Prediction of biomarkers associated with membranous nephropathy: Bioinformatic analysis and experimental validation.

Membranous nephropathy (MN), the most prevalent form of nephrotic syndrome in non-diabetic adults globally, is currently the second most prevalent and fastest-increasing primary glomerular disease in China. Numerous renal disorders are developed partly due to ferroptosis. However, its relationship to the pathogenesis of MN has rarely been investigated in previous studies; actually, ferroptosis is closely linked to the immune microenvironment and inflammatory response, which might affect the entire process of MN development. In this study, we aimed to identify ferroptosis-related genes that are potentially related to immune cell infiltration, which can further contribute to MN pathogenesis. The microarray datasets were downloaded from the Gene Expression Omnibus (GEO) database. Ferroptosis-related differentially expressed genes (FDEGs) were identified, which were further used for functional enrichment analysis. The common genes identified using the Least Absolute Shrinkage and Selection Operator (LASSO) logistic regression algorithm and the support vector machine recursive feature elimination (SVM-RFE) algorithm were used to identify the characteristic genes related to ferroptosis. The feasibility of the 7 genes as a distinguishing factor was assessed using the receiver operating characteristic (ROC) curve, with the area under the curve (AUC) score serving as the evaluation metric. Gene set enrichment analysis (GSEA) and correlation analysis of these genes were further performed. The correlation between the expression of these genes and immune cell infiltration inferred by single sample gene set enrichment analysis (ssGSEA) algorithm was explored. As a result, 7 genes, including NR1D1, YTHDC2, EGR1, ZFP36, RRM2, RELA and PDK4, which were most relevant to immune cell infiltration, were identified to be potential diagnostic genes in MN patients. Next, the signature genes were validated with other GEO datasets. In the subsequent steps, we conducted quantitative real-time fluorescence PCR (qRT-PCR) analysis and immunohistochemistry (IHC) method on the cationic bovine serum albumin (C-BSA) induced membranous nephropathy (MN) rat model and the passive Heymann nephritis (pHN) rat model to examine characteristic genes. Finally, we analysed the mRNA expression patterns of hub genes in MN patients and normal controls using the Nephroseq V5 online platform. In concise terms, our study successfully identified biomarkers specific to MN patients and delved into the potential interplay between these markers and immune cell infiltration. This knowledge bears significance for the diagnosis and prospective treatment strategies for individuals affected by MN.

Identification of hub genes and their correlation with immune infiltrating cells in membranous nephropathy: an integrated bioinformatics analysis.

BACKGROUND: Membranous nephropathy (MN) is a chronic glomerular disease that leads to nephrotic syndrome in adults. The aim of this study was to identify novel biomarkers and immune-related mechanisms in the progression of MN through an integrated bioinformatics approach. METHODS: The microarray data were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between MN and normal samples were identified and analyzed by the Gene Ontology analysis, the Kyoto Encyclopedia of Genes and Genomes analysis and the Gene Set Enrichment Analysis (GSEA) enrichment. Hub The hub genes were screened and identified by the weighted gene co-expression network analysis (WGCNA) and the least absolute shrinkage and selection operator (LASSO) algorithm. The receiver operating characteristic (ROC) curves evaluated the diagnostic value of hub genes. The single-sample GSEA analyzed the infiltration degree of several immune cells and their correlation with the hub genes. RESULTS: We identified a total of 574 DEGs. The enrichment analysis showed that metabolic and immune-related functions and pathways were significantly enriched. Four co-expression modules were obtained using WGCNA. The candidate signature genes were intersected with DEGs and then subjected to the LASSO analysis, obtaining a total of 6 hub genes. The ROC curves indicated that the hub genes were associated with a high diagnostic value. The CD4+ T cells, CD8+ T cells and B cells significantly infiltrated in MN samples and correlated with the hub genes. CONCLUSIONS: We identified six hub genes (ZYX, CD151, N4BP2L2-IT2, TAPBP, FRAS1 and SCARNA9) as novel biomarkers for MN, providing potential targets for the diagnosis and treatment.

