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1.
Exp Dermatol ; 2021 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-34331820

RESUMO

The mammalian target of rapamycin inhibitor (mTOR-I) Rapamycin, a drug widely used in kidney transplantation, exerts important anti-cancer effects, particularly in Kaposi's Sarcoma (KS), through several biological interactions. In this in vivo and in vitro study, we explored whether the activation of the autophagic pathway through the low-affinity receptor for nerve growth factor, p75NTR , may have a pivotal role in the anti-cancer effect exerted by Rapamycin in S. Our Kimmunohistochemistry results revealed a significant hyper-activation of the autophagic pathway in KS lesions. In vitro experiments on KS cell lines showed that Rapamycin exposure reduced cell viability by increasing the autophagic process, in the absence of apoptosis, through the transcriptional activation of p75NTR via EGR1. Interestingly, p75NTR gene silencing prevented the increase of the autophagic process and the reduction of cell viability. Moreover, p75NTR activation promoted the upregulation of phosphatase and tensin homolog (PTEN), a tumour suppressor that modulates the PI3K/Akt/mTOR pathway. In conclusion, our in vitro data demonstrated, for the first time, that in Kaposi's sarcoma, autophagy triggered by Rapamycin through p75NTR represented a major mechanism by which mTOR inhibitors may induce tumour regression. Additionally, it suggested that p75NTR protein analysis could be proposed as a new potential biomarker to predict response to Rapamycin in kidney transplant recipients affected by Kaposi's sarcoma.

2.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34068941

RESUMO

Diabetic nephropathy (DN) is the most frequent cause of end-stage renal disease. Tubulointerstitial accumulation of lysine 63 (K63)-ubiquitinated (Ub) proteins is involved in the progression of DN fibrosis and correlates with urinary miR-27b-3p downregulation. We explored the renoprotective effect of an inhibitor of K63-Ub (NSC697923), alone or in combination with the ACE-inhibitor ramipril, in vitro and in vivo. Proximal tubular epithelial cells and diabetic DBA/2J mice were treated with NSC697923 and/or ramipril. K63-Ub protein accumulation along with α-SMA, collagen I and III, FSP-1, vimentin, p16INK4A expression, SA-α Gal staining, Sirius Red, and PAS staining were measured. Finally, we measured the urinary albumin to creatinine ratio (uACR), and urinary miR-27b-3p expression in mice. NSC697923, both alone and in association with ramipril, in vitro and in vivo inhibited hyperglycemia-induced epithelial to mesenchymal transition by significantly reducing K63-Ub proteins, α-SMA, collagen I, vimentin, FSP-1 expression, and collagen III along with tubulointerstitial and glomerular fibrosis. Treated mice also showed recovery of urinary miR-27b-3p and restored expression of p16INK4A. Moreover, NSC697923 in combination with ramipril demonstrated a trend in the reduction of uACR. In conclusion, we suggest that selective inhibition of K63-Ub, when combined with the conventional treatment with ACE inhibitors, might represent a novel treatment strategy to prevent the progression of fibrosis and proteinuria in diabetic nephropathy and we propose miR-27b-3p as a biomarker of treatment efficacy.


Assuntos
Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/prevenção & controle , Fibrose/prevenção & controle , Lisina/química , Nitrofuranos/farmacologia , Ramipril/farmacologia , Sulfonas/farmacologia , Ubiquitinação , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Quimioterapia Combinada , Feminino , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Camundongos , Camundongos Endogâmicos DBA
3.
Methods Mol Biol ; 2325: 107-124, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34053054

RESUMO

Tissue microarray (TMA) is a smart technical innovation recently imposed in pathology research. This technology provides a high-throughput analysis of multiple tissues at the same time. The technique allows faster analysis and considerably reducing costs for the staining because many small representative tissue samples from hundreds of different cases are assembled on a single histologic slide. This versatile technique may improve conventional microscopic techniques to detect and characterize cytotoxic T lymphocytes (CTL). Immunohistochemistry (IHC) may be effectively employed in CTL characterization to identify the location and distribution of target antigens in tissues by staining with a specific antibody. The antibody may be conjugated to either a fluorescent or enzymatic label, and the location of the label seen through a microscope approximates the position of the target antigen.This article summarizes the technical aspects of tissue microarray construction and sectioning, advantages, application, and limitations associated with immunohistochemistry and immunofluorescence.


