3, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-18784209

RESUMO

Physiologic permeability of the glomerular capillary depends on the normal structure of podocyte foot processes forming a functioning slit diaphragm in between. Mutations in several podocyte genes as well as specific molecular pathways have been identified as the cause for progressive kidney failure with urinary protein loss. Podocyte injury is a hallmark of glomerular disease, which is generally displayed by the rearrangement of the podocyte slit diaphragm and the actin cytoskeleton. Recent studies demonstrate a unique role for the Ca(2+)-permeable ion channel protein TRPC6 as a regulator of glomerular ultrafiltration. In both genetic and acquired forms of proteinuric kidney disease, dysregulation of podocyte TRPC6 plays a pathogenic role. This article illustrates how recent findings add to emerging concepts in podocyte biology, particularly mechanosensation and signaling at the slit diaphragm.


Assuntos
Glomérulos Renais/fisiologia , Podócitos/fisiologia , Canais de Cátion TRPC/fisiologia , Animais , Membrana Basal/fisiologia , Caenorhabditis elegans/fisiologia , Capilares/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Nefropatias/genética , Nefropatias/fisiopatologia , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/fisiopatologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Síndrome Nefrótica/genética , Proteinúria/genética , Ratos , Canais de Cátion TRPC/deficiência , Canais de Cátion TRPC/genética , Canal de Cátion TRPC6
5.
Clin Nephrol Case Stud ; 3: 42-45, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-29043133

RESUMO

Lysozyme amyloidosis is an exceedingly rare hereditary autosomal dominant amyloidosis, which is characterized by the precipitation of lysozyme protein within the body, leading to multi-organ dysfunction. Herein, we present the case of a U.S. family affected by lysozyme amyloidosis. In particular, we report pericardial disease involvement leading to recurrent pericardial effusion, which to our knowledge has not been described yet. To our knowledge, we have also for the first time identified the amyloidogenic component of lysozyme amyloidosis via laser microdissection and mass spectrometry from a bone marrow biopsy. The diagnosis of this disease remains challenging as it can be easily mistaken for primary amyloidosis, which also presents with similar symptoms. Immunohistochemical staining of tissue for specific amyloidogenic proteins allows for an accurate diagnosis and should be performed in all amyloidosis patients in order to spare patients from potentially futile or harmful therapy. The widespread systemic involvement of lysozyme amyloidosis currently provides limited options for treatment, although kidney and/or liver transplantation appear to be promising palliative treatments. Practicing clinicians and researchers need to collect more information about this rare entity to further characterize the behavior of this disease and develop new potential treatment strategies.

6.
J Clin Invest ; 121(10): 3965-80, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21911934

RESUMO

Kidney podocytes are highly differentiated epithelial cells that form interdigitating foot processes with bridging slit diaphragms (SDs) that regulate renal ultrafiltration. Podocyte injury results in proteinuric kidney disease, and genetic deletion of SD-associated CD2-associated protein (CD2AP) leads to progressive renal failure in mice and humans. Here, we have shown that CD2AP regulates the TGF-ß1-dependent translocation of dendrin from the SD to the nucleus. Nuclear dendrin acted as a transcription factor to promote expression of cytosolic cathepsin L (CatL). CatL proteolyzed the regulatory GTPase dynamin and the actin-associated adapter synaptopodin, leading to a reorganization of the podocyte microfilament system and consequent proteinuria. CD2AP itself was proteolyzed by CatL, promoting sustained expression of the protease during podocyte injury, and in turn increasing the apoptotic susceptibility of podocytes to TGF-ß1. Our study identifies CD2AP as the gatekeeper of the podocyte TGF-ß response through its regulation of CatL expression and defines a molecular mechanism underlying proteinuric kidney disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Podócitos/citologia , Podócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Catepsina L/genética , Catepsina L/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Citoesqueleto/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/metabolismo , Peptídeo Hidrolases/metabolismo , Podócitos/efeitos dos fármacos , Proteinúria/etiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
7.
Pancreas ; 33(2): 148-55, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16868480

RESUMO

OBJECTIVES: Protein kinase C (PKC) is involved in cell growth, differentiation, and apoptosis. We investigated the effects of the PKC activator, the tetradecanylphorbol acetate (TPA), in human pancreatic cancer cells. METHODS: Cell proliferation was measured by thymidine incorporation. Expression of cell cycle proteins was investigated by Western blot. Real-time reverse transcriptase-polymerase chain reaction was used to measure p21 messenger RNA expression, whereas knockdown of its expression was accomplished with a specific small interferring RNA. Cell cycle phases were determined by flow cytometry. RESULTS: TPA time and concentration dependently inhibited thymidine incorporation in Panc-1 and CD18 cells and induced G2/M cell cycle arrest. The TPA decreased cyclin A and B expression, increased cyclin E, and markedly increased the expression of p21 at both the messenger RNA and protein levels. TPA-induced p21 expression and growth inhibition were blocked by the PKC inhibitor, bisindoylmaleimide. TPA induced extracellular signal-regulated kinase1/2 phosphorylation, whereas the MEK inhibitor, PD98059, blocked the TPA-induced p21 expression. Small interferring RNA targeted to p21 blocked TPA-induced p21 protein expression but not TPA-induced cell growth arrest. CONCLUSIONS: TPA-induced p21 expression is mediated by the MEK/ERK pathway but is not involved in TPA-induced growth inhibition. In contrast, cyclin A and cyclin B are likely involved in TPA-induced G2/M arrest because both proteins are involved in S phase and G2/M transition during cell proliferation.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Pancreáticas/patologia , Acetato de Tetradecanoilforbol/farmacologia , Divisão Celular , Linhagem Celular Tumoral , Ciclina A/metabolismo , Ciclina B/metabolismo , Ciclina B1 , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Flavonoides/farmacologia , Fase G2 , Humanos , Indóis/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Maleimidas/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo
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