肾移植术后肾功能延迟恢复的发病分子机制与诊断标志物研究

移植肾功能延迟恢复(DGF)为肾移植患者常见并发症之一,越来越多的研究开始关注肾移植患者术后DGF发生的新病理生理学机制以及潜在的诊断标志物。本研究对GEO数据库中肾脏移植手术患者的基因表达谱数据进行分析,通过差异表达基因分析发现了多个表达异常的转录因子和免疫相关基因,通过基因编码蛋白之间的相互作用网络分析进一步挖掘了疾病进展过程中的核心调控基因。通过结合加权基因共表达网络分析(WGCNA)和机器学习构建了肾移植术后DGF的预测模型。模型XGBoost的准确率能够达到82.4%,其受试者工作特征曲线下面积(AUC)为0.86,马修斯相关系数(MCC)为0.652,灵敏度(Sensitivity)及特异度(Specificity)则分别为0.789和0.867。对这些获得最优预测效能的特征基因进行检索发现,这些高区分度基因与肾功能密切相关。最后通过比对CMap数据库发现了多个潜在可用于疾病治疗的小分子化合物。本研究对肾移植术后DGF的病理生理学机制进行了多角度探索,为相关疾病的诊断和治疗提供了可靠的理论和实验依据。     

Fifteen hub genes associated with progression and prognosis of clear cell renal cell carcinoma identified by coexpression analysis

Renal cell carcinoma (RCC) is the most common type of renal tumor, and the clear cell renal cell carcinoma (ccRCC) is the most frequent subtype. In this study, our aim is to identify potential biomarkers that could effectively predict the prognosis and progression of ccRCC. First, we used The Cancer Genome Atlas (TCGA) RNA-sequencing (RNA-seq) data of ccRCC to identify 2370 differentially expressed genes (DEGs). Second, the DEGs were used to construct a coexpression network by weighted gene coexpression network analysis (WGCNA). Moreover, we identified the yellow module, which was strongly related to the histologic grade and pathological stage of ccRCC. Then, the functional annotation of the yellow module and single-samples gene-set enrichment analysis of DEGs were performed and mainly enriched in cell cycle. Subsequently, 18 candidate hub genes were screened through WGCNA and protein–protein interaction (PPI) network analysis. After verification of TCGA’s ccRCC data set, Gene Expression Omnibus (GEO) data set (GSE73731) and tissue validation, we finally identified 15 hub genes that can actually predict the progression of ccRCC. In addition, by using survival analysis, we found that patients of ccRCC with high expression of each hub gene were more likely to have poor prognosis than those with low expression. The receiver operating characteristic curve showed that each hub gene could effectively distinguish between localized and advanced ccRCC. In summary, our study indicates that 15 hub genes have great predictive value for the prognosis and progression of ccRCC, and may contribute to the exploration of the pathogenesis of ccRCC.     

CUBN as a novel locus for end-stage renal disease: insights from renal transplantation

Chronic kidney disease (CKD) is a complex disorder. As genome-wide association studies identified cubilin gene CUBN as a locus for albuminuria, and urinary protein loss is a risk factor for progressive CKD, we tested the hypothesis that common genetic variants in CUBN are associated with end-stage renal disease (ESRD) and proteinuria. First, a total of 1142 patients with ESRD, admitted for renal transplantation, and 1186 donors were genotyped for SNPs rs7918972 and rs1801239 (case-control study). The rs7918972 minor allele frequency (MAF) was higher in ESRD patients comparing to kidney donors, implicating an increased risk for ESRD (OR 1.39, p?=?0.0004) in native kidneys. Second, after transplantation recipients were followed for 5.8 [3.8-9.2] years (longitudinal study) documenting ESRD in transplanted kidneys--graft failure (GF). During post-transplant follow-up 92 (9.6%) cases of death-censored GF occurred. Donor rs7918972 MAF, representing genotype of the transplanted kidney, was 16.3% in GF vs 10.7% in cases with functioning graft. Consistently, a multivariate Cox regression analysis showed that donor rs7918972 is a predictor of GF, although statistical significance was not reached (HR 1.53, p?=?0.055). There was no association of recipient rs7918972 with GF. Rs1801239 was not associated with ESRD or GF. In line with an association with the outcome, donor rs7918972 was associated with elevated proteinuria levels cross-sectionally at 1 year after transplantation. Thus, we identified CUBN rs7918972 as a novel risk variant for renal function loss in two independent settings: ESRD in native kidneys and GF in transplanted kidneys.     