Sanqi Qushi Granule Alleviates Proteinuria and Podocyte Damage in NS Rat: A Network Pharmacology Study and in vivo Experimental Validation.

Background: Nephrotic syndrome (NS) and its numerous complications remain the leading causes of morbidity and mortality globally. Sanqi Qushi granule (SQG) is clinically effective in NS. However, its potential mechanisms have yet to be elucidated. Methods: A network pharmacology approach was employed in this study. Based on oral bioavailability and drug-likeness, potential active ingredients were picked out. After acquiring overlapping targets for drug genes and disease-related genes, a component-target-disease network and protein-protein interaction analysis (PPI) were constructed using Cytoscape, followed by GO and KEGG enrichment analyses. Adriamycin was injected into adult male Sprague-Dawley (SD) rats via the tail vein to establish NS model. Kidney histology, 24-hr urinary protein level, creatinine (Cr), blood urea nitrogen (BUN), triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL-C) level were assessed. Western blotting, immunohistochemistry, and TUNEL staining were applied. Results: In total, 144 latent targets in SQG acting on NS were screened by a network pharmacology study, containing AKT, Bax, and Bcl-2. KEGG enrichment analysis suggested that PI3K/AKT pathway was enriched primarily. In vivo validation results revealed that SQG intervention ameliorated urine protein level and podocyte lesions in the NS model. Moreover, SQG therapy significantly inhibited renal cells apoptosis and decreased the ratio of Bax/Bcl-2 protein expression. Moreover, we found that Caspase-3 regulated the PI3K/AKT pathway in NS rats, which mediated the anti-apoptosis effect. Conclusion: By combining network pharmacology with experimental verification in vivo, this work confirmed the treatment efficacy of SQG for NS. SQG protected podocyte from injury and inhibited kidney apoptosis in NS rats via the PI3K/AKT pathway at least partially.

A Network Pharmacology-Based Approach to Investigating the Mechanisms of Fushen Granule Effects on Intestinal Barrier Injury in Chronic Renal Failure.

PURPOSE: Fushen Granule (FSG) is a Chinese medicine prepared by doctors for treating patients with chronic renal failure, which is usually accompanied by gastrointestinal dysfunction. Here, we explore the protective effect of FSG on intestinal barrier injury in chronic renal failure through bioinformatic analysis and experimental verification. METHODS: In this study, information on the components and targets of FSG related to CRF is collected to construct and visualize protein-protein interaction networks and drug-compound-target networks using network pharmacological methods. DAVID is used to conduct gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Then, it is validated by in vitro experiments. In this study, the human intestinal epithelial (T84) cells are used and divided into four groups: control group, model group, FSG low-dose group, and FSG high-dose group. After the experiment, the activity of T84 cells is detected by a MTT assay, and the expressions of tight junction protein ZO-1, claudin-1, nuclear factor erythroid 2-related factor (Nrf2), heme oxygenase-1 (HO-1), malondialdehyde (MDA), and cyclooxygenase-2 (COX-2) are examined by immunofluorescence and/or western blotting. RESULTS: Eighty-six potential chronic renal failure-related targets are identified by FSG; among them, nine core genes are screened. Furthermore, GO enrichment analysis shows that the cancer-related signaling pathway, the PI3K-Akt signaling pathway, the HIF1 signaling pathway, and the TNF signaling pathway may play key roles in the treatment of CRF by FSG. The MTT method showed that FSG is not cytotoxic to uremic toxin-induced injured T84 cells. The results of immunofluorescence and WB indicate that compared with the control group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is decreased and protein expressions level of HO-1, MDA, and COX-2 is increased after urinary toxin treatment. Instead, compared with the model group, protein expressions level of ZO-1, claudin-1, and Nrf2 in T84 cells is increased and protein expressions level of HO-1, MDA, and COX-2 is decreased after FSG treatment. CONCLUSION: FSG had a protective effect on urinary toxin-induced intestinal epithelial barrier injury in chronic renal failure, and its mechanism may be related to the upregulation of Nrf2/HO-1 signal transduction and the inhibition of tissue oxidative stress and inflammatory responses. Screening CRF targets and identifying the corresponding FSG components by network pharmacological methods is a practical strategy to explain the mechanism of FSG in improving gastrointestinal dysfunction in CRF.

