Vitamin A excreted in the urine of canines is associated with a Tamm-Horsfall like protein

Under physiological conditions canines transport vitamin A in blood plasma primarily as retinyl esters bound to lipoproteins and excrete substantial amounts of vitamin A as retinol and retinyl esters with urine. In the aqueous environment of urine, the hydrophobic vitamin A has to be associated with a protein. This vitamin A-protein complex was purified to homogeneity, prepared by preparative ultracentrifugation (density 1.21 g/mL), native polyacrylamide gel electrophoresis (PAGE) and size exclusion chromatography. The vitamin A-protein complex has a high molecular mass of > 5,000 kDa under native conditions. SDS PAGE under reduced conditions revealed a single band with a molecular mass of about 100 kDa for the protein moiety. Peptides obtained after limited proteolysis with trypsin from the 100 kDa protein were characterised by MALDI-TOF mass spectrometry and showed amino acid sequence homology to the human Tamm-Horsfall Protein (THP). This was further confirmed by a positive immunoreaction of the isolated protein with crossreacting human THP antibodies. The localisation of THP in dog kidneys was determined by using immunohistology. The reaction was strong along the entire thick ascending limb ofthe Henle loop and distal convoluted tubule. Our data point to the possibility that THP functions as a novel carrier for vitamin A in the urine of canines.