Comparative proteomic analysis of laser microdissected human glomeruli and tubules in IgA nephropathy

Background：IgA nephropathy (IgAN) is the most common glomerulonephritis worldwide, and 30-40% of cases progress to end-stage kidney disease. Although the glomerular mesangium is the leading site of IgA deposition in IgAN, tubulointerstitial lesions vary among patients. A new workflow for laser microdissection (LMD) coupled with mass spectrometry (MS) was developed. Using this workflow for glomeruli and tubules, we attempted to detect molecules associated with the pathogenesis and disease progression in IgAN.
Methods：Using the treatment protocol for IgAN at Fujita Health University Hospital, patients with IgAN were divided into immunosuppressive (tonsillectomy and steroid pulse therapy, n=19) and conservative (n=14) therapy groups. Glomerular and tubular cross-sections were microdissected from stored formalin-fixed paraffin-embedded kidney biopsy tissues and control kidney tissues (n=10) purchased from OriGene Technologies. Samples were analyzed using Orbitrap FusionTM Tribrid mass spectrometer (liquid chromatography-mass spectrometry) coupled to EASY-nLC system, and the relative amounts of glomerular and tubular proteins were compared between the groups. The　data analysis was performed using Proteome Discoverer 2.4 and Epithelial-Mesenchymal Transition (EMT) Genes Database.
Results and Discussion：Glomerular proteomic analysis quantified 1807 proteins, of which 85 had significantly different abundances in patients with IgAN than in controls. Comparing the different treatment groups of patients with IgAN, 78 proteins showed significantly different abundances. These proteins are mainly a part of the complement system, such as factors H and H-related proteins. Several international genome-wide association studies have shown that these complement proteins are associated with IgAN. Therefore, these proteins are likely involved in IgAN pathogenesis and progression.
Tubular proteomic analysis quantified 2354 proteins, of which 73 were significantly differentially expressed between patients with IgAN and controls. When comparing the different IgAN treatment groups, 75 proteins showed significantly different abundances. These proteins include extracellular matrix and EMT-related proteins. In patients with IgAN, EMT induction in the tubules can cause fibrosis and loss of kidney function.
Conclusions：Using LMD-coupled MS, we demonstrated that the complement system in the glomerular lesion and the epithelial-mesenchymal transition system in the tubular interstitial lesion differed between different treatment groups. Investigating the relationship between these molecules and kidney prognosis is crucial for discovering new biomarkers to determine kidney prognosis.