**1. Introduction**

Cadmium (Cd) is an environmental pollutant that causes renal toxicity in animals and humans after chronic exposure in the diet [1]. Due to both the high affinity of Cd for sulfhydryl moieties in biomolecules within cells and the difficulty of excretion from cells, the biological half-life of Cd in the human kidney has been calculated to be more than 25 years [2]. The renal accumulation of Cd results in characteristic renal toxicity is known as Fanconi syndrome at the advanced stage [3–5]. Cd accumulation in the proximal tubules of the kidney has been believed to disturb the reabsorption of the luminal biomolecules, which are filtered through the glomerulus into proximal tubule epithelial cells (PTECs). Animals and humans exposed to Cd for a long time show increased urinary excretion of glucose, amino acids, and low-molecular-weight (LMW) proteins such as β2-microglobulin (β2-MG) and metallothionein (MT) [6–8]. Enhanced urinary levels of β2-MG have been used as sensitive and reliable indicators of Cd-induced renal tubular damage [9,10].

The reabsorption of luminal biomolecules including β2-MG and MT by PTECs is mediated by megalin-dependent endocytosis at the apical membrane of PTECs [11–14]. However, many studies on Cd cytotoxicity have focused on the mechanisms of cell lethality, including apoptosis caused by Cd [15–17], and only a few studies have examined Cd's direct effects on the efficiency of protein reabsorption by PTECs [18,19], especially under conditions where PTECs are surviving in the presence of Cd.

Recently, we developed an in vitro experimental system using mouse PTEC-derived S1, S2, and S3 cells, which maintain fundamental features of S1, S2, and S3 segment-specific expression of genes including metal transporters [20,21]. In the present study, we attempted to develop an in vitro experimental system for evaluating the endocytosis efficiency of LMW and high-molecular-weight (HMW) proteins into S1 and S2 cells derived from the S1 and S2 segments of proximal tubules where the reabsorption of glomerular-filtered proteins is highly active. To visualize and quantify the amounts of endocytosed proteins, we used fluorescent-labeled albumin, transferrin, β2-MG, and MT. Here, we show that flow cytometric analyses of the incorporation of fluorescent-labeled proteins into cultured PTECs can be used to quantitatively evaluate endocytosis efficiency. By using this in vitro assay system, we detected decreases in the endocytic uptakes of β2-MG and MT in cultured PTECs exposed to Cd.

#### **2. Materials and Methods**
