Background: Previous studies into the relevance of a putative circulating factor in the pathogenesis of minimal change nephrotic syndrome have opened the possibility that plasma hemopexin might be an important effector molecule in this disorder. Thus, intra renal infusion of isolated plasma hemopexin into rats induced minimal change like glomerular lesions and proteinuria. Both, in vivo and in vitro effects of the active isoform of hemopexin could be attributed to protease activity of this molecule. However, the question remained whether hemopexin per se rather than some contaminating plasma factor is responsible for the potential enzymatic activity of this molecule.
Methods: Recombinant hemopexin was prepared according to standard methods in Pichia pastoris and compared for its identity and protease activity with plasma hemopexin using Western blotting and various in vitro assays. Unilateral renal perfusion in anesthetized rats was used to test the proteinuria inducing capacity of recombinant hemopexin versus heat-inactivated recombinant hemopexin.
Results: The blotting results show identical 85 kD bands in both native as well as recombinant hemopexin. Incubation of kidney tissue with recombinant hemopexin resulted in loss of of glomerular ectoapyrase and sialoglycoproteins, as shown by immunohistochemistry, which effect can be inhibited with the serine protease inhibitor phenylmethanesulfonyl fluoride. Artificial substrates for serine proteases, like kallikrein or thrombin, are hydrolysed by recombinant hemopexin in vitro, and not by heat-inactivated recombinant hemopexin or saline. Unilateral kidney perfusion of recombinant hemopexin, in contrast to control Pichia transfection products or heat-inactivated recombinant hemopexin, followed by a protein marker showed significantly enhanced urinary protein leakage 5.0, 10.0, and 15.0 minutes after perfusion.
Conclusion: It is concluded that the hemopexin molecule as such can potentially act as a toxic protease, leading in the rat to proteinuria and glomerular alterations characteristic for minimal change nephrotic syndrome.