Activation of complement C1q and C3 in glomeruli might accelerate the progression of diabetic nephropathy: Evidence from transcriptomic data and renal histopathology

Abstract

Aims/introduction: It is not unclear whether the complement system is involved in the pathogenesis of diabetic nephropathy (DN). We explored the role of the complement system in glomeruli from patients with DN using integrated transcriptomic bioinformatics analysis and renal histopathology.

Materials and methods: Four datasets (GSE30528, GSE104948, GSE96804 and GSE99339) from the Gene Expression Omnibus database were integrated. We used a protein-protein interaction network and the Molecular Complex Detection App to obtain hub genes. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were carried out to identify significant pathways. We also investigated the associations of C1q and C3 deposition on renal histopathology with clinical data, pathological parameters and renal survival in DN patients.

Results: We identified 47 up- and 48 downregulated genes associated with DN. C3, C1QB and C1QA were found to be complement-related hub genes. The gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses identified complement activation and humoral immune response as the significant oncology terms, with C1QB and C3 positioned at the center of the pathway. Regarding renal histopathology, patients with both C1q and C3 deposition had more severe glomerular classes. Multivariate Cox proportional hazards regression showed that the deposition of glomerular C1q and C3 was an independent risk factor for kidney failure. Patients with high C1q, C3 or C4d expression in glomeruli were more likely to progress to kidney failure, whereas glomerular mannose-binding lectin was rare.

Conclusions: Complement activation is involved in the development of DN, and activation of the classical complement pathway in glomeruli might accelerate disease progression.

Keywords: Complement system; Diabetic nephropathy; Glomeruli.

Figure 1

Study flowchart. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MCODE, Molecular Complex Detection; PPI, protein–protein interaction.

Figure 2

Collection of differentially expressed genes (DEGs) and Kyoto Encyclopedia of Genes and Genomes pathway analysis in diabetic nephropathy. (a) Heat map of DEGs. Each column represents one dataset and each row represents one gene. Green represents a lower level of gene expression, red represents a higher expression level. (b) The upregulated DEGs enriched pathway analysis. (c) The downregulated DEGs enriched pathway analysis. PPI, protein–protein interaction. ECM, extracellular matrix.

Figure 3

(a) Gene Ontology annotation and (b) Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. ECM, extracellular matrix.

Figure 4

The event‐free survival probability of the Kaplan–Meier analysis. Kaplan–Meier curves comparison when dividing patients in strata of (a) glomerular C1q and C3 deposition pattern (with either or neither of C1q and C3 deposition; P = 0.0077); (b) C3 deposition versus without C3 deposition (P = 0.0345); and (c) C1q deposition versus without C1q deposition (P = 0.0018).

Figure 5

Risk factors for the composite renal outcome determined by (a) univariate or (b) multivariate Cox hazard analysis in diabetic nephropathy. CI, confidence interval; eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin; HR, hazard ratio; IFTA, interstitial fibrosis and tubular atrophy; RAAS, renin–angiotensin–aldosterone system.

Figure 6

Immunohistochemical staining for (a) C1q, (b) C3, (c) C4d and (d) mannose‐binding lectin (MBL) expression in the glomeruli (original magnification: ×200), and the expression levels of C1q, C3, C4d and MBL in the stable and progressive groups. The progressive group was defined as those with the occurrence of the composite renal outcome; on the contrary, the stable group referred to those who did not enter the endpoints. *P < 0.05 versus stable group. AOD, average optical density.

Figure 7

The gene expression levels of C1QA (a) and C3 (b) in glomeruli from patients with diabetic nephropathy when compared with those with other kidney diseases. *P < 0.05; **P < 0.01; ***P < 0.001. FSGS, focal segmental glomerulosclerosis; HT, hypertension; IgAN, IgA nephropathy; MCD, minimal change disease; MN, membranous nephropathy; SLE, systematic lupus erythematosus.