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1.
Front Med (Lausanne) ; 10: 1096869, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36844206

RESUMO

Introduction: Romani people have a high prevalence of kidney failure. This study examined a Romani cohort for pathogenic variants in the COL4A3, COL4A4, and COL4A5 genes that are affected in Alport syndrome (AS), a common cause of genetic kidney disease, characterized by hematuria, proteinuria, end-stage kidney failure, hearing loss, and eye anomalies. Materials and methods: The study included 57 Romani from different families with clinical features that suggested AS who underwent next-generation sequencing (NGS) of the COL4A3, COL4A4, and COL4A5 genes, and 83 family members. Results: In total, 27 Romani (19%) had autosomal recessive AS caused by a homozygous pathogenic c.1598G>A, p.Gly533Asp variant in COL4A4 (n = 20) or a homozygous c.415G>C, p.Gly139Arg variant in COL4A3 (n = 7). For p.Gly533Asp, 12 (80%) had macroscopic hematuria, 12 (63%) developed end-stage kidney failure at a median age of 22 years, and 13 (67%) had hearing loss. For p.Gly139Arg, none had macroscopic hematuria (p = 0.023), three (50%) had end-stage kidney failure by a median age of 42 years (p = 0.653), and five (83%) had hearing loss (p = 0.367). The p.Gly533Asp variant was associated with a more severe phenotype than p.Gly139Arg, with an earlier age at end-stage kidney failure and more macroscopic hematuria. Microscopic hematuria was very common in heterozygotes with both p.Gly533Asp (91%) and p.Gly139Arg (92%). Conclusion: These two founder variants contribute to the high prevalence of kidney failure in Czech Romani. The estimated population frequency of autosomal recessive AS from these variants and consanguinity by descent is at least 1:11,000 in Czech Romani. This corresponds to a population frequency of autosomal dominant AS from these two variants alone of 1%. Romani with persistent hematuria should be offered genetic testing.

2.
Front Med (Lausanne) ; 10: 1320054, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38170106

RESUMO

Background: Genetic focal segmental glomerulosclerosis (FSGS) is caused by pathogenic variants in a broad spectrum of genes that have a variable representation based on subjects' ethnicity and/or age. The most frequently mutated autosomal recessive gene in FSGS is NPHS2. In this study, we analyzed the spectrum of NPHS2 variants and their associated phenotype in Czech adult FSGS patients. Methods: A representative cohort of 234 adult patients with FSGS, derived from 225 families originating from all regions of Czechia, was analyzed by massively parallel sequencing. In this study, we focused on the comprehensive analysis of the NPHS2 gene. The histological classification of FSGS followed the Columbia classification. Results: We detected seven (3%) cases bearing homozygous or compound heterozygous pathogenic NPHS2 variants. A single pathogenic variant c.868G > A (p.Val290Met) was found in the majority of NPHS2-positive cases (86%; 6 out of 7) in histologically confirmed instances of FSGS. Its allele frequency among unrelated NPHS2-associated FSGS patients was 50% (6/12), and Haplotype analysis predicted its origin to be a result of a founder effect. There is an identical V290M-related haplotype on all V290M alleles spanning a 0,7 Mb region flanking NPHS2 in Central European FSGS populations. The phenotype of the p.Val290Met NPHS2-associated FSGS demonstrated a later onset and a much milder course of the disease compared to other NPHS2 pathogenic variants associated with FSGS. The mean age of the FSGS diagnosis based on kidney biopsy evaluation was 31.2 ± 7.46 years. In 50% of all cases, the initial disease manifestation of proteinuria occurred only in adulthood, with 83% of these cases not presenting with edemas. One-third (33%) of the studied subjects progressed to ESRD (2 out of 6) at the mean age of 35.0 ± 2.82 years. Conclusions: We identified the most prevalent pathogenic variant, p.Val290Met, in the NPHS2 gene among Czech adult FSGS patients, which has arisen due to a founder effect in Central Europe. The documented milder course of the disease associated with this variant leads to the underdiagnosis in childhood. We established the histopathological features of the NPHS2-associated adult FSGS cases based on the Columbia classification. This might improve patient stratification and optimize their treatment.

