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1.
Am J Physiol Renal Physiol ; 318(6): F1377-F1390, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32308020

RESUMO

Ste20-like kinase SLK is critical for embryonic development and may play an important role in wound healing, muscle homeostasis, cell migration, and tumor growth. Mice with podocyte-specific deletion of SLK show albuminuria and damage to podocytes as they age. The present study addressed the role of SLK in glomerular injury. We induced adriamycin nephrosis in 3- to 4-mo-old control and podocyte SLK knockout (KO) mice. Compared with control, SLK deletion exacerbated albuminuria and loss of podocytes, synaptopodin, and podocalyxin. Glomeruli of adriamycin-treated SLK KO mice showed diffuse increases in the matrix and sclerosis as well as collapse of the actin cytoskeleton. SLK can phosphorylate ezrin. The complex of phospho-ezrin, Na+/H+ exchanger regulatory factor 2, and podocalyxin in the apical domain of the podocyte is a key determinant of normal podocyte architecture. Deletion of SLK reduced glomerular ezrin and ezrin phosphorylation in adriamycin nephrosis. Also, deletion of SLK reduced the colocalization of ezrin and podocalyxin in the glomerulus. Cultured glomerular epithelial cells with KO of SLK showed reduced ezrin phosphorylation and podocalyxin expression as well as reduced F-actin. Thus, SLK deletion leads to podocyte injury as mice age and exacerbates injury in adriamycin nephrosis. The mechanism may at least in part involve ezrin phosphorylation as well as disruption of the cytoskeleton and podocyte apical membrane structure.


Assuntos
Citoesqueleto de Actina/enzimologia , Doxorrubicina , Glomerulosclerose Segmentar e Focal/enzimologia , Nefrose/enzimologia , Podócitos/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Citoesqueleto de Actina/patologia , Actinas/metabolismo , Albuminúria/induzido quimicamente , Albuminúria/enzimologia , Albuminúria/genética , Animais , Células Cultivadas , Proteínas do Citoesqueleto/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/genética , Glomerulosclerose Segmentar e Focal/patologia , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Nefrose/induzido quimicamente , Nefrose/genética , Nefrose/patologia , Fosfoproteínas/metabolismo , Fosforilação , Podócitos/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo
2.
Lab Invest ; 100(7): 945-958, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32203149

RESUMO

Podocyte injury and endoplasmic reticulum (ER) stress have been implicated in the pathogenesis of various glomerular diseases. ERdj3 (DNAJB11) and mesencephalic astrocyte-derived neurotrophic factor (MANF) are ER chaperones lacking the KDEL motif, and may be secreted extracellularly. Since podocytes reside in the urinary space, we examined if podocyte injury is associated with secretion of KDEL-free ER chaperones from these cells into the urine, and if chaperones in the urine reflect ER stress in glomerulonephritis. In cultured podocytes, ER stress increased ERdj3 and MANF intracellularly and in culture medium, whereas GRP94 (KDEL chaperone) increased only intracellularly. ERdj3 and MANF secretion was blocked by the secretory trafficking inhibitor, brefeldin A. Urinary ERdj3 and MANF increased in rats injected with tunicamycin (in the absence of proteinuria). After induction of passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrosis (PAN), there was an increase in glomerular ER stress, and appearance of ERdj3 and MANF in the urine, coinciding with the onset of proteinuria. Rats with PHN were treated with the chemical chaperone, 4-phenyl butyrate (PBA), starting at the time of disease induction, or after disease was established. In both protocols, 4-PBA reduced proteinuria and urinary ER chaperone secretion, compared with PHN rats treated with saline (control). In conclusion, urinary ERdj3 and MANF reflect glomerular ER stress. 4-PBA protected against complement-mediated podocyte injury and the therapeutic response could be monitored by urinary ERdj3 and MANF.


Assuntos
Estresse do Retículo Endoplasmático/fisiologia , Glomerulonefrite/urina , Proteínas de Choque Térmico HSP40/urina , Fatores de Crescimento Neural/urina , Animais , Células Cultivadas , Glomerulonefrite/metabolismo , Glomerulonefrite/fisiopatologia , Proteínas de Choque Térmico HSP40/metabolismo , Glomérulos Renais/citologia , Glomérulos Renais/efeitos dos fármacos , Masculino , Camundongos , Fatores de Crescimento Neural/metabolismo , Ratos , Ratos Sprague-Dawley , Tunicamicina/farmacologia
3.
Am J Physiol Renal Physiol ; 315(1): F186-F198, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29187370

