# Decoding the Mechanism behind the Pathogenesis of the Focal Segmental Glomerulosclerosis

Authors: Xiao Zhu; Liping Tang; Jingxin Mao; Yasir Hameed; Jingyu Zhang; Ning Li; Danny Wu; Yongmei Huang; Chen Li
Journal: Computational and Mathematical Methods in Medicine
 (2022)

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## Abstract

Focal segmental glomerulosclerosis (FSGS) is a chronic glomerular disease associated with podocyte injury which is named after the pathologic features of the kidney. The aim of this study is to decode the key changes in gene expression and regulatory network involved in the formation of FSGS. Integrated network analysis included Gene Expression Omnibus (GEO) datasets to identify differentially expressed genes (DEGs) between FSGS patients and healthy donors. Bioinformatics analysis was used to identify the roles of the DEGs and included the development of protein-protein interaction (PPI) networks, Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and the key modules were assured. The expression levels of DEGs were validated using the additional dataset. Eventually, transcription factors and ceRNA networks were established to illuminate the regulatory relationships in the formation of FSGS. 1130 DEGs including 475 upregulated genes and 655 downregulated genes with functional enrichment analysis were determined. Further analysis uncovered that the validated hub genes were defined as candidate genes, including Complement C3a Receptor 1 (C3AR1), C-C Motif Chemokine Receptor 1(CCR1), C-X3-C Motif Chemokine Ligand 1 (CX3CL1), Melatonin Receptor 1A (MTNR1A), and Purinergic Receptor P2Y13 (P2RY13). More importantly, we identified transcription factors and mRNA-miRNA-lncRNA regulatory networks associated with the candidate genes. The candidate genes and regulatory networks discovered in this study can help to comprehend the molecular mechanism of FSGS and supply potential targets for the diagnosis and therapy of FSGS.


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## Body

## 1. Introduction

Focal segmental glomerulosclerosis (FSGS) is a common glomerular disease, accounting for 40% of adult cases and 20% of children, with the major cause of the steroid-resistant nephrotic syndrome and end-stage renal disease [1]. The incidence of the disease has gradually increased in recent years and the prognosis is relatively poor, with 50% of patients gradually progressing to end-stage renal disease within 5 to 10 years, accounting for about 20% of dialysis patients [2]. In 1970, the international children’s kidney disease research collaboration formally proposed FSGS as a separate clinicopathological syndrome [3]. The main pathological manifestations of FSGS are focal and segmental sclerosis of the affected glomeruli, corresponding tubular atrophy, and interstitial fibrosis [4]. FSGS is clinically characterized by varying degrees of proteinuria, which may be accompanied by nephrotic syndromes such as microscopic hematuria, hypertension, or impaired tubular function, and is prone to complications such as infection and deep vein thrombosis [5]. The pathogenesis of FSGS is not fully understood and is currently thought to be caused by a range of podocyte injury factors, including circulating factor-mediated podocyte injury; podocyte gene mutations; viral infections such as human immunodeficiency virus, Epstein-Barr virus, and cytomegalovirus; nephrotoxic drug injuries such as heroin, alpha interferon, rapamycin, and calcium-regulated phosphatase inhibitors; and dysplastic factors such as very low birth weight, congenital nephrogenic unitopia with compensatory hypertrophy, and unilateral renal hypoplasia [6]. In recent years, there has been an increasing trend in renal biopsy detection rates of FSGS reported around the world, and the incidence of FSGS varies by region worldwide [7]. In the Americas, FSGS has become a major pathological type of glomerular disease; in the Middle East, South Asia, and Europe, the prevalence is high; and in other parts of Asia, the prevalence is increasing year by year, although it is not a major pathological type [8].The noncoding RNAs (ncRNAs) serve as functional regulators that are transcribed from DNA but not translated into proteins [9]. The miRNAs, circRNAs, and lncRNAs are involved in the ncRNA epigenetic regulation. Generally, ncRNAs function to regulate gene expression at the transcriptional and posttranscriptional level. In the last decade, ncRNA has drawn more and more consideration in the chronic kidney disease from both bench and clinic sides [10]. Previous studies have disclosed the emerging roles of ncRNA and related message RNA (mRNA) in the development of various kidney diseases, including chronic kidney disease [11]. Chen et al. revealed that retinoic acid receptor responder protein 1, which is mainly restricted to podocytes, upregulates in the glomerular of the chronic nephrosis patients. Inversely, lower Rarres1 improved Adriamycin-induced nephropathy in the inducible RARRES1 knockdown mice [12]. By using a rodent FSGS model induced by Adriamycin, Qi et al. suggested that miR-150 inhibitor exerts protective effect on the glomerular via ameliorating renal fibrosis and inflammation [13]. The ncRNAs also serve as a diagnostic biomarker for FSGS [14] and can value the progression of chronic kidney disease [15]. Nevertheless, few studies have clarified systematically the regulatory network and mechanism in the pathogenesis of focal segmental glomerulosclerosis.For the clear knowledge of the biomarkers, we screened microarray data of mRNAs in glomeruli samples from FSGS patients and healthy donors in the current study. After identification and validation, hub different expression genes (DFGs), transcription factors (TFs), and competing endogenous RNA (ceRNA) network were reconstructed, respectively, which were exploited to illustrate the regulatory mechanism. Our output discriminated FSGS-related genes with high credibility in the patients and empowered a more comprehensive approach to identify diagnosis and therapeutic targets for FSGS.


