RESUMO
INTRODUCTION: Accumulating evidence has disclosed that IgA nephropathy (IgAN) could present shortly after the second dose of COVID-19 mRNA vaccine. However, the undying mechanism remains unclear and we aimed to investigate the potential molecular mechanisms. METHODS: We downloaded gene expression datasets of COVID-19 mRNA vaccination (GSE201535) and IgAN (GSE104948). Weighted Gene Co-Expression Network Analysis (WGCNA) was performed to identify co-expression modules related to the second dose of COVID-19 mRNA vaccination and IgAN. Differentially expressed genes (DEGs) were screened, and a transcription factor (TF)-miRNA regulatory network and protein-drug interaction were constructed for the shared genes. RESULTS: WGCNA identified one module associated with the second dose of COVID-19 mRNA vaccine and four modules associated with IgAN. Gene ontology (GO) analyses revealed enrichment of cell cycle-related processes for the COVID-19 mRNA vaccine hub genes and immune effector processes for the IgAN hub genes. We identified 74 DEGs for the second dose of COVID-19 mRNA vaccine and 574 DEGs for IgAN. Intersection analysis with COVID-19 vaccine-related genes led to the identification of two shared genes, TOP2A and CEP55. The TF-miRNA network analysis showed that hsa-miR-144 and ATF1 might regulate the shared hub genes. CONCLUSIONS: This study provides insights into the common pathogenesis of COVID-19 mRNA vaccination and IgAN. The identified pivotal genes may offer new directions for further mechanistic studies of IgAN secondary to COVID-19 mRNA vaccination.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Glomerulonefrite por IGA , Glomerulonefrite por IGA/genética , Humanos , Vacinas contra COVID-19/efeitos adversos , COVID-19/prevenção & controle , COVID-19/complicações , MicroRNAs/genética , Redes Reguladoras de Genes , Vacinas de mRNA , SARS-CoV-2 , Vacinação/efeitos adversosRESUMO
OBJECTIVE: This study aims to explore the association between glomerular mammalian target of rapamycin complex 1 (mTORC1) pathway activation and crescents' degree in lupus nephritis (LN) patients. METHODS: A total of 159 biopsy-proven LN patients were enrolled in this retrospective study. The clinical and pathological data of them were collected at the time of renal biopsy. mTORC1 pathway activation was measured by immunohistochemistry, expressed by the mean optical density (MOD) of p-RPS6 (ser235/236), and multiplexed immunofluorescence. The association of mTORC1 pathway activation with clinico-pathological features especially renal crescentic lesions, and the composite outcomes in LN patients was further analyzed. RESULTS: mTORC1 pathway activation could be detected in the crescentic lesions and was positively correlated with the percentage of crescents (r = 0.479, P < 0.001) in LN patients. Subgroup analysis showed mTORC1 pathway was more activated in patients with cellular or fibrocellular crescentic lesions (P < 0.001), but not fibrous crescentic lesions (P = 0.270). The optimal cutoff value of the MOD of p-RPS6 (ser235/236) was 0.0111299 for predicting the presence of cellular-fibrocellular crescents in >7.39% of the glomeruli by the receiver operating characteristic curve. Cox regression survival analysis showed that mTORC1 pathway activation was an independent risk factor for the worse outcome (defined by composite endpoints of death, end-stage renal disease and a decrease of >30% in eGFR from baseline). CONCLUSION: Activation of mTORC1 pathway was closely associated with the cellular-fibrocellular crescentic lesions and could be a prognostic marker in LN patients.
