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Is 100KF an Isoform of Hemopexin? Immunochemical Characterization of the Vasoactive Plasma Factor 100KF

PO KAM CHEUNG^*, BEN STULP^*, STEPHAN IMMENSCHUH, THEO BORGHUIS^*, JULIUS F. W. BALLER^* and WINSTON W. BAKKER^*

^* Department of Pathology, University of Groningen, The Netherlands
Department of Internal Medicine, Georg-August-University, Göttingen, Germany.

Correspondence to Dr. Winston W. Bakker, Department of Pathology, University of Groningen, P. O. Box 30.001, 9700 RB Groningen, The Netherlands. Phone: +31 50 3619527; Fax: +31 50 3632510; E-mail: w.w.bakker{at}path.azg.nl

Abstract

Abstract. The human vasoactive plasma factor 100KF has been proposed to play a role in minimal change disease in relapse. Since preliminary data suggested similarity between 100KF and the human plasma glycoprotein hemopexin (Hx), this study was conducted to compare 100KF with purified Hx for sequence homology, immunostaining properties in Western and dot-blot assays, ability to affect glomerular ecto-ATPase and glomerular polyanions in vitro, as well as their glomerular permeability increasing effect following alternate perfusion into the rat kidney ex vivo. 100KF was purified from normal pooled plasma according to standard chromatographic techniques, and from the same batch Hx was prepared using affinity chromatography. A second batch of Hx was prepared directly from human serum according to a standard protocol. (For comparison, additional Hx samples obtained from other centers were also included in the study.) The results show: (1) 100% homology of 100KF with plasma Hx after internal sequence analysis; (2) positive staining of the eluate with both monoclonal and polyclonal anti-Hx IgG as well as anti-100KF IgG in dot-blot assays, and similar bands on Western blotting using the same antibodies; (3) affection of glomerular polyanions and glomerular ecto-ATPase after incubation of kidney tissue with either 100KF or Hx (1.5 respectively 1.0 mg/ml; 1.0 h, 37°C), as detected by computerized histochemical quantification; and (4) significant enhancement of urinary protein leakage after Hx perfusion followed by diluted rat serum into the rat kidney ex vivo (Hx: 210.65 ± 49.79 µg protein leakage per min versus heat-inactivated Hx control: 112.2 ± 49.18 µg per min [both n = 6]). From these data and from the observation that both Hx and 100KF activity can be inhibited by serine protease inhibitors but not by broad spectrum collagenase inhibitors, it is concluded that Hx may be closely related or identical to the active moiety of 100KF.

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Copyright © 2008 by the American Society of Nephrology. Online ISSN: 1533-3450 Print ISSN: 1046-6673