RESUMO
Human genetic and in vivo animal studies have helped to define the critical importance of podocytes for kidney function in health and disease. However, as in any other research area, by default these approaches do not allow for mechanistic studies. Such mechanistic studies require the availability of cells grown ex vivo (i.e., in culture) with the ability to directly study mechanistic events and control the environment such that specific hypotheses can be tested. A seminal breakthrough came about a decade ago with the documentation of differentiation in culture of primary rat and human podocytes and the subsequent development of conditionally immortalized differentiated podocyte cell lines that allow deciphering the decisive steps of differentiation and function of 'in vivo' podocytes. Although this paper is not intended to provide a comprehensive review of podocyte biology, nor their role in proteinuric renal diseases or progressive glomerulosclerosis, it will focus specifically on several aspects of podocytes in culture. In particular, we will discuss the scientific and research rationale and need for cultured podocytes, how podocyte cell-culture evolved, and how cultured podocytes are currently being used to uncover novel functions of podocytes that can then be validated in vivo in animal or human studies. In addition, we provide a detailed description of how to properly culture and characterize podocytes to avoid potential pitfalls.
Assuntos
Técnicas de Cultura de Células/tendências , Podócitos/citologia , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Humanos , Nefropatias/patologia , Nefropatias/fisiopatologia , Camundongos , Fenótipo , Podócitos/fisiologia , RatosRESUMO
Podocyte injury and loss contribute to progressive glomerulosclerosis. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear hormone receptor, which we have found to be increased in podocytes in a variety of kidney diseases. It is not known if PPAR-gamma contributes to renal injury or if it serves as a countermeasure to limit renal injury during disease progression. We tested these possibilities utilizing the puromycin aminonucleoside (PAN) model of renal injury in immortalized mouse podocytes. The cultured podocytes expressed PPAR-gamma mRNA at baseline but this was decreased by PAN. Pioglitazone, a pharmacologic agonist of PPAR-gamma, increased both PPAR-gamma mRNA and activity in injured podocytes, as assessed by a reporter plasmid assay. Further, pioglitazone significantly decreased PAN-induced podocyte apoptosis and necrosis while restoring podocyte differentiation. The PPAR-gamma agonist significantly restored expression of the cyclin-dependent kinase inhibitor p27 and the antiapoptotic molecule Bcl-xL while significantly decreasing proapoptotic caspase-3 activity. Pioglitazone tended to decrease PAN-induced transforming growth factor-beta (TGF-beta) mRNA expression. Our study shows that PPAR-gamma is normally expressed by podocytes and its activation is protective against PAN-induced apoptosis and necrosis. We postulate that this protective effect may be mediated in part by effects on p27 and TGF-beta expression.
Assuntos
Apoptose/efeitos dos fármacos , Glomérulos Renais/patologia , PPAR gama/agonistas , Podócitos/efeitos dos fármacos , Podócitos/patologia , Animais , Inibidores de Caspase , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Camundongos , Necrose , PPAR gama/metabolismo , Pioglitazona , Plasmídeos/efeitos dos fármacos , Podócitos/metabolismo , Puromicina Aminonucleosídeo/toxicidade , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Tiazolidinedionas/farmacologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Class 3 semaphorins are guidance proteins that play crucial roles during development. Semaphorins 3A (sema 3A) and 3F are expressed by podocytes in vivo throughout ontogeny and their function is unknown. Here we examined the expression of class 3 semaphorins (3A, 3B, 3C, 3D, 3E, and 3F) and their receptors (neuropilins 1 and 2, plexins A1, A2, A3, B2, and D1) in undifferentiated and differentiated mouse podocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). All class 3 semaphorins, neuropilins 1 and 2 are expressed by undifferentiated and differentiated podocytes at similar levels. Differentiated podocytes expressed 2-4-fold higher plexin A1, A2, and A3 mRNA levels than undifferentiated podocytes. To examine semaphorin regulation, we exposed podocytes to recombinant sema 3A. Sema 3A decreased semaphorin 3B, plexin A1, A3, and D1 >/=50% and reduced plexin A2 mRNA to undetectable levels. To identify sema 3A function in podocytes, we examined whether sema 3A regulates slit diaphragm proteins and podocyte survival. Sema 3A induced a dose-response podocin downregulation and decreased its interaction with CD2-associated protein and nephrin, as determined by Western analysis and co-immunoprecipitation. To evaluate sema 3A role in podocyte survival, we quantified podocyte apoptosis using a caspase 3 activity marker. Sema 3A induced a 10-fold increase in podocyte apoptosis and significantly decreased the activity of the Akt survival pathway. Our data indicate that (1) immortalized podocytes in culture have a functional autocrine semaphorin system that is regulated by differentiation and ligand availability; (2) sema 3A signaling regulates the expression and interactions of slit-diaphragm proteins and decreases podocyte survival.