## 2. Materials and Methods

### 2.1. Data Processing and Screening of Differentially Expressed Genes (DEGs)

The overall study design is illustrated in Figure1. The expression profiling datasets GSE108109, GSE104066, and GSE104948, organized from Homo sapiens, were obtained from the publicly available Gene Expression Omnibus (GEO) [16]. These datasets were based on the GPL19983 [HuGene-2_1-st] Affymetrix Human Gene 2.1 ST Array [HuGene21st_Hs_ENTREZG_19.0.0] platform or GPL22945 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array [CDF: Brainarray HGU133Plus2_Hs_ENTREZG_v19] platform (Table 1). The interactive web tool GEO2R is employed to screen of differentially expressed genes (DEGs) from selected datasets (GSE108109 and GSE104066) individually [17]. The DEGs between FSGS samples and living donor samples under the following threshold were retained, an adjusted P value < 0.05 and a ∣logfoldchange∣>1, as the mRNA were decoded for the following analysis.Figure 1
The workflow of the study design.Table 1
The summary of included microarray sets.
SeriesPlatformSample sizeFSGSGlomeruliSpeciesFSGSLiving donorGSE108109 [74]GPL1998311130636Homo sapiensGSE104066 [75]GPL199837648654Homo sapiensGSE104948 [74]GPL2294571101626Homo sapiens


### 2.2. DEG Functional and Pathway Enrichment Analysis

Gene Ontology (GO) function [18] and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment [19] analysis of overlapping genes were analyzed by utilizing the Metascape, an online enrichment analysis tool for gene annotation [20]. Three categorized enrichments, biological process, cellular component, molecular function, are involved in the Gene Ontology (GO) functional enrichment. The adjusted P<0.05 and an enriched gene count>5 were set as criteria for significance for the identification of GO terms and KEGG pathways, with the top 20 terms visualized.


### 2.3. Integration of protein-protein interaction (PPI) network and identification of the most significant modules and hub genes

The Search Tool for the Retrieval of Interacting Genes (STRING) is a web tool to predict interactions between the proteins of candidate genes [21]. The shared DEGs were mapped to STRING to evaluate the interaction, and the PPI pairs that confidence score of v≥0.9 were considered significant. Cytoscape software was used to visualize and construct PPI network [22]. The hub genes were identified by CytoHubba [23] with 5 chosen methods, degree method (Deg), density of maximum neighborhood component (DMNC), edge percolated component (EPC), maximum neighborhood component (MNC), and maximal clique centrality (MCC). To investigate more specific regulatory relationship of proteins, the molecular complex detection (MCODE) [24] was carried out to screen the crucial clustering modules in the PPI network with the default setting.


### 2.4. Validation of Hub Genes by Separated Datasets

To verify the robustness of hub genes, a separated dataset GSE104948 was utilized to validate the differential expression of these hub genes. GraphPad Prism software was used to calculate and generate bar plots.


### 2.5. Integration of Validated Hub Gene-Transcription Factor Network

Transcription factors (TF) are essential for the regulation of gene expression and are, as a consequence, found in all living organisms. NetworkAnalyst is an integrated platform for TFs-gene interaction in numerous species to identify the vital TFs with validated hub genes [25]. In addition, the ENCODE database that produced the TFs-gene network is included in the NetworkAnalyst. The validated hub gene-TF network was also visualized by Cytoscape.


### 2.6. The miRNA Prediction and Validated Hub Gene-miRNA Network Construction

The modulations between the miRNAs and 5 validated hub genes of FSGS were built by Cytoscape via 5 miRNA databases, namely, TargetScan [26], DIANA-microT [27], miRDB [28], miRWalk [29], and miRmap [30]. The overlapping miRNAs were involved in more than 3 databases selected as the predicted results.


### 2.7. The Associated lncRNA Prediction and ceRNA Network Construction

According to the regulatory relationship, lncRNAs can act as miRNA sponges, affecting their regulatory effect on mRNAs. The interactions between lncRNAs and miRNAs that are related to 5 validated hub genes were predicted by the starBase database [31]. Afterward, based on the acquired lncRNA-miRNA pairs, the competing endogenous RNA (ceRNA) network was mapped by bonding lncRNA-miRNA-mRNA pairs and visualized via the Cytoscape.


### 2.8. Statistical Analysis

SPSS 18.0 software was employed to perform statistical analysis. The significant differences between the two groups were evaluated by Student’st-test. Data are expressed as mean±S.D. The P value < 0.05 was considered as significant. All authors take full responsibility for the integrity of the data.


## 2.1. Data Processing and Screening of Differentially Expressed Genes (DEGs)

The overall study design is illustrated in Figure1. The expression profiling datasets GSE108109, GSE104066, and GSE104948, organized from Homo sapiens, were obtained from the publicly available Gene Expression Omnibus (GEO) [16]. These datasets were based on the GPL19983 [HuGene-2_1-st] Affymetrix Human Gene 2.1 ST Array [HuGene21st_Hs_ENTREZG_19.0.0] platform or GPL22945 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array [CDF: Brainarray HGU133Plus2_Hs_ENTREZG_v19] platform (Table 1). The interactive web tool GEO2R is employed to screen of differentially expressed genes (DEGs) from selected datasets (GSE108109 and GSE104066) individually [17]. The DEGs between FSGS samples and living donor samples under the following threshold were retained, an adjusted P value < 0.05 and a ∣logfoldchange∣>1, as the mRNA were decoded for the following analysis.Figure 1
The workflow of the study design.Table 1
The summary of included microarray sets.
SeriesPlatformSample sizeFSGSGlomeruliSpeciesFSGSLiving donorGSE108109 [74]GPL1998311130636Homo sapiensGSE104066 [75]GPL199837648654Homo sapiensGSE104948 [74]GPL2294571101626Homo sapiens


## 2.2. DEG Functional and Pathway Enrichment Analysis

Gene Ontology (GO) function [18] and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment [19] analysis of overlapping genes were analyzed by utilizing the Metascape, an online enrichment analysis tool for gene annotation [20]. Three categorized enrichments, biological process, cellular component, molecular function, are involved in the Gene Ontology (GO) functional enrichment. The adjusted P<0.05 and an enriched gene count>5 were set as criteria for significance for the identification of GO terms and KEGG pathways, with the top 20 terms visualized.


