Absence of Increased α1-Microglobulin in IgA Nephropathy Proteinuria

Shuji Morita,Masao Katoh,Hiromichi Suzuki,Masanori Naitou,Masashi Hiramoto,Masatoshi Yuri,Hirokazu Okada,Yoshihiko Kanno,Hiroyuki Yokota,Atsushi Ichikawa

doi:10.1074/mcp.m600336-mcp200

Shuji Morita, Masao Katoh

Open Access

https://doi.org/10.1074/mcp.m600336-mcp200

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Abstract

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using two-dimensional DIGE. Most of the 172 spots identified in the urine were serum proteins, and their amounts in IgA nephropathy urine were much higher than those in normal urine; this can be explained as proteinuria caused by glomerular dysfunction. However, only alpha(1)-microglobulin, also one of the major serum proteins, in IgA nephropathy urine was not higher in amount than that in normal urine. We confirmed using ELISA analysis that the amounts of transferrin and albumin in IgA nephropathy and diabetic nephropathy urine were much higher than those in normal urine, whereas the amount of alpha(1)-microglobulin in IgA nephropathy urine was not higher than that in normal urine and was much lower than that in diabetic nephropathy urine. Approximately 50% of alpha(1)-microglobulin forms a complex with IgA in serum. These results suggest that alpha(1)-microglobulin in IgA nephropathy urine is a characteristic protein and might be a biomarker for IgA nephropathy and that alpha(1)-microglobulin might have a relationship with IgA nephropathy pathology.

Highlights

To search for biomarkers of IgA nephropathy, protein profiles of urine samples from patients with IgA nephropathy and normal volunteers were compared using twodimensional DIGE
Renal function evaluated by estimated GFR in the patients with IgA nephropathy mostly remained in the normal range, and most of the patients in both groups were classified as having early stage chronic kidney disease [11, 12]
IgA nephropathy was diagnosed by positive immunofluorescence staining of glomerular IgA deposition

Summary

EXPERIMENTAL PROCEDURES

Materials and Reagents—IPG strip gels, 1-(5-carboxypentyl)-1Јpropylindocarbocyanine halide (Cy3) N-hydroxysuccinimidyl ester, 1-(5-carboxypentyl)-1Ј-methylindodicarbocyanine halide (Cy5) N-hydroxysuccinimidyl ester, and other 2D DIGE-related materials were purchased from GE Healthcare. The primary anti-human ␣1-microglobulin antibody (Cortex Biochem CR3021SP) diluted 1:5000 in 0.1 M borate buffer (pH 9.5) was added into the wells of a 96-well microplate and incubated overnight at 4 °C. After washing the wells with PBS-Tween buffer (8.1 mM Na2HPO4, 1.5 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, and 0.05% Tween 20), Block Ace reagent was added for postcoating and incubated for 1 h at 37 °C. After washing the wells with the PBS-Tween buffer, centrifuged and diluted urine samples and ␣1-microglobulin standard were added into the wells and incubated for 1 h at 37 °C. The secondary mouse anti-human ␣1-microglobulin antibody (AgriSera AS01-013) diluted 1:5000 in the PBSTween buffer was added and incubated for 1 h at 37 °C. Peroxidase-conjugated anti-mouse immunoglobulin antibody (Invitrogen AMI4404) was added and incubated for 1 h at 37 °C.

RESULTS

Cystatin C

DISCUSSION