3.
Kidney Int ; 94(6): 1111-1126, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30385042

RESUMO

Insufficient podocyte regeneration after injury is a central pathomechanism of glomerulosclerosis and chronic kidney disease. Podocytes constitutively secrete the chemokine CXCL12, which is known to regulate homing and activation of stem cells; hence we hypothesized a similar effect of CXCL12 on podocyte progenitors. CXCL12 blockade increased podocyte numbers and attenuated proteinuria in mice with Adriamycin-induced nephropathy. Similar studies in lineage-tracing mice revealed enhanced de novo podocyte formation from parietal epithelial cells in the setting of CXCL12 blockade. Super-resolution microscopy documented full integration of these progenitor-derived podocytes into the glomerular filtration barrier, interdigitating with tertiary foot processes of neighboring podocytes. Quantitative 3D analysis revealed that conventional 2D analysis underestimated the numbers of progenitor-derived podocytes. The 3D analysis also demonstrated differences between juxtamedullary and cortical nephrons in both progenitor endowment and Adriamycin-induced podocyte loss, with more robust podocyte regeneration in cortical nephrons with CXCL12 blockade. Finally, we found that delayed CXCL12 inhibition still had protective effects. In vitro studies found that CXCL12 inhibition uncoupled Notch signaling in podocyte progenitors. These data suggest that CXCL12-driven podocyte-progenitor feedback maintains progenitor quiescence during homeostasis, but also limits their intrinsic capacity to regenerate lost podocytes, especially in cortical nephrons. CXCL12 inhibition could be an innovative therapeutic strategy in glomerular disorders.


Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Quimiocina CXCL12/antagonistas & inibidores , Glomerulosclerose Segmentar e Focal/tratamento farmacológico , Regeneração/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Aptâmeros de Nucleotídeos/uso terapêutico , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Doxorrubicina/toxicidade , Retroalimentação Fisiológica/efeitos dos fármacos , Glomerulosclerose Segmentar e Focal/induzido quimicamente , Glomerulosclerose Segmentar e Focal/complicações , Humanos , Imageamento Tridimensional , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Confocal/métodos , Podócitos/efeitos dos fármacos , Podócitos/patologia , Proteinúria/tratamento farmacológico , Proteinúria/etiologia , Células-Tronco/fisiologia , Resultado do Tratamento
4.
Sci Rep ; 7(1): 2775, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28584258

RESUMO

Cathepsin(Cat)-S processing of the invariant chain-MHC-II complex inside antigen presenting cells is a central pathomechanism of autoimmune-diseases. Additionally, Cat-S is released by activated-myeloid cells and was recently described to activate protease-activated-receptor-(PAR)-2 in extracellular compartments. We hypothesized that Cat-S blockade targets both mechanisms and elicits synergistic therapeutic effects on autoimmune tissue injury. MRL-(Fas)lpr mice with spontaneous autoimmune tissue injury were treated with different doses of Cat-S inhibitor RO5459072, mycophenolate mofetil or vehicle. Further, female MRL-(Fas)lpr mice were injected with recombinant Cat-S with/without concomitant Cat-S or PAR-2 blockade. Cat-S blockade dose-dependently reversed aberrant systemic autoimmunity, e.g. plasma cytokines, activation of myeloid cells and hypergammaglobulinemia. Especially IgG autoantibody production was suppressed. Of note (MHC-II-independent) IgM were unaffected by Cat-S blockade while they were suppressed by MMF. Cat-S blockade dose-dependently suppressed immune-complex glomerulonephritis together with a profound and early effect on proteinuria, which was not shared by MMF. In fact, intravenous Cat-S injection induced severe glomerular endothelial injury and albuminuria, which was entirely prevented by Cat-S or PAR-2 blockade. In-vitro studies confirm that Cat-S induces endothelial activation and injury via PAR-2. Therapeutic Cat-S blockade suppresses systemic and peripheral pathomechanisms of autoimmune tissue injury, hence, Cat-S is a promising therapeutic target in lupus nephritis.


Assuntos
Doenças Autoimunes/etiologia , Doenças Autoimunes/patologia , Autoimunidade/efeitos dos fármacos , Catepsinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Animais , Doenças Autoimunes/tratamento farmacológico , Catepsinas/efeitos adversos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Inibidores Enzimáticos/farmacocinética , Feminino , Glomérulos Renais/efeitos dos fármacos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Nefrite Lúpica/tratamento farmacológico , Nefrite Lúpica/etiologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Camundongos , Camundongos Endogâmicos MRL lpr , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptor PAR-2/genética , Receptor PAR-2/metabolismo
5.
Cell Death Dis ; 7(11): e2482, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27882940