## 2.3. Integration of protein-protein interaction (PPI) network and identification of the most significant modules and hub genes

The Search Tool for the Retrieval of Interacting Genes (STRING) is a web tool to predict interactions between the proteins of candidate genes [21]. The shared DEGs were mapped to STRING to evaluate the interaction, and the PPI pairs that confidence score of v≥0.9 were considered significant. Cytoscape software was used to visualize and construct PPI network [22]. The hub genes were identified by CytoHubba [23] with 5 chosen methods, degree method (Deg), density of maximum neighborhood component (DMNC), edge percolated component (EPC), maximum neighborhood component (MNC), and maximal clique centrality (MCC). To investigate more specific regulatory relationship of proteins, the molecular complex detection (MCODE) [24] was carried out to screen the crucial clustering modules in the PPI network with the default setting.


## 2.4. Validation of Hub Genes by Separated Datasets

To verify the robustness of hub genes, a separated dataset GSE104948 was utilized to validate the differential expression of these hub genes. GraphPad Prism software was used to calculate and generate bar plots.


## 2.5. Integration of Validated Hub Gene-Transcription Factor Network

Transcription factors (TF) are essential for the regulation of gene expression and are, as a consequence, found in all living organisms. NetworkAnalyst is an integrated platform for TFs-gene interaction in numerous species to identify the vital TFs with validated hub genes [25]. In addition, the ENCODE database that produced the TFs-gene network is included in the NetworkAnalyst. The validated hub gene-TF network was also visualized by Cytoscape.


## 2.6. The miRNA Prediction and Validated Hub Gene-miRNA Network Construction

The modulations between the miRNAs and 5 validated hub genes of FSGS were built by Cytoscape via 5 miRNA databases, namely, TargetScan [26], DIANA-microT [27], miRDB [28], miRWalk [29], and miRmap [30]. The overlapping miRNAs were involved in more than 3 databases selected as the predicted results.


## 2.7. The Associated lncRNA Prediction and ceRNA Network Construction

According to the regulatory relationship, lncRNAs can act as miRNA sponges, affecting their regulatory effect on mRNAs. The interactions between lncRNAs and miRNAs that are related to 5 validated hub genes were predicted by the starBase database [31]. Afterward, based on the acquired lncRNA-miRNA pairs, the competing endogenous RNA (ceRNA) network was mapped by bonding lncRNA-miRNA-mRNA pairs and visualized via the Cytoscape.


## 2.8. Statistical Analysis

SPSS 18.0 software was employed to perform statistical analysis. The significant differences between the two groups were evaluated by Student’st-test. Data are expressed as mean±S.D. The P value < 0.05 was considered as significant. All authors take full responsibility for the integrity of the data.


## 3. Results

### 3.1. DEGs between the FSGS Patients and Living Donors

DEGs in the glomeruli tissues of patients with FSGS were identified compared with healthy donors. In total, it is reported in Figure2(a), 624 upregulated and 764 downregulated genes were identified between FSGS patients and healthy donors in the GSE108109. Meanwhile, 494 upregulated and 702 downregulated genes were identified between FSGS patients and healthy donors in the GSE104066. Moreover, the top DEGs could discriminate between the patients with FSGS and the healthy donors in Figure 2(b). The intersection of upregulated genes or downregulated genes from two datasets (GSE108109 and GSE104066) is shown in the Venn diagram (Figure 2(c)); among these overlapping genes, 475 genes were upregulated genes and 655 genes were downregulated genes, and these consistently changed mRNAs were selected as overlapped genes for the next further analysis.Figure 2
DEGs between FSGS patients and healthy donors from GSE104066 and GSE108109, respectively. (a) Volcano plots. (b) Heatmaps. (c) Venn diagrams indicate overlap of upregulated genes and downregulated DEGs, respectively.
(a)(b)(c)


### 3.2. GO and KEGG Annotations of DEGs

The pathogenesis of FSGS is complicated, and comprehending the functions of the DEGs can indicate the novel bench studies and clinic treatments. Each part of GO enrichments is displayed in Figures3(a)–3(c) (upregulated DEGs) and Figures 3(e)–3(g) (downregulated DEGs). For biological process (BP) enrichment analysis, the results showed that the upregulated DEGs strongly took part in the vasculature development, blood vessel development, positive regulation of cellular component movement, positive regulation of cell motility, and positive regulation of cell migration (Figure 3(a)). The downregulated genes were strongly involved in monocarboxylic acid metabolic process, alpha-amino acid metabolic process, cellular amino acid metabolic process, organic acid catabolic process, and alpha-amino acid catabolic process (Figure 3(e)). For cell component (CC) enrichment analysis, the present study showed that the upregulated DEGs were mainly involved in extracellular matrix, external encapsulating structure, side of membrane, collagen-containing extracellular matrix, and endocytic vesicle (Figure 3(b)). The downregulated DEGs mainly revolved in apical plasma membrane, apical part of the cell, brush border membrane, basolateral plasma membrane, and basal plasma membrane (Figure 3(f)). In addition, in the enrichment analysis of molecular function (MF), upregulated DEGs are significantly enriched in cell adhesion molecule binding, integrin binding, growth factor binding, protein homodimerization activity, and actin binding (Figure 3(c)). For downregulated DEGs, they significantly take part in sodium ion transmembrane transporter activity, solute-sodium symporter activity, butyrate-CoA ligase activity, secondary active transmembrane transporter activity, and bitter taste receptor activity (Figure 3(g)).Figure 3
The functional and pathway enrichment analysis of the overlap DEGs between the FSGS patients and healthy donors. (a) Molecular function, (b) biological process, (c) cellular component, and (d) KEGG of overlap upregulated genes. (e) Molecular function, (f) biological process, (g) cellular component, and (h) KEGG of overlap downregulated genes.
(a)(b)(c)(d)(e)(f)(g)(h)Moreover, the KEGG pathway enrichment analysis by upregulated DEGs and downregulated DEGs was listed, respectively. Among the pathways, AGE-RAGE signaling pathway in diabetic complications, cytokine-cytokine receptor interaction, Epstein-Barr virus infection, chemokine signaling pathway, and TNF signaling pathway are involved in the mechanism of upregulated DEGs (Figure3(d)). On the side, downregulated DEGs were mainly enriched in drug metabolism-cytochrome P450, retinol metabolism, drug metabolism, metabolism of xenobiotics by cytochrome P450, and taste transduction (Figure 3(h)).


