title: Development of enzyme-linked immunosorbent assays for urinary thiazide-sensitive Na-Cl cotransporter measurement.
abstract: The Na-Cl cotransporter (NCC) in the distal convoluted tubules in kidney is known to be excreted in urine. However, its clinical significance has not been established because of the lack of quantitative data on urinary NCC. We developed highly sensitive enzyme-linked immunosorbent assays (ELISAs) for urinary total NCC (tNCC) and its active form, phosphorylated NCC (pNCC). We first measured the excretion of tNCC and pT55-NCC in urinary exosomes in pseudohypoaldosteronism type II (PHAII) patients since PHAII is caused by NCC activation. Highly increased excretion of tNCC and pNCC was observed in PHAII patients. In contrast, the levels of tNCC and pNCC in the urine of patients with Gitelman's syndrome were not detectable or very low, indicating that both assays could specifically detect the changes in urinary NCC excretion caused by the changes of NCC activity in the kidney. Then, to test whether these assays could be feasible for a more general patient population, we measured tNCC and pNCC in the urine of outpatients with different clinical backgrounds. Although urinary protein levels >30 mg/dl interfered with our ELISA, we could measure urinary pNCC in all patients without proteinuria. Thus we established highly sensitive and quantitative assays for urinary NCC, which could be valuable tools for estimating NCC activity in vivo.
authors.affiliation: Dept. of Nephrology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental Univ., 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan. suchida.kid@tmd.ac.jp.
authors.forename: Kiyoshi
authors.initials: K
authors.lastname: Isobe
authors.forename: Takayasu
authors.initials: T
authors.lastname: Mori
authors.forename: Takako
authors.initials: T
authors.lastname: Asano
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kawaguchi
authors.forename: Shigeaki
authors.initials: S
authors.lastname: Nonoyama
authors.forename: Naonori
authors.initials: N
authors.lastname: Kumagai
authors.forename: Fumiaki
authors.initials: F
authors.lastname: Kamada
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Morimoto
authors.forename: Matsuhiko
authors.initials: M
authors.lastname: Hayashi
authors.forename: Eisei
authors.initials: E
authors.lastname: Sohara
authors.forename: Tatemitsu
authors.initials: T
authors.lastname: Rai
authors.forename: Sei
authors.initials: S
authors.lastname: Sasaki
authors.forename: Shinichi
authors.initials: S
authors.lastname: Uchida
mesh_terms: D000368:Aged; D000818:Animals; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D055354:Exosomes / Q000737:chemistry; D005260:Female; D053579:Gitelman Syndrome / Q000652:urine; D006801:Humans; D008297:Male; D051379:Mice; D008875:Middle Aged; D010766:Phosphorylation; D011546:Pseudohypoaldosteronism / Q000652:urine; D049993:Sodium Chloride Symporter Inhibitors; D064486:Solute Carrier Family 12, Member 3 / Q000652:urine*
chemical_list: D049993:Sodium Chloride Symporter Inhibitors; D064486:Solute Carrier Family 12, Member 3
keywords: WNK kinase; exosome; hypertension; pseudohypoaldosteronism type II