Assuntos
Comunicação Autócrina , Proteínas de Membrana/metabolismo , Podócitos/citologia , Proteínas/metabolismo , Semaforinas/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Células Cultivadas , Proteínas do Citoesqueleto , Regulação para Baixo , Peptídeos e Proteínas de Sinalização Intracelular , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Camundongos , Fosforilação/efeitos dos fármacos , Podócitos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Imunológicos/metabolismo , Semaforina-3A/genética , Semaforina-3A/farmacologia , Semaforina-3A/fisiologia , Semaforinas/genéticaRESUMO
BACKGROUND: The mature podocyte is a terminally differentiated cell with a limited proliferative capacity. The precise cell cycle proteins necessary for establishing podocyte quiescence during development or permitting podocyte cell cycle re-entry in disease states have not been fully defined. Accordingly, we studied the role of the cyclin dependent kinase (CDK)-inhibitor p57Kip2 (p57) in modulating these processes. METHODS: The expression of p57 protein in relation to markers of DNA synthesis was examined in developing mouse kidneys, and in the passive Heymann nephritis (PHN) and anti-glomerular antibody models of glomerular disease by immunohistochemistry. The role of p57 in glomerulogenesis was explored by examining renal tissue from embryonic p57-/- mice, and the expression of p21, p27 and p57 protein and mRNA was examined in podocytes in vitro. RESULTS: The de novo expression of p57 during glomerulogenesis coincides with the cessation of podocyte proliferation, and the establishment of a mature phenotype, and p57 is expressed exclusively in podocytes in mature glomeruli. However, p57 knockout mice have normal glomerular podocyte development. In addition, mRNA but not protein levels of p57 increased upon differentiation of podocytes in vitro. There was a marked decrease in p57 expression in both animal models of podocyte injury. This was diffuse in PHN, whereas in the murine model, loss of expression of p57 occurred predominantly in proliferating podocytes, expressing proliferating cell nuclear antigen (PCNA). CONCLUSION: Despite the de novo expression of p57 protein coinciding with the cessation of primitive podocyte proliferation during glomerulogenesis, embryonic p57-/- mice glomeruli were histologically normal. Cultured podocytes did not require changes in p57 protein levels to undergo differentiation. These data suggest that p57 alone is not required for podocyte differentiation, and that other cell cycle regulators may play a role. Furthermore, although injury to mature podocytes in experimental glomerular disease is associated with a decrease in p57, the levels of all three members of the Cip/Kip family of CDK inhibitors appear to determine the capability of podocytes to proliferate.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Glomerulonefrite/metabolismo , Rim/embriologia , Rim/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Senescência Celular , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , DNA/biossíntese , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Glomerulonefrite/imunologia , Glomerulonefrite/patologia , Temperatura Alta , Rim/patologia , Glomérulos Renais/embriologia , Camundongos , Camundongos Knockout/genética , Distribuição Tecidual , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologiaRESUMO
CD2AP, an adapter protein containing multiple SH3 domains, plays a critical role in kidney function. Mice lacking CD2AP die soon after birth because of kidney failure. In the kidney, CD2AP is expressed in glomerular podocytes, which suggests that it may play a role in a specialized adhesion complex known as the slit diaphragm. One of the major components of the slit diaphragm is nephrin, a podocyte-specific protein. Here we demonstrate that CD2AP localizes to the slit diaphragm in podocytes using immunoelectron microscopy and that nephrin and CD2AP co-immunoprecipitate from a podocyte cell line. The specificity of this interaction was verified by mapping studies, which demonstrated that a novel domain at the C terminus of CD2AP interacts with the C-terminal portion of the nephrin cytoplasmic domain. These studies lend further support to the idea that CD2AP plays a role in the structural integrity of the slit diaphragm.