### 3.3. PPI Network Analysis, Most Significant Modules and Hub Gene Identification for Suggesting Therapy

To illustrate the PPI network of these overlapped DEGs, the analysis was constructed through the STRING platform and was visualized by Cytoscape (Figure4). We totally found 820 paired interactions and 175 upregulated and 80 downregulated genes involved in this network (Supplementary file 1-2).Figure 4
The PPI network of overlap DEGs between the FSGS patients and healthy donors.The subnetwork of genes acts crucial roles in the whole integrated networks. Module analysis on the PPI network through the molecular complex detection (MCODE) plugin revealed top 10 significant clusters, which are shown in Figure5(a). Cluster 1 with the highest cluster score of 22 includes Apelin Receptor (APLNR), Complement C3a Receptor 1 (C3AR1), Complement C5a Receptor 1 (C5AR1), C-C Motif Chemokine Ligand 5 (CCL5), C-C Motif Chemokine Receptor 1 (CCR1), C-X3-C Motif Chemokine Ligand 1 (CX3CL1), C-X-C Motif Chemokine Ligand 16 (CXCL16), G Protein Subunit Gamma 2 (GNG2), G Protein Subunit Gamma 7 (GNG7), Kininogen 1 (KNG1), Melatonin Receptor 1A (MTNR1A), Purinergic Receptor P2Y13 (P2RY13), Prostaglandin E Receptor 3 (PTGER3), Sphingosine-1-Phosphate Receptor 4 (S1PR4), and bitter taste receptor family (TAS2R10, TAS2R14, TAS2R19, TAS2R20, TAS2R3, TAS2R31, TAS2R4, and TAS2R5). Additionally, the overlapped DEGs were imported into CytoHubba plugin of Cytoscape, which was used to screen out top 20 crucial genes through 5 different algorithms, including degree method (Deg), density of maximum neighborhood component (DMNC), edge percolated component (EPC), maximum neighborhood component (MNC), and maximal clique centrality (MCC). Subsequently, 14 common genes were selected by overlapping the 20 genes, as shown in Table 2; these 14 common genes are also involved in the gene cluster 1. To learn the tissues that these 14 genes express, we screened the database and the literature. The TAS2R family genes are excluded since the proteins that are encoded by these genes are specifically expressed in the taste receptor cells of the tongue and palate epithelia. Ultimately, C3AR1, C5AR1, CCR1, CX3CL1, CXCL16, MTNR1A, P2RY13, PTGER3, and S1PR4 are identified as the hub genes (Figure 5(b)).Figure 5
Identification of the most significant modules and hub genes. (a) Top 10 modules of DEGs between the FSGS patients and healthy donors. The subclusters 1-10 were extracted from PPI networks. (b) 14 hub genes were identified by overlapping the first 20 genes in the 5 classification methods of CytoHubba.
(a)(b)Table 2
The top hub genes ranking in the CytoHubba.
Rank methods in CytoHubbaShared top hub genesDegreeDMNCEPCMCCMNCKNG1APLNRGNG2APLNRGNG2TAS2R5GNG2CX3CL1KNG1CX3CL1GNG7TAS2R4GNG7TAS2R10C3AR1TAS2R10KNG1TAS2R3C3AR1TAS2R3GNG7TAS2R3S1PR4TAS2R19EGFTAS2R4TAS2R10TAS2R4APLNRTAS2R10S1PR4TAS2R5TAS2R14TAS2R5CX3CL1S1PR4CCL5CXCL16C5AR1CXCL16TAS2R10PTGER3APLNRCCR1TAS2R19CCR1TAS2R3P2RY13CX3CL1C3AR1CCL5C3AR1TAS2R4MTNR1ATAS2R10MTNR1AS1PR4MTNR1ATAS2R5CXCL16TAS2R3P2RY13TAS2R31P2RY13CXCL16CX3CL1TAS2R4C5AR1CXCL16C5AR1CCR1CCR1TAS2R5PTGER3P2RY13PTGER3C3AR1C5AR1CXCL16TAS2R19TAS2R3TAS2R19MTNR1AC3AR1CCR1TAS2R31CX3CL1TAS2R31P2RY13MTNR1ATAS2R20CCR1TAS2R20C5AR1P2RY13TAS2R14MTNR1ATAS2R14PTGER3C5AR1CCL5TAS2R4CCL5TAS2R19PTGER3S1PR4PTGER3S1PR4TAS2R31TAS2R19ACTN1TAS2R5ACTN1TAS2R20


### 3.4. Validation of Hub Gene Expression

All the 9 hub genes underwent expression validation in GSE104948 (GPL22945). GraphPad Prism was used to perform Student’st-test statistical analyses and draw boxplots (Figure 6). Consistent with our previous predictions, C3AR1, CCR1, CX3CL1, and P2RY13 were significantly upregulated in FSGS compared to living donors (P<0.05), while MTNR1A was significantly downregulated in FSGS compared to the living donors (P<0.05). However, PTGER3, S1PR4, C5AR1, and CXCL16 showed no significance between FSGS samples and healthy donors in dataset GSE104948 (GPL22945), which may be affected by the small sample size. As a result, we found that C3AR1, CCR1, CX3CL1, MTNR1A, and P2RY13 exhibited accordantly significant expression changes in these three datasets.Figure 6
The mRNA expression of the 9 hub genes was validated by the GSE104948 dataset.


