RESUMO
Unlike classical protein kinase A, with separate catalytic and regulatory subunits, EPACs are single chain multi-domain proteins containing both catalytic and regulatory elements. The importance of cAMP-Epac-signaling as an energy provider has emerged over the last years. However, little is known about Epac1 signaling in chronic kidney disease. Here, we examined the role of Epac1 during the progression of glomerulonephritis (GN). We first observed that total genetic deletion of Epac1 in mice accelerated the progression of nephrotoxic serum (NTS)-induced GN. Next, mice with podocyte-specific conditional deletion of Epac1 were generated and showed that NTS-induced GN was exacerbated in these mice. Gene expression analysis in glomeruli at the early and late phases of GN showed that deletion of Epac1 in podocytes was associated with major alterations in mitochondrial and metabolic processes and significant dysregulation of the glycolysis pathway. In vitro, Epac1 activation in a human podocyte cell line increased mitochondrial function to cope with the extra energy demand under conditions of stress. Furthermore, Epac1-induced glycolysis and lactate production improved podocyte viability. To verify the in vivo therapeutic potential of Epac1 activation, the Epac1 selective cAMP mimetic 8-pCPT was administered in wild type mice after induction of GN. 8-pCPT alleviated the progression of GN by improving kidney function with decreased structural injury with decreased crescent formation and kidney inflammation. Importantly, 8-pCPT had no beneficial effect in mice with Epac1 deletion in podocytes. Thus, our data suggest that Epac1 activation is an essential protective mechanism in GN by reprogramming podocyte metabolism. Hence, targeting Epac1 activation could represent a potential therapeutic approach.
Assuntos
AMP Cíclico , Glomerulonefrite , Fatores de Troca do Nucleotídeo Guanina , Reprogramação Metabólica , Podócitos , Animais , Humanos , Masculino , Camundongos , Linhagem Celular , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Metabolismo Energético/efeitos dos fármacos , Glomerulonefrite/patologia , Glomerulonefrite/metabolismo , Glomerulonefrite/genética , Glomerulonefrite/prevenção & controle , Glicólise , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Podócitos/metabolismo , Podócitos/patologia , Transdução de SinaisRESUMO
BACKGROUND: Without a viable cure, chronic kidney disease is a global health concern. Inflammatory damage in and around the renal tubules dictates disease severity and is contributed to by multiple cell types. Activated in response to danger associated molecular patterns (DAMPs) including ATP, the NOD-like receptor protein-3 (NLRP3) inflammasome is integral to this inflammation. In vivo, we have previously observed that increased expression of Connexin 43 (Cx43) is linked to inflammation in chronic kidney disease (CKD) whilst in vitro studies in human proximal tubule cells highlight a role for aberrant Cx43 hemichannel mediated ATP release in tubule injury. A role for Cx43 hemichannels in priming and activation of the NLRP3 inflammasome in tubule epithelial cells remains to be determined. METHODS: Using the Nephroseq database, analysis of unpublished transcriptomic data, examined gene expression and correlation in human CKD. The unilateral ureteral obstruction (UUO) mouse model was combined with genetic (tubule-specific Cx43 knockout) and specific pharmacological blockade of Cx43 (Peptide5), to explore a role for Cx43-hemichannels in tubule damage. Human primary tubule epithelial cells were used as an in vitro model of CKD. RESULTS: Increased Cx43 and NLRP3 expression correlates with declining glomerular filtration rate and increased proteinuria in biopsies isolated from patients with CKD. Connexin 43-tubule deletion prior to UUO protected against tubular injury, increased expression of proinflammatory molecules, and significantly reduced NLRP3 expression and downstream signalling mediators. Accompanied by a reduction in F4/80 macrophages and fibroblast specific protein (FSP1+) fibroblasts, Cx43 specific hemichannel blocker Peptide5 conferred similar protection in UUO mice. In vitro, Peptide5 determined that increased Cx43-hemichannel activity primes and activates the NLRP3 inflammasome via ATP-P2X7 receptor signalling culminating in increased secretion of chemokines and cytokines, each of which are elevated in individuals with CKD. Inhibition of NLRP3 and caspase 1 similarly decreased markers of tubular injury, whilst preventing the perpetual increase in Cx43-hemichannel activity. CONCLUSION: Aberrant Cx43-hemichannel activity in kidney tubule cells contributes to tubule inflammation via activation of the NLRP3 inflammasome and downstream paracrine mediated cell signalling. Use of hemichannel blockers in targeting Cx43-hemichannels is an attractive future therapeutic target to slow or prevent disease progression in CKD. Video Abstract.
