**3. Results**

#### *3.1. Fluorescence Imaging of Endocytic Uptakes of the Labeled Proteins into Mouse PTECs*

We first confirmed the incorporation of fluorescent-labeled proteins into mouse PTECs with confocal and fluorescence microscopes. As shown in Figure 1A, the green fluorescent signals of FITC and Alexa488 were clearly detected in S1 cells 30 min after the addition of FITC-labeled albumin, β2-MG, MT, and Alexa-labeled transferrin. The yellow fluorescent signals observed in the cells indicate the overlapping of the red fluorescence of the antibody against EEA1 and the green fluorescence of the proteins, suggesting the incorporation of the labeled proteins into the early endosomes.

**Figure 1.** Fluorescence imaging of endocytosed proteins in mouse proximal tubule epithelial cell (PTEC)-derived S1 cells. (**A**) S1 cells were incubated with 50 μg/mL fluorescein isothiocyanate (FITC)-albumin, Alexa-transferrin, FITC-β2-MG, or FITC-MT (green) for 30 min and then fixed with paraformaldehyde for immunofluorescence labeling with anti-EEA1 (red). Yellow staining demonstrates the colocalization of fluorescent-labeled proteins and early endosomes. (**B**) S1 cells were incubated with Alexa-transferrin for 1, 5, 10, 15, and 30 min. The localization of Alexa-transferrin (green) and early endosomes stained with anti-EEA1 (red) was visualized by confocal microscopy. Bars, 5 μm.

Figure 1B shows the time-dependent changes in the fluorescent signals of transferrin that showed the strongest fluorescent intensities. The green signals of transferrin began to be detected within cells 1 min after the addition of the protein. Yellow fluorescent signals, indicative of the incorporation of transferrin into the early endosomes, began to appear at 10 min and then increased up to 30 min. Thus, the results of the fluorescence imaging provided evidence for endocytic incorporation of the labeled proteins into S1 cells.

#### *3.2. Quantification of Endocytic Uptakes of the Labeled Proteins into Mouse PTECs*

Next, we set up a quantification system for determining the endocytosis efficiency of each protein into the cells by using flow cytometry. The cells were cultured with each fluorescent-labeled protein for 30 min, washed, harvested, and applied to flow cytometry. As shown in Figure 2, the percentages of the cell population in lower-right section in the quadrants were used as the indicator of endocytosis efficiency (%). Based on the results of preliminary experiments, the amounts of the labeled proteins were decided to be 25 μg/mL for albumin and transferrin and 50 μg/mL for β2-MG and MT as the optimal conditions for incorporation. We used both S1 and S2 cells derived from the S1 and S2 segments of mouse proximal tubules, respectively, since both cell lines showed similar expression levels of megalin, cubilin, and transferrin receptor, which are essential for endocytosis in PTECs (Figure S1). We determined the time-dependent changes in endocytosis efficiency for each protein (Figures S2 and S3). Although these data are obtained by preliminary experiments, it was shown that the uptake rates of albumin and transferrin during 30 min were almost the same between S1 and S2 cells while those of β2-MG and MT were lower into S2 cells than into S1 cells. Since most proteins showed maximal uptakes at 30 min, the effects of Cd exposure on the endocytic uptakes of these proteins were examined 30 min after the addition of the labeled proteins in the subsequent experiments.

**Figure 2.** Evaluation of endocytosis efficiency of the fluorescent-labeled proteins by using flow cytometry. The cells were cultured with each of the fluorescent-labeled protein for 0 or 30 min and applied to flow cytometry. Typical quadrant data of each protein in S1 cells were shown here. X-axis indicates fluorescent intensity and y-axis indicates side scattering. The untreated cells (0 min) showing the auto-fluorescence were gated to be the lower-left section in the quadrants. The cell populations in the lower-right section were expressed as the percentage of total cells and used as the indicator of endocytosis efficiency (%) in the subsequent experiments.

The high e fficiency of transferrin incorporation into S1 and S2 cells may be partially caused by the expression of transferrin receptor in these cells (Figure S1). It is known that the transferrin receptor in PTECs is expressed in the basolateral [22] and apical [23] membranes, whereas megalin and cubilin are expressed at the apical membrane [24], suggesting that both uptake systems for transferrin contribute to the highly e fficient uptake into the endosomes in S1 cells.

#### *3.3. E*ff*ects of Cd Exposure on the Endocytic Uptakes of the Labeled Proteins into Mouse PTECs*

Before examining the e ffects of Cd exposure on the endocytic uptakes of the labeled proteins into S1 and S2 cells, we checked the lethal toxicity of Cd in S1 and S2 cells using the alamarBlue assay (Figure 3). Based on the results of this assay, we selected sublethal doses of Cd, as indicated by the arrows in Figure 3, for the subsequent endocytosis experiments. We also attempted to use much higher doses of Cd (5 μM Cd for 3 days and 1 μM Cd for 6 days), but S1 and S2 cells could not survive these concentrations of Cd when cultured in 6-well plates for endocytosis experiments. The discrepancy in cytotoxicity between the 96-well (alamarBlue ® assay) and 6-well plates may be attributable to the di fferences in cell density. Therefore, in the endocytosis experiments we used 10 and 15 μM Cd for 1 day, 1 and 3 μM Cd for 3 days, and 0.1 and 0.5 μM Cd for 6 days. Under these conditions, very few cells were found to be detached from the plates at the end of Cd exposure, and the three-times washing of the cells with ice-cold PBS before harvesting did not result in the detachment of the cells. Thus, the effects of Cd on the endocytosis e fficiencies in the following experiments were carried out with the cells including least populations of dead cells.