RESUMO

SLK is essential for embryonic development and may play a key role in wound healing, tumor growth, and metastasis. Expression and activation of SLK are increased in kidney development and during recovery from ischemic acute kidney injury. Overexpression of SLK in glomerular epithelial cells/podocytes in vivo induces injury and proteinuria. Conversely, reduced SLK expression leads to abnormalities in cell adhesion, spreading, and motility. Tight regulation of SLK expression thus may be critical for normal renal structure and function. We produced podocyte-specific SLK-knockout mice to address the functional role of SLK in podocytes. Mice with podocyte-specific deletion of SLK showed reduced glomerular SLK expression and activity compared with control. Podocyte-specific deletion of SLK resulted in albuminuria at 4-5 mo of age in male mice and 8-9 mo in female mice, which persisted for up to 13 mo. At 11-12 mo, knockout mice showed ultrastructural changes, including focal foot process effacement and microvillous transformation of podocyte plasma membranes. Mean foot process width was approximately twofold greater in knockout mice compared with control. Podocyte number was reduced by 35% in knockout mice compared with control, and expression of nephrin, synaptopodin, and podocalyxin was reduced in knockout mice by 20-30%. In summary, podocyte-specific deletion of SLK leads to albuminuria, loss of podocytes, and morphological evidence of podocyte injury. Thus, SLK is essential to the maintenance of podocyte integrity as mice age.


Assuntos
Albuminúria/enzimologia , Glomérulos Renais/enzimologia , Podócitos/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores Etários , Albuminúria/genética , Albuminúria/fisiopatologia , Animais , Adesão Celular , Células Cultivadas , Colágeno/metabolismo , Feminino , Predisposição Genética para Doença , Glomérulos Renais/fisiopatologia , Glomérulos Renais/ultraestrutura , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Fenótipo , Podócitos/ultraestrutura , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Ratos , Proteínas Repressoras/metabolismo , Fatores Sexuais , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Proteínas WT1
4.
Am J Physiol Renal Physiol ; 315(4): F954-F966, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29873512

RESUMO

Mutations in α-actinin-4 (actinin-4) result in hereditary focal segmental glomerulosclerosis (FSGS) in humans. Actinin-4 mutants induce podocyte injury because of dysregulation of the cytoskeleton and proteotoxicity. Injury may be associated with endoplasmic reticulum (ER) stress and polyubiquitination of proteins. We assessed if the chemical chaperone 4-phenylbutyrate (4-PBA) can ameliorate the proteotoxicity of an actinin-4 mutant. Actinin-4 K255E, which causes FSGS in humans (K256E in the mouse), showed enhanced ubiquitination, accelerated degradation, aggregate formation, and enhanced association with filamentous (F)-actin in glomerular epithelial cells (GECs). The mutant disrupted ER function and stimulated autophagy. 4-PBA reduced actinin-4 K256E aggregation and its tight association with F-actin. Transgenic mice that express actinin-4 K256E in podocytes develop podocyte injury, proteinuria, and FSGS in association with glomerular ER stress. Treatment of these mice with 4-PBA in the drinking water over a 10-wk period significantly reduced albuminuria and ER stress. Another drug, celastrol, which enhanced expression of ER and cytosolic chaperones in GECs, tended to reduce actinin-4 aggregation but did not decrease the tight association of actinin-4 K256E with F-actin and did not reduce albuminuria in actinin-4 K256E transgenic mice. Thus, chemical chaperones, such as 4-PBA, may represent a novel therapeutic approach to certain hereditary glomerular diseases.


Assuntos
Actinina/genética , Glomérulos Renais/lesões , Mutação/genética , Proteostase/genética , Citoesqueleto de Actina/metabolismo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Glomerulosclerose Segmentar e Focal/metabolismo , Glomérulos Renais/metabolismo , Camundongos Transgênicos , Podócitos/metabolismo , Proteinúria/metabolismo
5.
J Biol Chem ; 291(28): 14468-82, 2016 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-27226532

RESUMO

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria.


Assuntos
Glomerulonefrite/genética , Fosfolipases A2 do Grupo VI/genética , Glomérulos Renais/patologia , Podócitos/patologia , Envelhecimento , Animais , Autofagia , Células Cultivadas , Estresse do Retículo Endoplasmático , Glomerulonefrite/patologia , Glomérulos Renais/metabolismo , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/patologia , Fosfolipases A2 Independentes de Cálcio/genética , Podócitos/metabolismo , Proteinúria/genética , Proteinúria/patologia
6.
Biochim Biophys Acta ; 1863(9): 2147-55, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27216364