Assuntos
Imunofluorescência/métodos , Imuno-Histoquímica/métodos , Linfócitos T Citotóxicos/metabolismo , Análise Serial de Tecidos/métodos , Anticorpos , Antígenos , Corantes Fluorescentes , Humanos , Inclusão em Parafina/métodos , Coloração e Rotulagem/métodos
4.
Aging (Albany NY) ; 13(8): 10920-10933, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33875620

RESUMO

Pentraxins are a family of evolutionarily conserved pattern recognition molecules with pivotal roles in innate immunity and inflammation, such as opsonization of pathogens during bacterial and viral infections. In particular, the long Pentraxin 3 (PTX3) has been shown to regulate several aspects of vascular and tissue inflammation during solid organ transplantation. Our study investigated the role of PTX3 as possible modulator of Complement activation in a swine model of renal ischemia/reperfusion (I/R) injury. We demonstrated that I/R injury induced early PTX3 deposits at peritubular and glomerular capillary levels. Confocal laser scanning microscopy revealed PTX3 deposits co-localizing with CD31+ endothelial cells. In addition, PTX3 was associated with infiltrating macrophages (CD163), dendritic cells (SWC3a) and myofibroblasts (FSP1). In particular, we demonstrated a significant PTX3-mediated activation of classical (C1q-mediated) and lectin (MBL-mediated) pathways of Complement. Interestingly, PTX3 deposits co-localized with activation of the terminal Complement complex (C5b-9) on endothelial cells, indicating that PTX3-mediated Complement activation occurred mainly at the renal vascular level. In conclusion, these data indicate that PTX3 might be a potential therapeutic target to prevent Complement-induced I/R injury.


Assuntos
Injúria Renal Aguda/imunologia , Proteína C-Reativa/metabolismo , Ativação do Complemento , Rim/irrigação sanguínea , Traumatismo por Reperfusão/imunologia , Componente Amiloide P Sérico/metabolismo , Injúria Renal Aguda/patologia , Animais , Biópsia , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Rim/imunologia , Rim/patologia , Traumatismo por Reperfusão/patologia , Sus scrofa
5.
Front Immunol ; 12: 605212, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868226

RESUMO

Sepsis-induced acute kidney injury (AKI) is a frequent complication in critically ill patients, refractory to conventional treatments. Aberrant activation of innate immune system may affect organ damage with poor prognosis for septic patients. Here, we investigated the efficacy of polymethyl methacrylate membrane (PMMA)-based continuous hemofiltration (CVVH) in modulating systemic and tissue immune activation in a swine model of LPS-induced AKI. After 3 h from LPS infusion, animals underwent to PMMA-CVVH or polysulfone (PS)-CVVH. Renal deposition of terminal complement mediator C5b-9 and of Pentraxin-3 (PTX3) deposits were evaluated on biopsies whereas systemic Complement activation was assessed by ELISA assay. Gene expression profile was performed from isolated peripheral blood mononuclear cells (PBMC) by microarrays and the results validated by Real-time PCR. Endotoxemic pigs presented oliguric AKI with increased tubulo-interstitial infiltrate, extensive collagen deposition, and glomerular thrombi; local PTX-3 and C5b-9 renal deposits and increased serum activation of classical and alternative Complement pathways were found in endotoxemic animals. PMMA-CVVH treatment significantly reduced tissue and systemic Complement activation limiting renal damage and fibrosis. By microarray analysis, we identified 711 and 913 differentially expressed genes with a fold change >2 and a false discovery rate <0.05 in endotoxemic pigs and PMMA-CVVH treated-animals, respectively. The most modulated genes were Granzyme B, Complement Factor B, Complement Component 4 Binding Protein Alpha, IL-12, and SERPINB-1 that were closely related to sepsis-induced immunological process. Our data suggest that PMMA-based CVVH can efficiently modulate immunological dysfunction in LPS-induced AKI.


Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/terapia , Ativação do Complemento/efeitos dos fármacos , Hemofiltração , Lipopolissacarídeos/efeitos adversos , Polimetil Metacrilato/administração & dosagem , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/mortalidade , Animais , Biomarcadores , Proteína C-Reativa/genética , Proteína C-Reativa/metabolismo , Modelos Animais de Doenças , Fibrose , Expressão Gênica , Hemofiltração/efeitos adversos , Hemofiltração/métodos , Humanos , Imuno-Histoquímica , Mediadores da Inflamação , Testes de Função Renal , Diálise Renal , Sepse/complicações , Componente Amiloide P Sérico/genética , Componente Amiloide P Sérico/metabolismo , Suínos , Resultado do Tratamento
7.
Nephrol Dial Transplant ; 36(3): 452-464, 2021 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-33200215

