Identification of inflammatory biomarkers in IgA nephropathy using the NanoString technology: a validation study in Caucasians

Abstract

Abstract Objective and design Immunoglobulin A nephropathy (IgAN) is a kidney disease characterized by the accumulation of IgA deposits in the glomeruli of the kidney, leading to inflammation and damage to the kidney. The inflammatory markers involved in IgAN remain to be defined. Gene expression analysis platforms, such as the NanoString nCounter system, are promising screening and diagnostic tools, especially in oncology. Still, their role as a diagnostic and prognostic tool in IgAN remains scarce. In this study, we aimed to validate the use of NanoString technology to identify potential inflammatory biomarkers involved in the progression of IgAN. Subjects A total of 30 patients with biopsy-proven IgAN and 7 cases of antineutrophil cytoplasmic antibody (ANCA)-associated pauci-immune glomerulonephritis were included for gene expression measurement. For the immunofluorescence validation experiments, a total of 6 IgAN patients and 3 controls were included. Methods Total RNA was extracted from formalin-fixed paraffin-embedded kidney biopsy specimens, and a customized 48-plex human gene CodeSet was used to study 29 genes implicated in different biological pathways. Comparisons in gene expression were made between IgAN and ANCA-associated pauci-immune glomerulonephritis patients to delineate an expression profile specific to IgAN. Gene expression was compared between patients with low and moderate risk of progression. Genes for which RNA expression was associated with disease progression were analyzed for protein expression by immunofluorescence and compared with controls. Results IgAN patients had a distinct gene expression profile with decreased expression in genes IL-6, INFG, and C1QB compared to ANCA patients. C3 and TNFRSF1B were identified as potential biomarkers for IgAN progression in patients early in their disease course. Protein expression for those 2 candidate genes was upregulated in IgAN patients compared to controls. Expression of genes implicated in fibrosis (PTEN, CASPASE 3, TGM2, TGFB1, IL2, and TNFRSF1B) was more pronounced in IgAN patients with severe fibrosis compared to those with none. Conclusions Our findings validate our NanoString mRNA profiling by examining protein expression levels of two candidate genes, C3 and TNFRSF1B, in IgAN patients and controls. We also identified several upregulated mRNA transcripts implicated in the development of fibrosis that may be considered fibrotic markers within IgAN patients.