ULTRAMICRODETERMINATION OF VASOPRESSIN-REGULATED UREA TRANSPORTER PROTEIN IN MICRODISSECTED RENAL TUBULES

The vasopressin-regulated urea transporter (VRUT) is a 97-kDa protein (also called ''UT-1'') responsible for facilitated urea transport acro ss the apical plasma membrane of inner medullary collecting duct (IMCD ) cells. To determine the abundance of VRUT protein in collecting duct cells of the rat, we designed a sensitive fluorescence-based enzyme-l inked immunosorbent assay capable of detecting <5 fmol of VRUT protein . In collecting duct segments, measurable VRUT was found in microdisse cted IMCD segments but not in other portions of the collecting duct. I n the mid-IMCD, the measured level averaged 5.3 fmol/mm tubule length, corresponding to similar to 5 million copies of VRUT per cell. Thus V RUT is extremely abundant in the IMCD, accounting, in part, for the ex tremely high urea permeability of this segment. Feeding a low-protein diet (8% protein) markedly decreased urea clearance but did not alter the quantity of VRUT protein in the IMCD. Thus increased urea transpor t across the collecting duct with dietary protein restriction is not a consequence of increased expression of VRUT. Based on urea fluxes mea sured in the IMCD and our measurements of the number of copies of VRUT , we estimate a turnover number of greater than or equal to 0.3-1 x 10 (5) s. In view of the large magnitude of this value and previously rep orted biophysical properties of urea transport in collecting ducts, we hypothesize that the VRUT may function as a channel rather than a car rier.