Cadmium Uptake by Primary Cultures of Rat Renal Cortical Epithelial Cells: Influence of Cell Density and Other Metal Ions

Abstract

The uptake and accumulation of 1-5 μM cadmium (Cd) was studied in primary cultures of rat renal cortical epithelial cells under protein-free conditions at 4 or 37°C for up to 30 min. The cells were isolated from female rats by collagenase digestion and cultured for 3-7 days. Confluency of the culture, monitored morphologically as well as by total protein content, was achieved on Day 5. Cd accumulation at 1 μM concentration demonstrated an inverse relationship to the cell density; the Cd level in Day 5 culture was only 45% of that in Day 3 culture. In subconfluent cultures (Days 3 and 4) the Cd accumulation was temperature sensitive; on Day 3 the cells accumulated one-third less Cd at 4°C than at 37°C. In comparison, the confluent cells (Day 5) had the same Cd accumulation regardless of the incubation temperature. In these cells, preincubation with cyanide also had no significant effect on Cd accumulation, implying a lack of energy requirement for Cd uptake. As the transport of Cd may involve processes that exist for the essential metal ions, the effect of 30 μM zinc (Zn) and copper (Cu) on the accumulation of 1 μM Cd was studied in Day 5 cultures. Coincubation with Zn caused a 16% reduction in Cd levels at 37°C and even greater reduction (44% of control) at 4°C. Similarly, Cu inhibited Cd accumulation by 26 and 45% at 37 and 4°C, respectively, as compared to the temperature-matched controls. The V max for the initial Cd uptake (1 min) was 125 pmol/mg protein/mm and the K m was 7 μM. Both Zn and Cu exhibited competitive inhibition kinetics and doubled the K m for Cd uptake. The K i for Zn and Cu was 23 and 30 μM, respectively. Mercury (Hg) and lead (Pb) were also tested for their ability to affect Cd accumulation. As compared to the controls, 1 μM Hg caused an 11% reduction in Cd level at 37°C. In contrast, 1 μM Pb enhanced Cd accumulation by 20%. However, neither Hg nor Pb had any significant effect on Cd accumulation at 4°C. All four metals had no significant effect on the efflux of Cd from the cells. Thus, these metals affected Cd accumulation by changing its uptake rather than its efflux. The results of Cd accumulation at varying cell densities demonstrated that as the proximal renal epithelial cells became confluent, and supposedly formed tight junctions and developed brush border, the Cd uptake became more restricted than under subconfluent culture conditions where the uptake was apparently through the basolateral membrane. Lack of effect of cyanide and lower temperature on Cd accumulation in confluent cultures further suggests that Cd transport through the apical membrane might occur by diffusion. However, under subconfluent culture conditions, where Cd accumulation is temperature sensitive, the transport through the basolateral membrane could possibly occur via ion channels or carriers.