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1.
Am J Physiol Renal Physiol ; 300(4): F1026-42, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21289056

RESUMO

Podocytes are considered terminally differentiated cells in the mature kidney under normal conditions. In the face of injury, podocytes may proceed along several possible pathways, including dedifferentiation and proliferation, persistent cell cycle arrest, hypertrophy, apoptosis, or necrosis. There is mounting evidence that transdifferentiation into a dysregulated phenotype may also be a potential cell fate. We have previously reported that the transcript of SM22α, an actin-binding protein considered one of the earliest markers of smooth muscle differentiation, is upregulated nearly 70-fold in glomeruli of rats with passive Heymann nephritis (PHN). In contrast, the SM22α transcript is absent in normal adult rat glomeruli. The purpose of this study was to define SM22α's expression during kidney development and its role in glomerular diseases characterized by podocyte injury and proteinuria. During glomerulogenesis and podocyte differentiation, SM22α was expressed in glomeruli. This expression disappeared with glomerular maturation. Along with SM22α induction in PHN, confirmed at both mRNA and protein levels, SM22α was also induced across a broad range of proteinuric diseases, including experimental animal models (puromycin aminonucleoside nephropathy, adriamycin nephropathy, passive nephrotoxic nephritis, and diet-induced obesity) and human diseases (collapsing glomerulopathy, diabetic nephropathy, classic focal segmental glomerulosclerosis, IgA nephropathy, minimal-change disease, membranous nephropathy, and membranoproliferative glomerulonephritis). Crescentic glomerulonephritis was induced in SM22α +/+ and SM22α -/- mice by intraperitoneal injection of sheep anti-rabbit glomeruli antibody 12.5 mg/20 g body wt × 2 doses (n = 12-15/group), with mice euthanized at 7 and 14 days. Compared with SM22α -/- mice, SM22α +/+ mice demonstrated worse disease by histopathological parameters. In addition, there was greater apoptosis (cleaved caspase-3 immunostaining), fewer podocytes (Wilms' tumor-1 immunostaining), and less proliferation (Ki-67 immunostaining) in diseased SM22α +/+ mice. Furthermore, there was decreased activation of Erk1/2 in diseased SM22α +/+ mice. We conclude that the de novo expression of SM22α in glomerular epithelial cells affects the course of crescentic glomerulonephritis.


Assuntos
Nefropatias/metabolismo , Glomérulos Renais/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Podócitos/metabolismo , Proteinúria/metabolismo , Animais , Apoptose , Western Blotting , Humanos , Imuno-Histoquímica , Nefropatias/patologia , Glomérulos Renais/patologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Podócitos/patologia , Proteinúria/patologia
2.
Nephron Exp Nephrol ; 112(2): e43-58, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19390219

RESUMO

BACKGROUND: Passive Heymann nephritis (PHN), the best characterized animal model of experimental membranous nephropathy, is characterized by subepithelial immune deposits, podocyte foot processes effacement and massive proteinuria beginning 4 days following disease induction. Although single genes involved in PHN have been studied, no whole genome-wide expression analysis of kidney tissue has been performed. METHODS: Microarray analysis was performed to identify gene expression changes in PHN rat kidneys during the onset of proteinuria. RESULTS: Our results showed that 234 transcripts were differentially expressed in diseased animals compared to controls. Genes exclusively upregulated in diseased animals were mainly required for cell structure and motility, immunity and defense, cell cycle, and developmental processes. The single most increased gene was transgelin (Tagln) showing a 70-fold upregulation in animals with PHN. Protein-protein interaction analysis revealed the following four processes of major relevance in disease manifestation: (i) DNA damage and repair; (ii) changes in the extracellular matrix; (iii) deregulation of cytokines and growth factors, as well as (iv) rearrangements of the cytoskeleton. CONCLUSION: We show for the first time the complex interplay between multiple different genes in experimental membranous nephropathy, supporting a role for genomic approaches to better understanding and defining specific disease processes.