Assuntos
Conexina 43 , Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Insuficiência Renal Crônica , Animais , Humanos , Camundongos , Trifosfato de Adenosina/metabolismo , Conexina 43/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
Microvasculature consisting of endothelial cells and pericytes is the main site of injury during antibody-mediated rejection (ABMR) of renal grafts. Little is known about the mechanisms of activation of pericytes in this pathology. We have found recently that activation of Notch3, a mediator of vascular smooth muscle cell proliferation and dedifferentiation, promotes renal inflammation and fibrosis and aggravates progression of renal disease. Therefore, we studied the pericyte expression of Notch3 in 49 non-selected renal graft biopsies (32 for clinical cause, 17 for graft surveillance). We analysed its relationship with patients' clinical and morphological data, and compared with the expression of partial endothelial mesenchymal transition (pEndMT) markers, known to reflect endothelial activation during ABMR. Notch3 was de novo expressed in pericytes of grafts with ABMR, and was significantly correlated with the microcirculation inflammation scores of peritubular capillaritis and glomerulitis and with the expression of pEndMT markers. Notch3 expression was also associated with graft dysfunction and proteinuria at the time of biopsy and in the long term. Multivariate analysis confirmed pericyte expression of Notch3 as an independent risk factor predicting graft loss. These data suggest that Notch3 is activated in the pericytes of renal grafts with ABMR and is associated with poor graft outcome.
Assuntos
Rejeição de Enxerto , Pericitos , Receptor Notch3 , Anticorpos , Biomarcadores/análise , Biópsia , Células Endoteliais/imunologia , Células Endoteliais/patologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Pericitos/imunologia , Pericitos/patologia , Receptor Notch3/biossíntese , Receptor Notch3/imunologiaRESUMO
BACKGROUND: Polycystic kidney disease (PKD) is a genetic disorder affecting millions of people worldwide that is characterized by fluid-filled cysts and leads to end-stage renal disease (ESRD). The hallmarks of PKD are proliferation and dedifferentiation of tubular epithelial cells, cellular processes known to be regulated by Notch signaling. METHODS: We found increased Notch3 expression in human PKD and renal cell carcinoma biopsies. To obtain insight into the underlying mechanisms and the functional consequences of this abnormal expression, we developed a transgenic mouse model with conditional overexpression of the intracellular Notch3 (ICN3) domain specifically in renal tubules. We evaluated the alterations in renal function (creatininemia, BUN) and structure (cysts, fibrosis, inflammation) and measured the expression of several genes involved in Notch signaling and the mechanisms of inflammation, proliferation, dedifferentiation, fibrosis, injury, apoptosis and regeneration. RESULTS: After one month of ICN3 overexpression, kidneys were larger with tubules grossly enlarged in diameter, with cell hypertrophy and hyperplasia, exclusively in the outer stripe of the outer medulla. After three months, mice developed numerous cysts in proximal and distal tubules. The cysts had variable sizes and were lined with a single- or multilayered, flattened, cuboid or columnar epithelium. This resulted in epithelial hyperplasia, which was observed as protrusions into the cystic lumen in some of the renal cysts. The pre-cystic and cystic epithelium showed increased expression of cytoskeletal filaments and markers of epithelial injury and dedifferentiation. Additionally, the epithelium showed increased proliferation with an aberrant orientation of the mitotic spindle. These phenotypic tubular alterations led to progressive interstitial inflammation and fibrosis. CONCLUSIONS: In summary, Notch3 signaling promoted tubular cell proliferation, the alignment of cell division, dedifferentiation and hyperplasia, leading to cystic kidney diseases and pre-neoplastic lesions.
