AQP2 in human urine is predominantly localized to exosomes with preserved water channel activities

Autor: Saki Mikami, Tadashi Yamamoto, Keiko Yamamoto, Yuko Miyazawa, Tatsuya Saito, Masaki Sakai, Kenichi Ishibashi, Sei Sasaki
Rok vydání: 2018
Předmět:
Zdroj: Clinical and Experimental Nephrology. 22:782-788
ISSN: 1437-7799
1342-1751
DOI: 10.1007/s10157-018-1538-6
Popis: AQP2 water channel is critical for urinary concentration in the kidney. Interestingly, AQP2 is abundantly excreted in the urine as extracellular vesicles (EVs), which is known to be a useful biomarker for water-balance disorders although the character of AQP2-enriched EVs is poorly understood including water channel function. Human urine EVs were obtained by a differential centrifugation method. AQP2-bearing EVs were isolated by immunoprecipitation with an AQP2-specific antibody, and the proteins in the EVs were analyzed by LC–MS/MS proteomic analysis. Osmotic water permeability (Pf) of the AQP2-rich EVs was measured by a stopped-flow method monitoring scattered light intensity in response to outwardly directed osmotic gradient. Sequential centrifugation of human urine showed that AQP2 was present predominantly (80%) in low-density EVs (160,000 g), whereas negligible amount in high-density EVs (17,000 g). Proteomic analysis of the AQP2-bearing EVs identified 137 proteins, mostly in the endosome pathway, including the components of ESCRT (endosomal sorting complex required transporter)-I, II, III. Pf value of the 160,000 g EVs was 4.75 ± 0.38 × 10−4 cm s−1 (mean ± SE) with the activation energy of 3.51 kcal mol−1 which was inhibited with 0.3 mM HgCl2 by 63%, suggesting a channel-mediated water transport. Moreover, Pf value showed a significant correlation with the abundance of AQP2 protein in EVs. Taken together, AQP2 is localized predominantly to urinary exosomes with preserved water channel activities.
Databáze: OpenAIRE
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