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

Abstract

Several transporters have been localized along the nephron by physiological methods or immunocytochemistry. However, the actual abundance of these molecules has not been established. To accomplish this goal, we have developed a fluorescence-based ELISA method and have 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) or purified AQP-CHIP standards (0-200 fmol) were utilized in a fluorescence ELISA protocol after covalent immobilization on epoxy-activated Sepharose beads. The lower limit of detection was 2.4 fmol of AQP-CHIP. Preabsorption with excess purified AQP-CHIP or use of nonimmune serum eliminated the signal. In proximal segments, the measured AQP-CHIP was linearly related to tubule length (1-10 mm). The measured AQP-CHIP was (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; type 2 DTL, 86.5 +/- 19.5; type 3 DTL, 43.0 +/- 11.2. In thin ascending limbs, thick ascending limbs, distal convoluted tubules, connecting tubules, and collecting ducts, the AQP-CHIP signal was indistinguishable from zero. Based on the unit water conductance of single CHIP molecules, our calculations show that the content of AQP-CHIP is sufficient to explain water permeability measured in isolated proximal tubules and DTL segments.