RESUMO

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is the most frequent primary glomerulonephritis. The role of the microbiota and mucosal immunity in the pathogenesis of IgAN remains a key element. To date, the hypothetical relationship between commensal bacteria, elevated tumour necrosis factor (TNF) superfamily member 13 [also known as B-cell activating factor (BAFF)] levels, perturbed homoeostasis of intestinal-activated B cells and intestinal IgA class switch has not been clearly shown in IgAN patients. METHODS: We studied the intestinal-renal axis connections, analysing levels of BAFF, TNF ligand superfamily member 13 (APRIL) and intestinal-activated B cells in IgAN patients, healthy subjects (HSs) and patients with non-IgA glomerulonephritides. RESULTS: IgAN patients had increased serum levels of BAFF cytokine, correlating with higher amounts of five specific microbiota metabolites, and high APRIL cytokine serum levels. We also found that subjects with IgAN have a higher level of circulating gut-homing (CCR9+ ß7 integrin+) regultory B cells, memory B cells and IgA+ memory B cells compared with HSs. Finally, we found that IgAN patients had high levels of both total plasmablasts (PBs) and intestinal-homing PBs. Interestingly, PBs significantly increased in IgAN but not in patients with other glomerulonephritides. CONCLUSIONS: Our results demonstrate a significant difference in the amount of intestinal-activated B lymphocytes between IgAN patients and HSs, confirming the hypothesis of the pathogenic role of intestinal mucosal hyperresponsiveness in IgAN. The intestinal-renal axis plays a crucial role in IgAN and several factors may contribute to its complex pathogenesis and provide an important area of research for novel targeted therapies to modulate progression of the disease.


Assuntos
Linfócitos B/imunologia , Microbioma Gastrointestinal/imunologia , Glomerulonefrite por IGA/complicações , Imunidade nas Mucosas/imunologia , Imunoglobulina A/sangue , Inflamação/patologia , Mucosa Intestinal/imunologia , Adulto , Linfócitos B/metabolismo , Linfócitos B/patologia , Estudos de Casos e Controles , Citocinas/metabolismo , Feminino , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Masculino , Pessoa de Meia-Idade
8.
Am J Transplant ; 21(2): 838-845, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33091234

RESUMO

IgA nephropathy (IgAN) is a frequent cause of chronic kidney disease (CKD) and progressive renal impairment. A native renal biopsy diagnosis of IgAN is a predictor of graft loss, with a relative risk of 47% but it is difficult to predict graft survival and progressive allograft dysfunction in these patients. Deletion of complement factor H-related genes 1 and 3 (delCFHR3-1) has been associated with a decreased risk of developing IgAN on native kidneys, but the impact on the graft in IgAN-transplanted patients is unknown. We hypothesized that delCFHR3-1 is also associated with the processes that influence graft survival in transplant recipients with IgAN and tested whether cellular senescence is involved in mediating graft damage. We found that patients carrying two copies of CFHR1-3 had a worse outcome (P = .000321) and presented increased FHR1 deposits at glomerular and tubulointerstitial level associated with higher expression of the senescence marker p16INK4a (P = .001) and tubulointerstitial fibrosis (P = .005). Interestingly, FHR1 deposits were associated with increased complement activation as demonstrated by C5b-9 deposits. These data support both the role of FHR1 in mediating complement activation and tubular senescence, and suggest the possibility of genotyping delCFHR3-1 to predict graft survival in IgAN-transplanted patients.


Assuntos
Glomerulonefrite por IGA , Transplante de Rim , Senescência Celular , Sobrevivência de Enxerto , Humanos , Rim , Transplante de Rim/efeitos adversos
9.
Sci Rep ; 10(1): 18400, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33110136

RESUMO

Pentraxin 3 (PTX3) is an essential component of the innate immune system and a recognized modulator of Complement cascade. The role of Complement system in the pathogenesis of prostate cancer has been largely underestimated. The aim of our study was to investigate the role of PTX3 as possible modulator of Complement activation in the development of this neoplasia. We performed a single center cohort study; from January 2017 through December 2018, serum and prostate tissue samples were obtained from 620 patients undergoing prostate biopsy. A group of patients with benign prostatic hyperplasia (BPH) underwent a second biopsy within 12-36 months demonstrating the presence of a prostate cancer (Group A, n = 40) or confirming the diagnosis of BPH (Group B, N = 40). We measured tissue PTX3 protein expression together with complement activation by confocal microscopy in the first and second biopsy in group A and B patients. We confirmed that that PTX3 tissue expression in the first biopsy was increased in group A compared to group B patients. C1q deposits were extensively present in group A patients co-localizing and significantly correlating with PTX3 deposits; on the contrary, C1q/PTX3 deposits were negative in group B. Moreover, we found a significantly increased expression of C3a and C5a receptors within resident cells in group A patient. Interestingly, C1q/PTX3 deposits were not associated with activation of the terminal Complement complex C5b-9; moreover, we found a significant increase of Complement inhibitor CD59 in cancer tissue. Our data indicate that PTX3 might play a significant pathogenic role in the development of this neoplasia through recruitment of the early components of Complement cascade with hampered activation of terminal Complement pathway associated with the upregulation of CD59. This alteration might lead to the PTX3-mediated promotion of cellular proliferation, angiogenesis and insensitivity to apoptosis possible leading to cancer cell invasion and migration.