RESUMO

Murine double minute-2 (MDM2) is an E3-ubiquitin ligase and the main negative regulator of tumor suppressor gene p53. MDM2 has also a non-redundant function as a modulator of NF-kB signaling. As such it promotes proliferation and inflammation. MDM2 is highly expressed in the unchallenged tubular epithelial cells and we hypothesized that MDM2 is necessary for their survival and homeostasis. MDM2 knockdown by siRNA or by genetic depletion resulted in demise of tubular cells in vitro. This phenotype was completely rescued by concomitant knockdown of p53, thus suggesting p53 dependency. In vivo experiments in the zebrafish model demonstrated that the tubulus cells of the larvae undergo cell death after the knockdown of mdm2. Doxycycline-induced deletion of MDM2 in tubular cell-specific MDM2-knockout mice Pax8rtTa-cre; MDM2f/f caused acute kidney injury with increased plasma creatinine and blood urea nitrogen and sharp decline of glomerular filtration rate. Histological analysis showed massive swelling of renal tubular cells and later their loss and extensive tubular dilation, markedly in proximal tubules. Ultrastructural changes of tubular epithelial cells included swelling of the cytoplasm and mitochondria with the loss of cristae and their transformation in the vacuoles. The pathological phenotype of the tubular cell-specific MDM2-knockout mouse model was completely rescued by co-deletion of p53. Tubular epithelium compensates only partially for the cell loss caused by MDM2 depletion by proliferation of surviving tubular cells, with incomplete MDM2 deletion, but rather mesenchymal healing occurs. We conclude that MDM2 is a non-redundant survival factor for proximal tubular cells by protecting them from spontaneous p53 overexpression-related cell death.


Assuntos
Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/prevenção & controle , Células Epiteliais/patologia , Túbulos Renais Proximais/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Animais , Morte Celular , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Deleção de Genes , Técnicas de Silenciamento de Genes , Larva , Camundongos Knockout , Fenótipo , Proteína Supressora de Tumor p53/metabolismo , Peixe-Zebra
6.
JCI Insight ; 1(17): e87877, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27777973

RESUMO

To derive new insights in diabetic complications, we integrated publicly available human protein-protein interaction (PPI) networks with global metabolic networks using metabolomic data from patients with diabetic nephropathy. We focused on the participating proteins in the network that were computationally predicted to connect the urine metabolites. MDM2 had the highest significant number of PPI connections. As validation, significant downregulation of MDM2 gene expression was found in both glomerular and tubulointerstitial compartments of kidney biopsy tissue from 2 independent cohorts of patients with diabetic nephropathy. In diabetic mice, chemical inhibition of MDM2 with Nutlin-3a led to reduction in the number of podocytes, increased blood urea nitrogen, and increased mortality. Addition of Nutlin-3a decreased WT1+ cells in embryonic kidneys. Both podocyte- and tubule-specific MDM2-knockout mice exhibited severe glomerular and tubular dysfunction, respectively. Interestingly, the only 2 metabolites that were reduced in both podocyte and tubule-specific MDM2-knockout mice were 3-methylcrotonylglycine and uracil, both of which were also reduced in human diabetic kidney disease. Thus, our bioinformatics tool combined with multi-omics studies identified an important functional role for MDM2 in glomeruli and tubules of the diabetic nephropathic kidney and links MDM2 to a reduction in 2 key metabolite biomarkers.


Assuntos
Nefropatias Diabéticas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Biologia de Sistemas , Albuminúria , Animais , Biologia Computacional , Diabetes Mellitus Experimental/metabolismo , Humanos , Glomérulos Renais/fisiopatologia , Túbulos Renais/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Podócitos
7.
Am J Pathol ; 186(6): 1442-53, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27102769

RESUMO

Rapidly progressive glomerulonephritis is characterized by glomerular necroinflammation and crescent formation. Its treatment includes unspecific and toxic agents; therefore, the identification of novel therapeutic targets is required. The E3-ubiquitin ligase murine double minute (MDM)-2 is a nonredundant element of NF-κB signaling and the negative regulator of tumor suppressor gene TP53-mediated cell cycle arrest and cell death. We hypothesized that the MDM2 would drive crescentic glomerulonephritis by NF-κB-dependent glomerular inflammation and by p53-dependent parietal epithelial cell hyperproliferation. Indeed, the pre-emptive MDM2 blockade by nutlin-3a ameliorated all aspects of crescentic glomerulonephritis. MDM2 inhibition had identical protective effects in Trp53-deficient mice, with the exception of crescent formation, which was not influenced by nutlin-3a treatment. In vitro experiments confirmed the contribution of MDM2 for induction of NF-κB-dependent cytokines in murine glomerular endothelial cells and for p53-dependent parietal epithelial cell proliferation. To evaluate MDM2 blockade as a potential therapeutic intervention in rapidly progressive glomerulonephritis, we treated mice with established glomerulonephritis with nutlin-3a. Delayed onset of nutlin-3a treatment was equally protective as the pre-emptive treatment in abrogating crescentic glomerulonephritis. Together, the pathogenic effects of MDM2 are twofold, that is, p53-independent NF-κB activation increasing intraglomerular inflammation and p53-dependent parietal epithelial cell hyperplasia and crescent formation. We therefore propose MDM2 blockade as a potential novel therapeutic strategy in rapidly progressive glomerulonephritis.