Assuntos
Glomérulos Renais/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Sítios de Ligação/genética , Linhagem Celular , Proteínas do Citoesqueleto , Junções Intercelulares/metabolismo , Glomérulos Renais/citologia , Glomérulos Renais/ultraestrutura , Proteínas de Membrana , Camundongos , Camundongos Knockout , Microscopia Imunoeletrônica , Mutação , Testes de Precipitina , Ligação Proteica , Proteínas/química , Proteínas/genéticaRESUMO
NPHS2 was recently identified as a gene whose mutations cause autosomal recessive steroid-resistant nephrotic syndrome. Its product, podocin, is a new member of the stomatin family, which consists of hairpin-like integral membrane proteins with intracellular NH(2)- and COOH-termini. Podocin is expressed in glomerular podocytes, but its subcellular distribution and interaction with other proteins are unknown. Here we show, by immunoelectron microscopy, that podocin localizes to the podocyte foot process membrane, at the insertion site of the slit diaphragm. Podocin accumulates in an oligomeric form in lipid rafts of the slit diaphragm. Moreover, GST pull-down experiments reveal that podocin associates via its COOH-terminal domain with CD2AP, a cytoplasmic binding partner of nephrin, and with nephrin itself. That podocin interacts with CD2AP and nephrin in vivo is shown by coimmunoprecipitation of these proteins from glomerular extracts. Furthermore, in vitro studies reveal direct interaction of podocin and CD2AP. Hence, as with the erythrocyte lipid raft protein stomatin, podocin is present in high-order oligomers and may serve a scaffolding function. We postulate that podocin serves in the structural organization of the slit diaphragm and the regulation of its filtration function.
Assuntos
Glomérulos Renais/química , Proteínas de Membrana/química , Proteínas/química , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Clonagem Molecular , Proteínas do Citoesqueleto , Humanos , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/análise , Proteínas de Membrana/genética , Camundongos , Dados de Sequência MolecularRESUMO
We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with alpha-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A-sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein-myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Diferenciação Celular , Linhagem Celular , Expressão Gênica , Proteínas de Fluorescência Verde , Resposta ao Choque Térmico , Calefação , Humanos , Proteínas Luminescentes/genética , Camundongos , Proteínas dos Microfilamentos/classificação , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Dados de Sequência Molecular , Proteínas Musculares/classificação , Proteínas Musculares/genética , Proteínas Musculares/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Liso/citologia , Músculo Liso/metabolismo , Mutagênese , Miocárdio/metabolismo , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Transporte Proteico , RNA Mensageiro , Proteínas Recombinantes de Fusão/genética , Homologia de Sequência de Aminoácidos , Estresse Fisiológico , Tiazóis/farmacologia , TiazolidinasRESUMO
NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-O-acetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions.
