MECHANISM AND REGULATION OF RIBOFLAVIN UPTAKE BY HUMAN RENAL PROXIMALTUBULE EPITHELIAL-CELL LINE HK-2

Riboflavin (RF), a water-soluble vitamin, is essential for normal cell ular functions, growth, and development. Normal RF body homeostasis de pends on intestinal absorption and recovery of the filtered vitamin in renal tubules. The mechanism and cellular regulation of the RF renal reabsorption process, especially in the human situation, are poorly un derstood. The aim of this study was therefore to address these issues, using a recently established human normal renal epithelial cell line, HK-2, as a model. Uptake of RF by HK-2 cells was found to be 1) linea r with time for 5 min of incubation and occurring with minimal metabol ic alterations, 2) temperature dependent, 3) Na+ independent, 4) satur able as a function of concentration [apparent Michaelis constant (K-m) of 0.67 +/- 0.21 mu M and maximal velocity (V-max) of 10.05 +/- 0.87 pmol.mg protein(-1).3 min(-1)], 5) inhibited by structural analogs and anion transport inhibitors, and Bi energy dependent. Protein kinase C -, protein kinase A-, and protein tyrosine kinase-mediated pathways we re found to have no role in regulating RF uptake. On the other hand, a Ca2+/calmodulin-mediated pathway appeared to play a role in the regul ation of RF uptake by HK-2 cells via an effect on the V-max, as well a s on the apparent K-m of the RF uptake process. The uptake process of RF was also found to be adaptively regulated by the level of the subst rate in the growth medium, with the effect being mediated through chan ges in the apparent K-m and the V-max of the uptake process. These res ults demonstrate that RF uptake by the human-derived renal epithelial cell line HK-2 is via a carrier-mediated system that is temperature an d energy dependent and appears to be under the regulation of a Ca2+/ca lmodulin-mediated pathways and substrate level in the growth medium.