Assuntos
Glomerulonefrite/patologia , Imidazóis/farmacologia , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Animais , Western Blotting , Modelos Animais de Doenças , Glomerulonefrite/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/metabolismo
8.
Nat Commun ; 7: 10274, 2016 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-26817517

RESUMO

Crystals cause injury in numerous disorders, and induce inflammation via the NLRP3 inflammasome, however, it remains unclear how crystals induce cell death. Here we report that crystals of calcium oxalate, monosodium urate, calcium pyrophosphate dihydrate and cystine trigger caspase-independent cell death in five different cell types, which is blocked by necrostatin-1. RNA interference for receptor-interacting protein kinase 3 (RIPK3) or mixed lineage kinase domain like (MLKL), two core proteins of the necroptosis pathway, blocks crystal cytotoxicity. Consistent with this, deficiency of RIPK3 or MLKL prevents oxalate crystal-induced acute kidney injury. The related tissue inflammation drives TNF-α-related necroptosis. Also in human oxalate crystal-related acute kidney injury, dying tubular cells stain positive for phosphorylated MLKL. Furthermore, necrostatin-1 and necrosulfonamide, an inhibitor for human MLKL suppress crystal-induced cell death in human renal progenitor cells. Together, TNF-α/TNFR1, RIPK1, RIPK3 and MLKL are molecular targets to limit crystal-induced cytotoxicity, tissue injury and organ failure.


Assuntos
Apoptose , Oxalato de Cálcio/toxicidade , Pirofosfato de Cálcio/toxicidade , Nefropatias/fisiopatologia , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Ácido Úrico/toxicidade , Animais , Oxalato de Cálcio/química , Oxalato de Cálcio/metabolismo , Pirofosfato de Cálcio/química , Pirofosfato de Cálcio/metabolismo , Humanos , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Necrose , Fosforilação , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Ácido Úrico/química , Ácido Úrico/metabolismo
9.
J Am Soc Nephrol ; 27(6): 1635-49, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26567242

RESUMO

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia-induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68(+) intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage-derived circulating PAR2 agonist and mediator of endothelial dysfunction-related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases.


Assuntos
Catepsinas/fisiologia , Angiopatias Diabéticas/etiologia , Células Endoteliais/enzimologia , Receptor PAR-2/metabolismo , Animais , Catepsinas/antagonistas & inibidores , Células Cultivadas , Glomérulos Renais/citologia , Masculino , Camundongos , Microvasos , Prolina/análogos & derivados , Prolina/farmacologia , Urotélio/citologia
10.
Eur J Immunol ; 46(1): 223-9, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26531064

RESUMO

Neutrophil extracellular trap (NET) formation contributes to gout, autoimmune vasculitis, thrombosis, and atherosclerosis. The outside-in signaling pathway triggering NET formation is unknown. Here, we show that the receptor-interacting protein kinase (RIPK)-1-stabilizers necrostatin-1 or necrostatin-1s and the mixed lineage kinase domain-like (MLKL)-inhibitor necrosulfonamide prevent monosodium urate (MSU) crystal- or PMA-induced NET formation in human and mouse neutrophils. These compounds do not affect PMA- or urate crystal-induced production of ROS. Moreover, neutrophils of chronic granulomatous disease patients are shown to lack PMA-induced MLKL phosphorylation. Genetic deficiency of RIPK3 in mice prevents MSU crystal-induced NET formation in vitro and in vivo. Thus, neutrophil death and NET formation may involve the signaling pathway defining necroptosis downstream of ROS production. These data imply that RIPK1, RIPK3, and MLKL could represent molecular targets in gout or other crystallopathies.