Assuntos
Glomérulos Renais/metabolismo , Glomérulos Renais/ultraestrutura , Síndrome Nefrótica/metabolismo , Síndrome Nefrótica/patologia , Proteínas/metabolismo , Animais , Anticorpos , Anticorpos Monoclonais/farmacologia , Membrana Celular/efeitos dos fármacos , Colesterol/fisiologia , Detergentes/farmacologia , Resistência a Medicamentos , Retículo Endoplasmático/fisiologia , Feminino , Gangliosídeos/farmacologia , Imunoglobulina G/farmacologia , Lipídeos/fisiologia , Proteínas de Membrana , Fosforilação , Testes de Precipitina , Proteínas/fisiologia , Ratos , Ratos Sprague-Dawley , Tirosina/metabolismoRESUMO
Primary and secondary forms of focal segmental glomerulosclerosis (FSGS) are characterized by depletion of podocytes and constitute a central manifestation of chronic progressive glomerular diseases. Here we report that podocytes undergo apoptosis at early stages in the course of progressive glomerulosclerosis in TGF-beta1 transgenic mice. Apoptosis is associated with progressive depletion of podocytes and precedes mesangial expansion. Smad7 protein expression is strongly induced specifically in damaged podocytes of transgenic mice and in cultured murine podocytes treated with TGF-beta. TGF-beta1 and Smad7 each induce apoptosis in podocytes, and their coexpression has an additive effect. Activation of p38 MAP kinase and caspase-3 is required for TGF-beta-mediated apoptosis, but not for apoptosis induced by Smad7. Unlike TGF-beta, Smad7 inhibits nuclear translocation and transcriptional activity of the cell survival factor NF-kappaB. Our results suggest a novel functional role for Smad7 as amplifier of TGF-beta-induced apoptosis in podocytes and a new pathomechanism for podocyte depletion in progressive glomerulosclerosis.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Ligação a DNA/farmacologia , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Transativadores/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Animais , Caspase 3 , Caspases/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Mesângio Glomerular/patologia , Glomerulosclerose Segmentar e Focal/etiologia , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Proteína Smad7 , Fator de Crescimento Transformador beta/genética , Proteínas Quinases p38 Ativadas por MitógenoAssuntos
Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Proteinúria/enzimologia , Animais , Adesão Celular , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Expressão Gênica , Hormônio do Crescimento/genética , Humanos , Glomérulos Renais/química , Glomérulos Renais/enzimologia , Glomérulos Renais/ultraestrutura , Camundongos , Camundongos Transgênicos , Síndrome Nefrótica/congênito , Síndrome Nefrótica/enzimologia , Síndrome Nefrótica/genética , Fenótipo , Proteínas Serina-Treonina Quinases/análise , Puromicina/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , TransfecçãoRESUMO
Podocytes possess major processes containing microtubules (MTs) and intermediate filaments and foot processes containing actin filaments (AFs) as core cytoskeletal elements. Although the importance of these cytoskeletal elements for maintaining podocyte processes was previously shown, so far no data are available concerning the developmental regulation of podocyte process formation. A conditionally immortalized mouse podocyte cell line, which can be induced to develop processes similar to those found in vivo, was treated with various reagents to disrupt cytoskeletal elements or to inhibit protein phosphatases. MTs colocalized with vimentin intermediate filaments but not with AFs. After AF disassembly, major processes were maintained, whereas after depolymerization of MTs, podocytes lost their processes, rounded up, and maintained only actin-based peripheral projections. Suppression of MT elongation by nanomolar vinblastine or inhibition of serine/threonine phosphatase PP2A with okadaic acid abolished process formation. PP2A was expressed in undifferentiated but not in differentiated podocytes. One- and two-dimensional western blot analyses revealed a dose-dependent increase in serine/threonine phosphorylation after okadaic acid treatment. Hence, morphogenetic activity of MTs induces podocyte process formation via serine/threonine protein dephosphorylation by PP2A. These results may open new avenues for understanding the signaling mechanism underlying podocyte cytoskeleton alterations during development and in glomerular diseases.
