**5. Conclusions**

The aim of this study was to establish an in vitro assay system for evaluating the efficiency of endocytic uptakes of LMW and HMW proteins labeled with fluorescent moiety in cultured PTECs. Flowcytometric determinations of the uptakes of the fluorescent-labeled proteins into mouse S1 and S2 cells and human hRPTECs enabled us to find that a 3-day exposure to Cd resulted decreased endocytic uptakes of β2-MG and MT in all these cells. Thus, these cells, especially S1 cells, proved to be useful for the elucidation of the mechanisms of Cd-induced dysfunctions of renal tubular reabsorption. In future studies, this in vitro system can be used to investigate the effects of Cd exposure on the expression of megalin and cubilin at the apical membrane, the functions of mitochondria, which provide the energy for endocytosis, and other machineries required for the process of endocytosis. This system may also serve as a tool for studying the mechanisms of other toxicants causing renal tubular dysfunctions.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2305-6304/8/2/24/s1, Figure S1: Expression levels of the proteins involved in endocytosis in S1 and S2 cells, Figure S2: Time-dependent changes in endocytic uptakes of albumin, transferrin, β2-MG, and MT, Figure S3: Time-dependent changes in endocytic uptakes of albumin, transferrin, β2-MG, and MT, Figure S4: Effects of Cd on the endocytosis efficiencies of albumin and β2-MG into S1 cells.

**Author Contributions:** Conceptualization, H.F. and S.H.; cell biology experiments, H.F., H.Y., N.O. (Nobuki Otera), N.O. (Nanae Oka), and M.J.; fluorescence imaging, H.Y., N.O. (Nobuki Otera), and N.O. (Nanae Oka); synthesis and purification of recombinant proteins, H.F., N.O. (Nanae Oka), and M.J.; development of flow cytometric quantification of endocytosis, H.F. and H.Y.; writing and editing, H.F. and S.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was partly supported by JSPS KAKENHI Grant Numbers 19H05770 (H.F.) and 19H01081 (S.H. and H.F.), and by the Study of Health Effects of Heavy Metals organized by Ministry of the Environment, Japan (S.H.).

**Conflicts of Interest:** The authors declare no conflict of interest.