Cellular senescence in pretransplant renal biopsies predicts postoperative organ function

Older and marginal donors have been used to meet the shortfall in available organs for renal transplantation. Post-transplant renal function and outcome from these donors are often poorer than chronologically younger donors. Some organs, however, function adequately for many years. We have hypothesized that such organs are biologically younger than poorer performing counterparts. We have tested this hypothesis in a cohort of pre-implantation human renal allograft biopsies ( n  = 75) that have been assayed by real-time polymerase chain reaction for the expression of known markers of cellular damage and biological aging, including CDKN2A, CDKN1A, SIRT2 and POT1. These have been investigated for any associations with traditional factors affecting transplant outcome (donor age, cold ischaemic time) and organ function post-transplant (serum creatinine levels). Linear regression analyses indicated a strong association for serum creatinine with pre-transplant CDKN2A levels ( p  = 0.001) and donor age ( p  = 0.004) at 6 months post-transplant. Both these markers correlated significantly with urinary protein to creatinine ratios ( p  = 0.002 and p  = 0.005 respectively), an informative marker for subsequent graft dysfunction. POT1 expression also showed a significant association with this parameter ( p  = 0.05). Multiple linear regression analyses for CDKN2A and donor age accounted for 24.6% ( p  = 0.001) of observed variability in serum creatinine levels at 6 months and 23.7% ( p  = 0.001) at 1 year post-transplant. Thus, these data indicate that allograft biological age is an important novel prognostic determinant for renal transplant outcome.     

Intravenous renal cell transplantation for rats with acute and chronic renal failure

Acute kidney injury (AKI) and chronic renal failure (CKD) are the most challenging problems in nephrology. Multiple therapies have been attempted but these interventions have minimal effects on the eventual outcomes, and all too often the result is end-stage renal disease (ESRD). The only effective therapy for ESRD is renal transplantation but only a small fraction of patients receive transplants. In this work we introduce a novel approach to transplantation designed to regenerate kidneys afflicted by severe AKI or CKD: intravenous renal cell transplantation (IRCT) with adult rat primary renal cells reprogrammed to express the SAA gene localized and engrafted in kidneys of rat recipients that had severe AKI or CKD. IRCT significantly resolved renal dysfunction and limited kidney damage, inflammation, and fibrosis. Severe CKD was successfully improved by IRCT using kidney cells from donor rats or by renal cell self-donation in a form of autotransplantation. We propose that IRCT with adult primary renal cells reprogrammed to express the SAA gene can be used to effectively treat AKI and CKD.     

应用GSEA和WGCNA方法分析细菌性败血症宿主关键差异基因及意义

【目的】采用生物信息学方法分析公共数据库来源的细菌性败血症患者全血转录组学表达谱,探讨细菌败血症相关的宿主关键差异基因及意义。【方法】基于GEO数据库中GSE80496和GSE72829全血转录组基因数据集,采用GEO2R、基因集富集分析(GSEA)联用加权基因共表达网络分析(WGCNA)筛选细菌性败血症患者相比健康人群显著改变的差异基因,通过R软件对交集基因进行GO功能分析和KEGG富集分析。同时,通过String 11.0和Cytoscape分析枢纽基因,验证枢纽基因在数据集GSE72809(Health组52例,Definedsepsis组52例)全血标本中的表达情况,并探讨婴儿性别、月(胎)龄、出生体重、是否接触抗生素等因素与靶基因表达谱间的关系。【结果】分析GSE80496和GSE72829数据集分别筛选得到932个基因和319个基因,联合WGCNA枢纽模块交集得到与细菌性败血症发病相关的10个枢纽基因(MMP9、ITGAM、CSTD、GAPDH、PGLYRP1、FOLR3、OSCAR、TLR5、IL1RN和TIMP1);GSEA分析获得关键通路(氨基酸糖类-核糖代谢、PPAR信号通路、聚糖生物合成通路、自噬调控通路、补体、凝血因子级联反应、尼古丁和烟酰胺代谢、不饱和脂肪酸生物合成和阿尔兹海默症通路)及生物学过程(类固醇激素分泌、腺苷酸环化酶的激活、细胞外基质降解和金属离子运输)。【结论】本项研究通过GEO2R、GSEA联用WGCNA分析,筛选出与细菌性败血症发病相关的2个枢纽模块、10个枢纽基因以及一些关键信号通路和生物学过程,可为后续深入研究细菌性败血症致病机制奠定理论依据。     