Assuntos
Proteínas de Fase Aguda/metabolismo , Proteína C-Reativa/metabolismo , Ativação do Complemento/fisiologia , Neoplasias da Próstata/metabolismo , Componente Amiloide P Sérico/metabolismo , Estudos de Coortes , Humanos , Masculino
10.
Sci Rep ; 10(1): 15164, 2020 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938960

RESUMO

Primary IgA nephropathy (IgAN) diagnosis is based on IgA-dominant glomerular deposits and histological scoring is done on formalin-fixed paraffin embedded tissue (FFPE) sections using the Oxford classification. Our aim was to use this underexploited resource to extract RNA and identify genes that characterize active (endocapillary-extracapillary proliferations) and chronic (tubulo-interstitial) renal lesions in total renal cortex. RNA was extracted from archival FFPE renal biopsies of 52 IgAN patients, 22 non-IgAN and normal renal tissue of 7 kidney living donors (KLD) as controls. Genome-wide gene expression profiles were obtained and biomarker identification was carried out comparing gene expression signatures a subset of IgAN patients with active (N = 8), and chronic (N = 12) renal lesions versus non-IgAN and KLD. Bioinformatic analysis identified transcripts for active (DEFA4, TNFAIP6, FAR2) and chronic (LTB, CXCL6, ITGAX) renal lesions that were validated by RT-PCR and IHC. Finally, two of them (TNFAIP6 for active and CXCL6 for chronic) were confirmed in the urine of an independent cohort of IgAN patients compared with non-IgAN patients and controls. We have integrated transcriptomics with histomorphological scores, identified specific gene expression changes using the invaluable repository of archival renal biopsies and discovered two urinary biomarkers that may be used for specific clinical decision making.


Assuntos
Perfilação da Expressão Gênica/métodos , Glomerulonefrite por IGA/genética , Glomerulonefrite por IGA/patologia , Rim/metabolismo , Rim/patologia , Adulto , Idoso , Biomarcadores/urina , Biópsia , Estudos de Casos e Controles , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/urina , Quimiocina CXCL6/genética , Quimiocina CXCL6/urina , Doença Crônica , Estudos de Coortes , Feminino , Formaldeído , Glomerulonefrite por IGA/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Fixação de Tecidos
11.
Aging (Albany NY) ; 12(8): 7585-7602, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345771

RESUMO

Pentraxin-3 (PTX3) belongs to the pentraxine family, innate immune regulators involved in angiogenesis, proliferation and immune escape in cancer. Here, we evaluated PTX3 tissue expression and serum levels as biomarkers of clear cell renal cell carcinoma (ccRCC) and analyzed the possible role of complement system activation on tumor site. A 10-year retrospective cohort study including patients undergoing nephrectomy for ccRCC was also performed. PTX3 expression was elevated in both neoplastic renal cell lines and tissues, while it was absent in both normal renal proximal tubular cells (HK2) and normal renal tissues. Analysis of complement system activation on tumor tissues showed the co-expression of PTX3 with C1q, C3aR, C5R1 and CD59, but not with C5b-9 terminal complex. RCC patients showed higher serum PTX3 levels as compared to non-neoplastic patients (p<0.0001). Higher PTX3 serum levels were observed in patients with higher Fuhrman grade (p<0.01), lymph node (p<0.0001), and visceral metastases (p<0.001). Patients with higher PTX3 levels also showed significantly lower survival rates (p=0.002). Our results suggest that expression of PTX3 can affect the immunoflogosis in the ccRCC microenvironment, by activating the classical pathway of CS (C1q) and releasing pro-angiogenic factors (C3a, C5a). The up-regulation of CD59 also inhibits the complement-mediated cellular lysis.