Assuntos
Células Epiteliais/metabolismo , Túbulos Renais/metabolismo , Doenças Renais Policísticas/etiologia , Doenças Renais Policísticas/metabolismo , Receptor Notch3/metabolismo , Animais , Biomarcadores , Modelos Animais de Doenças , Suscetibilidade a Doenças , Células Epiteliais/patologia , Fibrose , Expressão Gênica , Imuno-Histoquímica , Neoplasias Renais/etiologia , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Túbulos Renais/patologia , Camundongos , Doenças Renais Policísticas/patologia , Receptor Notch3/genéticaRESUMO
[Figure: see text].
Assuntos
Antígeno CD146 , Glomerulonefrite , Animais , Antígeno CD146/genética , Antígeno CD146/metabolismo , Modelos Animais de Doenças , Descoberta de Drogas , Células Endoteliais/metabolismo , Fibrose/metabolismo , Fibrose/prevenção & controle , Técnicas de Inativação de Genes/métodos , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Glomerulonefrite/terapia , Inflamação/metabolismo , Inflamação/prevenção & controle , Junções Intercelulares/imunologia , Glomérulos Renais/imunologia , Camundongos , Regulação para CimaRESUMO
BACKGROUND: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. METHODS: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-ß1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/- mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. RESULTS: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-ß1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/- mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. CONCLUSION: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. Video Abstract In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-ß1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP]e), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability.
Assuntos
Trifosfato de Adenosina/metabolismo , Conexina 43/fisiologia , Túbulos Renais , Insuficiência Renal Crônica/metabolismo , Animais , Adesão Celular , Linhagem Celular , Humanos , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Camundongos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: The matricellular protein periostin has been associated with CKD progression in animal models and human biopsy specimens. Periostin functions by interacting with extracellular matrix components to drive collagen fibrillogenesis and remodeling or by signaling through cell-surface integrin receptors to promote cell adhesion, migration, and proliferation. However, its role in AKI is unknown. METHODS: We used mice with conditional tubule-specific overexpression of periostin or knockout mice lacking periostin expression in the renal ischemia-reperfusion injury model, and primary cultures of isolated tubular cells in a hypoxia-reoxygenation model. RESULTS: Tubular epithelial cells showed strong production of periostin during the repair phase of ischemia reperfusion. Periostin overexpression protected mice from renal injury compared with controls, whereas knockout mice showed increased tubular injury and deteriorated renal function. Periostin interacted with its receptor, integrin-ß1, to inhibit tubular cell cycle arrest and apoptosis in in vivo and in vitro models. After ischemia-reperfusion injury, periostin-overexpressing mice exhibited diminished expression of proinflammatory molecules and had more F4/80+ macrophages compared with knockout mice. Macrophages from periostin-overexpressing mice showed increased proliferation and expression of proregenerative factors after ischemia-reperfusion injury, whereas knockout mice exhibited the opposite. Coculturing a macrophage cell line with hypoxia-treated primary tubules overexpressing periostin, or treating such macrophages with recombinant periostin, directly induced macrophage proliferation and expression of proregenerative molecules. CONCLUSIONS: In contrast to the detrimental role of periostin in CKD, we discovered a protective role of periostin in AKI. Our findings suggest periostin may be a novel and important mediator of mechanisms controlling renal repair after AKI.
Assuntos
Injúria Renal Aguda , Moléculas de Adesão Celular/fisiologia , Proliferação de Células , Macrófagos/fisiologia , Injúria Renal Aguda/etiologia , Animais , Modelos Animais de Doenças , Rim/irrigação sanguínea , Masculino , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/patologiaRESUMO
Chronic kidney disease is an incurable to date pathology with a continuously growing incidence that contributes to the increase of the number of deaths worldwide. With currently no efficient prognostic or therapeutic options being available, the only possibility for treatment of end-stage renal disease is renal replacement therapy through dialysis or transplantation. Understanding the molecular mechanisms participating in the progression of renal diseases and uncovering the pathways implicated will permit the identification of novel and more efficient targets of therapy. Connexin43 was recently identified as a novel player in the development of chronic kidney disease. It was found de novo expressed and/or differentially localized in various renal cell populations during progression of renal disease, indicating an abnormal connexin signaling, both in patients and animal models. Subsequent in vivo studies demonstrated that connexin43 is involved in mediating inflammatory and fibrotic processes contributing to renal damage. Genetic, pharmaco-genetic or peptide-based inhibition of connexin43 in animal models and cell culture systems was successful in preventing the progression of the pathology and preserving the cell phenotypes. This review will summarize the recent advances on connexin43 in the field of kidney diseases and discuss the potential of future connexin43-based therapies against chronic kidney disease.