Assuntos
Biologia Computacional/métodos , Regulação da Expressão Gênica/genética , Glomerulonefrite Membranosa/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Citocinas/genética , Citocinas/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Dano ao DNA/genética , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Perfilação da Expressão Gênica/métodos , Glomerulonefrite Membranosa/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Masculino , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Proteinúria/genética , Proteinúria/metabolismo , Proteinúria/patologia , Ratos , Ratos Sprague-Dawley
3.
Mol Cell Neurosci ; 33(4): 401-11, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17055293

RESUMO

Activation of microglia by LPS leads to an induction of cytokine and NO release, reduced proliferation and increased outward K(+) conductance, the latter involving the activation of Kv1.5 and Kv1.3 channels. We studied the role of these channels for microglial function using two strategies to interfere with channel expression, a Kv1.5 knockout (Kv1.5(-/-)) mouse and an antisense oligonucleotide (AO) approach. The LPS-induced NO release was reduced by AO Kv1.5 and completely absent in the Kv1.5(-/-) animal; the AO Kv1.3 had no effect. In contrast, proliferation was augmented with both, loss of Kv1.3 or Kv1.5 channel expression. After facial nerve lesion, proliferation rate was higher in Kv1.5(-/-) animals as compared to wild type. Patch clamp experiments confirmed the reduction of the LPS-induced outward current amplitude in Kv1.5(-/-) microglia as well as in Kv1.5- or Kv1.3 AO-treated cells. Our study indicates that induction of K(+) channel expression is a prerequisite for the full functional spectrum of microglial activation.


Assuntos
Canal de Potássio Kv1.3/fisiologia , Canal de Potássio Kv1.5/fisiologia , Microglia/fisiologia , Animais , Encéfalo/citologia , Bromodesoxiuridina/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Embrião de Mamíferos , Doenças do Nervo Facial/metabolismo , Doenças do Nervo Facial/patologia , Imuno-Histoquímica/métodos , Canal de Potássio Kv1.3/química , Canal de Potássio Kv1.5/química , Canal de Potássio Kv1.5/genética , Lipopolissacarídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Óxido Nítrico/metabolismo , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Técnicas de Patch-Clamp/métodos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
4.
Am J Pathol ; 165(4): 1175-85, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15466384

RESUMO

Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in renal physiology, including the prevention of proteinuria. Cyclin-dependent kinase 5 (CDK5) has been shown to influence several cellular processes in other terminally differentiated cells, in particular neurons. In this study, we examined the role of CDK5 in podocyte differentiation, proliferation, and morphology. In conditionally immortalized mouse podocytes in culture, CDK5 increased in association with podocyte differentiation. During mouse glomerulogenesis in vivo, CDK5 expression was predominantly detected in podocytes from the capillary loop stage to maturation and persisted in the podocytes of adult glomeruli. In contrast, CDK5 was markedly decreased in the proliferating and dedifferentiated podocytes of mice with anti-glomerular basement membrane nephritis and in human immunodeficiency virus transgenic mice. p35, the activator of CDK5, was also detected in podocytes and the p35/CDK5 complex was active. Cell fractionation studies showed that active p35/CDK5 was mainly localized to the plasma membrane. Specific inhibition of CDK5 in differentiated cultured podocytes, either pharmacologically or with siRNA, induced shape changes, with cellular elongation and loss of process formation compared to the characteristic arborized phenotype. These data suggest a role for CDK5 as a regulator of podocyte differentiation, proliferation, and morphology.


