Bakker Winston W, Borghuis Theo, Harmsen Martin C, van den Berg Anke, Kema Ido P, Niezen Klary E, Kapojos Jola J
Department of Pathology and Laboratory Medicine, and Department of Pediatrics, University Hospital Groningen, Groningen, The Netherlands.
Kidney Int. 2005 Aug;68(2):603-10. doi: 10.1111/j.1523-1755.2005.00438.x.
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.
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.
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.
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.
先前关于一种假定的循环因子在微小病变肾病综合征发病机制中的相关性研究表明，血浆血红素结合蛋白可能是该疾病中的一种重要效应分子。因此，将分离的血浆血红素结合蛋白肾内注射到大鼠体内可诱发类似微小病变的肾小球损伤和蛋白尿。血红素结合蛋白活性异构体的体内和体外效应都可归因于该分子的蛋白酶活性。然而，问题仍然存在，即究竟是血红素结合蛋白本身而非某些污染的血浆因子导致了该分子的潜在酶活性。
按照毕赤酵母中的标准方法制备重组血红素结合蛋白，并使用蛋白质印迹法和各种体外试验将其与血浆血红素结合蛋白的同一性和蛋白酶活性进行比较。在麻醉大鼠中进行单侧肾灌注，以测试重组血红素结合蛋白与热灭活重组血红素结合蛋白诱导蛋白尿的能力。
印迹结果显示天然和重组血红素结合蛋白中均有相同的85 kD条带。免疫组织化学显示，重组血红素结合蛋白与肾组织孵育导致肾小球外核苷酸焦磷酸酶和唾液糖蛋白丢失，丝氨酸蛋白酶抑制剂苯甲基磺酰氟可抑制该效应。丝氨酸蛋白酶的人工底物，如激肽释放酶或凝血酶，在体外可被重组血红素结合蛋白水解，而热灭活重组血红素结合蛋白或生理盐水则不能。与对照毕赤酵母转染产物或热灭活重组血红素结合蛋白相比，重组血红素结合蛋白单侧肾灌注后，灌注5.0、10.0和15.0分钟后，蛋白质标志物显示尿蛋白渗漏显著增加。
得出结论，血红素结合蛋白分子本身可能作为一种毒性蛋白酶，在大鼠中导致微小病变肾病综合征特征性的蛋白尿和肾小球改变。