Assuntos
Falência Renal Crônica , Nefrite Lúpica , Humanos , Nefrite Lúpica/patologia , Estudos Retrospectivos , Glomérulos Renais/patologia , Rim/patologia , Falência Renal Crônica/complicaçõesRESUMO
The current study was initiated to comprehensively evaluate renal NLRP3 inflammasome pathway activation in lupus nephritis (LN) patients and their clinicopathological significances based on a Chinese LN cohort. We found that the expressions of NLRP3, ASC, caspase-1, IL-1ß and IL-18 were all significantly higher in the kidneys of LN patients and were predominantly expressed in glomerular mesangial cells, podocytes, renal tubular epithelial cells and macrophages. The expressions of NLRP3, ASC, caspase-1 and IL-1ß were positively correlated to SLEDAI scores and several renal pathological activity indices, while the expression of NLRP3 was negatively associated with chronicity scores. Moreover, the foot process width was positively correlated with glomerular caspase-1 levels, and several podocyte injury markers were decreased significantly in LN patients with higher caspase-1 expression compared with those with lower expression. Our findings indicated that renal NLRP3 inflammasome was activated in LN patients and correlated with disease activity, which needs further explorations.
Assuntos
Nefrite Lúpica , Humanos , Nefrite Lúpica/patologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Rim/patologia , Caspase 1/metabolismoRESUMO
OBJECTIVE: This study was initiated to evaluate mammalian target of rapamycin (mTOR) activation in renal tissue of LN patients. METHODS: This retrospective study included 187 LN patients, 20 diabetic nephropathy (DN) patients, 10 minimal change disease (MCD) patients and 10 normal controls (NCs). Seven of 187 LN patients had repeated renal biopsies. mTORC1/2 activation was evaluated by immunohistochemistry and multiplexed immunofluorescence. The association of mTORC1/2 activation with the clinicopathologic indices and prognostic outcomes was analysed among 187 LN patients. Proteomics was performed in renal biopsies of 20 LN patients. Proteomics was employed to comprehensively evaluate the impact of mTOR activation on intrarenal gene expression. RESULTS: mTORC1/2 was significantly activated in podocytes, mesangial cells, endothelial cells and tubular epithelial cells of LN patients as compared with those with MCD or NC. The glomerular mTORC1 activation was higher in LN patients compared with DN patients. mTORC1, but not mTORC2, activation strongly correlated with serum albumin, complement C3, proteinuria and the following pathological biomarkers of LN: crescent formation, interstitial inflammation and fibrosis. Moreover, mTORC1 activation was identified as a prognostic marker in LN patients. Bioinformatic analyses of proteomics and immunohistochemical data unveiled increased complement activation, antigen presentation and phagocytosis in LN patients with mTORC1 activation. CONCLUSION: Renal mTORC1 activation could be a biomarker to reveal disease activity and predict clinical prognosis in LN patients.
Assuntos
Nefrite Lúpica , Biomarcadores/metabolismo , Células Endoteliais/metabolismo , Humanos , Rim/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Prognóstico , Estudos RetrospectivosRESUMO
The cyclic GMP-AMP synthase (cGAS), upon cytosolic DNA stimulation, catalyzes the formation of the second messenger 2'3'-cGAMP, which then binds to stimulator of interferon genes (STING) and activates downstream signaling. It remains to be elucidated how the cGAS enzymatic activity is modulated dynamically. Here, we reported that the ER ubiquitin ligase RNF185 interacted with cGAS during HSV-1 infection. Ectopic-expression or knockdown of RNF185 respectively enhanced or impaired the IRF3-responsive gene expression. Mechanistically, RNF185 specifically catalyzed the K27-linked poly-ubiquitination of cGAS, which promoted its enzymatic activity. Additionally, Systemic Lupus Erythematosus (SLE) patients displayed elevated expression of RNF185 mRNA. Collectively, this study uncovers RNF185 as the first E3 ubiquitin ligase of cGAS, shedding light on the regulation of cGAS activity in innate immune responses.