RESUMO

Expression and activation of SLK increases during renal ischemia-reperfusion injury. When highly expressed, SLK signals via c-Jun N-terminal kinase and p38 to induce apoptosis, and it exacerbates apoptosis induced by ischemia-reperfusion injury. Overexpression of SLK in glomerular epithelial cells (GECs)/podocytes in vivo induces injury and proteinuria. In response to various stresses, cells enhance expression of chaperones or heat shock proteins (e.g. Hsp70), which are involved in the folding and maturation of newly synthesized proteins, and can refold denatured or misfolded proteins. We address the interaction of SLK with the heat shock factor 1 (HSF1)-Hsp70 pathway. Increased expression of SLK in GECs (following transfection) induced HSF1 transcriptional activity. Moreover, HSF1 transcriptional activity was increased by in vitro ischemia-reperfusion injury (chemical anoxia/recovery) and heat shock, and in both instances was amplified further by SLK overexpression. HSF1 binds to promoters of target genes, such as Hsp70 and induces their transcription. By analogy to HSF1, SLK stimulated Hsp70 expression. Hsp70 was also enhanced by anoxia/recovery and was further amplified by SLK overexpression. Induction of HSF1 and Hsp70 was dependent on the kinase activity of SLK, and was mediated via polo-like kinase-1. Transfection of constitutively active HSF1 enhanced Hsp70 expression and inhibited SLK-induced apoptosis. Conversely, the proapoptotic action of SLK was augmented by HSF1 shRNA, or the Hsp70 inhibitor, pifithrin-µ. In conclusion, increased expression/activity of SLK activates the HSF1-Hsp70 pathway. Hsp70 attenuates the primary proapoptotic effect of SLK. Modulation of chaperone expression may potentially be harnessed as cytoprotective therapy in renal cell injury.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Células COS , Proteínas de Ciclo Celular/metabolismo , Morte Celular , Hipóxia Celular , Chlorocebus aethiops , Células Epiteliais/metabolismo , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico , Glomérulos Renais/citologia , Mutação/genética , Proteínas Proto-Oncogênicas/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Ativação Transcricional/genética , Quinase 1 Polo-Like
7.
J Biol Chem ; 290(5): 3009-20, 2015 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-25492867

RESUMO

Injury of visceral glomerular epithelial cells (GECs) causes proteinuria in many glomerular diseases. We reported previously that calcium-independent phospholipase A2γ (iPLA2γ) is cytoprotective against complement-mediated GEC injury. Because iPLA2γ is localized at the endoplasmic reticulum (ER), this study addressed whether the cytoprotective effect of iPLA2γ involves the ER stress unfolded protein response (UPR). In cultured rat GECs, overexpression of the full-length iPLA2γ, but not a mutant iPLA2γ that fails to associate with the ER, augmented tunicamycin-induced activation of activating transcription factor-6 (ATF6) and induction of the ER chaperones, glucose-regulated protein 94 (GRP94) and glucose-regulated protein 78 (GRP78). Augmented responses were inhibited by the iPLA2γ inhibitor, (R)-bromoenol lactone, but not by the cyclooxygenase inhibitor, indomethacin. Tunicamycin-induced cytotoxicity was reduced in GECs expressing iPLA2γ, and the cytoprotection was reversed by dominant-negative ATF6. GECs from iPLA2γ knock-out mice showed blunted ATF6 activation and chaperone up-regulation in response to tunicamycin. Unlike ATF6, the two other UPR pathways, i.e. inositol-requiring enzyme 1α and protein kinase RNA-like ER kinase pathways, were not affected by iPLA2γ. Thus, in GECs, iPLA2γ amplified activation of the ATF6 pathway of the UPR, resulting in up-regulation of ER chaperones and cytoprotection. These effects were dependent on iPLA2γ catalytic activity and association with the ER but not on prostanoids. Modulating iPLA2γ activity may provide opportunities for pharmacological intervention in glomerular diseases associated with ER stress.


Assuntos
Fator 6 Ativador da Transcrição/metabolismo , Estresse do Retículo Endoplasmático/fisiologia , Fosfolipases A2 Independentes de Cálcio/metabolismo , Fator 6 Ativador da Transcrição/genética , Animais , Células COS , Chlorocebus aethiops , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático/genética , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Fosfolipases A2 Independentes de Cálcio/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tunicamicina/farmacologia
8.
Biochim Biophys Acta ; 1853(10 Pt A): 2539-52, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26094769

RESUMO

Expression and activity of the Ste20-like kinase, SLK, are increased during kidney development and recovery from ischemia-reperfusion injury. SLK mediates apoptosis in various cells, and can regulate cell cycle progression and cytoskeletal remodeling. In cells, SLK is detected in a high molecular mass complex, suggesting that SLK is a dimer/oligomer, or is in tight association with other proteins. To better understand the regulation, localization and function of SLK, we sought to identify proteins in this high molecular mass complex. Analysis by mass spectroscopy identified the nucleoporin, translocated promoter region (Tpr), and the cytoskeletal protein, α-actinin-4, as potential SLK-interacting proteins. Using a protein complementation assay, we showed that the 350 amino acid C-terminal, coiled-coil domain of SLK was responsible for homodimerization, as well as interaction with Tpr and α-actinin-4. The association of SLK with Tpr and α-actinin-4, respectively, was confirmed by co-immunoprecipitation. Subsets of total cellular SLK colocalized with Tpr at the nuclear envelope, and α-actinin-4 in the cytoplasm. Expression of Tpr attenuated activation-specific autophosphorylation of SLK, and blocked SLK-induced apoptosis and AP-1 activity. In contrast to the effect of Tpr, autophosphorylation of SLK was not affected by α-actinin-4. Thus, SLK interacts with Tpr and α-actinin-4 in cells, and these protein-protein interactions may control the subcellular localization and the biological activity of SLK.