Assuntos
Conexina 43/metabolismo , Insuficiência Renal Crônica/patologia , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Humanos , Rim/metabolismo , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Oligonucleotídeos Antissenso/uso terapêutico , Peptídeos/química , Peptídeos/metabolismo , Peptídeos/uso terapêutico , Podócitos/citologia , Podócitos/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismoRESUMO
Chronic kidney disease, the end result of most renal and some systemic diseases, is a common condition where renal function is compromised due to fibrosis. During renal fibrosis, calreticulin, a multifunctional chaperone of the endoplasmic reticulum (ER) is up-regulated in tubular epithelial cells (TECs) both in vitro and in vivo. Proteomic analysis of cultured TECs overexpressing calreticulin led to the identification of the family of 14-3-3 proteins as key proteins overexpressed as well. Furthermore, an increased expression in the majority of 14-3-3 family members was observed in 3 different animal models of renal pathologies: the unilateral ureteric obstruction, the nephrotoxic serum administration and the ischaemia-reperfusion. In all these models, the 14-3-3σ isoform (also known as stratifin) was predominantly overexpressed. As in all these models ischaemia is a common denominator, we showed that the ischaemia-induced transcription factor HIF1α is specifically associated with the promoter region of the 14-3-3σ gene. Finally, we evaluated the expression of the family of 14-3-3 proteins and specifically 14-3-3σ in biopsies from IgA nephropathy and membranous nephropathy patients. These results propose an involvement of 14-3-3σ in renal pathology and provide evidence for the first time that hypoxia may be responsible for its altered expression.
Assuntos
Proteínas 14-3-3/genética , Biomarcadores Tumorais/genética , Exorribonucleases/genética , Glomerulonefrite por IGA/genética , Glomerulonefrite Membranosa/genética , Insuficiência Renal Crônica/genética , Traumatismo por Reperfusão/genética , Obstrução Ureteral/genética , Proteínas 14-3-3/metabolismo , Animais , Biomarcadores Tumorais/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Linhagem Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Exorribonucleases/metabolismo , Fibrose , Regulação da Expressão Gênica , Glomerulonefrite por IGA/metabolismo , Glomerulonefrite por IGA/patologia , Glomerulonefrite Membranosa/metabolismo , Glomerulonefrite Membranosa/patologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Proteômica/métodos , Insuficiência Renal Crônica/metabolismo , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologiaRESUMO
Acute kidney injury is a major risk factor for subsequent chronic renal and/or cardiovascular complications. Previous studies have shown that Notch3 was de novo expressed in the injured renal epithelium in the early phases of chronic kidney disease. Here we examined whether Notch3 is involved in the inflammatory response and the epithelial cell damage that typifies ischemic kidneys using Notch3 knockout mice and mice with short-term activated Notch3 signaling (N3ICD) in renal epithelial cells. After ischemia/reperfusion, N3ICD mice showed exacerbated infiltration of inflammatory cells and severe tubular damage compared to control mice. Inversely, Notch3 knockout mice were protected against ischemia/reperfusion injury. Renal macrophages derived from Notch3 knockout mice failed to activate proinflammatory cytokines. Chromatin immunoprecipitation analysis of the Notch3 promoter identified NF-κB as the principal inducer of Notch3 in ischemia/reperfusion. Thus, Notch3 induced by NF-κB in the injured epithelium sustains a proinflammatory environment attracting activated macrophages to the site of injury leading to a rapid deterioration of renal function and structure. Hence, targeting Notch3 may provide a novel therapeutic strategy against ischemia/reperfusion and acute kidney injury by preservation of epithelial structure and disruption of proinflammatory signaling.