Assuntos
Armadilhas Extracelulares/metabolismo , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Transdução de Sinais/imunologia , Animais , Western Blotting , Armadilhas Extracelulares/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fosforilcolina/análogos & derivados , Fosforilcolina/toxicidade , Ácidos Polimetacrílicos/toxicidade , Proteínas Quinases/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Ácido Úrico/toxicidade
11.
Histol Histopathol ; 30(11): 1271-82, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26062755

RESUMO

Murine double minute (MDM)-2 is an intracellular molecule with diverse biological functions. It was first described to limit p53-mediated cell cycle arrest and apoptosis, hence, gain of function mutations are associated with malignancies. This generated a rationale for MDM2 being a potential therapeutic target in cancer therapy. Meanwhile, several additional functions and pathogenic roles of MDM2 have been identified that either enforce therapeutic MDM2 blockade or raise caution about potential side effects. MDM2 is also required for organ development and tissue homeostasis because unopposed p53 activation leads to p53-overactivation-dependent cell death, referred to as podoptosis. Podoptosis is caspase-independent and, therefore, different from apoptosis. The mitogenic role of MDM2 is also needed for wound healing upon tissue injury, while MDM2 inhibition impairs re-epithelialization upon epithelial damage. In addition, MDM2 has p53-independent transcription factor-like effects in nuclear factor-kappa beta (NFκB) activation. Therefore, MDM2 promotes tissue inflammation and MDM2 inhibition has potent anti-inflammatory effects in tissue injury. Here we review the biology of MDM2 in the context of tissue development, homeostasis, and injury and discuss how the divergent roles of MDM2 could be used for certain therapeutic purposes. MDM2 blockade had mostly anti-inflammatory and anti-mitotic effects that can be of additive therapeutic efficacy in inflammatory and hyperproliferative disorders such as certain cancers or lymphoproliferative autoimmunity, such as systemic lupus erythematosus or crescentic glomerulonephritis.


Assuntos
Inflamação/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Regeneração , Animais , Morte Celular , Humanos , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Mutação , NF-kappa B/metabolismo , Neoplasias/genética , Neoplasias/patologia , Organogênese , Proteínas Proto-Oncogênicas c-mdm2/genética , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo
12.
J Am Soc Nephrol ; 26(10): 2399-413, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25644111

RESUMO

Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.


Assuntos
Vasos Sanguíneos/patologia , Armadilhas Extracelulares/fisiologia , Glomerulonefrite/complicações , Histonas/fisiologia , Animais , Bovinos , Armadilhas Extracelulares/efeitos dos fármacos , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/etiologia , Histonas/efeitos dos fármacos , Camundongos , Necrose/etiologia , Índice de Gravidade de Doença
13.
Nephrol Dial Transplant ; 30(10): 1622-30, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25538161

RESUMO

Proper control of the cell cycle is mandatory during homeostasis and disease. The balance of p53 and MDM2 integrates numerous signalling pathways to regulate the cell cycle, which is executed by multiple proteins including the cyclins, cyclin kinases and cyclin kinase inhibitors. Mutations or environmental factors that affect cell cycle control can lead to inappropriate hyperplasia or cancer as well as to cell loss and tissue atrophy. Normal kidney function is maintained largely by post-mitotic quiescent cells in the G0 phase with a low turnover. Early cell cycle activation during kidney injury contributes to cell death via mitotic catastrophe, i.e. death via mitosis, e.g. of cell with significant DNA damage. At later stages, cell cycle entry supports tissue regeneration and functional reconstitution via cell hypertrophy and/or cell proliferation. It is of note that so-called proliferation markers such as Ki67, PCNA or BrdU identify only cell cycle entry without telling whether this results in cell hypertrophy, cell division or mitotic catastrophe. With this in mind, some established concepts on kidney injury and regeneration are to be re-evaluated. Here, we discuss the components and functional roles of p53/MDM2-mediated cell cycle regulation in kidney homeostasis and disease.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Rim/fisiologia , Animais , Humanos
14.
J Am Soc Nephrol ; 26(7): 1513-23, 2015 07.
Artigo em Inglês | MEDLINE | ID: mdl-25349197