Assuntos
Imunidade Inata/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Proteínas Mitocondriais/imunologia , Nucleotidiltransferases/imunologia , Ubiquitina-Proteína Ligases/imunologia , Adolescente , Adulto , Células Cultivadas , Feminino , Herpes Simples/imunologia , Herpesvirus Humano 1 , Humanos , Immunoblotting , Imunoprecipitação , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , Adulto JovemRESUMO
A1CF (apobec-1 complementation factor) acts as a component of the apolipoprotein-B messenger RNA editing complex. Previous researches mainly focused on its post-transcriptional cytidine to uridine RNA editing. However, few study reported its role in progression of breast carcinoma cells. Wound healing assay and flow cytometry were applied to detect the migration and apoptosis; western blot, real-time polymerase chain reaction, and dual-luciferase assays were applied to investigate the potential regulation mechanism of A1CF-mediated cell migration and apoptosis. Knockdown of A1CF decreased cell migration and enhanced cell apoptosis in MCF7 cells in vitro. Western blot analysis showed that knockdown of A1CF decreased Dickkopf1 but increased c-Myc and ß-catenin expression, and overexpression of A1CF can get opposite results. Knockdown of Dickkopf1 in A1CF-overexpressed cells decreased cell migration and enhanced cell apoptosis compared with A1CF-overexpressed cells. Luciferase-fused 3' untranslated region of human Dickkopf1 activity was highly upregulated in A1CF-overexpressed MCF7 cells, but this upregulation can be inhibited by mutating conserved binding motifs of Dickkopf1 3' untranslated region. A1CF played a crucial role in cell migration and survival through affecting 3' untranslated region of Dickkopf1 to upregulate its expression in MCF7 cells.
Assuntos
Desaminase APOBEC-1/genética , Neoplasias da Mama/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Ligação a RNA/genética , Regiões 3' não Traduzidas , Apoptose/genética , Neoplasias da Mama/patologia , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Células MCF-7 , Ligação Proteica , Edição de RNA/genética , beta Catenina/genéticaRESUMO
The transforming growth factor-ß (TGFß) family signaling pathways play an important role in regulatory cellular networks and exert specific effects on developmental programs during embryo development. However, the function of TGFß signaling pathways on the early kidney development remains unclear. In this work, we aim to detect the underlying role of TGFß type II receptor (TßRII) in vitro, which has a similar expression pattern as the crucial regulator Six2 during early kidney development. Firstly, the 5-ethynyl-2'-deoxyuridine (EdU) assay showed knock down of TßRII significantly decreased the proliferation ratio of metanephric mesenchyme (MM) cells. Additionally, real-time Polymerase Chain Reaction (PCR) and Western blot together with immunofluorescence determined that the mRNA and protein levels of Six2 declined after TßRII knock down. Also, Six2 was observed to be able to partially rescue the proliferation phenotype caused by the depletion of TßRII. Moreover, bioinformatics analysis and luciferase assay indicated Smad3 could transcriptionally target Six2. Further, the EdU assay showed that Smad3 could also rescue the inhibition of proliferation caused by the knock down of TßRII. Taken together, these findings delineate the important function of the TGFß signaling pathway in the early development of kidney and TßRII was shown to be able to promote the expression of Six2 through Smad3 mediating transcriptional regulation and in turn activate the proliferation of MM cells.
Assuntos
Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Proliferação de Células , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Humanos , Rim/embriologia , Rim/metabolismo , Proteínas do Tecido Nervoso/genética , Organogênese/genética , Fenótipo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/deficiência , Receptores de Fatores de Crescimento Transformadores beta/genética , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Apobec-1 complementation factor (A1CF) is a heterogeneous nuclear ribonuceloprotein (hnRNP) and mediates apolipoprotein-B mRNA editing. A1CF can promote the regeneration of the liver by post-transcriptionally stabilizing Interleukin-6 (IL-6) mRNA. It also contains two transcriptional variants-A1CF64 and A1CF65, distinguished by the appearance of a 24-nucleotide motif which contributes to the corresponding eight-amino acid motif of EIYMNVPV. For the first time, we demonstrated that the EIYMNVPV motif was essential for A1CF nucleus localization, A1CF deficient of the EIYMNVPV motif, A1CF (-8aa) showed cytoplasm distribution. More importantly, we found that A1CF (-8aa), but not its full-length counterpart, can promote proliferation of MDA-MB-231 cells accompanied with increased level of IL-6 mRNA. Furthermore, silencing of IL-6 attenuated A1CF (-8aa)-induced proliferation in MDA-MB-231 cells. In conclusion, notably, these findings suggest that A1CF (-8aa) promoted proliferation of MDA-MB-231 cells in vitro viewing IL-6 as a target. Thus, the EIYMNVPV motif could be developed as a potential target for basal-like breast cancer therapy.