Assuntos
Actinina/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Actinina/genética , Animais , Células COS , Chlorocebus aethiops , Humanos , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas/genética
9.
Am J Physiol Renal Physiol ; 311(5): F1035-F1046, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27630065

RESUMO

Kidney cell injury may be associated with protein misfolding and induction of endoplasmic reticulum (ER) stress. Examples include complement-induced glomerular epithelial cell (GEC)/podocyte injury in membranous nephropathy and ischemia-reperfusion injury. Renal cell injury can also result from mutations in integral proteins, which lead to their misfolding and accumulation. Certain nephrin missense mutants misfold, accumulate in the ER, and induce ER stress. We examined if enhancement of ubiquitin-proteasome system function may facilitate proteostasis and confer protection against injury. Ubiquitin-specific protease 14 (Usp14) is reported to retard proteasomal protein degradation. Thus inhibition of Usp14 may enhance degradation of misfolded proteins and attenuate cell injury. In GEC, the reporter proteins GFPu (a "misfolded" protein) and CD3δ (an ER-associated degradation substrate) undergo time-dependent proteasomal degradation. Complement did not affect degradation of CD3δ-yellow fluorescent protein (YFP), but accelerated degradation of GFPu, and the Usp14-directed inhibitor IU1 further accelerated this degradation. Conversely, overexpression of Usp14 reduced degradation of GFPu and CD3δ-YFP. In 293T cells, IU1 did not enhance degradation of disease-associated nephrin missense mutants I171N and S724C, whereas overexpression of Usp14 reduced degradation. IU1 was cytoprotective after injury induced by the ER stressor tunicamycin and in vitro ischemia-reperfusion, but did not affect complement-induced cytotoxicity. In conclusion, Usp14 controls proteasomal degradation of some misfolded proteins. In addition, a Usp14-directed inhibitor reduces cytotoxicity in the context of global protein misfolding during certain types of renal cell injury.


Assuntos
Células Epiteliais/metabolismo , Glomérulos Renais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteínas do Sistema Complemento/metabolismo , Células HEK293 , Humanos , Proteólise , Traumatismo por Reperfusão/metabolismo
10.
J Biol Chem ; 288(6): 3871-85, 2013 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-23258543

RESUMO

In experimental membranous nephropathy, complement C5b-9-induces glomerular epithelial cell (GEC) injury and proteinuria. The effects of C5b-9 are mediated via signaling pathways, including calcium-independent phospholipase A(2)γ (iPLA(2)γ), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The iPLA(2)γ pathway is cytoprotective. This study addresses the mechanisms of iPLA(2)γ activation. iPLA(2)γ activity was monitored by quantifying prostaglandin E(2) (PGE(2)) production. In GECs, iPLA(2)γ localized at the endoplasmic reticulum and mitochondria. Complement-mediated production of PGE(2) was amplified in GECs that overexpress iPLA(2)γ, compared with control cells, and was blocked by the iPLA(2)γ inhibitor bromoenol lactone in both iPLA(2)γ-overexpressing and control GECs. In GECs that overexpress iPLA(2)γ, complement-mediated PGE(2) production was reduced by inhibitors of MAP/ERK kinase 1 (MEK1) and p38 but not JNK. In COS-1 cells that overexpress iPLA(2)γ and cyclooxygenase-1, PGE(2) production was induced by co-expression of constitutively active MEK1 or MAPK-interacting kinase 1 (MNK1) as well as by stimulation with epidermal growth factor (EGF) + ionomycin. Complement- and EGF + ionomycin-stimulated iPLA(2)γ activity was attenuated by the S511A/S515A double mutation. Moreover, complement and EGF + ionomycin enhanced phosphorylation of Ser-511. Thus, complement-mediated activation of iPLA(2)γ is mediated via ERK and p38 pathways, and phosphorylation of Ser-511 and/or Ser-515 plays a key role in the catalytic activity and signaling of iPLA(2)γ. Defining the mechanisms by which complement activates iPLA(2)γ provides opportunities for development of novel therapeutic approaches to GEC injury and proteinuria.


