Nearly 25% of patients with FSGS develop recurrent disease following renal transplantation. A plasma factor has been isolated from these patients which increases glomerular permeability to protein in vitro. NO modulates renal hemodynamics and extracellular matrix production. Therefore, we studied whether the FSGS plasma factor alters NO synthesis by RMC.

RMC were cultured in DMEM containing IFN-gamma (50 U/ml) and LPS (10 mcg/ml). Test media contained either (a) no further additives; (b) control plasma; or (c) plasma from patients with recurrent FSGS. Plasma was precipitated with 70% ammonium sulfate and then added to media at a concentration of 20 μicroliter (2 ng protein)/ml. NO production was assayed by the Griess reaction. Cell viability was determined by adding MTS reagent and measuring absorbance at 490 nm. iNOS protein content was assessed by immunoblotting and mRNA abundance by Northern analysis.

Data (mean±SEM) for RMC incubated in control media with no additives or with control plasma were virtually identical and were combined for further analysis. Exposure to the FSGS factor for 24 h enhanced RMC proliferation from 0.97±0.06 to 1.74±0.10 (n=8) (P<0.05). RMC proliferation was unchanged after 48 h incubation with the FSGS factor. In contrast, NO production (nmol/well) was reduced from 0.36±0.09 to 0.09±0.02 after 24 h, and from 1.98±0.54 to 0.12±0.03 after 48 h(P<0.05). Indomethacin (0.1-1.0 mcM) did not reverse the inhibitory effect of the FSGS factor on NO synthesis. Changes in NO production were paralleled by alterations in steady state iNOS mRNA abundance at 24 h and iNOS protein content at 48 h.

We conclude that the FSGS factor reduces iNOS gene expression and protein synthesis and suppresses NO production in cultured RMC. Because of the time-course and the inability of indomethacin to reverse the effect, alterations in NO metabolism induced by the FSGS factor are unlikely to contribute to the acute alterations in glomerular permeability. Instead, the effect of the plasma factor on iNOS activity may play a role in the progressive glomerulosclerosis observed in recurrent disease post-transplantation.