OBJECTIVES: To gain insights into the mechanisms of Cd-induced albuminuria, we investigated effects of Cd on the expression of megalin and chloride channel 5 (ClC5), two key players in albumin-receptor--mediated endocytosis.
METHODS: We used quantitative polymerase chain reaction, Western blotting, the albumin endocytosis assay, and confocal microscopy to evaluate effects of Cd on the expression and regulation of megalin and ClC5 in cultured LLC-PK1 cells, a pig proximal tubular cell model.
RESULTS: Ten micromolar cadmium chloride ([CdCl.sub.2]) caused a significant time- and dose-dependent decrease in both mRNA and protein levels of megalin and ClC5, whereas no changes resulted from exposure to other divalent metals (zinc chloride, manganese chloride, magnesium chloride, and nickel chloride).
CONCLUSIONS: We found that Cd reduced the transcriptional expression of megalin and ClC5 and, at the same time, increased the degradation of megalin and ClC5 proteins via the lysosomal pathway in an in vitro model of renal proximal tubular cells.
KEY WORDS: albuminuria, cadmium, ClC5, heavy metals, megalin, nephrotoxicity, renal proximal tubules.
Albumin endocytosis requires a macromolecular protein complex formed by the megalin/cubilin scavenger receptor [Na.sup.+]-[H.sup.+] exchanger isoform 3 (NHE3), vacuolar proton-ATPase (v-[H.sup.+]-ATPase), and chloride channel 5 (ClC5).
To take this one step further, we examined whether Cd exposure affects the expression of the two other proteins involved in renal albumin endocytosis: megalin and ClC5. In proximal tubule cells, megalin, a 600-kDa transmembrane glycoprotein, interacts synergistically with cubulin as scavenger for the luminal uptake of a large number of proteins, including albumin (Verroust et al.
Cd down-regulates gene expression of megalin and ClC5 in proximal tubule cells.
[CdCl.sub.2] specifically reduces megalin and ClC5 transcript levels.
Time course of [CdCl.sub.2]-induced down-regulation of megalin and ClC5 protein expression.
Cd-induced down-regulation of megalin and ClC5 proteins is due to an increase of their lysosomal degradation.