Identification of biomarkers and construction of a microRNA­mRNA regulatory network for clear cell renal cell carcinoma using integrated bioinformatics analysis.

With the recent research development, the importance of microRNAs (miRNAs) in renal clear cell carcinoma (CCRCC) has become widely known. The purpose of this study is to screen out the potential biomarkers of renal clear cell carcinoma (CCRCC) by microarray analysis. The miRNA chip (GSE16441) and mRNA chip (GSE66270) related to CCRCC were downloaded from the Gene Expression Omnibus (GEO) database. After data filtering and pretreating, R platform and a series of analysis tools (funrich3.1.3, string, Cytoscape_ 3.2.1, David, etc.) were used to analyze chip data and identify the specific and highly sensitive biomarkers. Finally, by constructing the miRNA -mRNA interaction network, it was determined that five miRNAs (hsa-mir-199a-5p, hsa-mir-199b-5p, hsa-mir-532-3p and hsa-mir-429) and two key genes (ETS1 and hapln1) are significantly related to the overall survival rate of patients.

Efficacy of Chinese Herbal Injections for the Treatment of Primary Nephrotic Syndrome: A Bayesian Network Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Considering the adverse reactions and side effects of immunosuppressive and cytotoxic drugs for the treatment of Primary Nephrotic Syndrome (PNS) and the extensive exploration of Chinese herbal injections (CHIs), systematic evaluation of the efficacy of different CHIs in the treatment of PNS is a key imperative. In this study, we performed a network meta-analysis to investigate the efficacy of CHIs in the treatment of PNS. METHODS: A systematic literature review including studies published from the establishment of each database to May 28, 2020, was conducted in PubMed, the Cochrane Library, Embase, Web of Science, the Chinese Biological Medicine Literature Service System (CBM), the China National Knowledge Infrastructure (CNKI) database, the Chinese Scientific Journal Database (VIP), and the Wanfang Database (WF).Two evaluators independently screened the literature, extracted data and the Cochrane Reviewer's Handbook 5.1 method was used to evaluate the quality of included studies. The differences in efficacy of different CHIs were compared and ranked using Stata 16.0 software. Surface under the cumulative ranking curve (SUCRA) probability values were applied to rank the examined treatments. Clustering analysis was performed to compare the effects of CHIs between two different outcomes. RESULTS: A total of 41 eligible randomized controlled trials involving 2879 patients and nine CHIs were included. Nine CHIs were Xiangdan injection (XDI), Huangqi injection (HQI), Shenkang injection (SKI), Danshen injection (DSI), Yinxingdamo injection (YXI), Dengzhanhuasu injection (DZI), Danhong injection (DHI), Shuxuetong injection (SXI), Chuanxiongqin injection (CXI). The results of the network meta-analysis showed that: with Western medical (WM) treatment as a co-intervention, in terms of improving the total clinical effectiveness and serum albumin level, DHI was the most likely to be the best choice for treatment (SUCRA = 82.2%); YXI had the highest probability of being the best option in terms of reducing 24-h urinary protein excretion (SUCRA = 97.8%); in cholesterol-lowering comparisons, the SUCRA value allows for the most likely to be the best treatment is DZI (SUCRA = 84.5%). SXI was the most effective CHIs in terms of lowering serum triglycerides (SUCRA = 85.6%), whereas on the reducing fibrinogen side, the efficacy of CXI was significant (SUCRA = 67.6%). The result cluster analysis indicated that YXI and DHI were the best interventions with respect to total clinical effectiveness, 24-h urinary protein excretion and serum albumin. CONCLUSIONS: CHIs were found to be superior to WM alone in the treatment of PNS and may be beneficial for patients with PNS. WM+YXI and WM+DHI had the potential to be the best CHI with respect to the total clinical effectiveness, 24-h urinary protein excretion and serum albumin. However, more well-designed randomized controlled trials are still warranted.