Assuntos
Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Glomerulonefrite Membranosa/enzimologia , Fosfolipases A2 do Grupo VI/metabolismo , Glomérulos Renais/enzimologia , Substituição de Aminoácidos , Animais , Células COS , Ionóforos de Cálcio/farmacologia , Linhagem Celular , Chlorocebus aethiops , Complexo de Ataque à Membrana do Sistema Complemento/farmacologia , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 1/metabolismo , Dinoprostona/genética , Dinoprostona/metabolismo , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Fator de Crescimento Epidérmico/farmacologia , Glomerulonefrite Membranosa/genética , Glomerulonefrite Membranosa/patologia , Fosfolipases A2 do Grupo VI/genética , Humanos , Fatores Imunológicos/metabolismo , Fatores Imunológicos/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ionomicina/farmacologia , Glomérulos Renais/lesões , Glomérulos Renais/patologia , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Mutação de Sentido Incorreto , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteinúria/enzimologia , Proteinúria/genética , Ratos
11.
Am J Physiol Renal Physiol ; 307(5): F634-47, 2014 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-25056348

RESUMO

The unfolded protein response and endoplasmic reticulum-associated degradation (ERAD) contribute to injury in renal glomerular diseases, including those mediated by complement C5b-9. In the present study, we address the role of protein-tyrosine phosphatase 1B (PTP1B) in complement-mediated glomerular injury and ERAD. In glomerular epithelial cells (GECs)/podocytes and PTP1B-deficient mouse embryonic fibroblasts exposed to complement, inhibition/deletion of PTP1B reduced ERAD, as monitored by the ERAD reporter CD3δ. Overexpression of PTP1B produced an effect similar to PTP1B deficiency on ERAD in complement-treated GECs. Complement-mediated cytotoxicity was reduced after PTP1B overexpression and tended to be reduced after PTP1B inhibition. PTP1B enhanced the induction of certain ERAD components via the inositol-requiring-1α branch of the unfolded protein response. PTP1B knockout mice with anti-glomerular basement membrane glomerulonephritis had decreased proteinuria and showed less podocyte loss and endoplasmic reticulum dysfunction compared with wild-type littermates. These results imply that endogenous levels of PTP1B are tightly regulated and that both overexpression and inhibition can affect ERAD. The cytoprotective effects of PTP1B deletion in cultured cells and in anti-glomerular basement membrane nephritis suggest that PTP1B may potentially be a therapeutic target in complement-mediated diseases.


Assuntos
Proteínas do Sistema Complemento/fisiologia , Degradação Associada com o Retículo Endoplasmático/fisiologia , Glomerulonefrite/prevenção & controle , Glomerulonefrite/fisiopatologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Animais , Células Cultivadas , Proteínas do Sistema Complemento/farmacologia , Modelos Animais de Doenças , Degradação Associada com o Retículo Endoplasmático/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células Epiteliais/fisiologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibroblastos/fisiologia , Glomerulonefrite/patologia , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/patologia , Glomérulos Renais/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Podócitos/efeitos dos fármacos , Podócitos/patologia , Podócitos/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/deficiência , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Ratos , Resposta a Proteínas não Dobradas/fisiologia
12.
Physiol Rep ; 12(1): e15897, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38163671

RESUMO

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.


Assuntos
Nefrose , Podócitos , Camundongos , Animais , Podócitos/metabolismo , Paxilina/metabolismo , Vinculina/metabolismo , Talina/genética , Talina/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Doxorrubicina/toxicidade , Proteínas Serina-Treonina Quinases/metabolismo
13.
Sci Rep ; 14(1): 11718, 2024 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-38778209

RESUMO

Protein misfolding in the endoplasmic reticulum (ER) of podocytes contributes to the pathogenesis of glomerular diseases. Protein misfolding activates the unfolded protein response (UPR), a compensatory signaling network. We address the role of the UPR and the UPR transducer, inositol-requiring enzyme 1α (IRE1α), in streptozotocin-induced diabetic nephropathy in mice. Diabetes caused progressive albuminuria in control mice that was exacerbated in podocyte-specific IRE1α knockout (KO) mice. Compared to diabetic controls, diabetic IRE1α KO mice showed reductions in podocyte number and synaptopodin. Glomerular ultrastructure was altered only in diabetic IRE1α KO mice; the major changes included widening of podocyte foot processes and glomerular basement membrane. Activation of the UPR and autophagy was evident in diabetic control, but not diabetic IRE1α KO mice. Analysis of human glomerular gene expression in the JuCKD-Glom database demonstrated induction of genes associated with the ER, UPR and autophagy in diabetic nephropathy. Thus, mice with podocyte-specific deletion of IRE1α demonstrate more severe diabetic nephropathy and attenuation of the glomerular UPR and autophagy, implying a protective effect of IRE1α. These results are consistent with data in human diabetic nephropathy and highlight the potential for therapeutically targeting these pathways.


