Integrative analysis of COL6A3 in lupus nephritis: insights from single-cell transcriptomics and proteomics

Abstract

Introduction: Lupus nephritis (LN), a severe complication of systemic lupus erythematosus (SLE), presents significant challenges in patient management and treatment outcomes. The identification of novel LN-related biomarkers and therapeutic targets is critical to enhancing treatment outcomes and prognosis for patients.

Methods: In this study, we analyzed single-cell expression data from LN (n=21) and healthy controls (n=3). A total of 143 differentially expressed genes were identified between the LN and control groups. Then, proteomics analysis of LN patients (n=9) and control (SLE patients without LN, n=11) revealed 55 differentially expressed genes among patients with LN and control group. We further utilizes protein-protein interaction network and functional enrichment analyses to elucidate the pivotal role of COL6A3 in key signaling pathways. Its diagnostic value is evaluate through its correlation with disease progression and renal function metrics, as well as Receiver Operating Characteristic Curve (ROC) analysis. Additionally, immunohistochemistry and qPCR experiments were performed to validate the expression of COL6A3 in LN.

Results: By comparison of single-cell and proteomics data, we discovered that COL6A3 is significantly upregulated, highlighting it as a critical biomarker of LN. Our findings emphasize the substantial involvement of COL6A3 in the pathogenesis of LN, particularly noting its expression in mesangial cells. Through comprehensive protein-protein interaction network and functional enrichment analyses, we uncovered the pivotal role of COL6A3 in key signaling pathways including integrin-mediated signaling pathways, collagen-activated signaling pathways, and ECM-receptor interaction, suggesting potential therapeutic targets. The diagnostic utility is confirmed by its correlation with disease progression and renal function metrics of the glomerular filtration rate. ROC analysis further validates the diagnostic value of COL6A3, with the area under the ROC values of 0.879 in the in-house cohort, and 0.802 and 0.915 in tubular and glomerular external cohort samples, respectively. Furthermore, immunohistochemistry and qPCR experiments were consistent with those obtained from the single-cell RNA sequencing and proteomics studies.

Discussion: These results proved that COL6A3 is a promising biomarker and therapeutic target, advancing personalized medicine strategies for LN.

Keywords: COL6A3; biomarkers; diagnosis; kidney disease; lupus nephritis; proteomics; single-cell RNA sequencing; systemic lupus erythematosus.

Figure 1

Workflow of this study. We began with single-cell RNA sequencing analysis of 21 lupus nephritis (LN) patients and 3 healthy controls to identify differentially expressed genes (DEGs). This was followed by proteomics analysis to distinguish serum protein expression between SLE patients without LN (n=11) and LN patients (n=9), identifying differentially expressed proteins (DEPs). Gene ontology and pathway analyses of DEPs were conducted. Comparative analysis of DEGs and DEPs leaded to the identification of COL6A3 as a potential biomarker. The association between COL6A3 expression and renal function was evaluated, alongside its diagnostic value using receiver operating characteristic (ROC) curve analysis. Lastly, analyses including protein-protein interaction (PPI), functional enrichment, and gene set enrichment for COL6A3 were performed to elucidate its role in LN.

Figure 2

Single-cell RNA sequencing (scRNA-seq) analysis of LN. T-distributed stochastic neighbor embedding (t-SNE) plots display the major cell types in kidney and skin samples from LN patients and healthy controls (A–C), and detailed cell type analysis within kidney (D–F). (A) T-SNE analysis of the scRNA-seq data showing six major cell types in the kidney and skin samples. (B) T-SNE analysis of two groups, including the kidney and skin samples of healthy control and LN patients. (C) T-SNE analysis of the kidney and skin samples from each healthy control and LN patient. (D) T-SNE analysis of the scRNA-seq data showing five major cell types in the kidney samples (LN group: n=21, healthy group: n=3). (E) T-SNE analysis of two groups, including the kidney samples of healthy control and LN patients. (F) T-SNE analysis of the kidney samples from each healthy control and LN patient.

Figure 3

Identification of differentially expressed genes of renal scRNA-seq data in LN. (A) A volcano plot showed the upregulated and downregulated DEGs between LN and healthy controls. (B) A heatmap showed the upregulated and downregulated DEGs between LN and healthy controls. (C) A heatmap showed the upregulated and (D) downregulated DEGs between LN and healthy controls in five major cell types.

Figure 4

Proteomics analysis of LN. (A) Differentially expressed proteins (DEPs) in control (SLE without LN, n=11), and LN patients (n=9). (B) Venn diagrams showing the overlap of upregulated DEPs and DEGs, indicating shared and unique molecular features. (C) Venn diagrams showing non overlap of upregulated DEPs and DEGs. (D) The expression of COL6A3 in five major cell types of renal sample by scRNA-seq analysis. (E) The expression of COL6A3 in proteomics data. (F) The relationship between COL6A3 expression and estimated glomerular filtration rate (GFR) in our internal cohort. (G) The area under the receiver operating characteristic curve (AUC) for COL6A3 in our internal cohort. (H) The mRNA expression level of COL6A3 in LN (n=3) and control (healthy volunteers, n=3) examined by Real-time PCR analysis. (I) Hematoxylin and eosin (HE), periodic acid-Schiff (PAS), Masson’s trichrome (MASSON), Picrosirius red (PSA), and periodic acid silver methenamine (PASM) staining of LN and control (IgA nephropathy) tissue. (J) Protein expression of COL6A3 in LN and control (IgA nephropathy, n=3) tissue analyzed by immunohistochemical staining.

Figure 5

External validation of the clinical significance of COL6A3. Expression in tubulointerstitium (A) and glomeruli (B) from the Ju cohort (15) and its relationship with GFR (C, D). (E, F) Diagnostic performance of AUC results in the Ju cohort.