Markers of cellular senescence in zero hour biopsies predict outcome in renal transplantation

Although chronological donor age is the most potent predictor of long-term outcome after renal transplantation, it does not incorporate individual differences of the aging-process itself. We therefore hypothesized that an estimate of biological organ age as derived from markers of cellular senescence in zero hour biopsies would be of higher predictive value. Telomere length and mRNA expression levels of the cell cycle inhibitors CDKN2A (p16INK4a) and CDKN1A (p21WAF1) were assessed in pre-implantation biopsies of 54 patients and the association of these and various other clinical parameters with serum creatinine after 1 year was determined. In a linear regression analysis, CDKN2A turned out to be the best single predictor followed by donor age and telomere length. A multiple linear regression analysis revealed that the combination of CDKN2A values and donor age yielded even higher predictive values for serum creatinine 1 year after transplantation. We conclude that the molecular aging marker CDKN2A in combination with chronological donor age predict renal allograft function after 1 year significantly better than chronological donor age alone.     

Correlation of zinc finger protein 2, a prognostic biomarker,with immune infiltrates in liver cancer

Purpose: The expression and clinical value of zinc finger protein 2 gene (ZIC2) in hepatocellular carcinoma (HCC) were analyzed by mining gene information from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases.Methods: Gene chip data sets were retrieved from GEO and TCGA and screened for differentially expressed genes in HCC. Gene expression profile interaction analysis (GEPIA) and Kaplan–Meier curves were used to analyze the relationship between differentially expressed genes (DEGs) and survival and prognosis in patients with HCC. Moreover, the Genecards database was used to extract ZIC2-related proteins and to analyze the physiological process of protein enrichment. Furthermore, the relationships between ZIC2 gene and tumor cell immune invasion and that between immune cell infiltration and the 5-year survival rate were studied using the tumor immune evaluation resource (TIMER) database.Results: Datasets from GEO and TCGA revealed that ZIC2 was differentially expressed in HCC tissues and normal tissues (P<0.05). High ZIC2 expression was associated with overall survival (OS) and progress-free survival in HCC patients. Overall, 25 ZIC2 related proteins, including Gli3, PRKDC, and rnf180 were identified and protein enrichment analysis indicated these were associated with four types of cell components, six types of cell functions, and eight types of biological processes. ZIC2 was positively correlated with immune infiltration cells in patients with HCC, and higher expression of ZIC2 mRNA CD4+T cells is associated with a better 5-year survival.Conclusion: ZIC2 gene may be used as an immune response marker in liver cancer to predict the prognosis of HCC.     

肾透明细胞癌Caki-1细胞系差异表达基因的生物信息学分析

目的比较肾透明细胞癌Caki-1细胞系与正常肾上皮细胞系ASE-5063中的差异表达基因（DEGs）,寻找潜在的肾透明细胞癌特异性分子标志物。 方法利用GEO数据库自带的GEO2R在线分析工具分析基因芯片GSE78179,将筛选出的DEGs分别导入Metascape、STRING以及Cytoscape进行综合分析并筛选出核心基因。最后使用FunRich等软件对筛选出的核心基因进行GO和KEGG富集分析。 结果共筛选出562个DEGs,其中上调基因345个,下调基因217个。进一步使用MCODE筛选出36个关键基因,GO功能分析发现这些基因与细胞粘附分子活性、趋化因子活性、细胞通讯和信号转导等密切相关;KEGG通路富集结果则表明差异基因主要集中在趋化因子信号通路、TNF信号通路以及NF-κB信号通路等多种与肿瘤相关的通路上。 结论运用生物信息学方法筛选出肾透明细胞癌Caki-1细胞系中DEGs,其中数个核心基因广泛参与多种肿瘤的病理进程,但尚未在肾透明细胞癌有相关研究报道,提示其可能是治疗肾透明细胞癌的潜在靶点。     

Differential immunogenicity and clinical relevance of kidney compartment specific antigens after renal transplantation