Assuntos
Autofagia , Diabetes Mellitus Experimental , Nefropatias Diabéticas , Endorribonucleases , Camundongos Knockout , Podócitos , Proteínas Serina-Treonina Quinases , Resposta a Proteínas não Dobradas , Animais , Podócitos/metabolismo , Podócitos/patologia , Endorribonucleases/metabolismo , Endorribonucleases/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Camundongos , Autofagia/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Humanos , Masculino , Estresse do Retículo Endoplasmático , Albuminúria/genética , Albuminúria/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Deleção de Genes , Retículo Endoplasmático/metabolismo
14.
J Biol Chem ; 287(8): 5446-58, 2012 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-22203681

RESUMO

Expression and activation of the Ste20-like kinase, SLK, is increased during kidney development and recovery from ischemic acute kidney injury. SLK promotes apoptosis, and it may regulate cell survival during injury or repair. This study addresses the role of phosphorylation in the regulation of kinase activity. We mutated serine and threonine residues in the putative activation segment of the SLK catalytic domain and expressed wild type (WT) and mutant proteins in COS-1 or glomerular epithelial cells. Compared with SLK WT, the T183A, S189A, and T183A/S189A mutants showed reduced in vitro kinase activity. SLK WT, but not mutants, increased activation-specific phosphorylation of c-Jun N-terminal kinase (JNK) and p38 kinase. Similarly, SLK WT stimulated activator protein-1 reporter activity, but activation of activator protein-1 by the three SLK mutants was ineffective. To test if homodimerization of SLK affects phosphorylation, the cDNA encoding SLK amino acids 1-373 (which include the catalytic domain) was fused with a cDNA for a modified FK506-binding protein, Fv (Fv-SLK 1-373). After transfection, the addition of AP20187 (an FK506 analog) induced regulated dimerization of Fv-SLK 1-373. AP20187-stimulated dimerization enhanced the kinase activity of Fv-SLK 1-373 WT. In contrast, kinase activity of Fv-SLK 1-373 T183A/S189A was weak and was not enhanced after dimerization. Finally, apoptosis was increased after expression of Fv-SLK 1-373 WT but not T183A/S189A. Thus, phosphorylation of Thr-183 and Ser-189 plays a key role in the activation and signaling of SLK and could represent a target for novel therapeutic approaches to renal injury.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Animais , Biocatálise , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Ativação Enzimática , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Mutagênese , Mutação , Fosforilação , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estrutura Quaternária de Proteína , Ratos , Fator de Transcrição AP-1/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
15.
Biochim Biophys Acta ; 1823(5): 1007-16, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22426620

RESUMO

In experimental membranous nephropathy, complement C5b-9 induces sublethal glomerular epithelial cell (GEC) injury and proteinuria. C5b-9 also activates mechanisms that restrict injury or facilitate recovery. The ubiquitin-proteasome system (UPS) selectively degrades damaged or abnormal proteins, while misfolded proteins in the endoplasmic reticulum (ER) undergo ER-associated degradation (ERAD). In this study, we investigated the effect of complement on the UPS and ERAD. We monitored UPS function by transfection of rat GECs with a UPS reporter, GFP(u) (CL1 degron fused with green fluorescent protein). By analogy, CD3δ-yellow fluorescent protein (YFP) was employed as a reporter of ERAD. We demonstrated decreased GFP(u) levels in GECs after incubation with antibody and complement, compared with control. Using C8-deficient serum with or without purified C8, cycloheximide (an inhibitor of protein synthesis), and the proteasome inhibitor, MG132, we confirmed that the decrease of GFP(u) was mediated by C5b-9, and subsequent proteasomal degradation of the reporter. Inhibition of the c-Jun N-terminal kinase attenuated the effect of complement on GFP(u) degradation. Complement, however, increased the level of CD3δ-YFP in GECs, implying an impairment of ERAD, likely due to an overabundance of misfolded proteins in the ER. The overall ubiquitination of proteins was enhanced in complement-treated GECs and in glomeruli of rats with experimental membranous nephropathy, although ubiquitin mRNA was unchanged in GECs. Proteasome inhibition with MG132 increased the cytotoxic effect of complement in GECs. Complement-stimulated UPS function, by accelerating removal of damaged proteins, may be a novel mechanism to limit complement-induced injury.


Assuntos
Proteínas do Sistema Complemento/metabolismo , Degradação Associada com o Retículo Endoplasmático , Células Epiteliais/metabolismo , Glomérulos Renais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Ciclina A/metabolismo , Estresse do Retículo Endoplasmático , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Glomerulonefrite Membranosa/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Proteólise , Ratos , Ubiquitinação
16.
Front Med (Lausanne) ; 10: 1122328, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36993805

