Reabsorption of yellow fluorescent protein in the Rana temporaria kidney by receptor-mediated endocytosis

The absorption of yellow fluorescent protein (YFP) and the expression of the endocytic receptors, megalin and cubilin, were investigated in the renal proximal tubules (PT) in Rana temporaria frogs after parenteral YFP injections using confocal microscopy and immunohistochemistry. The dynamics of YFP absorption was analyzed in 2 h after injection. The logarithmic time dependence of the accumulation of YFP-containing endocytic vesicles in PT cells and the completion of absorption process 90-120 min after injection were revealed. In contrast to a pronounced megalin and cubilin expression in 15-30 min after YFP injection, the immunolabeled endocytic receptors were not detected in PT cells even in 2 h. The reinjection of YFP led to an appearance of apical endocytic vesicles containing megalin or cubilin colocalized with YFP. At the same time, a decrease in YFP uptake associated with the reduction in the number of receptor-containing vesicles was demonstrated, suggesting an insufficiency of megalin and cubilin expression. The decreased absorption capacity of PT cells after YFP reinjection was similar to that found previously in conditions of competitive absorption of green fluorescent protein (GFP) and YFP injected in different order (Prutskova, Seliverstova, 2013). The data represent yet another demonstration of the putative mechanism that restricts the tubular protein reabsorption in the frog kidney; they also indicate the involvement of megalin and cubilin in the uptake and vesicular transport of YFP.