### 3.5. TF-Gene Interactions

In order to explore transcriptional signatures of the 5 validated hub genes, the TF-validated hub gene networks are found in Figure7. The network covers 25 TF-validated hub gene pairs totally. CX3CL1 was modulated by 20 TFs, C3AR1 was modulated by 3 TFs, and MTNR1A was modulated by 2 TFs. No TFs regulate more than one validated hub genes in the network. There were no predicted TF of CCR1 and P2RY13.Figure 7
The network of validated hub gene-TF. The red round rectangle nodes are the validated hub genes, and yellow V-shaped nodes are the TFs.


### 3.6. Exploration of the miRNA-mRNA Network

The regulatory network that involved the predicted miRNA-validated hub gene pairs is shown in Figure8, which revealed that one validated hub gene was modulated by multiple miRNAs. Subsequently, we obtained 57 target miRNAs of 5 validated hub genes and identified 58 mRNA-miRNA pairs. miRNA-6124 could affect mRNA CX3CL1 and CCR1 at the same time. No other miRNAs regulate more than one validated hub genes in the network.Figure 8
Network of predicted miRNAs, showing relationships between 57 miRNAs and 5 validated hub genes (mRNAs). The red rectangle nodes are the validated hub genes (mRNAs), and the green ellipse nodes are miRNAs.


### 3.7. Construction of the lncRNA-Associated ceRNA Network

To identify correlations in the ceRNA network, coexpressed lncRNA-miRNA pairs and circRNA-miRNA pairs were screened. Afterward, 18 lncRNAs all associated with target miRNAs were obtained. Finally, the lncRNA-miRNA-mRNA ceRNA networks were constructed, respectively, including 4 mRNAs, 11 miRNAs, and 18 lncRNAs (Figure9). We observed that merely the upregulated mRNAs (P2RY13, CX3CL1, CCR1, and C3AR1) are involved in both ceRNA networks; there were no predicted lncRNAs of miRNAs that target MTNR1A which is downregulated in the FSGS.Figure 9
Interaction of RNAs in the FSGS-associated ceRNA network. The red rectangle nodes are the validated hub genes (mRNAs), the green ellipse nodes are the miRNAs, and the blue diamond nodes are the lncRNAs.


## 3.1. DEGs between the FSGS Patients and Living Donors

DEGs in the glomeruli tissues of patients with FSGS were identified compared with healthy donors. In total, it is reported in Figure2(a), 624 upregulated and 764 downregulated genes were identified between FSGS patients and healthy donors in the GSE108109. Meanwhile, 494 upregulated and 702 downregulated genes were identified between FSGS patients and healthy donors in the GSE104066. Moreover, the top DEGs could discriminate between the patients with FSGS and the healthy donors in Figure 2(b). The intersection of upregulated genes or downregulated genes from two datasets (GSE108109 and GSE104066) is shown in the Venn diagram (Figure 2(c)); among these overlapping genes, 475 genes were upregulated genes and 655 genes were downregulated genes, and these consistently changed mRNAs were selected as overlapped genes for the next further analysis.Figure 2
DEGs between FSGS patients and healthy donors from GSE104066 and GSE108109, respectively. (a) Volcano plots. (b) Heatmaps. (c) Venn diagrams indicate overlap of upregulated genes and downregulated DEGs, respectively.
(a)(b)(c)


## 3.2. GO and KEGG Annotations of DEGs

The pathogenesis of FSGS is complicated, and comprehending the functions of the DEGs can indicate the novel bench studies and clinic treatments. Each part of GO enrichments is displayed in Figures3(a)–3(c) (upregulated DEGs) and Figures 3(e)–3(g) (downregulated DEGs). For biological process (BP) enrichment analysis, the results showed that the upregulated DEGs strongly took part in the vasculature development, blood vessel development, positive regulation of cellular component movement, positive regulation of cell motility, and positive regulation of cell migration (Figure 3(a)). The downregulated genes were strongly involved in monocarboxylic acid metabolic process, alpha-amino acid metabolic process, cellular amino acid metabolic process, organic acid catabolic process, and alpha-amino acid catabolic process (Figure 3(e)). For cell component (CC) enrichment analysis, the present study showed that the upregulated DEGs were mainly involved in extracellular matrix, external encapsulating structure, side of membrane, collagen-containing extracellular matrix, and endocytic vesicle (Figure 3(b)). The downregulated DEGs mainly revolved in apical plasma membrane, apical part of the cell, brush border membrane, basolateral plasma membrane, and basal plasma membrane (Figure 3(f)). In addition, in the enrichment analysis of molecular function (MF), upregulated DEGs are significantly enriched in cell adhesion molecule binding, integrin binding, growth factor binding, protein homodimerization activity, and actin binding (Figure 3(c)). For downregulated DEGs, they significantly take part in sodium ion transmembrane transporter activity, solute-sodium symporter activity, butyrate-CoA ligase activity, secondary active transmembrane transporter activity, and bitter taste receptor activity (Figure 3(g)).Figure 3
The functional and pathway enrichment analysis of the overlap DEGs between the FSGS patients and healthy donors. (a) Molecular function, (b) biological process, (c) cellular component, and (d) KEGG of overlap upregulated genes. (e) Molecular function, (f) biological process, (g) cellular component, and (h) KEGG of overlap downregulated genes.
(a)(b)(c)(d)(e)(f)(g)(h)Moreover, the KEGG pathway enrichment analysis by upregulated DEGs and downregulated DEGs was listed, respectively. Among the pathways, AGE-RAGE signaling pathway in diabetic complications, cytokine-cytokine receptor interaction, Epstein-Barr virus infection, chemokine signaling pathway, and TNF signaling pathway are involved in the mechanism of upregulated DEGs (Figure3(d)). On the side, downregulated DEGs were mainly enriched in drug metabolism-cytochrome P450, retinol metabolism, drug metabolism, metabolism of xenobiotics by cytochrome P450, and taste transduction (Figure 3(h)).


