Yuko Miyazawa1, Saki Mikami1, Keiko Yamamoto2, Masaki Sakai1, Tatsuya Saito1, Tadashi Yamamoto2, Kenichi Ishibashi1, Sei Sasaki3,4. 1. Department of Pathophysiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, 204-8588, Japan. 2. Biofluid Biomarker Center, Niigata University, Niigata, 950-2181, Japan. 3. Department of Pathophysiology, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo, 204-8588, Japan. ssasaki.kid@tmd.ac.jp. 4. Department of Nephrology, Tokyo Medical and Dental University, Tokyo, 113-8519, Japan. ssasaki.kid@tmd.ac.jp.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Taken together, AQP2 is localized predominantly to urinary exosomes with preserved water channel activities.
BACKGROUND:AQP2water 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. METHODS: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. RESULTS: 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. CONCLUSION: Taken together, AQP2 is localized predominantly to urinary exosomes with preserved water channel activities.
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