Distribution of the alphaGal- and the non-alphaGal T-antigens in the pig kidney: potential targets for rejection in pig-to-man xenotransplantation

Carbohydrate antigens, present on pig vascular endothelial cells, seem to be the prime agents responsible for graft rejection, and although genetically modified animals that express less amounts of carbohydrate antigen are available, it is still useful to decide the localization of the reactive xenoantigens in organs contemplated for xenotransplantation. Here we compare the distribution in pig kidney of antigens important in xenograft destruction, namely the Galalpha1-3Gal (alphaGal) glycans, with the localization of the T-antigen (Galbeta1-3GalNAc). The alpha-galactose-specific lectin Griffonia simplicifolia isolectin 1B4 was used to detect the Galalpha1-3Gal glycans, whereas Arachis hypogaea (PNA) lectin and a monoclonal antibody (3C9) detected T-antigen. In addition, two vascular markers (anti-caveolin-1 and anti-von Willebrand factor) served to identify vascular structures of the kidney. Both conventional fluorescence and confocal microscopy were used to distinguish lectin and immunohistochemical staining. On the basis of fluorescence signals, the results indicate that the carbohydrate antigens are heterogeneously distributed in the pig kidney. alphaGal epitopes were sparse in the capillary loops forming the glomeruli and in the capillaries surrounding the convoluted tubules, but showed stronger staining in capillaries surrounding the limbs of Henle. In addition, the brush border and basement membranes of the convoluted tubules strongly reacted with the GS1-B4-lectin. Finally, the Galalpha1-3Gal glycans were also present on epithelial cells of the large collecting tubules. Regarding the T-antigen, PNA and 3C9 reacted with different glomerular cells, whereas both reacted strongly with the endothelial cells lining the large kidney vessels. Human serum incubation of pig kidney sections, in which the alphaGal epitopes were blocked by unconjugated GS1-B4, showed staining of the same vascular structures as were obtained after incubation with the T-antigen-detecting agents. The study thus proves a complex spatial distribution of carbohydrate antigens relevant for xenotransplantation of pig kidney.