Assuntos
Proteína C-Reativa/genética , Carcinoma de Células Renais/genética , DNA de Neoplasias/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Componente Amiloide P Sérico/genética , Proteínas de Fase Aguda , Biomarcadores Tumorais/metabolismo , Proteína C-Reativa/biossíntese , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Feminino , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Masculino , Prognóstico , Estudos Retrospectivos , Componente Amiloide P Sérico/biossíntese , Microambiente Tumoral
12.
Sci Rep ; 9(1): 11357, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31388051

RESUMO

Diabetic Nephropathy (DN) is a chronic complication of diabetes and the primary cause of end stage renal disease. Differential diagnosis for DN requires invasive histological investigation, thus there is need for non-invasive biomarkers to discriminate among different histological lesions in diabetic patients. With the aim to identify a pattern of differentially expressed miRNAs in kidney biopsies of DN patients, we assayed miRNA expression in kidney biopsies from DN patients, diabetic patients with membranous nephropathy and patients with normal histology. Nine miRNAs were differentially expressed among the three groups, and 2 miRNAs (miR-27b-3p and miR-1228-3p) showed interaction with an ubiquitin-conjugating E2 enzyme variant (UBE2v1). UBE2v1 mediates the formation of lysine 63-linked ubiquitin chains, a mechanism we previously showed as involved in DN kidney fibrosis. Both miRNAs were validated as down-regulated in biopsies and urines of DN patients, possibly affected by DNA methylation. Interestingly, the urinary levels of both miRNAs could also discriminate among different degrees of renal fibrosis. Finally, we showed that the combined urinary expression of both miRNAs was also able to discriminate DN patients from other glomerulonephritides in diabetic patients. In conclusion we identified two miRNAs potentially useful as candidate biomarkers of tubular-interstitial fibrosis in diabetic patients with DN.


Assuntos
Nefropatias Diabéticas/complicações , Fibrose/urina , Nefropatias/urina , Rim/metabolismo , MicroRNAs/urina , Adulto , Idoso , Biomarcadores/urina , Progressão da Doença , Feminino , Fibrose/etiologia , Fibrose/genética , Fibrose/metabolismo , Regulação da Expressão Gênica , Humanos , Nefropatias/etiologia , Nefropatias/genética , Nefropatias/metabolismo , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade
13.
FASEB J ; 33(10): 10753-10766, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31268775

RESUMO

Endothelial dysfunction is a hallmark of LPS-induced acute kidney injury (AKI). Endothelial cells (ECs) acquired a fibroblast-like phenotype and contributed to myofibroblast generation through the endothelial-to-mesenchymal transition (EndMT) process. Of note, human adult renal stem/progenitor cells (ARPCs) enhance the tubular regenerative mechanism during AKI but little is known about their effects on ECs. Following LPS exposure, ECs proliferated, decreased EC markers CD31 and vascular endothelial cadherin, and up-regulated myofibroblast markers, collagen I, and vimentin. The coculture with ARPCs normalized the EC proliferation rate and abrogated the LPS-induced EndMT. The gene expression analysis showed that most of the genes modulated in LPS-stimulated ARPCs belong to cell activation and defense response pathways. We showed that the ARPC-specific antifibrotic effect is exerted by the secretion of CXCL6, SAA4, and BPIFA2 produced after the anaphylatoxin stimulation. Next, we investigated the molecular signaling that underlies the ARPC protective mechanism and found that renal progenitors diverge from differentiated tubular cells and ECs in myeloid differentiation primary response 88-independent pathway activation. Finally, in a swine model of LPS-induced AKI, we observed that activated ARPCs secreted CXCL6, SAA4, and BPIFA2 as a defense response. These data open new perspectives on the treatment of both sepsis- and endotoxemia-induced AKI, suggesting an underestimated role of ARPCs in preventing endothelial dysfunction and novel strategies to protect the endothelial compartment and promote kidney repair.-Sallustio, F., Stasi, A., Curci, C., Divella, C., Picerno, A., Franzin, R., De Palma, G., Rutigliano, M., Lucarelli, G., Battaglia, M., Staffieri, F., Crovace, A., Pertosa, G. B., Castellano, G., Gallone, A., Gesualdo, L. Renal progenitor cells revert LPS-induced endothelial-to-mesenchymal transition by secreting CXCL6, SAA4, and BPIFA2 antiseptic peptides.


Assuntos
Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Células-Tronco Adultas/patologia , Quimiocina CXCL6/metabolismo , Células Endoteliais/patologia , Proteínas e Peptídeos Salivares/metabolismo , Proteína Amiloide A Sérica/metabolismo , Injúria Renal Aguda/genética , Células-Tronco Adultas/efeitos dos fármacos , Células-Tronco Adultas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Lipopolissacarídeos/toxicidade , Fator 88 de Diferenciação Mieloide/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Regeneração/fisiologia , Transdução de Sinais/efeitos dos fármacos , Sus scrofa
14.
Aging (Albany NY) ; 11(13): 4382-4406, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31284268