RESUMO

Background: Human glomerulonephritis (GN)-membranous nephropathy (MN), focal segmental glomerulosclerosis (FSGS) and IgA nephropathy (IgAN), as well as diabetic nephropathy (DN) are leading causes of chronic kidney disease. In these glomerulopathies, distinct stimuli disrupt metabolic pathways in glomerular cells. Other pathways, including the endoplasmic reticulum (ER) unfolded protein response (UPR) and autophagy, are activated in parallel to attenuate cell injury or promote repair. Methods: We used publicly available datasets to examine gene transcriptional pathways in glomeruli of human GN and DN and to identify drugs. Results: We demonstrate that there are many common genes upregulated in MN, FSGS, IgAN, and DN. Furthermore, these glomerulopathies were associated with increased expression of ER/UPR and autophagy genes, a significant number of which were shared. Several candidate drugs for treatment of glomerulopathies were identified by relating gene expression signatures of distinct drugs in cell culture with the ER/UPR and autophagy genes upregulated in the glomerulopathies ("connectivity mapping"). Using a glomerular cell culture assay that correlates with glomerular damage in vivo, we showed that one candidate drug - neratinib (an epidermal growth factor receptor inhibitor) is cytoprotective. Conclusion: The UPR and autophagy are activated in multiple types of glomerular injury. Connectivity mapping identified candidate drugs that shared common signatures with ER/UPR and autophagy genes upregulated in glomerulopathies, and one of these drugs attenuated injury of glomerular cells. The present study opens the possibility for modulating the UPR or autophagy pharmacologically as therapy for GN.

17.
Biochim Biophys Acta Mol Basis Dis ; 1868(6): 166391, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35304860

RESUMO

Glomerular diseases involving podocyte/glomerular epithelial cell (GEC) injury feature protein misfolding and endoplasmic reticulum (ER) stress. Inositol-requiring enzyme 1α (IRE1α) mediates chaperone production and autophagy during ER stress. We examined the role of IRE1α in selective autophagy of the ER (reticulophagy). Control and IRE1α knockout (KO) GECs were incubated with tunicamycin to induce ER stress and subjected to proteomic analysis. This showed IRE1α-dependent upregulation of secretory pathway mediators, including the coat protein complex II component Sec23B. Tunicamycin enhanced expression of Sec23B and the reticulophagy adaptor reticulon-3-long (RTN3L) in control, but not IRE1α KO GECs. Knockdown of Sec23B reduced autophagosome formation in response to ER stress. Tunicamycin stimulated colocalization of autophagosomes with Sec23B and RTN3L in an IRE1α-dependent manner. Similarly, during ER stress, glomerular α5 collagen IV colocalized with RTN3L and autophagosomes. Degradation of RTN3L and collagen IV increased in response to tunicamycin, and the turnover was blocked by deletion of IRE1α; thus, the IRE1α pathway promotes RTN3L-mediated reticulophagy and collagen IV may be an IRE1α-dependent reticulophagy substrate. In experimental glomerulonephritis, expression of Sec23B, RTN3L, and LC3-II increased in glomeruli of control mice, but not in podocyte-specific IRE1α KO littermates. In conclusion, during ER stress, IRE1α redirects a subset of Sec23B-positive vesicles to deliver RTN3L-coated ER fragments to autophagosomes. Reticulophagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.


Assuntos
Endorribonucleases/metabolismo , Podócitos , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Autofagia/fisiologia , Retículo Endoplasmático/metabolismo , Endorribonucleases/genética , Inositol/metabolismo , Camundongos , Podócitos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteômica , Transdutores , Resposta a Proteínas não Dobradas
18.
Biochim Biophys Acta ; 1803(4): 468-81, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20138193

RESUMO

Induction of endoplasmic reticulum (ER) stress by the complement membrane attack complex is enhanced by activation of cytosolic phospholipase A(2)-alpha (cPLA(2)). To address mechanisms by which cPLA(2) may modulate ER stress, we produced a mutant cPLA(2), containing an ER targeting domain (cPLA(2)-ERmut). After transfection and fractionation of COS-1 cells, cPLA(2)-ERmut was present mainly in the membrane fraction, whereas wild type (wt) cPLA(2) was principally in the cytosol. By fluorescence microscopy, cPLA(2)-ERmut was enriched in a perinuclear distribution under basal conditions, colocalizing with the ER protein, calnexin, while cPLA(2)-wt was mainly cytosolic. Both forms of cPLA(2) transiently expressed in COS cells showed basal phosphorylation at serine(505), which correlates with catalytic activity. Expression of cPLA(2)-wt was approximately 5-fold greater, compared with cPLA(2)-ERmut, but both enzymes produced comparable increases in free arachidonic acid, implying that cPLA(2)-ERmut effectively hydrolyzed ER membrane phospholipids. Although transfection of cPLA(2)-ERmut or wt did not induce ER stress independently, cPLA(2)-ERmut and wt enhanced the induction of ER stress by tunicamycin, dithiothreitol and ionomycin (monitored by induction of grp94 and C/EBP homologous protein-10), and the effect was dependent on the catalytic activity. cPLA(2)-ERmut enhanced production of superoxide. Induction of ER stress in tunicamycin-treated cells expressing cPLA(2)-ERmut was attenuated in the presence of the antioxidant, N-acetyl cysteine, and reduced glutathione, and was exacerbated by dl-buthionine-(S,R)-sulfoximine (which depletes glutathione). Expression of cPLA(2)-ERmut exacerbated tunicamycin-induced apoptosis. Thus, induction of ER stress is facilitated by the activation of cPLA(2) at the ER. The mechanism involves ER membrane phospholipid hydrolysis, and accumulation of reactive oxygen species.