## 3.3. PPI Network Analysis, Most Significant Modules and Hub Gene Identification for Suggesting Therapy

To illustrate the PPI network of these overlapped DEGs, the analysis was constructed through the STRING platform and was visualized by Cytoscape (Figure4). We totally found 820 paired interactions and 175 upregulated and 80 downregulated genes involved in this network (Supplementary file 1-2).Figure 4
The PPI network of overlap DEGs between the FSGS patients and healthy donors.The subnetwork of genes acts crucial roles in the whole integrated networks. Module analysis on the PPI network through the molecular complex detection (MCODE) plugin revealed top 10 significant clusters, which are shown in Figure5(a). Cluster 1 with the highest cluster score of 22 includes Apelin Receptor (APLNR), Complement C3a Receptor 1 (C3AR1), Complement C5a Receptor 1 (C5AR1), C-C Motif Chemokine Ligand 5 (CCL5), C-C Motif Chemokine Receptor 1 (CCR1), C-X3-C Motif Chemokine Ligand 1 (CX3CL1), C-X-C Motif Chemokine Ligand 16 (CXCL16), G Protein Subunit Gamma 2 (GNG2), G Protein Subunit Gamma 7 (GNG7), Kininogen 1 (KNG1), Melatonin Receptor 1A (MTNR1A), Purinergic Receptor P2Y13 (P2RY13), Prostaglandin E Receptor 3 (PTGER3), Sphingosine-1-Phosphate Receptor 4 (S1PR4), and bitter taste receptor family (TAS2R10, TAS2R14, TAS2R19, TAS2R20, TAS2R3, TAS2R31, TAS2R4, and TAS2R5). Additionally, the overlapped DEGs were imported into CytoHubba plugin of Cytoscape, which was used to screen out top 20 crucial genes through 5 different algorithms, including degree method (Deg), density of maximum neighborhood component (DMNC), edge percolated component (EPC), maximum neighborhood component (MNC), and maximal clique centrality (MCC). Subsequently, 14 common genes were selected by overlapping the 20 genes, as shown in Table 2; these 14 common genes are also involved in the gene cluster 1. To learn the tissues that these 14 genes express, we screened the database and the literature. The TAS2R family genes are excluded since the proteins that are encoded by these genes are specifically expressed in the taste receptor cells of the tongue and palate epithelia. Ultimately, C3AR1, C5AR1, CCR1, CX3CL1, CXCL16, MTNR1A, P2RY13, PTGER3, and S1PR4 are identified as the hub genes (Figure 5(b)).Figure 5
Identification of the most significant modules and hub genes. (a) Top 10 modules of DEGs between the FSGS patients and healthy donors. The subclusters 1-10 were extracted from PPI networks. (b) 14 hub genes were identified by overlapping the first 20 genes in the 5 classification methods of CytoHubba.
(a)(b)Table 2
The top hub genes ranking in the CytoHubba.
Rank methods in CytoHubbaShared top hub genesDegreeDMNCEPCMCCMNCKNG1APLNRGNG2APLNRGNG2TAS2R5GNG2CX3CL1KNG1CX3CL1GNG7TAS2R4GNG7TAS2R10C3AR1TAS2R10KNG1TAS2R3C3AR1TAS2R3GNG7TAS2R3S1PR4TAS2R19EGFTAS2R4TAS2R10TAS2R4APLNRTAS2R10S1PR4TAS2R5TAS2R14TAS2R5CX3CL1S1PR4CCL5CXCL16C5AR1CXCL16TAS2R10PTGER3APLNRCCR1TAS2R19CCR1TAS2R3P2RY13CX3CL1C3AR1CCL5C3AR1TAS2R4MTNR1ATAS2R10MTNR1AS1PR4MTNR1ATAS2R5CXCL16TAS2R3P2RY13TAS2R31P2RY13CXCL16CX3CL1TAS2R4C5AR1CXCL16C5AR1CCR1CCR1TAS2R5PTGER3P2RY13PTGER3C3AR1C5AR1CXCL16TAS2R19TAS2R3TAS2R19MTNR1AC3AR1CCR1TAS2R31CX3CL1TAS2R31P2RY13MTNR1ATAS2R20CCR1TAS2R20C5AR1P2RY13TAS2R14MTNR1ATAS2R14PTGER3C5AR1CCL5TAS2R4CCL5TAS2R19PTGER3S1PR4PTGER3S1PR4TAS2R31TAS2R19ACTN1TAS2R5ACTN1TAS2R20


## 3.4. Validation of Hub Gene Expression

All the 9 hub genes underwent expression validation in GSE104948 (GPL22945). GraphPad Prism was used to perform Student’st-test statistical analyses and draw boxplots (Figure 6). Consistent with our previous predictions, C3AR1, CCR1, CX3CL1, and P2RY13 were significantly upregulated in FSGS compared to living donors (P<0.05), while MTNR1A was significantly downregulated in FSGS compared to the living donors (P<0.05). However, PTGER3, S1PR4, C5AR1, and CXCL16 showed no significance between FSGS samples and healthy donors in dataset GSE104948 (GPL22945), which may be affected by the small sample size. As a result, we found that C3AR1, CCR1, CX3CL1, MTNR1A, and P2RY13 exhibited accordantly significant expression changes in these three datasets.Figure 6
The mRNA expression of the 9 hub genes was validated by the GSE104948 dataset.


