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  • Title: Proteomic analysis of whole glomeruli in patients with IgA nephropathy using microsieving.
    Author: Kojima S, Koitabashi K, Iizuka N, Okamoto K, Arito M, Sato T, Kurokawa MS, Suematsu N, Shibagaki Y, Yasuda T, Kimura K, Kato T.
    Journal: Am J Nephrol; 2014; 39(1):36-45. PubMed ID: 24434790.
    Abstract:
    BACKGROUND: To promote understanding of immunoglobulin A nephropathy (IgAN) pathophysiology, we tried to elucidate glomerular protein profiles in IgAN, using microsieving that we established recently to isolate glomeruli from renal biopsy samples and proteomic approaches. METHODS: Glomeruli were isolated from renal biopsy samples of patients with IgAN (n = 5) and with minimal change nephrotic syndrome (MCNS; n = 5) using microsieving. Proteins extracted from the isolated glomeruli were separated by 2-dimensional differential gel electrophoresis (2D-DIGE). Proteins with different amounts between the two groups were identified by mass spectrometry. One of the identified proteins, α-actinin-4 (ACTN4), was further analyzed by Western blotting, RT-polymerase chain reaction (PCR), and immunohistochemistry. RESULTS: By 2D-DIGE, 72 out of the detected 1,170 protein spots showed significantly different intensity between the two groups (p < 0.05). Thirty-four out of the 72 protein spots showed more than 1.5-fold or less than 1/1.5-fold intensity, out of which 16 protein spots were successfully identified. No microbial protein was identified. ACTN4 molecules with a low molecular weight of approximately 77 kDa were found to increase in the IgAN group. Lack of an N-terminal part of ACTN4 was demonstrated by Western blotting. No defect of mRNA for ACTN4 was evidenced by RT-PCR. Predominant existence of ACTN4 in capillary walls of glomeruli of IgAN patients was demonstrated by immunohistochemistry in glomerular sections of patients with IgAN. CONCLUSION: Use of microsieving enabled us to biochemically analyze glomerular proteins in renal biopsy samples from patients with glomerular diseases. With this method, we demonstrated skewed glomerular protein profiles in IgAN.
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