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

Abstract

-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 of the 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. l'environnement aqueux de l'urine, la vitamine A hydrophobe doit être associée à une protéine. Ce complexe protéine-vitamine A a été purifié et homogénéisé, préparé par ultracentrifugation (1,21 g/mL de densité), électrophorèse sur gel de polyacrylamide natif (PAGE) et chromatographie d'exclusion de taille. Le complexe protéine-vitamine A a une masse moléculaire élevée supérieure à 5 000 kDa dans les conditions natives. Le SDS PAGE effectué dans des conditions réduites a révélé une bande unique avec une masse moléculaire d'environ 100 kDa pour la partie protéique. Les peptides obtenus à partir de la protéine de 100 kDa, après protéolyse limitée avec de la trypsine, ont été caractérisés par spectrométrie de masse MALDI-TOF et ont montré une homologie de séquence en acides aminés avec la protéine Tamm-Horsfall (PTH) humaine. Ceci a été confirmé par une immuno-réaction positive entre la protéine isolée et les anticorps présentant une réaction croisée avec la PTH humaine. La localisation de la PTH dans le rein de chien a été déterminée par immunohistologie. La réaction a été forte tout le long de la section ascendante épaisse de l'anse de Henle, et du tubule contourné distal. Nos résultats suggèrent que la PTH fonctionne comme un nouveau transporteur pour la vitamine A dans l'urine des canidés.vitamine A / canidés / rein / urine / protéine Tamm-Horsfall

Localization of Canine, Feline, and Mouse Renal Membrane Proteins

“…31,36 In previous reports, immunohistochemical staining was most intense at the apical margins of cells in the TAL. 31,36 Within the DCT, positive cells appeared intermittently and had a homogenous cytoplasmic expression. 31,36 Similar findings have been found in humans.…”

“…31,36 Humans reabsorb retinol, but dogs (and it is thought carnivores in general) excrete retinol and retinyl esters in their urine. 31,36 These products are hydrophilic and need a carrier in urine, which has recently been shown to be a Tamm-Horsfall-like protein. 36 In the study of this canine phenomenon, THP has been localized to the luminal cells of the TAL of the loop of Henle in the outer medulla and the cells composing the cortical DCT.…”

“…31,36 These products are hydrophilic and need a carrier in urine, which has recently been shown to be a Tamm-Horsfall-like protein. 36 In the study of this canine phenomenon, THP has been localized to the luminal cells of the TAL of the loop of Henle in the outer medulla and the cells composing the cortical DCT. 31,36 In previous reports, immunohistochemical staining was most intense at the apical margins of cells in the TAL.…”

“…36 In the study of this canine phenomenon, THP has been localized to the luminal cells of the TAL of the loop of Henle in the outer medulla and the cells composing the cortical DCT. 31,36 In previous reports, immunohistochemical staining was most intense at the apical margins of cells in the TAL. 31,36 Within the DCT, positive cells appeared intermittently and had a homogenous cytoplasmic expression.…”

Alterações morfofuncionais renais em cães Golden Retriever Distróficos (GRMD)

“…O predomínio de proteínas na região correspondente ao peso molecular da albumina foi descrito nos primeiros estudos de eletroforese em gel de poliacrilamida das proteínas urinárias em cães hígidos, sendo que a presença de proteínas de diferentes pesos moleculares na urina de cães sadios é normal, mas em pequenas quantidades (Finco 1995). A proteína de Tamm-Horsfall, proveniente dos rins, é um constituinte normal da urina de cães sadios (Schweigert et al 2002, Forterre et al 2004) na sua forma nativa esta proteína é um polímero de peso > 5000 kDa, que sofre dissociação em monômeros de peso em torno de 100 kDa durante a desnaturação por SDS-PAGE (Schweigert et al 2002). Portanto a banda protéica que aparece na faixa de proteínas de alto peso molecular, não precisa ser necessariamente interpretada como um componente patológico de origem glomerular, mas deve ser visto como um constituinte normal da urina (Raila et al 2007).…”