Cheung P K, Stulp B, Immenschuh S, Borghuis T, Baller J F, Bakker W W
Department of Pathology, University of Groningen, The Netherlands.
J Am Soc Nephrol. 1999 Aug;10(8):1700-8. doi: 10.1681/ASN.V1081700.
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 degrees 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 microg protein leakage per min versus heat-inactivated Hx control: 112.2+/-49.18 microg 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.
有人提出人类血管活性血浆因子100KF在微小病变病复发中起作用。由于初步数据表明100KF与人类血浆糖蛋白血红素结合蛋白（Hx）相似，本研究旨在比较100KF与纯化的Hx在序列同源性、蛋白质免疫印迹和斑点印迹分析中的免疫染色特性、体外影响肾小球外ATP酶和肾小球多阴离子的能力，以及它们在大鼠离体肾脏交替灌注后增加肾小球通透性的作用。按照标准色谱技术从正常混合血浆中纯化100KF，并使用亲和色谱法从同一批次中制备Hx。根据标准方案直接从人血清中制备第二批Hx。（为作比较，研究中还纳入了从其他中心获得的额外Hx样本。）结果显示：（1）内部序列分析后100KF与血浆Hx具有100%的同源性；（2）在斑点印迹分析中，洗脱液用单克隆和多克隆抗Hx IgG以及抗100KF IgG染色均呈阳性，并且使用相同抗体进行蛋白质免疫印迹时出现相似条带；（3）通过计算机组织化学定量检测，用100KF或Hx（分别为1.5和1.0 mg/ml；37℃孵育1.0小时）孵育肾组织后，肾小球多阴离子和肾小球外ATP酶受到影响；（4）离体将Hx灌注大鼠肾脏后再灌注稀释的大鼠血清，尿蛋白漏出显著增加（Hx：每分钟210.65±49.79μg蛋白漏出，而热灭活Hx对照：每分钟112.2±49.18μg [n均为6]）。从这些数据以及Hx和100KF活性均可被丝氨酸蛋白酶抑制剂抑制但不能被广谱胶原酶抑制剂抑制这一观察结果得出结论，Hx可能与100KF的活性部分密切相关或相同。