Assuntos
Glomérulos Renais/citologia , Microtúbulos/ultraestrutura , Fosfoproteínas Fosfatases/fisiologia , Urotélio/citologia , Citoesqueleto de Actina/ultraestrutura , Animais , Western Blotting , Linhagem Celular , Inibidores Enzimáticos/farmacologia , Imuno-Histoquímica , Filamentos Intermediários/ultraestrutura , Glomérulos Renais/enzimologia , Glomérulos Renais/ultraestrutura , Camundongos , Microscopia de Contraste de Fase , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Urotélio/enzimologia , Urotélio/ultraestruturaRESUMO
Synaptopodin, a 100 kD protein, associated with the actin cytoskeleton of the postsynaptic density and dendritic spines, is thought to play a role in modulating actin-based shape and motility of dendritic spines during formation or elimination of synaptic contacts. Temporal lobe epilepsy in humans and in rats shows neuronal damage, aberrant sprouting of hippocampal mossy fibers and subsequent synaptic remodeling processes. Using kainic acid (KA) induced epilepsy in rats, the postictal hippocampal expression of synaptopodin was analyzed by in situ hybridization (ISH) and immunohistochemistry. Sprouting of mossy fibers was visualized by a modified Timm's staining. ISH showed elevated levels of Synaptopodin mRNA in perikarya of CA3 principal neurons, dentate granule cells and in surviving hilar neurons these levels persisted up to 8 weeks after seizure induction. Synaptopodin immunoreactivity in the dendritic layers of CA3, in the hilus and in the inner molecular layer of the dentate gyrus (DG) was initially reduced. Eight weeks after KA treatment Synaptopodin protein expression returned to control levels in dendritic layers of CA3 and in the entire molecular layer of the DG. The recovery of protein expression was accompanied by simultaneous supra- and infragranular mossy fiber sprouting. Postictal upregulation of Synaptopodin mRNA levels in target cell populations of limbic epilepsy-elicited damage and subsequent Synaptopodin protein expression largely co-localized with remodeling processes as demonstrated by mossy fiber sprouting. It may thus represent a novel postsynaptic molecular correlate of hippocampal neuroplasticity.
Assuntos
Epilepsia do Lobo Temporal/metabolismo , Hipocampo/metabolismo , Proteínas dos Microfilamentos/genética , Neurônios/patologia , Animais , Dendritos/patologia , Giro Denteado/metabolismo , Giro Denteado/patologia , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/patologia , Hipocampo/patologia , Imuno-Histoquímica , Hibridização In Situ , Ácido Caínico , Masculino , Proteínas dos Microfilamentos/análise , Fibras Nervosas/patologia , Neurônios/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Transcrição GênicaRESUMO
Dendritic spines are dynamic structures that rapidly remodel their shape and size. These morphological adaptations are regulated by changes in synaptic activity, and result from rearrangements of the postsynaptic cytoskeleton. A cytoskeletal molecule preferentially found in mature spines is the actin-associated protein synaptopodin. It is strongly expressed by spine-bearing neurons in the olfactory bulb, striatum, cerebral cortex, and hippocampus. In the hippocampus, principal cells express synaptopodin mRNA and sort the protein to the spine compartment. Within the spine microdomain, synaptopodin is preferentially located in the spine neck and is closely associated with the spine apparatus. On the basis of these data we hypothesize that synaptopodin could affect spine motility by bundling actin filaments in the spine neck. In addition, it could link the actin cytoskeleton of spines to intracellular calcium stores, i.e., the spine apparatus and the smooth endoplasmic reticulum.
Assuntos
Actinas/fisiologia , Dendritos/fisiologia , Proteínas dos Microfilamentos/fisiologia , Animais , Cálcio/metabolismo , Dendritos/metabolismo , Retículo Endoplasmático Liso/metabolismo , Hipocampo/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Plasticidade Neuronal/fisiologia , RNA Mensageiro/metabolismo , Distribuição TecidualRESUMO
BACKGROUND: Mature podocytes are growth-arrested because of the expression of cyclin-dependent kinase inhibitors. Under pathological conditions, podocytes may undergo mitosis, but not cell division. Exceptions to this rule are collapsing glomerulopathies (CGs), including HIV-associated nephropathy (HIVAN) and idiopathic CG, where podocytes undergo a dysregulation of their differentiated phenotype and proliferate. METHODS: To shed light on the mechanism underlying podocyte proliferation in CG, we analyzed the expression of the proliferation marker Ki-67, cyclins (A, D1), cyclin-dependent kinase inhibitors (p27, p57), and podocyte differentiation marker synaptopodin in eight cases of HIVAN and two cases of idiopathic CG. Normal fetal and adult kidneys served as controls. RESULTS: Both HIVAN and idiopathic CG showed a marked reduction in the expression of p27, p57, and cyclin D1 (absent in 69, 62, and 80% of all glomeruli, respectively). Cyclin A and Ki-67 were expressed in 11 and 29% of all glomeruli. Moreover, there was partial loss of synaptopodin and cyclin D1 expression in nonaffected glomeruli. CONCLUSIONS: The loss of p27 and p57 leading to expression of cyclin A may account for the activation of podocyte proliferation in CG. Furthermore, the loss of cyclin D1 from histologically normal glomeruli suggests a possible role of cyclin D1 in mediating the dysregulation of the podocyte cell cycle in CG. These novel findings offer insight into the molecular regulation of mature podocyte differentiation. Podocyte proliferation in CG provides evidence in support of a previously underestimated plasticity of mature podocytes.
