**4. Discussion**

Disturbances in the tubular reabsorption of glomerular-filtered biomolecules by PTECS in kidney are a hallmark of Cd-induced nephrotoxicity. The urinary excretion of β2-MG has been particularly widely used as an indicator of renal tubular dysfunction among residents of Cd-polluted areas [6,9,10]. However, the precise mechanisms underlying Cd-induced dysfunctions of tubular reabsorption of LMW proteins by PTECs have not been fully investigated. This is partly due to the lack of a proper in vitro experimental system.

In the present study, we developed an in vitro experimental system for evaluating the endocytosis efficiency of fluorescent-labeled proteins into cultured PTECs derived from mouse and human kidney. The endocytic uptakes of the labeled proteins and their cellular localization were confirmed by fluorescence imaging (Figure 1). Flowcytometric analyses of the fluorescent-labeled proteins have enabled us to quantitatively evaluate the uptake rates of labeled proteins into S1 and S2 cells (Figure 2). The exposure of these cells to sublethal doses of Cd for 3 days resulted in significant decreases in the endocytic uptakes of β2-MG and MT (Figure 5), but not in those of albumin or transferrin (Figure 4). These results demonstrated that the assay system developed in this study permitted the detection of Cd-induced declines in renal reabsorption of LMW proteins in cultured PTECs. The reason for the absence of the e ffects of Cd after 6-day exposure remains unknown. Possibly, more complicated factors are involved in the 6-day exposure than the 3-day exposure experiments. Although future studies are required for the mechanisms of Cd-induced decreases in the LMW protein uptakes, this assay system may be useful for screening other renal toxicants that may cause damage in tubular reabsorption.

To date, epidemiological studies in humans and experimental studies in animals have linked increases in urinary excretion of LMW proteins such as β2-MG with the loss of functional PTECs and nephrons in the kidney at the advanced stage of Cd nephrotoxicity [25,26]. Most mechanistic studies on Cd cytotoxicity have focused on the molecular pathways leading to Cd-associated cell death and not on the direct e ffects of Cd on the endocytic uptakes of LMW proteins in living PTECs [15–17]. However, the e ffects of moderately higher, but not lethal, doses of Cd on the reabsorption e fficiencies of LMW proteins by the surviving PTECs remain unclear. The results of this study demonstrated that a 3-day exposure to Cd resulted in significant declines in the endocytic uptakes of β2-MG and MT under the conditions in which nonlethal doses of Cd were used. This could not be ascribed simply to the increase in dead cells after Cd exposure, since no e ffects were observed in the endocytosis efficiencies of albumin or transferrin (Figure 4) and only the surviving cells that were not detached from the plates during Cd exposure were used for flow cytometry analyses. Although a few studies have investigated the e ffects of Cd on the interactions of LMW proteins with megalin/cubilin systems in cultured renal cells [18,19], the present study utilized flow cytometry for quantitatively evaluating the endocytosis e fficiency of the proteins and showed the e ffects of Cd on the uptakes of LMW proteins in cultured PTECs.

Although many epidemiological studies undertaken in Cd-polluted areas have demonstrated that urinary excretion of β2-MG is an excellent biomarker for renal tubular dysfunctions [6,9,10], recent evidence suggested that β2-MG plays much broader roles as a biomarker not only for tubular dysfunctions, but also for glomerular dysfunctions as well as non-renal diseases [27]. The results of our in vitro study added a piece of evidence that the decreased incorporation of β2-MG into renal tubular cells is involved in Cd-induced kidney damages. Future studies are required to test whether the decrease in the β2-MG incorporation into renal tubular cells is inducible specifically by Cd, or commonly by other renal toxicants using this assay system.

Compared with the uptakes of β2-MG and MT, those of albumin and transferrin by S1 cells appear to be far less sensitive to Cd toxicity (Figure 4). Many human studies have suggested that the urinary excretion of β2-MG, a LMW protein, reflects damage in renal tubular reabsorption, whereas the urinary excretion of albumin, an HMW protein, may reflect the dysfunction of glomeruli [28–30], although recent evidence has indicated that tubular reabsorption of albumin should not be ignored [31]. The results of this study also demonstrated that substantial amounts of albumin can be taken up by cultured

PTECs. Although the differences in the endocytic pathways between HMW albumin and LMW β2-MG remains unclear, the higher sensitivity of β2-MG than albumin in this in vitro assay system may reflect the in vivo observation that β2-MG is the most sensitive indicator for renal tubular damage caused by Cd exposure [28,29]. Regarding the comparisons between HMW and LMW proteins, the differences in molecular weights, i.e., albumin and MT, could have affected the endocytosis efficiency because we used 25 μg/mL albumin and 50 μg/mL MT as the optimal conditions, not based on a molar basis, in this assay.

The strengths and limitations of this study should be noted. (1) The use of flow cytometry enabled the quantitative evaluation of the endocytosis efficiencies of fluorescent-labeled proteins, (2) the use of both mouse- and human-derived PTECs enabled the confirmation of reproducibility of Cd effects, and (3) the use of sublethal doses of Cd enabled the sensitive detection of the effects of Cd on the PTECs that survived the lethal toxicity of Cd. However, the observed effects of Cd on the endocytosis efficiencies of β2-MG and MT were not so marked, though statistically significant, and the involvements of Cd cytotoxicity on the results of flow cytometry may not be completely excluded. Since the in vivo dysfunctions of renal tubular reabsorption generally occur under conditions where a large part of nephrons and PTECs is lost [25,26], the in vitro effects of renal toxicants on the endocytosis efficiency in the living PTECs may reflect only a part of the whole events of renal dysfunction. Nevertheless, this in vitro assay system may provide a useful tool for the future screening of other renal toxicants and for more detailed mechanistic studies.

For future applications of this assay system to the detection of possible renal toxicants damaging tubular reabsorption, the merits and disadvantages of this system should be discussed here. Since mouse S1 and S2 cells and human hRPTECs are all immortalized cell lines, they can provide reproducible and reliable results compared with the primary cultured PTECs prepared freshly from the proximal tubules of kidney. In this study, both mouse and human PTECs showed decreases in endocytic uptakes of β2-MG and MT after the 3-day exposure to Cd. However, approximately ten times higher concentrations of Cd were required in hRPTECs than in S1 and S2 cells to produce similar detrimental effects (Figure 6) due to the high Cd resistance of hRPTECs, which is the major drawback of using hRPTECs. S2 cells showed a lower efficiency of endocytic uptakes of β2-MG and MT than S1 cells, whereas the uptake efficiencies of albumin and transferrin were similar to those of S1 cells (Figure 2). Although both S1 and S2 cells showed significant decreases in endocytic uptakes of β2-MG and MT (Figure 5) when exposed to Cd for 3 days, S1 cells may be more preferable for screening renal toxicants that affect the endocytic uptakes of LMW proteins, since the uptake efficiencies of LMW proteins under unexposed conditions are stable and reliable in S1 cells. We are now planning a screening study using S1 cells to test whether other renal toxicants affect the endocytic uptakes of β2-MG.
