Cadmium Impairs Albumin Reabsorption by Down-regulating Megalin and ClC5 Channels in Renal Proximal Tubule Cells

BACKGROUND: Cadmium (Cd) is a potent nephrotoxicant that impairs the reabsorptive and secretory functions of the renal proximal tubule, leading to albuminuria. 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 (CdCl2) 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). After inhibiting protein synthesis using cycloheximide (CHX), we found that levels of both megalin and ClC5 were lower in Cd-challenged cells than in cells treated with Cd or CHX only, which is consistent with reduced translation and/or post--translational down-regulation. Moreover, Cd-induced degradation of megalin and ClC5 was abolished by the lysosomal pathway inhibitor bafilomycin A-1 but not by the proteasome system blocker MG-132, suggesting that the enhanced proteolysis was occurring via lysosomes. Using confocal microscopy, we observed a remarkable reduction of fluoroisothiocyanate (FITC)-labeled albumin uptake after Cd exposure. 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. Overall, these results provide valuable insights into the molecular mechanisms by which Cd impairs luminal protein reabsorption by renal proximal tubules.