Assuntos
Injúria Renal Aguda/patologia , Túbulos Renais/patologia , Receptor Notch3/metabolismo , Traumatismo por Reperfusão/complicações , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/imunologia , Animais , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Epitélio/metabolismo , Epitélio/patologia , Humanos , Túbulos Renais/imunologia , Túbulos Renais/metabolismo , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Regiões Promotoras Genéticas/genética , Receptor Notch3/genéticaRESUMO
Background Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein.Methods We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin.Results Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and α-actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well.Conclusions Palladin has an important role in podocyte function in vitro and in vivo.
Assuntos
Actinas/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Podócitos/metabolismo , Animais , Citoesqueleto , Feminino , Adesões Focais , Expressão Gênica , Inativação Gênica , Humanos , Glomérulos Renais/patologia , Masculino , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Morfolinos/farmacologia , Podócitos/patologia , RNA Mensageiro/metabolismo , Vinculina/genética , Vinculina/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
De novo expression in the kidney of periostin, a protein involved in odontogenesis and osteogenesis, has been suggested as a biomarker of renal disease. In this study, we investigated the mechanism(s) of induction and the role of periostin in renal disease. Using a combination of bioinformatics, reporter assay, and chromatin immunoprecipitation analyses, we found that NFκB and other proinflammatory transcription factors induce periostin expression in vitro and that binding of these factors on the periostin promoter is enriched in glomeruli during experimental GN. Mice lacking expression of periostin displayed preserved renal function and structure during GN. Furthermore, delayed administration of periostin antisense oligonucleotides in wild-type animals with GN reversed already established proteinuria, diminished tissue inflammation, and improved renal structure. Lack of periostin expression also blunted the de novo renal expression of integrin-ß3 and phosphorylation of focal adhesion kinase and AKT, known mediators of integrin-ß3 signaling that affect cell motility and survival, observed during GN in wild-type animals. In vitro, recombinant periostin increased the expression of integrin-ß3 and the concomitant phosphorylation of focal adhesion kinase and AKT in podocytes. Notably, periostin and integrin-ß3 were highly colocalized in biopsy specimens from patients with inflammatory GN. These results demonstrate that interplay between periostin and renal inflammation orchestrates inflammatory and fibrotic responses, driving podocyte damage through downstream activation of integrin-ß3 signaling. Targeting periostin may be a novel therapeutic strategy for treating CKD.
Assuntos
Moléculas de Adesão Celular/fisiologia , Integrina beta3/fisiologia , Nefropatias/etiologia , NF-kappa B/fisiologia , Animais , Feminino , Glomerulonefrite/complicações , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de SinaisRESUMO
The main hallmark of chronic kidney disease (CKD) is excessive inflammation leading to interstitial tissue fibrosis. It has been recently reported that NOV/CCN3 could be involved in kidney damage but its role in the progression of nephropathies is poorly known. NOV/CCN3 is a secreted multifunctional protein belonging to the CCN family involved in different physiological and pathological processes such as angiogenesis, inflammation and cancers. The purpose of our study was to determine the role of NOV/CCN3 in renal inflammation and fibrosis related to primitive tubulointerstitial injury. After unilateral ureteral obstruction (UUO), renal histology and real-time PCR were performed in NOV/CCN3-/- and wild type mice. NOV/CCN3 mRNA expression was increased in the obstructed kidneys in the early stages of the obstructive nephropathy. Interestingly, plasmatic levels of NOV/CCN3 were strongly induced after 7 days of UUO and the injection of recombinant NOV/CCN3 protein in healthy mice significantly increased CCL2 mRNA levels. Furthermore, after 7 days of UUO NOV/CCN3-/- mice displayed reduced proinflammatory cytokines and adhesion markers expression leading to restricted accumulation of interstitial monocytes, in comparison with their wild type littermates. Consequently, in NOV/CCN3-/- mice interstitial renal fibrosis was blunted after 15 days of UUO. In agreement with our experimental data, NOV/CCN3 expression was highly increased in biopsies of patients with tubulointerstitial nephritis. Thus, the inhibition of NOV/CCN3 may represent a novel target for the progression of renal diseases.
