Kidney whole-transcriptome profiling in primary antiphospholipid syndrome reveals complement, interferons and NETs-related gene expression

Abstract

Objective: Pathogenesis of antiphospholipid syndrome (APS) remains poorly elucidated. We aimed to evaluate for the first time kidney transcriptome profiles in primary APS vs systemic lupus erythematosus (SLE) and control subjects.

Methods: We performed RNA sequencing on archival formalin-fixed paraffin-embedded kidney biopsies from APS (n = 4), SLE (n = 5) and control (n = 3) individuals, differential gene expression analysis (DGEA) and enrichment analysis using gene ontology (GO) and CORUM, KEGG and Reactome pathway databases.

Results: Two-dimensional projection showed a distinct gene profile in primary APS vs control kidneys samples, but similar to SLE. DGEA in APS vs controls returned 276 upregulated and 217 downregulated genes, while the comparison between APS and SLE identified 75 upregulated and 111 downregulated genes. In 276 upregulated genes, enriched GO terms were (innate) immune response, inflammatory response, leucocyte and lymphocyte activation, cytokine production and T cell activation. CORUM and KEGG revealed complement-related genes (C3, C4A, C4B). Expression levels showed logFC values of 2.25 (P = 1.58e-05) for C3, 2.17 (P = 2.69e-06) for C4A and 2.135 (P = 3.7e-06) for C4B in APS vs controls, without differences between APS and SLE. Interferon (IFN) alpha/beta signalling was revealed by Reactome. Expression levels of nine IFN-regulated genes found upregulated in APS vs control kidneys (P-values ≤ 0.001 for all). Examining neutrophil-extracellular traps (NETs)-related gene expression, 13 of 15 upregulated NETs-related genes exhibited higher expression in APS vs controls but not vs SLE.

Conclusion: Complement, interferon and NETs-related genes are highly expressed in APS kidney tissues, similarly to SLE, pointing out the role of innate immunity in APS nephropathy pathogenesis and potential treatment targets.

Keywords: antiphospholipid syndrome; complement; innate immunity; interferon; nephropathy; neutrophils.

Figure 1.

RNA sequencing in kidney tissues of primary antiphospholipid syndrome (APS), systemic lupus erythematosus (SLE) and control individuals. A. Dimensionality reduction of all kidney biopsy samples. Principle Component Analysis was applied in order to depict the samples in the 2D space. Each dot corresponds to a sample. The smaller the distance between each sample pair, the greater the similarity of the gene expression profiles. B. Illustration of the relationship between statistical significance and magnitude of fold change for each gene in a comparative analysis between APS and control kidneys (B1), and between APS and SLE (B2). Each point represents an individual gene. The x-axis displays the log2 fold change, indicating the magnitude of the change in gene expression. Positive values signify upregulation, while negative values represent downregulation. The y-axis represents the -log10 of the meta P-value, reflecting the statistical significance of the observed changes. Higher values indicate greater significance. The significance threshold is set at meta P-value of 0.05 and log2FC >|1.5|, indicating with green colour the downregulated and with red colour the upregulated genes. C. Enrichment analysis results for upregulated genes in APS versus control kidney samples. Selected terms are presented. The x-axis represents the enriched terms, the y-axis shows the precision, the size of the points indicates the intersection size, and the colour corresponds to the -log10 of the P-value of enrichment (red and blue colour indicates higher and lower significance, respectively). D. Comparison of enriched terms in APS versus controls and APS versus SLE kidney samples. The bar plots illustrate the intersection of enriched terms among the upregulated genes in the comparisons between APS versus controls and APS versus SLE. In the color scheme, red bars represent the APS versus controls comparison, and the blue bars represent the APS versus SLE comparison. The y-axis denotes the enriched terms, while the x-axis represents the number of genes intersecting with each term. E. Complement-related genes expression in APS, SLE and control kidney samples. Heatmap representation that displays the z-score-scaled mean normalized expression values of the three complement-related genes (C3, C4A, C4B) found up-regulated in APS samples in the differential expression analysis. F. Interferon-regulated genes expression in APS, SLE and control kidney samples. Heatmap representation that displays the z-score-scaled mean normalized expression values of the nine interferon-regulated genes found upregulated in APS samples in the differential expression analysis. G. NETs-related gene expression in APS, SLE and control kidney samples. Heatmap representation that displays the z-score-scaled mean normalized expression values of the 15 neutrophil extracellular traps (NETs)-related genes that were found to be expressed in kidney specimens of patients with APS