RESUMO

Epigenetic mechanisms, such as DNA methylation, affect tubular maladaptive response after Acute Kidney Injury (AKI) and accelerate renal aging. Upon ischemia/reperfusion (I/R) injury, Complement activation leads to C5a release that mediates damage; however, little is known about the effect of C5a-C5a Receptor (C5aR) interaction in Renal Tubular Epithelial Cells (RTEC).Through a whole-genome DNA methylation analysis in cultured RTEC, we found that C5a induced aberrant methylation, particularly in regions involved in cell cycle control, DNA damage and Wnt signaling. The most represented genes were BCL9, CYP1B1 and CDK6. C5a stimulation of RTEC led to up-regulation of SA-ß Gal and cell cycle arrest markers such as p53 and p21. C5a increased also IL-6, MCP-1 and CTGF gene expression, consistent with SASP development. In accordance, in a swine model of renal I/R injury, we found the increased expression of Wnt4 and ßcatenin correlating with SA-ß Gal, p21, p16 and IL-6 positivity. Administration of Complement Inhibitor (C1-Inh), antagonized SASP by reducing SA-ß Gal, p21, p16, IL-6 and abrogating Wnt4/ßcatenin activation.Thus, C5a affects the DNA methylation of genes involved in tubular senescence. Targeting epigenetic programs and Complement may offer novels strategies to protect tubular cells from accelerated aging and to counteract progression to Chronic Kidney Disease.


Assuntos
Injúria Renal Aguda/metabolismo , Complemento C5a/fisiologia , Metilação de DNA , Túbulos Renais/metabolismo , Traumatismo por Reperfusão/metabolismo , Injúria Renal Aguda/etiologia , Células Cultivadas , Senescência Celular , Quinase 6 Dependente de Ciclina/metabolismo , Citocromo P-450 CYP1B1/metabolismo , Epigênese Genética , Células Epiteliais/metabolismo , Humanos , Traumatismo por Reperfusão/etiologia , Fatores de Transcrição/metabolismo , Via de Sinalização Wnt
15.
Int J Mol Sci ; 20(15)2019 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-31357597

RESUMO

During sepsis, the increased synthesis of circulating lipopolysaccharide (LPS)-binding protein (LBP) activates LPS/TLR4 signaling in renal resident cells, leading to acute kidney injury (AKI). Pericytes are the major source of myofibroblasts during chronic kidney disease (CKD), but their involvement in AKI is poorly understood. Here, we investigate the occurrence of pericyte-to-myofibroblast trans-differentiation (PMT) in sepsis-induced AKI. In a swine model of sepsis-induced AKI, PMT was detected within 9 h from LPS injection, as evaluated by the reduction of physiologic PDGFRß expression and the dysfunctional α-SMA increase in peritubular pericytes. The therapeutic intervention by citrate-based coupled plasma filtration adsorption (CPFA) significantly reduced LBP, TGF-ß, and endothelin-1 (ET-1) serum levels, and furthermore preserved PDGFRß and decreased α-SMA expression in renal biopsies. In vitro, both LPS and septic sera led to PMT with a significant increase in Collagen I synthesis and α-SMA reorganization in contractile fibers by both SMAD2/3-dependent and -independent TGF-ß signaling. Interestingly, the removal of LBP from septic plasma inhibited PMT. Finally, LPS-stimulated pericytes secreted LBP and TGF-ß and underwent PMT also upon TGF-ß receptor-blocking, indicating the crucial pro-fibrotic role of TLR4 signaling. Our data demonstrate that the selective removal of LBP may represent a therapeutic option to prevent PMT and the development of acute renal fibrosis in sepsis-induced AKI.


Assuntos
Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Proteínas de Fase Aguda/metabolismo , Proteínas de Transporte/metabolismo , Transdiferenciação Celular , Glicoproteínas de Membrana/metabolismo , Miofibroblastos/metabolismo , Pericitos/metabolismo , Receptor 4 Toll-Like/metabolismo , Injúria Renal Aguda/patologia , Animais , Biópsia , Transdiferenciação Celular/genética , Células Cultivadas , Modelos Animais de Doenças , Endotoxinas/efeitos adversos , Fibrose , Imuno-Histoquímica , Modelos Biológicos , Miofibroblastos/citologia , Suínos
16.
Stem Cells Int ; 2019: 6795845, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31089331