Assuntos
Nefropatias/patologia , Rim/patologia , Nefrite Intersticial/patologia , Proteína Sobre-Expressa em Nefroblastoma/metabolismo , Animais , Biomarcadores/metabolismo , Fibrose/metabolismo , Fibrose/patologia , Regulação da Expressão Gênica , Humanos , Nefropatias/genética , Nefropatias/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Nefrite Intersticial/genética , Nefrite Intersticial/metabolismo , Proteína Sobre-Expressa em Nefroblastoma/sangue , Proteína Sobre-Expressa em Nefroblastoma/genética , Insuficiência Renal Crônica/patologia , Obstrução Ureteral/metabolismoRESUMO
The incidence and prevalence of chronic kidney disease represents an important problem for public health. In renal diseases, the main histologic alterations derive from the development of renal fibrosis which results from the loss of the balance between pro- and anti-fibrotic factors. Tyrosine kinase receptors (RTKs) and matricellular proteins (MPs) are nowadays studied as potential modulators of renal injury. RTKs regulate cell cycle, migration, metabolism and cellular differentiation. Discoidin domain receptor-1 (DDR-1) is an RTK that has been extensively studied in cancer, and lung and renal diseases. It modulates inflammatory recruitment, extracellular matrix deposition and fibrosis; in renal diseases, it appears to act independently of the underlying disease. MPs regulate cell-matrix interactions and matrix accumulation, cellular adhesion and migration, and expression of inflammatory cells. Periostin is an MP, mainly studied in bone, heart, lung and cancer. Several studies demonstrated that it mediates cell-matrix interactions, migration of inflammatory cells and development of fibrosis. Recently, it has been reported in several nephropathies. In this review, we discuss the potential pathological roles of DDR-1 and periostin focussing on the kidney in both experimental models and human diseases.
Assuntos
Moléculas de Adesão Celular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Insuficiência Renal Crônica/metabolismo , Receptor com Domínio Discoidina 1 , HumanosRESUMO
Notch3 expression is found in the glomerular podocytes of patients with lupus nephritis or focal segmental GN but not in normal kidneys. Here, we show that activation of the Notch3 receptor in the glomeruli is a turning point inducing phenotypic changes in podocytes promoting renal inflammation and fibrosis and leading to disease progression. In a model of rapidly progressive GN, Notch3 expression was induced by several-fold in podocytes concurrently with disease progression. By contrast, mice lacking Notch3 expression were protected because they exhibited less proteinuria, uremia, and inflammatory infiltration. Podocyte outgrowth from glomeruli isolated from wild-type mice during the early phase of the disease was higher than outgrowth from glomeruli of mice lacking Notch3. In vitro studies confirmed that podocytes expressing active Notch3 reorganize their cytoskeleton toward a proliferative/migratory and inflammatory phenotype. We then administered antisense oligodeoxynucleotides targeting Notch3 or scramble control oligodeoxynucleotides in wild-type mice concomitant to disease induction. Both groups developed chronic renal disease, but mice injected with Notch3 antisense had lower values of plasma urea and proteinuria and inflammatory infiltration. The improvement of renal function was accompanied by fewer deposits of fibrin within the glomeruli and by decreased peritubular inflammation. Finally, abnormal Notch3 staining was observed in biopsy samples of patients with crescentic GN. These results demonstrate that abnormal activation of Notch3 may be involved in the progression of renal disease by promoting migratory and proinflammatory pathways. Inhibiting Notch3 activation could be a novel, promising approach to treat GN.
Assuntos
Regulação da Expressão Gênica , Glomerulosclerose Segmentar e Focal/genética , Glomérulos Renais/patologia , Podócitos/patologia , Receptores Notch/genética , Ativação Transcricional/genética , Análise de Variância , Animais , Nitrogênio da Ureia Sanguínea , Células Cultivadas , Modelos Animais de Doenças , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Imuno-Histoquímica , Glomérulos Renais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos/metabolismo , Proteinúria/fisiopatologia , RNA Mensageiro/análise , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Receptor Notch3 , Valores de Referência , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Ovinos , Regulação para CimaRESUMO
Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well-established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up-regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up-regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin-induced) and heart fibrosis (desmin-null) were examined. In the lung model, calreticulin expression was up-regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF-beta, a well known major contributing factor in several fibrotic processes, was found to up-regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.