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 across 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-linked immunosorbent assay capable of detecting <5 fmol of VRUT protein. In collecting duct segments, measurable VRUT was found in microdissected IMCD segments but not in other portions of the collecting duct. In the mid-IMCD, the measured level averaged 5.3 fmol/mm tubule length, corresponding to approximately 5 million copies of VRUT per cell. Thus VRUT is extremely abundant in the IMCD, accounting, in part, for the extremely 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 transport across the collecting duct with dietary protein restriction is not a consequence of increased expression of VRUT. Based on urea fluxes measured in the IMCD and our measurements of the number of copies of VRUT, we estimate a turnover number of > or = 0.3-1 x 10(5) s. In view of the large magnitude of this value and previously reported biophysical properties of urea transport in collecting ducts, we hypothesize that the VRUT may function as a channel rather than a carrier.
Kishore
B K
BK
Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Terris
J
J
Fernández-Llama
P
P
Knepper
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D001692:Biological Transport; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D018753:Diet, Protein-Restricted; D004210:Dissection; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007679:Kidney Medulla / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D014508:Urea / Q000378:metabolism*; D014556:Urine / Q000737:chemistry; D000096982:Urea Transporters
D000906:Antibodies; D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010446:Peptide Fragments; D014508:Urea; D000096982:Urea Transporters