Assuntos
Núcleo Celular/metabolismo , Interleucina-6/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Regulação para Cima , Motivos de Aminoácidos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citoplasma/metabolismo , Cães , Humanos , Células Madin Darby de Rim Canino , Proteínas de Ligação a RNA/genéticaRESUMO
Apobec-1 complementation factor (A1CF) is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, which participates in site-specific posttranscriptional RNA editing of apolipoprotein B (apoB) transcript. The posttranscriptional editing of apoB mRNA by A1CF in the small intestine is required for lipid absorption. Apart from the intestine, A1CF mRNA is also reported to be highly expressed in the kidneys. However, it is remained unknown about the functions of A1CF in the kidneys. The aim of this paper is to explore the potential functions of A1CF in the kidneys. Our results demonstrated that in C57BL/6 mice A1CF was weakly expressed in embryonic kidneys from E15.5dpc while strongly expressed in mature kidneys after birth, and it mainly existed in the tubules of inner cortex. More importantly, we identified A1CF negatively regulated the process of epithelial-mesenchymal transition (EMT) in kidney tubular epithelial cells. Our results found ectopic expression of A1CF up-regulated the epithelial markers E-cadherin, and down-regulated the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in NRK52e cells. In addition, knockdown of A1CF enhanced EMT contrary to the overexpression effect. Notably, the two A1CF variants led to the similar trend in the EMT process. Taken together, these data suggest that A1CF may be an antagonistic factor to the EMT process of kidney tubular epithelial cells.
Assuntos
Movimento Celular , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Túbulos Renais Proximais/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular , Células Epiteliais/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas/genética , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
Nephron progenitor cells surround around the ureteric bud tips (UB) and inductively interact with the UB to originate nephrons, the basic units of renal function. This process is determined by the internal balance between self-renewal and consumption of the nephron progenitor cells, which is depending on the complicated regulation networks. It has been reported that Zeb1 regulates the proliferation of mesenchymal cells in mouse embryos. However, the role of Zeb1 in nephrons generation is not clear, especially in metanephric mesenchyme (MM). Here, we detected cell proliferation, apoptosis and migration in MM cells by EdU assay, flow cytometry assay and wound healing assay, respectively. Meanwhile, Western and RT-PCR were used to measure the expression level of Zeb1 and Six2 in MM cells and developing kidney. Besides, the dual-luciferase assay was conducted to study the molecular relationship between Zeb1 and Six2. We found that knock-down of Zeb1 decreased cell proliferation, migration and promoted cell apoptosis in MM cells and Zeb1 overexpression leaded to the opposite data. Western-blot and RT-PCR results showed that knock-down of Zeb1 decreased the expression of Six2 in MM cells and Zeb1 overexpression contributed to the opposite results. Similarly, Zeb1 promoted Six2 promoter reporter activity in luciferase assays. However, double knock-down of Zeb1 and Six2 did not enhance the apoptosis of MM cells compared with control cells. Nevertheless, double silence of Zeb1 and Six2 repressed cell proliferation. In addition, we also found that Zeb1 and Six2 had an identical pattern in distinct developing phases of embryonic kidney. These results indicated that there may exist a complicated regulation network between Six2 and Zeb1. Together, we demonstrate Zeb1 promotes proliferation and apoptosis and inhibits the migration of MM cells, in association with Six2.