## 3.5. TF-Gene Interactions

In order to explore transcriptional signatures of the 5 validated hub genes, the TF-validated hub gene networks are found in Figure7. The network covers 25 TF-validated hub gene pairs totally. CX3CL1 was modulated by 20 TFs, C3AR1 was modulated by 3 TFs, and MTNR1A was modulated by 2 TFs. No TFs regulate more than one validated hub genes in the network. There were no predicted TF of CCR1 and P2RY13.Figure 7
The network of validated hub gene-TF. The red round rectangle nodes are the validated hub genes, and yellow V-shaped nodes are the TFs.


## 3.6. Exploration of the miRNA-mRNA Network

The regulatory network that involved the predicted miRNA-validated hub gene pairs is shown in Figure8, which revealed that one validated hub gene was modulated by multiple miRNAs. Subsequently, we obtained 57 target miRNAs of 5 validated hub genes and identified 58 mRNA-miRNA pairs. miRNA-6124 could affect mRNA CX3CL1 and CCR1 at the same time. No other miRNAs regulate more than one validated hub genes in the network.Figure 8
Network of predicted miRNAs, showing relationships between 57 miRNAs and 5 validated hub genes (mRNAs). The red rectangle nodes are the validated hub genes (mRNAs), and the green ellipse nodes are miRNAs.


## 3.7. Construction of the lncRNA-Associated ceRNA Network

To identify correlations in the ceRNA network, coexpressed lncRNA-miRNA pairs and circRNA-miRNA pairs were screened. Afterward, 18 lncRNAs all associated with target miRNAs were obtained. Finally, the lncRNA-miRNA-mRNA ceRNA networks were constructed, respectively, including 4 mRNAs, 11 miRNAs, and 18 lncRNAs (Figure9). We observed that merely the upregulated mRNAs (P2RY13, CX3CL1, CCR1, and C3AR1) are involved in both ceRNA networks; there were no predicted lncRNAs of miRNAs that target MTNR1A which is downregulated in the FSGS.Figure 9
Interaction of RNAs in the FSGS-associated ceRNA network. The red rectangle nodes are the validated hub genes (mRNAs), the green ellipse nodes are the miRNAs, and the blue diamond nodes are the lncRNAs.


