Proteomic analysis of whole glomeruli in patients with IgA nephropathy using microsieving.

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.