RESUMO

Toll-like receptors (TLRs) represent one of the bridges that regulate the cross-talk between the innate and adaptive immune systems. TLRs interact with molecules shared and preserved by the pathogens of origin but also with endogenous molecules (damage/danger-associated molecular patterns (DAMPs)) that derive from injured tissues. This is probably why TLRs have been found to be expressed on several kinds of stem/progenitor cells (SCs). In these cells, the role of TLRs in the regulation of the basal motility, proliferation, differentiation processes, self-renewal, and immunomodulation has been demonstrated. In this review, we analyze the many different functions that the TLRs assume in SCs, pointing out that they can have different effects, depending on the background and on the kind of ligands that they recognize. Moreover, we discuss the TLR involvement in the response of SC to specific tissue damage and in the reparative processes, as well as how the identification of molecules mediating the differential function of TLR signaling could be decisive for the development of new therapeutic strategies. Considering the available studies on TLRs in SCs, here we address the importance of TLRs in sensing an injury by stem/progenitor cells and in determining their behavior and reparative activity, which is dependent on the conditions. Therefore, it could be conceivable that SCs employed in therapy could be potentially exposed to TLR ligands, which might modulate their therapeutic potential in vivo. In this context, to modulate SC proliferation, survival, migration, and differentiation in the pathological environment, we need to better understand the mechanisms of action of TLRs on SCs and learn how to control these receptors and their downstream pathways in a precise way. In this manner, in the future, cell therapy could be improved and made safer.

17.
Front Immunol ; 9: 1002, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29875766

RESUMO

Pericytes are one of the principal sources of scar-forming myofibroblasts in chronic kidneys disease. However, the modulation of pericyte-to-myofibroblast transdifferentiation (PMT) in the early phases of acute kidney injury is poorly understood. Here, we investigated the role of complement in inducing PMT after transplantation. Using a swine model of renal ischemia/reperfusion (I/R) injury, we found the occurrence of PMT after 24 h of I/R injury as demonstrated by reduction of PDGFRß+/NG2+ cells with increase in myofibroblasts marker αSMA. In addition, PMT was associated with significant reduction in peritubular capillary luminal diameter. Treatment by C1-inhibitor (C1-INH) significantly preserved the phenotype of pericytes maintaining microvascular density and capillary lumen area at tubulointerstitial level. In vitro, C5a transdifferentiated human pericytes in myofibroblasts, with increased αSMA expression in stress fibers, collagen I production, and decreased antifibrotic protein Id2. The C5a-induced PMT was driven by extracellular signal-regulated kinases phosphorylation leading to increase in collagen I release that required both non-canonical and canonical TGFß pathways. These results showed that pericytes are a pivotal target of complement activation leading to a profibrotic maladaptive cellular response. Our studies suggest that C1-INH may be a potential therapeutic strategy to counteract the development of PMT and capillary lumen reduction in I/R injury.


Assuntos
Transdiferenciação Celular , Ativação do Complemento , Miofibroblastos/citologia , Pericitos/citologia , Traumatismo por Reperfusão/imunologia , Transdução de Sinais , eIF-2 Quinase/metabolismo , Injúria Renal Aguda/fisiopatologia , Animais , Proteína Inibidora do Complemento C1/farmacologia , Humanos , Rim , Camundongos , Camundongos Endogâmicos C57BL , Suínos
18.
J Allergy Clin Immunol ; 142(3): 883-891, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29729940

RESUMO

BACKGROUND: Hereditary angioedema (HAE) caused by C1-inhibitor deficiency is a lifelong illness characterized by recurrent acute attacks of localized skin or mucosal edema. Activation of the kallikrein/bradykinin pathway at the endothelial cell level has a relevant pathogenetic role in acute HAE attacks. Moreover, other pathways are involved given the variable clinical expression of the disease in different patients. OBJECTIVE: We sought to explore the involvement of other putative genes in edema formation. METHODS: We performed a PBMC microarray gene expression analysis on RNA isolated from patients with HAE during an acute attack and compared them with the transcriptomic profile of the same patients in the remission phase. RESULTS: Gene expression analysis identified 23 genes significantly modulated during acute attacks that are involved primarily in the natural killer cell signaling and leukocyte extravasation signaling pathways. Gene set enrichment analysis showed a significant activation of relevant biological processes, such as response to external stimuli and protein processing (q < 0.05), suggesting involvement of PBMCs during acute HAE attacks. Upregulation of 2 genes, those encoding adrenomedullin and cellular receptor for urokinase plasminogen activator (uPAR), which occurs during an acute attack, was confirmed in PBMCs of 20 additional patients with HAE by using real-time PCR. Finally, in vitro studies demonstrated the involvement of uPAR in the generation of bradykinin and endothelial leakage. CONCLUSIONS: Our study demonstrates the increase in levels of adrenomedullin and uPAR in PBMCs during an acute HAE attack. Activation of these genes usually involved in regulation of vascular tone and in inflammatory response might have a pathogenic role by amplifying bradykinin production and edema formation in patients with HAE.