RESUMO

Murine double minute-2 (MDM2), an E3 ligase that regulates the cell cycle and inflammation, is highly expressed in podocytes. In podocyte injury, MDM2 drives podocyte loss by mitotic catastrophe, but the function of MDM2 in resting podocytes has not been explored. Here, we investigated the effects of podocyte MDM2 deletion in vitro and in vivo. In vitro, MDM2 knockdown by siRNA caused increased expression of p53 and podocyte death, which was completely rescued by coknockdown of p53. Apoptosis, pyroptosis, pyronecrosis, necroptosis, ferroptosis, and parthanatos were excluded as modes of occurrence for this p53-overactivation-related cell death (here referred to as podoptosis). Podoptosis was associated with cytoplasmic vacuolization, endoplasmic reticulum stress, and dysregulated autophagy (previously described as paraptosis). MDM2 knockdown caused podocyte loss and proteinuria in a zebrafish model, which was consistent with the phenotype of podocyte-specific MDM2-knockout mice that also showed the aforementioned ultrastructual podocyte abnormalities before and during progressive glomerulosclerosis. The phenotype of both animal models was entirely rescued by codeletion of p53. We conclude that MDM2 maintains homeostasis and long-term survival in podocytes by preventing podoptosis, a p53-regulated form of cell death with unspecific features previously classified as paraptosis.


Assuntos
Autofagia/genética , Morte Celular/genética , Genes p53/fisiologia , Glomerulosclerose Segmentar e Focal/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Ativação Transcricional/genética , Análise de Variância , Animais , Western Blotting , Sobrevivência Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Genes p53/genética , Glomerulosclerose Segmentar e Focal/patologia , Glomerulosclerose Segmentar e Focal/fisiopatologia , Homeostase/genética , Imuno-Histoquímica , Testes de Função Renal , Camundongos , Camundongos Knockout , Microscopia Confocal , Podócitos/citologia , Podócitos/fisiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade , Ativação Transcricional/fisiologia , Peixe-Zebra
15.
J Am Soc Nephrol ; 25(5): 978-89, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24459235

RESUMO

AKI involves early Toll-like receptor (TLR)-driven immunopathology, and resolution of inflammation is needed for rapid regeneration of injured tubule cells. Notably, activation of TLRs also has been implicated in epithelial repair. We hypothesized that TLR signaling drives tubule regeneration after acute injury through the induction of certain ILs. Systematic screening in vitro identified IL-22 as a candidate proregeneratory factor in primary tubular cell recovery, and IL-22 deficiency or IL-22 blockade impaired post-ischemic tubular recovery after AKI in mice. Interstitial mononuclear cells, such as dendritic cells and macrophages, were the predominant source of IL-22 secretion, whereas IL-22 receptor was expressed by tubular epithelial cells exclusively. Depleting IL-22-producing cells during the healing phase impaired epithelial recovery, which could be rescued entirely by reconstituting mice with IL-22. In vitro, necrotic tubular cells and oxidative stress induced IL-22 secretion selectively through TLR4. Although TLR4 blockade during the early injury phase prevented tubular necrosis and AKI, TLR4 blockade during the healing phase suppressed IL-22 production and impaired kidney regeneration. Taken together, these results suggest that necrotic cell-derived TLR4 agonists activate intrarenal mononuclear cells to secrete IL-22, which accelerates tubular regeneration and recovery in AKI.


Assuntos
Injúria Renal Aguda/terapia , Interleucinas/biossíntese , Túbulos Renais/patologia , Regeneração/fisiologia , Traumatismo por Reperfusão/terapia , Receptor 4 Toll-Like/fisiologia , Urotélio/patologia , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucinas/metabolismo , Janus Quinases/metabolismo , Túbulos Renais/citologia , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Sistema Fagocitário Mononuclear/metabolismo , Sistema Fagocitário Mononuclear/patologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Fator de Transcrição STAT3/metabolismo , Urotélio/citologia , Interleucina 22
16.
J Pathol ; 230(3): 322-35, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23749457

