Endothelin-1 inactivating peptidase in the human kidney and urine
Endothelin-1 inactivating peptidase in the human kidney and urine
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DOI：
10.1097/00004872-200018040-00018
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发表时间：
2000-04-01
影响因子：
4.9
通讯作者：
Janas, RM
中科院分区：
文献类型：
--
作者：
Janas, J;Sitkiewicz, D;Janas, RM
目的从大鼠肾脏中分离到一种新的特异性内皮素1失活金属内肽酶（ET 1 peptidase）。在这项研究中，我们试图确定人类肾脏中是否存在相同或相似的肽酶，以及这种酶是否被排泄到尿液中。尿ET-1肽酶可作为肾内皮素系统生理和病理生理的一个间接指标。方法6例成人肾肿瘤患者的部分肾切除后的肾脏标本。采用差速离心、洗涤剂溶解膜蛋白、超滤和非变性凝胶电泳对酶进行纯化。在pH 5.5和37 ℃下，在浓度递增的未标记肽和抑制剂存在下，使用固定量的[I-125] ET-1作为底物，进行酶活性测定。用三氯乙酸沉淀法和高效液相色谱法测定其降解程度，并测定高血压患者尿中ET-1的降解活性，结果人肾ET-1肽酶与我们最近报道的大鼠ET-1肽酶具有相似的特性（J.Hypertens 1994; 12：1155 - 1162）。该酶是一种膜结合金属内肽酶，在非变性凝胶上具有低的电泳迁移率（R-f 0.08）;它是包含三个酶失活亚基的明显异源结构，其最适pH为5.5，对水解为两种主要降解产物的ET-1具有纳摩尔范围的亲和力（K（M）180 nmol/l），对大ET-1（K（M）11.5 μ mol/l）、内皮素11 - 21片段（K-M 15.3 μ mol/l）、内皮素拮抗剂Trp-Leu-Asp-lle-lle-Trp（K-M 3.1 μ mol/l）、胃泌素（K-M 2.2 μ mol/l）和胆囊收缩素（K-M 4.0 μ mol/l）的亲和力低10 - 100倍。P物质、神经肽Y、心钠素、缓激肽、血管紧张素II和脑啡肽是ET-1肽酶的不良底物（IC50 0.28 nmol/l），磷酰胺（IC50 0.55 nmol/l），菲咯啉（IC 50 11.5 μ mol/l），环孢菌素（IC50 400 μ mol/l），磷酸盐（IC50 1.2 mmol/l），柠檬酸（IC50 0.6 mmol/l）和苯胺萘磺酸我们的数据表明，三种ET-1降解肽酶的最佳活性分别在pH 4.5，5.5和7.0，排泄到尿液中。具有最适pH 4.5的酶是溶酶体来源的，而另外两种酶通过其最适pH对应于肾ET-1肽酶和中性内肽酶。我们发现统计学上的显著增加高血压患者和慢性肾功能衰竭患儿尿中溶酶体和ET-1肽酶活性与正常对照组和肾移植稳定患儿比较差异有显著性（P <0.001）。高特异性内皮素-1灭活金属内肽酶，其可能在肾和循环内皮素浓度的调节中起关键作用。该酶被排泄到尿液中，在高血压和慢性肾衰竭患者中其活性似乎增加;它可能作为肾内皮素系统的间接指标。J Hypertens 2000，18：475 - 483（C）Lippincott威廉姆斯& Wilkins.
Objective Recently, an apparently novel, specific endothelin-1 inactivating metalloendopeptidase (ET-1 peptidase) has been isolated from the rat kidney. In this study we attempted to determine whether the same or a similar peptidase is present in the human kidney, and whether the enzyme is excreted into the urine. The urinary ET-1 peptidase could serve as an indirect index of the renal endothelin system, both in physiology and pathophysiology.Methods Kidney specimens were obtained from part of nephrectomized kidneys unaffected by any neoplastic process from six adult patients. The enzyme was purified using differential centrifugation, detergent solubilization of the membrane proteins, ultrafiltration and nondenaturing gel electrophoresis. The enzyme activity assays were performed at pH 5.5 and 37 degrees C in the presence of increasing concentrations of unlabelled peptides and inhibitors using a fixed amount of [I-125]ET-1 as substrate. The degradation extent was quantified with trichloroacetic acid precipitation and high performance liquid chromatography, The degrading activity of ET-1 was determined in urine samples from adult patients with hypertension, children with chronic renal failure and those with stable renal allograft.Results ET-1 peptidase from the human kidney displays characteristics close to that of the rat ET-1 peptidase we have recently described (J. Hypertens 1994; 12:1155-1162). The enzyme, a membrane-bound metalloendopeptidase, exhibits low electro- phoretical mobility on nondenaturing gel (R-f 0.08); it is an apparently heterologous structure comprising three enzymatically inactive subunits, it has a pH optimum at 5.5, a nanomolar range affinity to the ET-1 (K(M)180 nmol/l) that is hydrolysed to two main degradation products, and a 10-100-fold lower affinity to big ET-1 (K(M)11.5 mu mol/l), endothelin 11 - 21 fragment(K-M 15.3 mu mol/l), endothelin antagonist Trp-Leu-Asp-lle-lle-Trp (K-M 3.1 mu mol/l), gastrin (K-M 2.2 mu mol/l) and cholecystokinin (K-M 4.0 mu mol/l). Substance P, neuropeptide Y, atrial natriuretic peptide, bradykinin, angiotensin II and enkephalin were poor substrates for the enzyme, The most powerful inhibitors of the ET-1 peptidase included thiorphan (IC50 0.28 nmol/l), phosphoramidon (IC50 0.55 nmol/l), phenanthroline (IC50 11.5 mu mol/l), cyclosporin (IC50 400 mu mol/l), phosphate (IC50 1.2 mmol/l), citrate (IC50 0.6 mmol/l) and aniline naphthalene sulphonic acid (IC50 0.25 mmol/l).Our data suggest that three ET-1 degrading peptidases with optimal activity at pH 4.5, 5.5 and 7.0, respectively, are excreted into the urine. The enzyme with a pH optimum 4.5 is of lysosomal origin whereas the two other enzymes correspond by their pH optima to the renal ET-1 peptidase and neutral endopeptidase. We have found statistically significant increases (P < 0.001) in the activity of both lysosomal and ET-1 peptidase in the urine in patients with hypertension and in children with chronic renal failure compared with healthy subjects or children with stable renal allograft.Conclusions Human kidney contains an acidic, highly specific endothelin-1 inactivating metalloendopeptidase that may have a key role in the regulation of concentrations of renal and circulating endothelins. The enzyme is excreted into the urine where its activity seems to be increased in patients with hypertension and chronic renal failure; it may potentially serve as an indirect index of the renal endothelin system. J Hypertens 2000, 18:475-483 (C) Lippincott Williams & Wilkins.