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1 Division of Nephrology, Indiana University School of Medicine, Indianapolis, IN, USA; Chemistry, Purdue University, West Lafayette, IN, USA
2 The Indiana Center for Biological Microscopy, Indianapolis, IN, USA
* To whom correspondence should be addressed. E-mail: bmolitor{at}iupui.edu.
Intra-vital 2-photon microscopy was used to follow the uptake and trafficking of fluorescent conjugates of folic acid in the rat kidney. Intravenously administered folate-linked dye molecules quickly filled the plasma volume but not cellular components of the blood. Glomerular filtration occurred immediately and binding to proximal tubule cells was seen within seconds. Fluorescence from a pH-insensitive conjugate of folic acid, folate-Texas Red (FTR), was readily observed on the apical surface of the proximal tubules and in multiple cellular compartments, but little binding or uptake could be detected in any other kidney cells. Fluorescence from a pH-sensitive conjugate of folic acid, folate-fluorescein, was only seen on the apical surface of proximal tubule cells, suggesting that internalized folate-conjugates are localized to acidic compartments. The majority of the conjugates internalized by proximal tubules accumulated within a lysosomal pool, as determined by colocalization studies. However, portions of the folate conjugate were also shown by electron microscopy to undergo transcytosis from apical to basal domains. Additional studies with colchicine, known to depolymerize microtubules and interrupt transcytosis, produced a marked reduction in endocytosis of FTR, with accumulation limited to the sub-apical region of the cell. No evidence of cytosolic release of either folate conjugate was observed, which may represent a key difference from the cytosolic deposition seen in neoplastic cells. Taken together, these data argue that folate conjugates (and by extrapolation physiological folates) bind to the apical surface of proximal tubule cells and are transported into and across the cells in endocytic compartments.
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