To evaluate the pathogenic role of non-HLA antibodies after organ transplantation, 81 unique serum samples from renal transplant patients were analyzed by protein array technology on integrative genomics approach (Li, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4148-53; Higgins, J. P.; et al. Mol. Biol. Cell 2004, 15 (2), 649-56), validated by ELISA, and the results correlated with clinical relevance with time post-transplantation or post-transplant graft function. There was a significant association of de novo non-HLA antibodies with time post-transplantation (n = 1,785) and decline in graft function over the subsequent year (n = 105). There was an enrichment of immunogenic antigens in the renal cortex (p = 0.01) with post-transplant time, and for glomerular specific targets (p = 0.02) with decline in graft function. Two targets with very strong correlation in each category (AGT and SPDYA) were validated by customized ELISA assays in independent patient sera and their localization confirmed by immunohistochemistry. In conclusion, defined profiles of these non-HLA antibodies to renal cortical proteins develop with increasing length of engraftment, and may reflect the increasing recognition of altered localization or exposure of renal tubular and interstitial proteins, affected by advancing chronic nonimmune graft injury. The panel of non-HLA antibodies to glomerular targets is most interesting, as these corresponding antigenic targets may be important pathways in functional graft injury and could provide novel targets for drug design.     

Timing, conditions, and complications of post-operative conception and pregnancy in female renal transplant recipients

The aim of this study was to explore the timing, conditions, and complications of post-operative conception and pregnancy among female renal transplant recipients in China. A cohort of 25 female renal transplant recipients who subsequently had successful pregnancies was randomly selected from eight organ transplantation centers in China. In this cohort, there were 38 post-transplant conceptions and 25 live births. The effects of conception and pregnancy on renal function as well as any effects of transplantation on delivery, prematurity, and maternal and infant health were investigated. Out of 38 conceptions after transplantation, seven ended in spontaneous abortion, six in artificial abortion, and 25 in single births, seven of which were premature (28%). The growth and development of all of the infants were normal. All the 25 received artificial (formula) feeding. Six patients had to return to hemodialysis therapy at 1–41 months after conception due to reduced function of the transplanted kidney. It appears best for female renal transplant recipients to wait at least for 2 years post-transplant before pregnancy. We found no significant effect on fetal growth and development. The incidence of premature births among female renal transplant recipients was high which might have an effect on transplant renal function and maternal health. Breast feeding is not considered suitable for these patients and was therefore not studied.     

SARS-CoV-2病毒感染潜在关键分子生物标志物及免疫浸润特征分析

应用生物信息学方法筛选新型冠状病毒肺炎（corona virus disease 2019,COVID-19）感染的潜在关键分子生物标志物并分析其免疫浸润特征。从GEO数据库下载GSE152418数据集,其中COVID-19患者17例,健康对照17例。用加权基因共表达网络分析（weighted gene co-expression network analysis,WGCNA）方法筛选出COVID-19最相关的模块基因。与差异基因取交集得到共同基因,进行功能及信号通路富集分析,构建蛋白互作网络筛选关键基因,构建关键基因的miRNATF-mRNA调控网络,用CIBERSORT算法预测样本免疫细胞浸润特征。差异分析得到2 049个差异基因。WGCNA分析7个模块中“土耳其蓝色”模块与COVID-19相关性最高（r=0.91,P<0.001）。模块中基因显著性和模块隶属度呈显著正相关（r=0.96,P<0.001）。得到共同基因766个,主要参与有丝分裂、微管结合、阳离子通道活性及卵母细胞减数分裂、细胞衰老等。蛋白互作网络筛选到前10位关键基因分别为CDK1、BUB1、CCNA2...     

Influence of recipient and donor IL-10, TNFA and INFG genotypes on the incidence of acute renal allograft rejection

Objective Transplantation of renal grafts is an established treatment for renal failure in a variety of medical conditions. Acute allograft rejection remains an important cause of morbidity after kidney transplantation, and has been shown to be a crucial determinant of long-term graft function. Although rejection is mediated by recipient lymphocytes, both donor and recipient factors contribute to the local environment that influences the severity of rejection response. Because cytokines are the main components of immune responses, we evaluate single nucleotide polymorphisms (SNP) of several cytokine genes that may influence the production of a given cytokine and therefore the features of immune reactions. Material and Methods The aim of this study was to determine the impact of the cytokine gene polymorphism of pro and anti-inflammatory cytokines on the development of acute allograft rejection, which could be used in pretransplant patient assessment. Three SNPs including IL-10 (−1082 G/A), TNFA (−308 G/A), and INFG (+874 T/A) were analyzed in 46 patients with acute allograft rejection, 54 patients with stable graft function and their kidney donors by PCR-ARMS method. Results We are unable to find statistically significant association between any of the studies polymorphisms and clinical outcomes. Conclusion We have found no evidence to suggest that either recipient or donor cytokines polymorphisms determine the incidence of acute rejection after renal transplantation. Our observation, however, is based on few cases, and this may mask a possible favorable effect. It is recommended that several functionally related genes should be tested in similar studies, since this approach has a higher chance to detect genetic risk factors than the screening of single genetic variants.     

