Absorption capacity of renal proximal tubular cells studied by combined injections of YFP and GFP in Rana temporaria L.

The capacity for protein reabsorption in the renal proximal tubule (PT) was studied in Rana temporaria frogs by separate, simultaneous and sequential introduction of yellow fluorescent protein (YFP) and green fluorescent protein (GFP). The uptake patterns of YFP and GFP in PT epithelial cells were investigated 15-120min after their bolus intravenous and intraperitoneal injection. As shown by confocal microscopy, the tubular uptake of YFP and GFP was time- and dose-dependent. These proteins are absorbed in similar way and can be accumulated in the same endocytic vesicles after their combined injections. When GFP was injected 30 and 90min before YFP, and vice versa, the number of vesicles with pre-injected protein increased and the percentage of vesicles with colocalized GFP and YFP reduced. At the same time, the uptake rate of a protein injected later progressively and significantly decreased. Subcellular localization of endocytic receptors, megalin and cubilin, in renal PT cells after intravenous YFP introduction were revealed by immunofluorescent microscopy. Colocalization of internalized YFP with megalin or cubilin in the endocytic vesicles was demonstrated. The data suggest the possibility of protein uptake by receptor-mediated endocytosis and the existence of a mechanism limiting the protein absorption rate in wintering frogs.