原发性高血压患者血浆和尿液细胞外囊泡中的糜蛋白酶活性

IF 3.2 Q1 UROLOGY & NEPHROLOGY
Sarfaraz Ahmad, Gagan Deep, Henry A Punzi, Yixin Su, Sangeeta Singh, Ashish Kumar, Shalini Mishra, Amit K Saha, Kendra N Wright, Jessica L VonCannon, Louis J Dell'Italia, Wayne J Meredith, Carlos M Ferrario
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引用次数: 0

摘要

背景:循环中的细胞外囊泡(EVs)携带核酸、蛋白质和代谢物等受保护的货物。在此,我们鉴定并验证了从原发性高血压患者血液和尿液中分离出的EVs中的糜蛋白酶、血管紧张素转换酶1(ACE)和2(ACE2)以及肾酶(NEP)的表面蛋白和酶活性:方法:对 34 名高血压患者的外周静脉血和点滴尿液进行处理,以分离血浆和尿液中的 EVs。免疫金标记和透射电子显微镜验证了血浆和尿液 EVs 表面存在外泌体标记蛋白 CD63。流式细胞术鉴定了血浆和尿液EV的CD63、CD9和CD81表面标记物。此外,还通过 Western 印迹分析了尿液中的外泌体 CD63、TSG101 和 Alix。尿液 EVs 不表达内质网蛋白 calnexin 和高尔基体蛋白 GM130。通过 HPLC 对 125I 底物 - 血管紧张素-(1-12)[Ang-(1-12)] 和血管紧张素 II (Ang II) - [各 1 nmol/L]的 Chymase、ACE、ACE2 和 NEP 活性进行量化。数据根据患者血压是否得到控制进行分析(第一组:结果;第二组:结果;第三组:结果):血浆和尿液中的 EVs 对 Ang-(1-12) 的糜蛋白酶活性明显高于 ACE、ACE2 和 NEP。此外,尿液 EVs 中的糜蛋白酶活性是血浆 EVs 中的 3 倍多。第二组患者的血浆和尿液 EV 中的糜蛋白酶活性都有所增加。第一组和第二组患者尿液 EV 中 ACE、ACE2 和 NEP 的酶活性没有可比性差异:这些研究表明,从高血压患者体内分离出的血浆和尿液 EVs 中,肾素血管紧张素系统酶的酶活性存在差异。在 EVs 中显示出相对较高的糜蛋白酶酶活性,进一步证实了之前关于高血压患者血浆 Ang-(1-12) 增高的发现。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Chymase Activity in Plasma and Urine Extracellular Vesicles in Primary Hypertension.

Background: Circulating extracellular vesicles (EVs) carry protected cargoes of nucleic acids, proteins, and metabolites. Here we identified and validated the surface proteins and enzymatic activity of chymase, angiotensin converting enzymes 1 (ACE) and 2 (ACE2), and neprilysin (NEP) in EVs isolated from the blood and urine of primary hypertensive patients.

Methods: Peripheral venous blood and spot urine from 34 hypertensive patients were processed to isolate plasma and urinary EVs. Immuno-gold labeling and transmission electron microscopy validated the presence of the exosomal marker protein CD63 on the surface of plasma and urinary EVs. Flow cytometry characterized plasma and urinary EVs for CD63, CD9, and CD81 surface markers. In addition, exosomal CD63, TSG101, and Alix were analyzed in urine by Western blotting. Urinary EVs did not express the endoplasmic reticulum protein calnexin and Golgi protein GM130. Chymase, ACE, ACE2, and NEP activities on 125I substrates ─ angiotensin-(1-12) [Ang-(1‒12)] and angiotensin II (Ang II) ─ [1 nmol/L each] were quantified by HPLC. Data were analyzed based on whether the patient's blood pressure was controlled (Group I: <140/80 mm Hg) or not controlled (Group II: ≥ 140/80 mm Hg).

Results: Chymase activity on Ang-(1‒12) was significantly higher in plasma and urinary EVs than in ACE, ACE2, and NEP. In addition, chymase activity in urine EVs was more than 3-fold higher than in plasma EVs. Chymase activity increased in plasma and urine EVs retrieved from Group II patients. No comparable differences were found in the enzymatic activities of ACE, ACE2, and NEP urinary EVs between Group I and Group II.

Conclusion: These studies reveal a differential enzymatic activity of renin angiotensin system enzymes in plasma and urine EVs isolated from hypertensive patients. Demonstrating a comparatively high chymase enzymatic activity in EVs expands a previously documented finding of increased plasma Ang-(1‒12) in hypertensive patients.

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来源期刊
Kidney360
Kidney360 UROLOGY & NEPHROLOGY-
CiteScore
3.90
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