Assuntos
Nefropatia Associada a AIDS/patologia , Glomérulos Renais/patologia , Proteínas Musculares , Adulto , Fatores Etários , Anticorpos Monoclonais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Ciclina A/análise , Ciclina A/imunologia , Ciclina A/metabolismo , Ciclina D1/análise , Ciclina D1/imunologia , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Cistos/patologia , Feminino , Feto/química , Feto/enzimologia , Feto/patologia , Humanos , Imunofenotipagem , Antígeno Ki-67/análise , Antígeno Ki-67/imunologia , Glomérulos Renais/química , Glomérulos Renais/enzimologia , Masculino , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/imunologia , Pessoa de Meia-Idade , Proteína de Ligação a Regiões Ricas em Polipirimidinas , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/imunologia , Ribonucleoproteínas/análise , Ribonucleoproteínas/imunologiaRESUMO
BACKGROUND: Human immunodeficiency virus-associated nephropathy (HIVAN) is the most common cause of renal failure in HIV-1-seropositive patients. Recent studies using an HIV-1 transgenic mouse model have demonstrated that expression of HIV-1 in the kidney is required for the development of HIVAN. What has remained unclear, however, is the renal cell type responsible for pathogenesis and the essential pathological process. METHODS: To address these issues, we used a transgenic murine model of HIVAN. We identified the cell types in kidney in which HIV transgene expression occurs using in situ hybridization. We evaluated evidence of proliferation by immunocytochemical analysis using an antibody to Ki-67 and cell type-specific markers, including WT-1, synaptopodin, Na+,K+-ATPase, adducin, and desmin. TUNEL assay was used to evaluate apoptosis. RESULTS: We found that glomerular and tubular epithelial cells express the HIV-1 transgene early in the disease process when renal architecture is well preserved. Transgene expression is lost, however, in tubular epithelial cells when they lose their differentiated cuboidal phenotype. In glomerular epithelial cells, dedifferentiation occurs with reduced expression of WT-1 and synaptopodin, in association with activation of desmin expression. Tubular microcysts also form with mislocalization of Na+,K+-ATPase expression to the lateral and apical cellular membranes. CONCLUSIONS: These studies support the hypothesis that the glomerular and renal epithelial cells are the primary targets of HIV-1 pathogenesis in the kidney. The essential pathologic process is dysregulation of the epithelial cell cycle with increased proliferation, apoptosis, cellular dedifferentiation, and altered cellular polarity.
Assuntos
Nefropatia Associada a AIDS/genética , Nefropatia Associada a AIDS/patologia , HIV-1/genética , Glomérulos Renais/patologia , Fatores Etários , Animais , Apoptose/genética , Biomarcadores , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Polaridade Celular/fisiologia , Proteínas de Ligação a DNA/análise , Modelos Animais de Doenças , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/enzimologia , Feto/química , Feto/enzimologia , Feto/patologia , Marcação In Situ das Extremidades Cortadas , Antígeno Ki-67/análise , Falência Renal Crônica/genética , Falência Renal Crônica/patologia , Falência Renal Crônica/virologia , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/análise , ATPase Trocadora de Sódio-Potássio/análise , Fatores de Transcrição/análise , Transgenes/fisiologia , Proteínas WT1RESUMO
It has recently been shown that formation of podocyte foot processes is dependent on a constant source of lipids and proteins (Simons M, Saffrich R, Reiser J, and Mundel P. J Am Soc Nephrol 10: 1633-1639, 1999). Here we characterize amino acid transport mechanisms in differentiated cultured podocytes and investigate whether it may be disturbed during podocyte injury. RT-PCR studies detected mRNA for transporters of neutral amino acids (ASCT1, ASCT2, and B(0/+)), cationic AA (CAT1 and CAT3), and anionic AA (EAAT2 and EAAT3). Alanine (Ala), asparagine, cysteine (Cys), glutamine (Gln), glycine (Gly), leucine (Leu), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), glutamic acid (Glu), arginine (Arg), and histidine (His) depolarized podocytes and increased their whole cell conductances. Depletion of extracellular Na(+) completely inhibited the depolarization induced by Ala, Gln, Glu, Gly, Leu, and Pro and decreased the depolarization induced by Arg and His, indicating the presence of Na(+)-dependent amino acid transport. Incubation of podocytes with 100 microg/ml puromycin aminonucleoside for 24 h significantly attenuated the effects induced by the various amino acids by approximately 70%. The data indicate the existence of different amino acid transporter systems in podocytes. Alteration of amino acid transport may participate in podocyte injury and disturbed foot process formation.
