Study of the in vitro effect on glomerular albumin permselectivity of serum before and after renal transplantation in focal segmental glomerulosclerosis.

BACKGROUND: The selective proteinuria observed in patients with focal segmental glomerulosclerosis (FSGS) suggests an abnormal loss of fixed anionic charges on the glomerular capillary wall. METHODS: In this article, we have studied the putative presence of such factor(s) by using a new in vitro assay to assess glomerular permselectivity by measuring glomerular volume variation (GVV) in isolated glomeruli after hypotonic stress. We randomly tested the serum GVV activity of 10 healthy donors and 143 patients before transplantation. Of the patients, 80 had FSGS, 26 membranous glomerulonephritis, 19 polycystic kidney disease, and 18 malformative uropathies. Moreover, we tested the pre- and posttransplantation serum of 14 patients with recurrence and 14 without recurrence. RESULTS: Serum GVV was significantly higher in patients with FSGS than in those with the other end-stage renal diseases studied (P<0.01) or in healthy donors (P<0.01). However, a wide distribution of serum GVV activity in patients with and without FSGS was observed. Statistically, pregraft GVV values were not predictive of the recurrence of FSGS after transplantation. Moreover, we observed a significant decrease in serum GVV activity after transplantation in patients without recurrence (P<0.01) compared to those who underwent a recurrence. CONCLUSIONS: These results reinforce the hypothesis of a circulating factor that alters glomerular albumin permselectivity in FSGS patients. However, the presence of this factor before transplantation did not appear to predict relapse of the disease after transplantation, as recently supported, although its activity seems to be down-regulated after transplantation in patients who do not experience recurrence of the disease.

Antihuman immunoglobulin affinity immunoadsorption strongly decreases proteinuria in patients with relapsing nephrotic syndrome.

Approximately 20 to 30% of patients with idiopathic nephrotic syndrome and focal glomerulosclerosis experience a relapse of their nephrotic syndrome after transplantation. Previously, it has been shown that ex vivo immunoadsorption on protein A strongly (although transiently) reduces proteinuria in relapsing patients. To investigate whether the factor(s) that give rise to albuminuria are bound directly to protein A in the immunoadsorption procedure or are part of a complex with Ig, four patients with relapse of focal glomerulosclerosis presenting as nephrotic syndrome after transplantation were treated, sequentially, using a (non-protein A) anti-Ig affinity column and a protein A column. This study reports that the effect on proteinuria of immunoadsorption using an anti-Ig immunoaffinity column is comparable in its magnitude and kinetics to that of immunoadsorption on protein A. The two procedures were also equally effective in depleting the relapsing patients' plasma of a factor capable of altering the albumin permselectivity of isolated glomeruli in vitro. This study demonstrates for the first time that immunoglobulins have a role in the nephrotic syndrome. In addition, the fact that the two different immunoadsorption procedures both resulted in the removal of the same putative albuminuric factor in these patients and that no autoreactivity of eluted immunoglobulins was observed on human tissues strongly suggests that the factor or factors that may be responsible for immediate nephrotic syndrome after transplantation are bound to an immunoglobulin. However, no firm evidence can be yet provided against a direct role of immunoglobulins.

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, albuminuric factor) present in their blood that alters glomerular permeability for albumin. METHODS: We used a sequential 50 and 70% ammonium sulfate (AS) precipitation of plasma from patients with relapsing FSGS and non-FSGS nephrotic syndrome (NS), in addition to plasma from healthy individuals, to obtain both an immunoglobulin (Ig)-rich fraction (50% AS precipitate) and a non-Ig fraction (70% AS supernatant). These fractions were injected intra-arterially or intravenously/intraperitoneally into Sprague-Dawley rats, and proteinuria (g protein/mmol creatinine) was measured for 24 hours. Ig fractions eluted from immunoadsorption onto protein A were also tested. A biochemical characterization was then carried out on the 70% AS supernatants by ultrafiltration on 30 and 50 kD cut-off membranes and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Differentially stained bands were sequenced. RESULTS: The 70% AS supernatants from FSGS patients induced proteinuria when injected intra-arterially into normal rats. This effect was significantly different (P < 0.05) from that observed when similar fractions were prepared from the plasma of patients suffering from non-FSGS NS, but was not different from that observed with fractions from healthy individuals and even with an injection of saline solution. Injections of other plasma fractions did not induce a significant proteinuria in the FSGS group versus the non-FSGS NS group. SDS-PAGE of 70% AS supernatants revealed a protein of 23 kD that was more concentrated in AS supernatants from FSGS plasma than the other plasma samples and that was identified by microsequencing as apolipoprotein A1. 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 concentration in the FSGS samples than in non-FSGS NS and healthy individuals samples. This band is likely to correspond to a candidate albuminuric factor recently reported by another group [1], and was identified by microsequencing as alpha1 acid glycoprotein or orosomucoid. Consequently, purified orosomucoid from the plasma of FSGS, non-FSGS NS patients, or healthy individuals 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.