Effect of plasma fractions from patients with focal and segmental glomerulosclerosis on rat proteinuria

Background. Patients suffering from focal and segmental glomerulosclerosis (FSGS) and in whom this disease recurs after transplantation are likely to have an active form of the disease and to have a factor(s) (such as, albumi nuric factor) present in their blood that alters glomerular permeability fo r albumin. Methods. We used a sequential 50 and 70% ammonium sulfate (AS) precipitatio n of plasma from patients with relapsing FSGS and non-FSGS nephrotic syndro me (NS), in addition to plasma from healthy individuals, to obtain both an immunoglobulin (Ig)-rich fraction (50% AS precipitate) and a non-Ig fractio n (70% AS supernatant). These fractions were injected intraarterially or in travenously/intraperitoneally into Sprague-Dawley rats, and proteinuria (g protein/mmol creatinine) was measured for 24 hours. Ig fractions eluted fro m immunoadsorption onto protein A were also tested. A biochemical character ization was then carried out on the 70% AS supernatants by ultrafiltration on 30 and 50 kD cut-off membranes and by sodium dodecyl sulfate-polyacrylam ide gel electrophoresis (SDS-PAGE). Differentially stained bands were seque nced. Results. The 70% AS supernatants from FSGS patients induced proteinuria whe n injected intra-arterially into normal rats. This effect was significantly different (P < 0.05) from that observed when similar fractions were prepar ed from the plasma of patients suffering from non-FSGS NS, but was not diff erent from that observed with fractions from healthy individuals and even w ith an injection of saline solution. Injections of other plasma fractions d id not induce a significant proteinuria in the FSGS group versus the non-FS GS NS group. SDS-PAGE of 70% AS supernatants revealed a protein of 23 kD th at was more concentrated in AS supernatants from FS GS plasma than the othe r plasma samples and that was identified by microsequencing as apolipoprote in Al. After sequential ultrafiltration of 70% AS supernatants on 30 and 50 kD cut-off membranes, a second band of 43 kD was found at a much higher co ncentration in the FSGS samples than in non-FSGS NS and healthy individuals samples. This band is likely to correspond to a candidate albuminuric fact or recently reported by another group [1], and was identified by microseque ncing as <alpha>(1) acid glycoprotein or orosomucoid. Consequently, purifie d orosomucoid from the plasma of FSGS, non-FSGS NS patients, or healthy ind ividuals was injected intra-arterially into rats. No differences were found between the proteinuria induced in each group. Conclusions. These data strongly suggest that in vivo injection of material into the rat is not a reliable model for testing plasma fraction activity and that the 43 kD orosomucoid is not likely to be the albuminuric factor.