Assuntos
Adrenomedulina/genética , Angioedemas Hereditários/genética , Ativador de Plasminogênio Tipo Uroquinase/genética , Doença Aguda , Adolescente , Adulto , Idoso , Células Cultivadas , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Células Jurkat , Leucócitos Mononucleares/imunologia , Masculino , Pessoa de Meia-Idade , Transcriptoma
19.
J Mol Med (Berl) ; 96(7): 645-659, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29806072

RESUMO

Diabetic nephropathy patients (DN) are characterized by increased lysine63 ubiquitination (Lys63-Ub) at the tubular level. Autophagy is deregulated under diabetic conditions, even though the molecular mechanisms and the consequences of this alteration need to be elucidated. The aim of this study was to investigate the link between Lys63-Ub and autophagy in DN and the involvement of these two processes in tubular cell fate. Immunohistochemistry of beclin-1, LC3, and p62 on kidney biopsies highlighted increased protein expression of all these autophagic factors at the tubular level in DN compared to other nephritis. Transmission electron microscopy confirmed the presence of diffuse vacuolization and autophago(lyso)somal structures in proximal tubular cells in DN. Accumulation of Lys63-Ub proteins in DN increased in accordance with the tubular damage and was associated to increased LC3 expression both in vivo and in vitro. Hyperglycemia (HG) induced LC3 and p62 protein expression in HK2 cells together with Lys63-ubiquitinated proteins, and the inhibition of HG-induced Lys63-Ub by NSC697923 inhibitor, significantly reduced both LC3 and p62 expression. Moreover, in DN, those tubules expressing LC3 showed increased caspase-3 expression, supporting the hypothesis that deregulated autophagy induces apoptosis of tubular cells. In vitro, we confirmed a tight association between impaired autophagy, Lys63-Ub, and apoptosis since Lys63-Ub inhibition by NSC697923 abrogated HG-induced cell death and LC3 silencing also blocked hyperglycemia-induced caspase-3 activation. Our data suggested that prolonged hyperglycemia in diabetic patients can impair autophagy as a consequence of Lys63-Ub protein accumulation, thus promoting intracellular autophagic vesicles increase, finally leading to tubular cell death in DN. KEY MESSAGES: In vivo autophagy is deregulated in diabetic patients with renal disease (DN). Accumulation of Lys63 ubiquitinated proteins is associated to autophagy deregulation. Accumulation of Lys63 ubiquitinated proteins correlated with apoptosis activation. Lys63 ubiquitination inhibition abrogated hyperglycemia-induced autophagy and apoptosis.


Assuntos
Autofagia , Nefropatias Diabéticas/metabolismo , Hiperglicemia/metabolismo , Lisina/metabolismo , Adulto , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Progressão da Doença , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Imunofluorescência , Expressão Gênica , Inativação Gênica , Humanos , Hiperglicemia/genética , Imuno-Histoquímica , Túbulos Renais/metabolismo , Túbulos Renais/patologia , Túbulos Renais/ultraestrutura , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Ubiquitinação
20.
Sci Rep ; 7(1): 8225, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28811645

RESUMO

Acute kidney injury (AKI) is a public health problem worldwide. Several therapeutic strategies have been made to accelerate recovery and improve renal survival. Recent studies have shown that human adult renal progenitor cells (ARPCs) participate in kidney repair processes, and may be used as a possible treatment to promote regeneration in acute kidney injury. Here, we show that human tubular ARPCs (tARPCs) protect physically injured or chemically damaged renal proximal tubular epithelial cells (RPTECs) by preventing cisplatin-induced apoptosis and enhancing proliferation of survived cells. tARPCs without toll-like receptor 2 (TLR2) expression or TLR2 blocking completely abrogated this regenerative effect. Only tARPCs, and not glomerular ARPCs, were able to induce tubular cell regeneration process and it occurred only after damage detection. Moreover, we have found that ARPCs secreted inhibin-A and decorin following the RPTEC damage and that these secreted factors were directly involved in cell regeneration process. Polysaccharide synthetic vesicles containing these molecules were constructed and co-cultured with cisplatin damaged RPTECs. These synthetic vesicles were not only incorporated into the cells, but they were also able to induce a substantial increase in cell number and viability. The findings of this study increase the knowledge of renal repair processes and may be the first step in the development of new specific therapeutic strategies for renal repair.


Assuntos
Células-Tronco Adultas/metabolismo , Decorina/biossíntese , Células Epiteliais/metabolismo , Inibinas/biossíntese , Túbulos Renais/metabolismo , Regeneração , Receptor 2 Toll-Like/metabolismo , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/metabolismo , Adulto , Células-Tronco Adultas/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Biomarcadores , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Cisplatino/farmacologia , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Humanos
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