**Figure 3.** Cytotoxicity of Cd in S1 and S2 cells. S1 (open circles) and S2 (closed circles) cells cultured in 96-well plates were incubated with the indicated concentrations of CdCl2 for 1, 3, and 6 days. Cell viability was determined by alamarBlue ® assay and expressed as a percentage of the nontreated cells. From these results, the Cd concentrations to be used in the subsequent experiments were determined (arrows). Data are presented as means ± SD (*n* = 4–6). Statistically significant di fference between S1 and S2 cells was indicated as \*\* *p* < 0.01.

After the exposure of S1 cells to Cd at these concentrations for 1, 3, and 6 days, the endocytic uptakes of the labeled albumin and transferrin into S1 cells were examined (Figure 4 and Figure S4). However, Cd exposure did not a ffect the endocytic uptake of either albumin or transferrin. On the other hand, the endocytic uptakes of β2-MG and MT were a ffected by Cd exposure depending on the exposure duration (Figure 5). The 1- and 3-day exposures of S1 and S2 cells to Cd resulted in statistically significant decreases in the endocytic uptake of β2-MG (Figure 5A), whereas only the 3-day exposure to Cd resulted in statistically significant decreases in endocytic uptakes of MT (Figure 5B and Figure S4). The 6-day exposure to Cd did not cause any significant decreases in endocytic uptakes of either β2-MG or MT in either cells.

**Figure 4.** Effects of Cd on the endocytosis efficiencies of albumin and transferrin into S1 cells. S1 cells were exposed to CdCl2 for 1, 3, and 6 days and then incubated with FITC-albumin (**A**) or Alexa-transferrin (**B**) for 30 min. The endocytosis efficiencies were determined by flow cytometry and expressed as percentages of the control cells (no exposure to Cd). Data are presented as means ± SD (*n* = 3–4).

#### *3.4. E*ff*ects of Cd Exposure on the Endocytic Uptakes of the Labeled Proteins into Human PTECs*

To test whether Cd exposure also affects endocytic uptakes of β2-MG and MT in human PTECs, we utilized hRPTECs, an immortalized cell line derived from human kidney PTECs. Since the effects of Cd on the endocytic uptakes of β2-MG and MT in mouse S1 and S2 cells were clearly detected after the 3-day exposure to Cd (Figure 5), hRPTECs were exposed to Cd for 3 days. Prior to the endocytosis experiment, we checked the sensitivity of hRPTECs to Cd. As shown in Figure 6A, hRPTECs were highly resistant to Cd compared with S1 or S2 cells. Therefore, we used 5 and 25 μM Cd for endocytosis experiments in hRPTECs. As shown in Figure 6B, the endocytic uptakes of both β2-MG and MT were significantly reduced by 3-day exposure to Cd.

**Figure 5.** Effects of Cd on the endocytosis efficiencies of β2-MG and MT into S1 and S2 cells. S1 and S2 cells were exposed to CdCl2 for 1, 3, and 6 days and then incubated with FITC-β2-MG (**A**) or FITC-MT (**B**) for 30 min. The endocytosis efficiencies were determined by flow cytometry and expressed as percentages of the control cells (no exposure to Cd). Open and closed columns represent S1 and S2 cells, respectively. Data are presented as means ± SD (*n* = 3–4). Statistical significance of the dose dependence determined by one-way ANOVA was detected in the following settings: day1-S1 cells (*p* < 0.05), day1-S2 cells (*p* < 0.05), day3-S1 cells (*p* < 0.05), and day3-S2 cells (*p* < 0.01) for β2-MG (**A**), and day3-S1 cells (*p* < 0.05) and day3-S2 cells (*p* < 0.05) for MT (**B**). Statistical significances compared with the control cells determined by Bonferroni multiple comparisons are indicated as \* *p* < 0.05, \*\* *p* < 0.01 (S1 cells) and # *p* < 0.05, ## *p* < 0.01 (S2 cells).

**Figure 6.** The effects of Cd exposure for 3 days on the endocytosis efficiencies of β2-MG and MT into hRPTEC human renal cells. (**A**) Cell viability was determined by alamarBlue assay and expressed as the percentages of the nontreated cells. From these results, the Cd concentrations to be used in the endocytosis experiment were determined (arrows). Data are presented as means ± SD (*n* = 4–6). (**B**) The cells were exposed to CdCl2 for 3 days and then incubated with FITC-β2-MG or FITC-MT for 30 min. The endocytosis efficiencies were determined by flow cytometry and expressed as percentages of the control cells (no exposure to Cd). Data are presented as means ± SD (*n* = 3–4). Statistical significance of the dose dependence determined by one-way ANOVA was detected in both β2-MG (*p* < 0.05) and MT (*p* < 0.05). Statistical significance compared with the control cells determined by Bonferroni multiple comparisons are indicated as \* *p* < 0.05.