Assuntos
Apoptose , Movimento Celular , Proliferação de Células , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sequência Conservada , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Proteínas de Homeodomínio/metabolismo , Humanos , Rim/crescimento & desenvolvimento , Mesoderma/citologia , Camundongos , Regiões Promotoras Genéticas , Ligação Proteica , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The metanephric mesenchyme (MM) cells are a subset of kidney progenitor cells and play an essential role in mesenchymal-epithelial transition (MET), the key step of nephron generation. Six2, a biological marker related to Wnt signaling pathway, promotes the proliferation, inhibits the apoptosis and maintains the un-differentiation of MM cells. Besides, LiCl is an activator of Wnt signaling pathway. However, the role of LiCl in cellular regulation of MM cells remains unclear, and the relationship between LiCl and Six2 in this process is also little known. Here, we performed EdU assay and flow cytometry assay to, respectively, detect the proliferation and apoptosis of MM cells treated with LiCl of increasing dosages. In addition, reverse transcription-PCR (RT-PCR) and Western-blot were conducted to measure the expression of Six2 and some maker genes of Wnt and bone-morphogenetic-protein (BMP) signaling pathway. Furthermore, luciferase assay was also carried out to detect the transcriptional regulation of Six2. Then we found LiCl promoted MM cell proliferation at low-concentration (10, 20, 30, and 40 mM). The expression of Six2 was dose-dependently increased in low-concentration (10, 20, 30, and 40 mM) at both mRNA and protein level. In addition, both of cell proliferation and Six2 expression in MM cells declined when dosage reached high-concentration (50 mM). However, Six2 knock-down converted the proliferation reduction at 50 mM. Furthermore, Six2 deficiency increased the apoptosis of MM cells, compared with negative control cells at relative LiCl concentration. However, the abnormal rise of apoptosis at 30 mM of LiCl concentration implies that it might be the reduction of GSK3ß that increased cell apoptosis. Together, these demonstrate that LiCl can induce the proliferation and apoptosis of MM cells coordinating with Six2.
Assuntos
Apoptose/genética , Proliferação de Células/genética , Proteínas de Homeodomínio/metabolismo , Cloreto de Lítio/farmacologia , Fatores de Transcrição/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Camundongos , Fatores de Transcrição/genética , Via de Sinalização WntRESUMO
Accumulating evidence demonstrated that miRNAs are highly involved in kidney fibrosis and Epithelial-Eesenchymal Transition (EMT), however, the mechanisms of miRNAs in kidney fibrosis are poorly understood. In this work, we identified that miR542-3p could promote EMT through down-regulating bone morphogenetic protein 7 (BMP7) expression by targeting BMP7 3'UTR. Firstly, real-time PCR results showed that miR542-3p was significantly up-regulated in kidney fibrosis in vitro and in vivo. Moreover, Western blot results demonstrated that miR542-3p may promote EMT in the NRK52e cell line. In addition, we confirmed that BMP7, which played a crucial role in anti-kidney fibrosis and suppressed the progression of EMT, was a target of miR542-3p through Dual-Luciferase reporter assay, as did Western blot analysis. The effects of miR542-3p on regulating EMT could also be suppressed by transiently overexpressing BMP7 in NRK52e cells. Taken together, miR542-3p may be a critical mediator of the induction of EMT via directly targeting BMP7.