Assuntos
Quinases Ciclina-Dependentes/biossíntese , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Glomérulos Renais/citologia , Glomérulos Renais/enzimologia , Fosfotransferases , Animais , Western Blotting , Diferenciação Celular , Divisão Celular , Membrana Celular/metabolismo , Células Cultivadas , Quinase 5 Dependente de Ciclina , Modelos Animais de Doenças , Ativação Enzimática/fisiologia , Imunofluorescência , Nefropatias/enzimologia , Nefropatias/metabolismo , Glomérulos Renais/crescimento & desenvolvimento , Camundongos , Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa
5.
Am J Pathol ; 164(4): 1417-24, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15039229

RESUMO

The proliferative response of podocytes to injury determines the histological phenotype. Moreover, an apparent lack of podocyte proliferation may underlie the development of glomerulosclerosis. Podocyte proliferation is closely linked with its state of differentiation. However, the mechanisms regulating these processes are not fully elucidated. Because D-type cyclins have been shown to be important in the regulation of proliferation and differentiation, we examined their expression in podocytes in vitro and in vivo. The glomerular expression of cyclins D1 and D3 was examined in vitro in cultured immortalized podocytes by immunostaining and Western blot analysis, and in embryonic mice and rats, the passive Heymann nephritis model of experimental membranous nephropathy in rats, and human immunodeficiency virus (HIV)-transgenic mice. Kidneys from cyclin D1 knockout mice were also examined. Cyclin D1 was abundant in cultured proliferating podocytes, but not in quiescent differentiated podocytes. In contrast, cyclin D3 was abundant in differentiated, but not proliferating podocytes. Cyclin D1 was expressed in embryonic mouse and rat glomeruli during the S- and comma-shaped stages, and was absent in podocytes at the capillary loop stage and in mature rodent glomeruli. Cyclin D1 protein increased after injury in passive Heymann nephritis rats and in HIV-transgenic mice. Cyclin D3 was constitutively and specifically expressed in podocytes in normal rodent glomeruli, and decreases during dedifferentiation and proliferation in HIV-transgenic mice. Kidneys from cyclin D1-/- mice were normal with the podocytes expressing specific differentiation markers. Cyclin D1 is not necessary for the terminal differentiation of podocytes, and expression coincides with cell-cycle entry. In contrast, cyclin D3 expression coincides with podocyte differentiation and quiescence.


Assuntos
Diferenciação Celular/fisiologia , Ciclinas/biossíntese , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Animais , Western Blotting , Divisão Celular/fisiologia , Células Cultivadas , Modelos Animais de Doenças , Embrião de Mamíferos , Glomerulonefrite/metabolismo , HIV/genética , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Ratos
6.
Kidney Int ; 65(1): 30-9, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14675034

RESUMO

BACKGROUND: Glomerular capillary hypertension, a common denominator in various forms of progressive glomerular disease, results in mechanical distention of the capillary tuft, and subsequent injury of the overlying podocyte layer. The mechanisms by which elevated intraglomerular pressure is translated into a maladaptive podocyte response remain poorly understood. Angiotensin II plays a central role in the pathogenesis of chronic renal injury, largely through its actions on the subtype 1 receptor. Accordingly, we have tested the hypothesis that mechanical strain up-regulates local angiotensin II in podocytes, thereby resulting in a progressive reduction in podocyte number. METHODS: Conditionally immortalized mouse podocytes were subjected to cyclical stretch of 10% amplitude. Nonstretched podocytes served as controls. Angiotensin II levels were measured in whole cell lysate by competitive enzyme-linked immunosorbent assay (ELISA). Expression of angiotensin II receptors (AT1R, AT2R) was measured by quantitative polymerase chain reaction (PCR) and Western blot analysis. Apoptosis was measured by Hoechst staining. Immunostaining for AT1R was performed in tissue sections from rats with 5/6 remnant kidney disease, a model of glomerular hypertension. RESULTS: Mechanical strain increased angiotensin II production in podocytes at 24, 48, and 72 hours (P < 0.05 vs. nonstretched controls). Stretching podocytes resulted in a fivefold increase in AT1R mRNA expression at 24 hours and a twofold increase in protein levels vs. controls (P < 0.05), and also an increase in transforming growth hormone-beta (TGF-beta) mRNA expression. AT1R staining was increased in a podocyte distribution in the 5/6 remnant kidney, consistent with our in vitro findings. Mechanical strain resulted in a 2.5-fold increase in apoptosis (P < 0.001 vs. nonstretched controls) in an angiotensin II-dependent fashion. CONCLUSION: Mechanical strain leads to up-regulation of the AT1R and increased angiotensin II production in conditionally immortalized podocytes. The resulting activation of a local tissue angiotensin system leads to an increase in podocyte apoptosis, mainly in an AT1R-mediated fashion.