RESUMO

Podocyte apoptosis as a pathway of podocyte loss is often suspected but rarely detected. To study podocyte apoptosis versus inflammatory forms of podocyte death in vivo, we targeted murine double minute (MDM)-2 for three reasons. First, MDM2 inhibits p53-dependent apoptosis; second, MDM2 facilitates NF-κB signalling; and third, podocytes show strong MDM2 expression. We hypothesized that blocking MDM2 during glomerular injury may trigger p53-mediated podocyte apoptosis, proteinuria, and glomerulosclerosis. Unexpectedly, MDM2 blockade in early adriamycin nephropathy of Balb/c mice had the opposite effect and reduced intra-renal cytokine and chemokine expression, glomerular macrophage and T-cell counts, and plasma creatinine and blood urea nitrogen levels. In cultured podocytes exposed to adriamycin, MDM2 blockade did not trigger podocyte death but induced G2/M arrest to prevent aberrant nuclear divisions and detachment of dying aneuploid podocytes, a feature of mitotic catastrophe in vitro and in vivo. Consistent with these observations, 12 of 164 consecutive human renal biopsies revealed features of podocyte mitotic catastrophe but only in glomerular disorders with proteinuria. Furthermore, delayed MDM2 blockade reduced plasma creatinine levels, blood urea nitrogen, tubular atrophy, interstitial leukocyte numbers, and cytokine expression as well as interstitial fibrosis. Together, MDM2-mediated mitotic catastrophe is a previously unrecognized variant of podocyte loss where MDM2 forces podocytes to complete the cell cycle, which in the absence of cytokinesis leads to podocyte aneuploidy, mitotic catastrophe, and loss by detachment. MDM2 blockade with nutlin-3a could be a novel therapeutic strategy to prevent renal inflammation, podocyte loss, glomerulosclerosis, proteinuria, and progressive kidney disease.


Assuntos
Doxorrubicina/toxicidade , Glomerulonefrite/patologia , Podócitos/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/efeitos dos fármacos , Criança , Progressão da Doença , Células Epiteliais/metabolismo , Feminino , Regulação da Expressão Gênica , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/tratamento farmacológico , Glomerulonefrite/fisiopatologia , Humanos , Imidazóis/farmacologia , Lactente , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Mitose/efeitos dos fármacos , Piperazinas/farmacologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/patologia , Proteinúria , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Adulto Jovem
17.
Nephrol Dial Transplant ; 28(8): 2028-37, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23476038

RESUMO

BACKGROUND: Tubular repair upon injury involves regeneration from either surviving tubular epithelial cells or from their surviving local progenitor cells; hence, compound screening with cell lines may be inadequate. Here, we demonstrate that the renal cell isolation procedure and subsequent outgrowth of tubular cells can mimic the renal injury phase and tubular cell regeneration from whichever surviving renal cells. METHODS: We set up assays to systematically screen and identify mediators of tubular survival and repair. RESULTS: Forty-eight hours after plating total kidney isolates from C57BL/6 mice, 69% of cells survived when prepared from 2-week-old pups, but only 4% of cells from 8-week-old mice, respectively. This poor survival was not modulated by co-incubation with any of 24 cytokines and growth factors, except for the Nrf2 agonist sulforaphane. In addition, only sulforaphane enhanced the regenerative outgrowth of tubular epithelial cells from the mixed population. Furthermore, sulforaphane enhanced wound closure upon scratching tubular epithelial cell monolayers in a dose-dependent manner. This process was associated with the induction of the tested Nrf2 target genes HO-1, NQO1 and murine-double minute 2 (MDM2). MDM2 blockade with nutlin-3a completely blocked the protective effects of sulforaphane on renal cell survival, outgrowth and wound closure. CONCLUSIONS: Together, renal cell isolation is a model of acute kidney injury (AKI). Primary tubular epithelial cell outgrowth represents a model of tubular regeneration. Nrf2 activation can enhance renal cell survival and tubular repair by inducing the cell cycle regulator MDM2.


Assuntos
Injúria Renal Aguda/metabolismo , Células Epiteliais/fisiologia , Túbulos Renais/fisiologia , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Regeneração/fisiologia , Injúria Renal Aguda/genética , Animais , Anticarcinógenos/farmacologia , Western Blotting , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Citometria de Fluxo , Isotiocianatos/farmacologia , Túbulos Renais/citologia , Túbulos Renais/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Regeneração/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfóxidos , Cicatrização/efeitos dos fármacos
18.
J Am Soc Nephrol ; 23(11): 1783-9, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22997256

RESUMO

Uromodulin/Tamm-Horsfall protein is not immunostimulatory in the tubular lumen, but through unknown mechanisms it can activate dendritic cells and promote inflammation in the renal interstitium. Here, we noted that uromodulin isolated from human urine aggregates to large, irregular clumps with a crystal-like ultrastructure. These uromodulin nanoparticles activated isolated human monocytes to express costimulatory molecules and to secrete the mature proinflammatory cytokines, including IL-1ß. Full release of IL-1ß in response to uromodulin depended on priming of pro-IL-1ß expression by Toll-like receptors, TNF-α, or IL-1α. In addition, uromodulin-induced secretion of mature IL-1ß depended on the NLRP3 inflammasome, its linker molecule ASC, and pro-IL-1ß cleavage by caspase-1. Activation of NLRP3 required phagocytosis of uromodulin particles into lysosomes, cathepsin leakage, oxidative stress, and potassium efflux from the cell. Taken together, these data suggest that uromodulin is a NLRP3 agonist handled by antigen-presenting cells as an immunostimulatory nanoparticle. Thus, in the presence of tubular damage that exposes the renal interstitium, uromodulin becomes an endogenous danger signal. The inability of renal parenchymal cells to secrete IL-1ß may explain why uromodulin remains immunologically inert inside the luminal compartment of the urinary tract.