Assuntos
Proteína Morfogenética Óssea 7/genética , Transição Epitelial-Mesenquimal/genética , MicroRNAs/genética , Interferência de RNA , Animais , Sítios de Ligação , Proteína Morfogenética Óssea 7/química , Linhagem Celular , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica , Humanos , Nefropatias/genética , Nefropatias/patologia , Camundongos , MicroRNAs/química , RNA Mensageiro/química , RNA Mensageiro/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
MicroRNAs (miRNAs) possess an important regulating effect among numerous renal diseases, while their functions in the process of epithelial-to-mesenchymal transition (EMT) after podocyte injury remain unclear. The purpose of our study is to identify the potential functions of miR-30a in EMT of podocytes and explore the underlying mechanisms of miR-30a in the impaired podocytes. The results revealed that downregulation of miR-30a in podocyte injury animal models and patients, highly induced the mesenchymal markers of EMT including Collagen I, Fibronectin and Snail. Furthermore, overexpression of miR-30a enhances epithelial markers (E-cadherin) but diminished mesenchymal markers (Collagen I, Fibronectin and Snail) in podocytes. In addition, we established miR-30a target NFATc3, an important transcription factor of Non-canonical Wnt signaling pathway. More importantly, our findings demonstrated that the augmentation of miR-30a level in podocytes inhibits the nuclear translocation of NFATc3 to protect cytoskeleton disorder or rearrangement. In summary, we uncovered the protective function of miR30a targeting NFATc3 in the regulation of podocyte injury response to EMT.
Assuntos
Transição Epitelial-Mesenquimal/fisiologia , MicroRNAs/biossíntese , Fatores de Transcrição NFATC/biossíntese , Podócitos/metabolismo , Transporte Ativo do Núcleo Celular/genética , Animais , Caderinas/biossíntese , Linhagem Celular , Colágeno Tipo I/biossíntese , Regulação para Baixo , Transição Epitelial-Mesenquimal/genética , Fibronectinas/biossíntese , Regulação Neoplásica da Expressão Gênica , Glomerulosclerose Segmentar e Focal/patologia , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Fatores de Transcrição NFATC/metabolismo , Fatores de Transcrição da Família Snail , Fatores de Transcrição/biossíntese , Via de Sinalização Wnt/fisiologiaRESUMO
Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2'-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3' untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development.
Assuntos
Apoptose , Proteínas de Homeodomínio/metabolismo , Rim/citologia , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas , Animais , Linhagem Celular , Proliferação de Células , Biologia Computacional , Regulação para Baixo , Proteínas de Homeodomínio/genética , Rim/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Néfrons/citologia , Néfrons/metabolismo , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
MicroRNAs (miRNAs) are small non-coding RNAs that down-regulate gene expression by binding to target mRNA for cleavage or translational repression, and play important regulatory roles in renal development. Despite increasing genes have been predicted to be miRNA targets by bioinformatic analysis during kidney development, few of them have been verified by experiment. The objective of our study is to identify the miRNAs targeting Six2, a critical transcription factor that maintains the mesenchymal progenitor pool via self-renewal (proliferation) during renal development. We initially analyzed the 3'UTR of Six2 and found 37 binding sites targeted by 50 putative miRNAs in the 3'UTR of Six2. Among the 50 miRNAs, miR-181b is the miRNAs predicted by the three used websites. In our study, the results of luciferase reporter assay, realtime-PCR and Western blot demonstrated that miR-181b directly targeted on the 3'UTR of Six2 and down-regulate the expression of Six2 at mRNA and protein levels. Furthermore, EdU proliferation assay along with the Six2 rescue strategy showed that miR-181b suppresses the proliferation of metanephric mesenchymal by targeting Six2 in part. In our research, we concluded that by targeting the transcription factor gene Six2, miR-181b inhibits the proliferation of metanephric mesenchymal cells in vitro and might play an important role in the formation of nephrons.