Identification of gene coexpression modules,hub genes,and pathways related to spinal cord injury using integrated bioinformatics methods

Spinal cord injury (SCI) is characterized by dramatic neurons loss and axonal regeneration suppression. The underlying mechanism associated with SCI-induced immune suppression is still unclear. Weighted gene coexpression network analysis (WGCNA) is now widely applied for the identification of the coexpressed modules, hub genes, and pathways associated with clinic traits of diseases. We performed this study to identify hub genes associated with SCI development. Gene Expression Omnibus (GEO) data sets GSE45006 and GSE20907 were downloaded and the significant correlativity and connectivity between them were detected using WGCNA. Three significant consensus modules, including 567 eigengenes, were identified from the master GSE45006 data following the preconditions of approximate scale-free topology for WGCNA. Further bioinformatics analysis showed these eigengenes were involved in inflammatory and immune responses in SCI. Three hub genes Rac2, Itgb2, and Tyrobp and one pathway “natural killer cell-mediated cytotoxicity” were identified following short time-series expression miner, protein-protein interaction network, and functional enrichment analysis. Gradually upregulated expression patterns of Rac2, Itgb2, and Tyrobp genes at 0, 3, 7, and 14 days after SCI were confirmed based on GSE45006 and GSE20907 data set. Finally, we found that Rac2, Itgb2, and Tyrobp genes might take crucial roles in SCI development through the “natural killer cell–mediated cytotoxicity” pathway.     

Differential coexpression networks in bronchiolitis and emphysema phenotypes reveal heterogeneous mechanisms of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with multiple molecular mechanisms. To investigate and contrast the molecular processes differing between bronchiolitis and emphysema phenotypes of COPD, we downloaded the GSE69818 microarray data set from the Gene Expression Omnibus (GEO), which based on lung tissues from 38 patients with emphysema and 32 patients with bronchiolitis. Then, weighted gene coexpression network analysis (WGCNA) and differential coexpression (DiffCoEx) analysis were performed, followed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes enrichment analysis (KEGG) analysis. Modules and hub genes for bronchiolitis and emphysema were identified, and we found that genes in modules linked to neutrophil degranulation, Rho protein signal transduction and B cell receptor signalling were coexpressed in emphysema. DiffCoEx analysis showed that four hub genes (IFT88, CCDC103, MMP10 and Bik) were consistently expressed in emphysema patients; these hub genes were enriched, respectively, for functions of cilium assembly and movement, proteolysis and apoptotic mitochondrial changes. In our re‐analysis of GSE69818, gene expression networks in relation to emphysema deepen insights into the molecular mechanism of COPD and also identify some promising therapeutic targets.     

Identification of Molecular Markers of Delayed Graft Function Based on the Regulation of Biological Ageing

IntroductionDelayed graft function is a prevalent clinical problem in renal transplantation for which there is no objective system to predict occurrence in advance. It can result in a significant increase in the necessity for hospitalisation post-transplant and is a significant risk factor for other post-transplant complications.MethodologyThe importance of microRNAs (miRNAs), a specific subclass of small RNA, have been clearly demonstrated to influence many pathways in health and disease. To investigate the influence of miRNAs on renal allograft performance post-transplant, the expression of a panel of miRNAs in pre-transplant renal biopsies was measured using qPCR. Expression was then related to clinical parameters and outcomes in two independent renal transplant cohorts.ResultsHere we demonstrate, in two independent cohorts of pre-implantation human renal allograft biopsies, that a novel pre-transplant renal performance scoring system (GRPSS), can determine the occurrence of DGF with a high sensitivity (>90%) and specificity (>60%) for donor allografts pre-transplant, using just three senescence associated microRNAs combined with donor age and type of organ donation.ConclusionThese results demonstrate a relationship between pre-transplant microRNA expression levels, cellular biological ageing pathways and clinical outcomes for renal transplantation. They provide for a simple, rapid quantitative molecular pre-transplant assay to determine post-transplant allograft function and scope for future intervention. Furthermore, these results demonstrate the involvement of senescence pathways in ischaemic injury during the organ transplantation process and an indication of accelerated bio-ageing as a consequence of both warm and cold ischaemia.