Assuntos
Proteínas de Transporte/imunologia , Imunidade Inata , Inflamassomos/imunologia , Interleucina-1beta/imunologia , Uromodulina/imunologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Células Cultivadas , Técnicas de Silenciamento de Genes , Humanos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/ultraestrutura , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nanopartículas/química , Nanopartículas/ultraestrutura , Multimerização Proteica , RNA Interferente Pequeno/genética , Uromodulina/química , Uromodulina/farmacologia , Uromodulina/ultraestrutura
19.
Kidney Int ; 81(12): 1199-211, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22297670

RESUMO

Murine double minute (MDM)-2, an E3 ubiquitin ligase, promotes cancer cell survival and growth, by degrading the cell cycle regulator p53. Antagonism of MDM2 by the small-molecule cis-imidazoline nutlin analogs is under current study for cancer therapy. To test whether MDM2 also promotes regenerative cell growth, we determined the effects of nutlin-3a on tubule cell healing during postischemic acute kidney injury (AKI). Treatment with nutlin-3a impaired tubular cell regeneration during postischemic AKI in wild-type mice in a p53-dependent manner; however, MDM2 blockade also prevented tubular necrosis by suppressing sterile inflammation during the early postischemic phase. This effect also occurred in p53 knockout mice, indicating a second, proinflammatory, p53-independent role for MDM2 in AKI. In vitro experiments confirmed that MDM2 is required to induce mRNA expression and secretion of NFκB-dependent cytokines upon Toll-like receptor stimulation by enhanced binding of NFκB to cytokine promoter-binding sites. Thus, MDM2 links inflammation and epithelial healing during AKI. These additional biological functions need to be regarded when considering MDM2 inhibition therapy.


Assuntos
Injúria Renal Aguda/enzimologia , Inflamação/enzimologia , Túbulos Renais/enzimologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Traumatismo por Reperfusão/enzimologia , Cicatrização , Injúria Renal Aguda/genética , Injúria Renal Aguda/patologia , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Proliferação de Células , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Imidazóis/administração & dosagem , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Injeções Intraperitoneais , Túbulos Renais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/metabolismo , Necrose , Piperazinas/administração & dosagem , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-mdm2/deficiência , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Mensageiro/metabolismo , Regeneração , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Fatores de Tempo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
20.
Neoplasia ; 14(12): 1097-101, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23308042

RESUMO

Murine double minute-2 (MDM2) is an intracellular molecule with multiple biologic functions. It serves as a negative regulator of p53 and thereby limits cell cycle arrest and apoptosis. Because MDM2 blockade suppresses tumor cell growth in vitro and in vivo, respective MDM2 inhibition is currently evaluated as anti-cancer therapy in clinical trials. However, the anti-proliferative effects of MDM2 inhibition also impair regenerative cell growth upon tissue injury. This was so far documented for tubular repair upon postischemic acute kidney injury and might apply to wound healing responses in general. Furthermore, MDM2 has numerous p53-independent effects. As a new entry, MDM2 was identified to act as a co-transcription factor for nuclear factor-kappa-light-enhancer of activated B cells (NF-κB) at cytokine promoters. This explains the potent anti-inflammatory effects of MDM2 inhibitors in vitro and in vivo. For example, the NF-κB-antagonistic and p53-agonistic activities of MDM2 inhibitors elicit potent therapeutic effects on experimental lymphoproliferative autoimmune disorders such as systemic lupus erythematosus. In this review, we discuss the classic p53-dependent, the recently discovered p53-independent, and the NF-κB-agonistic biologic functions of MDM2. We describe its complex regulatory role on p53 and NF-κB signaling and name areas of research that may help to foresee previously unexpected effects or potential alternative indications of therapeutic MDM2 blockade.


Assuntos
NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Animais , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Humanos , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/antagonistas & inibidores , Cicatrização
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