Table 1 Concentration of urinary protein and creatinine Urine protein (mg/ml) Urine creatinine (mg/dl) (A) First study IgAN 0.55 ± 0.06 133.6 ± 7.8 MN 2.97 ± 0.68 121.4 ± 14.2 SLE 2.99 ± 0.133 116.0 ± 18.6 FGS 2.37 ± 1.05 112.7 ± 13.9 MCNS 5.03 ± 1.42 77.6 ± 33.5 DMN
2.31 ± 1.05 62.7 ± 19.8 Other kidney diseases selleck chemical 1.60 ± 0.46 106.8 ± 16.5 (B) Second study IgAN (before treatment) 0.75 ± 0.17 134.9 ± 11.8 Inactive IgAN (after treatment) 0.63 ± 0.13 96.8 ± 16.9 Alport syndrome 1.55 ± 0.45 82.9 ± 10.7 Amyloidosis 0.71 ± 0.20 78.4 ± 13.3 MPGN 1.32 ± 0.25 111.3 ± 41.3 ANCA-related nephritis 1.37 ± 1.11 50.8 ± 3.4 TBMD 0.23 ± 0.11 124.1 ± 50.0 FGS 2.68 ± 1.46 128.1 ± 39.6 Lupus nephritis (SLE) 2.45 ± 1.71 187.4 ± 116.0 DMN 1.36 ± 0.24 76.4 ± 34.7 MN 1.63 ± 0.33 94.1 ± 17.9 Hypertensive nephrosclerosis 0.25 30.8 In
the second study (examination in other diseases groups—focused test to discriminate other diseases from IgAN), urine samples were obtained from various forms of biopsy-proven kidney disease patients exhibiting hematuria with or without proteinuria include IgAN (before treatment; 31 patients), and inactive IgAN; hematuria was no longer present after tonsillectomy with steroid pulse therapy (4 patients) [10–13], Alport syndrome (8 patients), amyloidosis (3 patients), membranoproliferative glomerulosclerosis (MPGN; 4 patients), anti-neutrophil cytoplasmic antibody (ANCA)-related nephritis (2 patients), thin basement membrane disease (TBMD; 2 patients), FGS (4 patients), SLE (2 patients), DMN (2 patients), MN (4 patients), and hypertensive nephrosclerosis (1 patient). Urinary MK-1775 in vivo protein and creatinine concentrations of each disease are shown in Table 1B. Reverse transcriptase Selleck TPX-0005 Immunoprecipitation (IP) method Anti-human IgA antibody (Cappel Co.)
was immobilized on Dynabeads® M-450 Epoxy (Invitrogen Co.) according to manufacturer’s instruction and blocked with bovine serum albumin (BSA). A Tris–HCl buffered (pH 7.5) urine sample containing 0.15 M sodium chloride (NaCl) was mixed with anti-IgA-immobilized beads or control beads (BSA-blocked beads) and incubated overnight at 4°C. After washing with phosphate-buffered saline (PBS), proteins were eluted from beads with 0.1 M citric acid buffer (pH 3.0) and dialyzed against 1/10 concentration of PBS containing 0.01% sodium azide (NaN3), and concentrated. Identification of proteins combined with IgA in urine Proteins recovered from the anti-IgA antibody affinity beads and control beads were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins of interest were analyzed according to the method of Katayama et al. [18]. Western blot analysis The 3 μl of protein solution prepared by IP was separated by SDS-PAGE, and the proteins were then electrophoretically blotted onto a nitrocellulose filter (BA85; Schleicher & Schuell).