## 4. Discussion

Although multiple factors are involved in the pathogenesis of FSGS, noteworthy primary FSGS is present in approximately 80% of cases [32]. Abundant evidence has confirmed that genetic factors play a vital part in the progression of FSGS; there are not only racial differences but also more than 18% of FSGS have familial aggregation [33]. Furthermore, mutations in the master effector gene take the leading role in the FSGS supposedly, which differs from other glomerular nephropathies [34]. Combined symptomatic therapy with hormone and cytotoxic drugs is most frequent in the FSGS, but the serious side effects caused by the high doses of hormone drugs are still inevitable [35]. Meanwhile, transplantation therapy involving endothelial progenitor cells and mesenchymal stem cells has received good achievements in animal experiments [36]. Blood purification is an emerging application for FSGS clinically, but large-scale controlled trials are still lacking [37]. Considering that the etiologies of the FSGS are not understood, the overall curative outcome is relatively poor compared with other pathological types.As the rapid development of sequencing and microarray that are widely applied in clinical diagnosis and bench medicine, this contributes to the essential biomarker filtrations and discovers in the combat with the various diseases [38]. Booming genes have been reported to be associated with the incidence of FSGS, with the majority of mutations causing before adulthood [39]. The discovery of FSGS-associated genes can deepen our knowledge and also has implications for therapy to prevent unnecessary hormone and immunosuppressive application. Especially, genetic sequencing for the suspected colony with gene mutations should be performed at an early stage of FSGS. In addition, gene sequencing also can be applied for the next generation in the family genetic counseling [40]. Comprehensive bioinformatics means supported in an analysis of how critical genes vary; totally, 88 FSGS patients and 28 healthy donors from 3 studies were selected in the present study that was initiated to discover the diagnostic biomarkers and therapeutic targets for FSGS. Initially, we manually extracted and overlapped all the DFGs from GSE108109 and GSE104066 and identified 1130 DEGs, including 475 upregulated DEGs and 655 downregulated DEGs.Then, we processed these up- and downregulated genes, respectively, for GO function and KEGG pathway enrichment analysis to clarify the role of these DEGs. The GO annotation demonstrated that upregulated DEGs in the FSGS were significantly enriched in vasculature development, blood vessel development, extracellular matrix, and cell adhesion molecule binding, which are consistent with previous evidence. Angiogenesis is the process of forming new blood vessels and requires the development and growth of endothelial cells which are the main cell type of the glomerulus. Leaky vessels, faulty blood vessels, and abnormal vessel growth play a role in the progression of FSGS [41]. The focal activation of parietal epithelial cells is initiated by loss of podocytes and forms cellular adhesions with the capillary tuft followingly [42]. FSGS is defined as the segmental or globular glomerulosclerosis due to the reduced number of podocytes and accumulation of extracellular matrix [43]. KEGG enrichment analysis revealed that AGE-RAGE is a significant signaling pathway; the receptor for advanced glycation end products (RAGE) was upregulated at the base of podocytes; especially, the endogenous secretory RAGE was highly detected in the serum of FSGS patients [44]. Kim and Dryer proved that αVβ3-integrin and RAGE in podocytes act as coreceptors for soluble urokinase plasminogen activator receptor (suPAR) signaling and that elevated expression leads to poor outcomes in chronic nephrosis in the recent report [45]. Additionally, the previous results are in line with downregulated KEGG analysis which showed that retinol metabolism is a remarkable pathway. Circulating retinol-binding protein 4, which is bound to retinol in the bloodstream, is high in children with steroid-resistant nephrotic syndrome-FSGS than first episode nephrotic syndrome [46].Fourteen overlapped hub genes include C3AR1, C5AR1, CCR1, CX3CL1, CXCL16, MTNR1A, P2RY13, PTGER3, S1PR4, TAS2R10, TAS2R19, TAS2R3, TAS2R4, and TAS2R5 which were identified by constructing the PPI network and weighing by CytoHubba and MCODE from Cytoscape. The training dataset (GSE104948) was used to verify the correlation between these overlapped hub genes and FSGS patients. We validated these data and came to the further conclusion that C3AR1, CCR1, CX3CL1, and P2RY13 are significantly high and MTNR1A is obviously low in the FSGS patients. In accordance with our findings, a recent report elucidated that the activation of complement in kidney cells with increased generation of C3 contributes to the induced podocyte injury in primary membranous nephropathy [47]. The emerging studies of chemokines and chemokine receptors showed a distinct perspective on the chronic nephropathy pandemic [48]. Theoretically, chemokines majorly act as a chemoattractant to guide the migration of leukocytes and immune cells in the respective anatomical locations [49]. Specifically, chemokines can effectively accelerate renal leukocyte trafficking and activation and subsequent local damage [50, 51]. Conclusively, various nephroses occur from the excessive infiltration of immune cells. BX471, the CCR1 antagonist, adequately weakened interstitial leukocyte accumulation in the murine FSGS model and the subsequent renal fibrosis [52]. It is worth mentioning that MRS2365 is a selective antagonist of the P2Y(13) receptor with the potential capacity to cure multiple allergic conditions [53]. Besides, melatonin affects the tubular via melatonin receptors (MTNRs) located in the kidney of mammals [54]. Huang et al. indicated that decreased MTNR1A occurs in the cytoplasm of tubular epithelial cells from experimental membranous nephropathy mice and blocking the MTNR1A receptor by luzindole causes further aggravating nephritis [55, 56]. These prior proofs are in accordance with our findings.A wide number of studies have indicated that the dysfunction of transcription factors is related to the onset or progression of FSGS [57–60]. All the 25 transcription factors found in our study are the participants in the progress of diverse renal diseases. It is worth noting that ATF3, which was found in the urine exosomes of the rat kidney injury model, aggravated podocyte injury [61, 62]. Ebf1 contributed to the development and maturation of glomerular as well as podocyte via the mediation of COX-2 expression and calcineurin/NFAT pathway [63, 64]. The inducible expression of SMAD4 is a critical segment in the process of podocyte apoptosis in chronic kidney diseases, as well as the development of kidney diseases [65, 66]. We definitely concentrate on the potentiality of these transcription factors being novel drug targets for FSGS through epigenetic modulation.FSGS is a heterogeneous group of glomerular disorders or podocytopathies, and accumulating studies indicated that dysregulation of microRNAs was involved in the process of the podocytes [67]. Upregulated expression of miR-378a-3p induced glomerular dysfunction in the proteinuric nephropathy, especially promoting podocyte effacement in the FSGS mammalian model [68]. Moreover, a comparative study pointed out that microRNAs in the circulating and urinary could act as potent biomarkers for diagnosis and also for therapy monitor [69]. Gebeshuber et al. reported that overexpressed miR-193a restrains the expression of the Wilms’ tumor protein (WT1) and its target genes, which impairs the homeostasis of podocytes, and ultimately causes FSGS, as well as increased expression of miR-193a was also found in the FSGS patients [70]. Existing reports described that there are intricate interactions among diverse RNA molecules, such as protein-coding messenger RNAs and noncoding RNAs (circRNAs, lncRNAs, and miRNAs). Crosstalk RNA molecules cooperate to generate a dynamic regulatory network functioning as competitive endogenous RNAs (ceRNAs) [71]. However, limited literature reports the ceRNA network in the podocyte or FSGS, thus showing a bright prospect. The P38/C/EBPβ pathway induced expression of long noncoding RNA LOC105374325, competitively binds miR-34c/miR-196a/b, and ulteriorly leads to increased levels of Bax and Bak in podocytes from individuals with FSGS [72]. In another study, circZNF609 caused podocyte injury in vivo and in vitro via decreasing miR-615-5p, WT1, and podocin expression [73]. Nevertheless, we are cautious of these competitive network analyses. To the best of our knowledge, there have been no studies on ceRNA networks in FSGS. In our study, ceRNA networks were built based on the interactions between lncRNA-miRNA and miRNA-mRNA. 18 lncRNA nodes, 11 miRNA nodes, and 4 mRNA nodes were included in the networks. Upon comprehensive analysis of the ceRNA network, some novel and crucial characteristics of FSGS were disclosed.The potential limitations of this study need to be considered. First, three datasets contain different populations of FSGS patients and controls, which may implicate the results. Additionally, because the data we analyzed were obtained from public databases, further experiments are necessary to validate the findings.


## 5. Conclusion

In summary, these findings provide new perspectives into the pathogenesis of FSGS and might ascertain potential diagnostic and therapeutic approaches for the following studies. Further analysis is needed to investigate the molecular mechanisms by which the five key genes affect the prognosis of patients with FSGS.



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*Source: 1941038-2022-04-19.xml*