Assuntos
Apoptose , Citosol/enzimologia , Retículo Endoplasmático/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Estresse Oxidativo , Animais , Antibacterianos/farmacologia , Western Blotting , Cálcio/metabolismo , Calnexina/metabolismo , Células Cultivadas , Chlorocebus aethiops , Imunofluorescência , Fosfolipases A2 do Grupo IV/genética , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Ionomicina/farmacologia , Ionóforos/farmacologia , Glomérulos Renais/enzimologia , Proteínas de Membrana/metabolismo , Mutação/genética , Fosfolipídeos/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Superóxidos/metabolismo , Tunicamicina/farmacologia
19.
Am J Physiol Renal Physiol ; 301(3): F554-64, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21677149

RESUMO

The expression and activation of the Ste20-like kinase, SLK, is increased during renal development and recovery from ischemic acute renal failure. SLK promotes apoptosis, and during renal injury and repair, transcriptional induction or posttranscriptional control of SLK may, therefore, regulate cell survival. SLK contains protein interaction (coiled-coil) domains, suggesting that posttranslational homodimerization may also modulate SLK activity. We therefore expressed coiled-coil regions in the C-terminal domain of SLK as fusion proteins and demonstrated their homodimerization. By gel-filtration chromatography, endogenous and heterologously expressed SLK were detected in a macromolecular protein complex. To test the role of homodimerization in kinase activation, we constructed a fusion protein consisting of the SLK catalytic domain (amino acids 1-373) and a modified FK506 binding protein, Fv (Fv-SLK 1-373). Addition of AP20187 (an analog of FK506) enhanced the homodimerization of Fv-SLK 1-373. In an in vitro kinase assay, the dimeric Fv-SLK 1-373 displayed greater kinase activity than the monomeric form. In cells expressing Fv-SLK 1-373, homodimerization increased activation-specific phosphorylation of the proapoptotic kinases, c-Jun N-terminal kinase and p38 kinase. Compared with the monomer, dimeric Fv-SLK 1-373 enhanced the activation of a Bax promoter-luciferase reporter. Finally, expression of Fv-SLK 1-373 induced apoptosis, and the effect was increased by homodimerization. Thus the activity, downstream signaling, and functional effects of SLK are enhanced by dimerization of the kinase domain.


Assuntos
Rim/metabolismo , Multimerização Proteica/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose/fisiologia , Células COS , Linhagem Celular , Chlorocebus aethiops , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Rim/citologia , Modelos Animais , Ratos , Transdução de Sinais/fisiologia
20.
Am J Physiol Renal Physiol ; 301(3): F496-508, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21159733

RESUMO

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in specific podocyte proteins. This study addresses mediation of GEC injury, focusing on endoplasmic reticulum (ER) stress. We studied signaling in cultured GECs in the presence or absence of the extracellular matrix (ECM). Adhesion to collagen supports cell survival, but adhesion to plastic (loss of contact with ECM) leads to apoptosis. Compared with collagen-adherent cells, GECs on plastic showed increased protein misfolding in the ER, and an adaptive-protective ER stress response, including increased expression of ER chaperones, increased phosphorylation of eukaryotic translation initiation factor-2α (eIF2α), and a reduction in protein synthesis. Activation of these ER stress pathways counteracted apoptosis. However, tunicamycin (a potent stimulator of ER stress) changed the ER stress response from protective to cytotoxic, as tunicamycin induced the proapoptotic ER stress gene, C/EBP homologous protein-10, and exacerbated apoptosis in GECs adherent to plastic, but not collagen. In GECs adherent to plastic, adaptive ER stress was associated with an increase in polyubiquitinated proteins and "choking" of the proteasome. Furthermore, pharmacological inhibition of the proteasome induced ER stress in GECs. Finally, we show that ER stress (induction of ER chaperones and eIF2α phosphorylation) was evident in experimental FSGS in vivo. Thus interactions of GECs with ECM may regulate protein folding and induction of the ER stress response. FSGS is associated with induction of ER stress. Enhancing protective aspects of the ER stress response may reduce apoptosis and possibly glomerulosclerosis.


Assuntos
Apoptose/fisiologia , Retículo Endoplasmático/fisiologia , Células Epiteliais/patologia , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Glomérulos Renais/patologia , Estresse Fisiológico/fisiologia , Animais , Células Cultivadas , Colágeno/metabolismo , Modelos Animais de Doenças , Células Epiteliais/fisiologia , Matriz Extracelular/fisiologia , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomérulos Renais/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ratos , Tunicamicina/efeitos adversos , Resposta a Proteínas não Dobradas/fisiologia
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