Assuntos
Angiotensina II/metabolismo , Glomérulos Renais/fisiologia , Mecanotransdução Celular/fisiologia , Vasoconstritores/metabolismo , Angiotensina II/farmacologia , Animais , Apoptose/fisiologia , Capilares/fisiologia , Linhagem Celular Transformada , Expressão Gênica/fisiologia , Hipertensão Renal/fisiopatologia , Glomérulos Renais/irrigação sanguínea , Glomérulos Renais/efeitos dos fármacos , Camundongos , RNA Mensageiro/análise , Ratos , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Circulação Renal/efeitos dos fármacos , Circulação Renal/fisiologia , Estresse Mecânico , Fator de Crescimento Transformador beta/genética , Regulação para Cima , Vasoconstritores/farmacologia
7.
Nephron Exp Nephrol ; 98(4): e114-23, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15627794

RESUMO

BACKGROUND: A decrease in podocyte number contributes to the development of glomerulosclerosis in diabetic nephropathy. Although podocytes have been detected in the urine in certain glomerular diseases, their viability is poorly understood. METHODS: Diabetes was induced in rats with streptozotocin. Urine was collected from control rats (given citrate), and rats with diabetic nephropathy, and cells obtained by centrifugation were resuspended in tissue culture media, and seeded onto collagen-coated tissue culture plates. Cells were grown under standard cell culture conditions ex vivo. Cell number was measured, the cell type in the urine was identified by immunostaining with specific antibodies, and morphology was assessed by light and electron microscopy. RESULTS: Within 24 h, cells obtained from the urine of diabetic rats attached to tissue culture plates ex vivo. Cells were not detected in the urine from control rats. All cells from diabetic rats stained positive for the podocyte-specific proteins synaptopodin, nephrin, podocin and Glepp-1 and negative for mesangial (OX-7), tubular (Tamm-Horsfall protein) and endothelial (RECA) cell antigens. The cell number increased daily, which is consistent with cell growth ex vivo. CONCLUSIONS: Rats with diabetic nephropathy shed podocytes into the urine that attach and grow ex vivo. These results are consistent with the detachment of viable podocytes in diabetes and add new perspectives into our understanding of development of glomerulosclerosis in diabetes mellitus.


Assuntos
Nefropatias Diabéticas/etiologia , Podócitos/citologia , Animais , Apoptose , Adesão Celular , Sobrevivência Celular , Células Cultivadas , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/patologia , Masculino , Podócitos/patologia , Podócitos/ultraestrutura , Ratos , Ratos Sprague-Dawley , Urina/citologia
8.
Kidney Int ; 64(4): 1222-31, 2003 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12969140

RESUMO

BACKGROUND: Podocyte loss contributes to the development of glomerulosclerosis. Although podocytes have been detected in the urine in certain glomerular diseases, the viability of detached cells is not known. METHODS: Urine was collected from rats with experimental membranous nephropathy [passive Heymann nephritis (PHN) model], centrifuged, and following resuspension in tissue culture media, cells were seeded onto collagen-coated tissue culture plates. Cells were grown under typical cell culture conditions. Cell number was measured, the cell type was identified by immunostaining with specific antibodies, and cell morphology was assessed by light and electron microscopy. RESULTS: Cells obtained in the urine from PHN rats were positive for synaptopodin, nephrin, podocin, WT-1, and GLEPP1 (podocyte-specific antigens). When grown ex vivo under cell culture conditions, cells obtained in the urine from PHN rats adhered to tissue culture plates, and expressed podocyte-specific proteins at the mRNA [reverse transcription-polymerase chain reaction (RT-PCR)] and protein (immunostaining) level. Cells did not stain with antibodies to mesangial (OX-7), tubular (Tamm-Horsfall protein) and endothelial (RECA) cells. Electron microscopy showed the presence of foot processes, and podocytes from PHN rats stained positive for C5b-9. Although podocyte number increased transiently during the first 5 days ex vivo, apoptosis increased significantly thereafter, reducing overall cell number. CONCLUSION: Rats with experimental membranous nephropathy shed podocytes into the urine that attach to tissue culture plates ex-vivo, and proliferate. These results suggest that detached podocytes are viable. These results add new perspectives into our understanding of podocyte loss in the development of glomerulosclerosis.