Assuntos
Proteínas de Homeodomínio/metabolismo , Células-Tronco Mesenquimais/citologia , MicroRNAs/metabolismo , Néfrons/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Proliferação de Células , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , MicroRNAs/genética , Dados de Sequência Molecular , Néfrons/citologia , RNA Mensageiro/metabolismo , Fatores de Transcrição/genéticaRESUMO
Since a 25% mortality rate occurred in critical Coronavirus disease 2019 (COVID-19) patients, investigating the potential drivers remains to be important. Here, the authors applied Weighted Gene Co-expression Network Analysis to identify the potential drivers in the blood samples of multiple COVID-19 expression profiles. The authors found that the darkslateblue module was significantly correlated with critical COVID-19, and Gene Ontology analysis indicated terms associated with the inflammation pathway and apoptotic process. The authors intersected differentially expressed genes, Maximal Clique Centrality calculated hub genes, and COVID-19 related genes in the Genecards dataset, and two genes, toll-like receptor 5 (TLR5) and acyl-CoA synthetase long chain family member 1 (ACSL1), were screened out. The Gene Set Enrichment Analysis further supports their core role in the inflammatory pathway. Furthermore, the cell-type identification by estimating relative subsets of RNA transcript demonstrated that TLR5 and ACSL1 were associated with neutrophil enrichment in critical COVID-19 patients. Collectively, the aurthors identified two hub genes that were strongly correlated with critical COVID-19. These may help clarify the pathogenesis and assist the immunotherapy development.
Assuntos
COVID-19 , Receptor 5 Toll-Like , Humanos , Apoptose , COVID-19/genética , Família , Perfilação da Expressão GênicaRESUMO
OBJECTIVE: This study was initiated to evaluate the mammalian target of the rapamycin (mTOR) signalling pathway involved in renal endothelial-podocyte crosstalk in patients with lupus nephritis (LN). METHODS: We compared the kidney protein expression patterns of 10 patients with LN with severe endothelial-podocyte injury and 3 patients with non-severe endothelial-podocyte injury on formalin-fixed paraffin-embedded kidney tissues using label-free liquid chromatography-mass spectrometry for quantitative proteomics analysis. Podocyte injury was graded by foot process width (FPW). The severe group was referred to patients with both glomerular endocapillary hypercellularity and FPW >1240 nm. The non-severe group included patients with normal endothelial capillaries and FPW in the range of 619~1240 nm. Gene Ontology (GO) enrichment analyses were performed based on the protein intensity levels of differentially expressed proteins in each patient. An enriched mTOR pathway was selected, and the activation of mTOR complexes in renal biopsied specimens was further verified in 176 patients with LN. RESULTS: Compared with those of the non-severe group, 230 proteins were upregulated and 54 proteins were downregulated in the severe group. Furthermore, GO enrichment analysis showed enrichment in the 'positive regulation of mTOR signalling' pathway. The glomerular activation of mTOR complex 1 (mTORC1) was significantly increased in the severe group compared with the non-severe group (p=0.034), and mTORC1 was located in podocytes and glomerular endothelial cells. Glomerular activation of mTORC1 was positively correlated with endocapillary hypercellularity (r=0.289, p<0.001) and significantly increased in patients with both endocapillary hypercellularity and FPW >1240 nm (p<0.001). CONCLUSIONS: Glomerular mTORC1 was highly activated in patients with both glomerular endocapillary hypercellularity and podocyte injury, which might be involved in podocytes to endothelial cells communication in lupus nephritis.
Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Podócitos , Humanos , Podócitos/metabolismo , Nefrite Lúpica/patologia , Células Endoteliais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
More recent studies suggested that metabolic disorders could contribute to the pathogenesis of systemic lupus erythematosus (SLE) and lupus nephritis (LN). The present work aimed at identifying metabolic reprogramming in the kidney of lupus nephritis via proteomics and investigating the potential regulatory mechanism. The proteomic studies on the renal biopsies revealed that the pentose phosphate pathway (PPP) was significantly enriched in the kidneys of LN patients compared with normal controls (NCs). Immunohistochemical stanning of glucose-6-phosphate dehydrogenase (G6PD), the key rate-limiting enzyme of PPP, verify the results of proteomics. We found that G6PD was highly expressed in the kidneys of LN patients and correlated with several clinicopathological indices. The univariate Cox regression analysis (HR, 95%CI, 1.877 (1.059-3.328), P = 0.031) and Kaplan-Meier (KM) analysis (P = 0.028) suggested that high G6PD expression in the tubulointerstitial area was a risk factor for worse prognosis. Moreover, the Gene set enrichment analysis (GSEA) demonstrated that the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway ranked first in the kidneys of LN patients with high G6PD expression and G6PD was co-localized with mTORC1 activation in the tubule. Immunoglobulin G (IgG) isolated from LN patients significantly activated the mTORC1 pathway and increased G6PD expression, G6PD activity, NADPH production, NADPH oxidase 2 (NOX2) expression, reactive oxygen species (ROS) production, and cell apoptosis in tubule cells in vitro. The above phenotypes were partially rescued after the addition of rapamycin or knock-down of G6PD. Overall, our study suggested that renal G6PD expression was associated with the overall enhanced disease activity and worse renal prognosis. mTORC1 activation might be involved in IgG-LN-induced tubular damage via PPP.
