Production of hemopexin by TNF- stimulated human mesangial cells

Plasma hemopexin has been shown to induce proteinuria after intrarenal infusion in rats, as well as glomerular alterations identical to those seen in corticosteroid-responsive nephrotic syndrome (CRNS). The question emerged whether also renal cells are potentially able to release hemopexin. Normal human mesangial cells (HMC) were incubated overnight in serum-free medium with or without tumor necrosis factor-alpha (TNF-alpha) (10 ng/mL). Parallel cultures were supplemented with prednisolone (10-3 mol/L). Concentrated supernatants were analyzed by Western blotting, using antihemopexin immunoglobulin G (IgG). Antitransferrin IgG served as control antibody. In addition, cytospins were stained using polyclonal or monoclonal antihemopexin IgG. A part of the cells was used for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR), to study hemopexin mRNA. Eighty five kD bands were exclusively detected by antihemopexin IgG in the Western blots in supernatants from TNF-alpha-stimulated cultures and to a lesser extent in prednisolone-treated cultures. Cells from TNF-alpha-stimulated cultures stain positive for hemopexin in contrast to those from prednisolone-treated or nonstimulated cultures. RT-PCR data suggest that mRNA for hemopexin is up-regulated in TNF-alpha-treated versus prednisolone-treated HMC. Stimulated HMC are able to release hemopexin in vitro in a corticosteroid-dependent manner. As preliminary data indicate that mesangial hemopexin is able to affect glomerular anionic sites, it is conceivable that stimulated mesangium may contribute to enhanced glomerular permeability in CRNS through local hemopexin release.