Assuntos
Glomerulonefrite Membranosa/patologia , Glomerulonefrite Membranosa/fisiopatologia , Rim/patologia , Rim/fisiopatologia , Animais , Adesão Celular , Contagem de Células , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Imunofluorescência , Glomerulonefrite Membranosa/urina , Rim/metabolismo , Rim/ultraestrutura , Masculino , Microscopia Eletrônica , Ratos , Ratos Sprague-Dawley , Coloração e Rotulagem , Urina/citologia
9.
Kidney Int ; 61(1): 40-50, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11786083

RESUMO

BACKGROUND: Mechanical stretch, a consequence of capillary glomerular hypertension, is thought to be the common final pathway for glomerulosclerosis in systemic hypertension, diabetes, reduced nephron number and focal segmental glomerulosclerosis. However, the effects of stretch on podocyte growth and the mechanisms that underlie this have not been elucidated. METHODS: Mouse podocyte growth (3H-thymidine, MTT-assay, FACS) was measured following the application of mechanical stretch created by vacuum. The expression of specific cell cycle regulatory proteins was examined by RNAse protection assay and Western blot analysis. Control cells were grown under similar conditions, but were not exposed to stretch. RESULTS: Mechanical stretch decreased DNA-synthesis (3H-thymidine incorporation) and cell number (MTT-assay) in podocytes at 24, 48 and 72 hours (P < 0.001 vs. control non-stretched cells), which was not due to apoptosis (Hoechst staining) nor cell detachment. Stretch decreased the mRNA and protein levels of cyclins D1, A and B1 within 24 hours. Stretching cells decreased the activity of Cdk2 (measured by histone H1 kinase assay) at 48 and 72 hours and Cdc2 at 72 hours. In contrast, stretch increased the protein levels of the cyclin dependent kinase inhibitors (CKI) p21Cip/Kip/Waf (p21) and p27Kip1 (p27) within the first 24 hours, and increased the mRNA levels of p57Kip2 (p57) at 72 hours. To examine the role of p21 in inhibiting proliferation induced by stretch, we studied p21-/- podocytes in culture. Stretch did not reduce proliferation in p21-/- podocytes (P> 0.05 vs. non-stretched podocytes; P < 0.001 vs. stretched p21+/+ podocytes). CONCLUSIONS: In contrast to mesangial cells, mechanical stretch decreases the growth of podocytes. This effect is mediated through the regulation of specific cell cycle regulatory proteins. These events may explain the apparent lack of podocyte proliferation in diseases correlated with capillary glomerular hypertension.


Assuntos
Quinases relacionadas a CDC2 e CDC28 , Glomérulos Renais/citologia , Glomérulos Renais/enzimologia , Animais , Western Blotting , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/genética , Divisão Celular/fisiologia , Células Cultivadas , Ciclina A/análise , Ciclina A/genética , Ciclina B/análise , Ciclina B/genética , Ciclina B1 , Ciclina D1/análise , Ciclina D1/genética , Ciclina D3 , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Inibidor de Quinase Dependente de Ciclina p57 , Quinases Ciclina-Dependentes/análise , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/análise , Ciclinas/genética , Citometria de Fluxo , Expressão Gênica/fisiologia , Técnicas In Vitro , Glomérulos Renais/química , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/análise , Estresse Mecânico , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/genética
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