Assuntos
Nefrite Lúpica , Via de Pentose Fosfato , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Imunoglobulina G , Nefrite Lúpica/genética , Nefrite Lúpica/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , ProteômicaRESUMO
OBJECTIVE: Proteomic approach was applied to identify candidate biomarkers of chronicity in patients with proliferative lupus nephritis (LN), and their clinicopathological significance and prognostic values were investigated. METHODS: This study recruited 10 patients with proliferative LN and 6 normal controls (NCs) with proteomic data to compare protein expression profiles, 58 patients with proliferative LN and 10 NCs to verify proteomic data by immunohistochemistry, and 14 patients with proliferative LN with urine samples to evaluate the urinary expression of the biomarker by western blot assay. The composite endpoints included end-stage renal disease and ≥50% reduction from baseline estimated glomerular filtration rate (eGFR). RESULTS: Proteomics detected 48 proteins upregulated in the group with chronicity index (CI) ≥1 compared with the CI=0 and NC groups. Further pathway analysis was enriched in 'other glycan degradation'. Neuraminidase 1 (NEU1), the most predominant protein in the pathway of other glycan degradation, was highly expressed in the kidney of patients with proliferative LN and could co-localise with podocyte, mesangial cells, endothelial cells and tubule cells. NEU1 expression in the tubulointerstitium area was significantly higher in the CI ≥1 group compared with the CI=0 and NC groups. Moreover, NEU1 expression was significantly correlated with serum creatinine value, eGFR and CI scores, respectively. Urinary NEU1 excretion in the CI ≥1 group was higher than in the CI=0 group and was also positively correlated with CI scores. Furthermore, the high expression of renal NEU1 was identified as an independent risk factor for renal prognosis by multivariate Cox regression analysis (HR, 6.462 (95% CI 1.025 to 40.732), p=0.047). CONCLUSIONS: Renal NEU1 expression was associated with pathological CI scores and renal outcomes in patients with proliferative LN.
Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , Biomarcadores , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Rim/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/diagnóstico , Neuraminidase/metabolismo , ProteômicaRESUMO
Pregnancy associated atypical hemolytic uremic syndrome (p-aHUS) is a disease with a triad of hemolytic anemia, thrombocytopenia and acute renal failure, which might be attributed to the uncontrolled complement activation. Herein, we sequenced a postpartum-aHUS patient and found the two missense variants of CD46, a novel mutation (c.403G > C, p.G135R) from her father and a once reported mutation (c.293C > T, p.T98I) without expressional and functional tests from her mother. The G135R mutation caused a significantly reduced membrane expression of CD46 in peripheral blood lymphocyte and renal cells. The T98I mutation caused a mild decrease membrane expression of CD46 in peripheral blood lymphocyte cells. Moreover, the expressed G135R protein was in precursor form, indicating that this mutant was retained intracellularly. The C3b binding ability of T98I mutant was slightly decreased while the C4b binding ability is not significantly changed. The cofactor ability of the two mutants for factor I in the degradation of C3b was demonstrated to be impaired. This study reported the first case of a four-generation postpartum-aHUS pedigree with isolated CD46 variants and the detailed disease progression, treatment, and prognosis provided more meaningful information for the understanding the disease.