Quantification of Aquaporin-CHIP water channel protein in microdissected renal tubules by fluorescence-based ELISA.

摘要: Several transporters have been localized along the nephron by physiological methods or immunocytochemistry. However, actual abundance of these molecules has not established. To accomplish this goal, we developed a fluorescence-based ELISA method and used it to quantitate Aquaporin-CHIP (AQP-CHIP) water channel protein in rat kidney tubules. Microdissected tubules (2 mm/sample, permeabilized with 0.5% Triton X-100) purified AQP-CHIP standards (0-200 fmol) were utilized fluorescence protocol after covalent immobilization on epoxy-activated Sepharose beads. The lower limit detection was 2.4 fmol AQP-CHIP. Preabsorption excess use nonimmune serum eliminated signal. In proximal segments, measured linearly related tubule length (1-10 mm). (mean +/- SE, fmol/mm): S-1 proximal, 10.8 2.1; S-2, 10.0 2.3; S-3, 21.3 3.1; type 1 thin descending limb (DTL), 12.9 4.6; 2 DTL, 86.5 19.5; 3 43.0 11.2. ascending limbs, thick distal convoluted tubules, connecting collecting ducts, signal indistinguishable from zero. Based unit conductance single CHIP molecules, our calculations show that content is sufficient explain permeability isolated DTL segments.