Assuntos
Aminoácidos/metabolismo , Rim/citologia , Rim/metabolismo , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , Primers do DNA/genética , Rim/efeitos dos fármacos , Potenciais da Membrana , Camundongos , Puromicina Aminonucleosídeo/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , Sódio/farmacologiaRESUMO
BACKGROUND: Effacement of podocyte foot processes occurs early in many glomerular diseases associated with proteinuria and is accompanied by a reorganization of the actin cytoskeleton. The molecular mechanisms regulating these structural changes are poorly understood. METHODS: To address these questions, we analyzed the effect of the polycation, protamine sulfate (PS), and puromycin aminonucleoside (PA) on the morphology, cytoskeleton, and tyrosine phosphorylation of differentiated process-bearing cultured podocytes. RESULTS: PS and PA induced similar profound morphological alterations, including retraction and detachment of podocyte processes from the extracellular matrix (ECM). The effects of PS occurred within six hours, whereas PA showed its most severe effects after 72 hours. Structural changes included reorganization of the actin cytoskeleton and focal contacts and were accompanied by an increase in tyrosine phosphorylation. The same effects were induced by application of vanadate, an inhibitor of protein tyrosine phosphatases (PTPs), suggesting that PTPs regulate podocyte process structure. Since disruption of the actin cytoskeleton with cytochalasin B protected the cells from PS-induced effacement and detachment, cytoplasmic PTPs were implicated in these events. Using reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated the expression of four cytoplasmic PTPs in podocytes: SHP-2, PTP-PEST, PTP-1B, and PTP-36. CONCLUSIONS: These studies indicate an important role for cytoplasmic PTPs as regulators of podocyte process dynamics. Future studies will aim at restoring the normal foot process architecture of podocytes in glomerular diseases associated with proteinuria by modulating the activity of cytoplasmic PTPs.
Assuntos
Glomérulos Renais/citologia , Proteínas Tirosina Fosfatases/fisiologia , Actinas/metabolismo , Animais , Células Cultivadas , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/fisiologia , Camundongos , Fosforilação , Protaminas/farmacologia , Puromicina Aminonucleosídeo/farmacologia , Tirosina/metabolismo , Vanadatos/farmacologiaRESUMO
Dendritic spines are sites of synaptic plasticity in the brain and are capable of remodeling their shape and size. However, little is known about the cellular mechanisms that regulate spine morphology and motility. Synaptopodin is a recently described actin-associated protein found in renal podocytes and dendritic spines (Mundel et al. J Cell Biol. [1997] 139:193-204), which is believed to play a role in spine plasticity. The present study analyzed the distribution of synaptopodin in the hippocampal formation. In situ hybridization histochemistry revealed a high constitutive expression of synaptopodin mRNA in the principal cell layers. Light microscopic immunohistochemistry showed that the protein is distributed throughout the hippocampal formation in a region- and lamina-specific manner. Postembedding immunogold histochemistry demonstrated that synaptopodin is exclusively present in dendrites and spines, specifically in the spine neck in close association with the spine apparatus. Spines lacking a spine apparatus are not immunoreactive for synaptopodin. These data suggest that synaptopodin links the spine apparatus to actin and may thus be involved in the actin-based plasticity of spines.