Cadmium decreases SGLT1 messenger RNA in mouse kidney cells.

Mouse renal cortical tubule cells in primary culture exposed to cadmium (Cd2+) develop decreased Na(+)-glucose cotransport activity as measured by uptake of the glucose analogue alpha-methyl-glucoside. RNA was isolated from kidney cell cultures, and after reversed transcription, the DNA was amplified with primers to rat SGLT1 (the high affinity isoform of the sodium glucose cotransporter) and mouse beta-actin. Only one product was identified after amplification with the rat SGLT1 primers, which on sequencing was 96% identical to rat SGLT1. Compared to beta-actin, the intensity of the SGLT1 message declined progressively as CdCl2 concentration in the medium increased from 0 to 10 microM. Similar decreases in SGLT1 mRNA were also observed as media zinc (Zn2+) concentrations rose from 0 to 75 microM or as copper (Cu) concentrations increased from 0 to 150 microM. Exposure to 8 microM Cd as Cd-metallothionein (Cd7-MT) also caused a fall in relative SGLT1 mRNA abundance, and at nearly identical internal Cd concentrations of 40-43 pmol/microgram DNA, both Cd7-MT and CdCl2 reduced SGLT1 mRNA to 33% of control. In general, the fall in SGLT1 mRNA was more rapid than the decline in Na(+)-dependent glucose uptake after cells were exposed to Cd2+. These findings suggest that the effects of Cd2+ and other metals on renal glucose transport are related to decreased expression of SGLT1 message.

Comparative effects of Cd2+ and Cd-metallothionein on cultured kidney tubule cells.

The effects of Ca2(+) and Cd-metallothionein on two cultured cells with proximal tubule characteristics, mouse kidney cortical cells and pig kidney LLC-PK1 cells, have been compared. Cd2+ inhibits Na(+)-glucose cotransport in LLC-PK1 cells and in the process decreases the number of binding sites for [3H]phlorizin, a competitive inhibitor of glucose for the Na(+)-glucose cotransporter. During 24 hr incubation and over a range of concentrations in the two cell types, only Cd2+ inhibited Na(+)-glucose cotransport even when approximately equal concentrations of intracellular Cd resulted from these treatments. Indeed, at low concentrations of Cd-metallothionein in mouse cells, transporter activity was elevated. Extension of incubations to 72 hr in mouse cells led to increased Cd uptake and reduction in cell density with both sources of Cd but only a progressive decline in Na(+)-glucose cotransport activity with Cd2+. Zn-metallothionein was without effect under comparable conditions. Both forms of Cd were accumulated by these cells, with the large majority of the metal ion localizing in metallothionein as a Cd, Zn-protein in LLC-PK1 cells. Under equal exposure conditions, the net uptake of Cd from Cd-metallothionein in the two cell types. It is evident that the mechanisms of toxicity of Cd2+ and Cd-metallothionein as well as their modes of uptake differ in these two cell types.

Cadmium inhibits glucose uptake in primary cultures of mouse cortical tubule cells.

We studied the effect of cadmium (Cd2+) on transport of alpha-methylglucoside in primary cultures of mouse kidney cortical tubule cells grown in defined medium. When cultured cells were exposed to Cd2+ concentrations from 0 to 6 microM for 24 h, uptake of alpha-methylglucoside was inhibited in a dose-dependent manner by up to 50%. By contrast, acute exposure of the cells to 7 microM Cd2+ for 60 min did not inhibit alpha-methylglucoside uptake. Increasing Cd2+ concentrations progressively decreased the Vmax of Na(+)-dependent glucose cotransport but not the Km for glucose. Cell ATP/ADP ratios of unexposed monolayers and of cells exposed to 4.5 microM Cd2+ for 24 h were 5.0 and 4.9, respectively (n = 3). Intracellular volume, lactate dehydrogenase activity, and cell Na+ and K+ concentrations were unaltered even after 24 h of exposure to 7 microM Cd2+. Untreated and Cd2+-treated monolayers preloaded with alpha-methylglucoside released the sugar analogue into the medium at nearly identical rates, indicating that Cd2+ did not alter cell permeability to glucose. Uptake of the amino acid analogue alpha-(methylamino)isobutyric acid was not affected by prior Cd2+ exposure. Whereas cell DNA content declined in Cd2(+)-exposed plates, both Na(+)-glucose and Na(+)-amino acid cotransport were enhanced at lower cell densities. Protein and DNA synthesis, estimated, respectively, by incorporation of [3H]leucine and [3H]thymidine into acid-insoluble material, were not significantly affected at 6 microM Cd2+. We conclude that after a lag time Cd2+ selectively inhibits renal Na(+)-dependent glucose transport despite an unchanged gradient for Na+ across the cell membrane.

Kinetic lability of zinc bound to metallothionein in Ehrlich cells.

Ehrlich ascites-tumour cells normally contain a large concentration of Zn-metallothionein. When cells are placed in culture media, containing or pretreated with the metal-ion-chelating resin Chelex-100, they stop growing, remain viable and lose zinc specifically from the metallothionein (MT) pool. The kinetics of loss of zinc are first-order and are very rapid, having a rate constant of greater than or equal to 0.6 h-1. MT protein labelled with 35S is biodegraded with a rate constant of 0.07-0.014 h-1 in control cells, 0.08 h-1 in cells exposed to the zinc-deficient medium and 0.12-0.18 h-1 in cells treated directly with Chelex. Over the 6 h period in which zinc is totally lost from Zn-MT there is relatively little decrease in MT-like protein as measured by cadmium-binding to the 10,000-Mr protein fraction. Other pools of zinc and 35S-labelled protein turn over more slowly. There is no loss of zinc from rat liver Zn-MT that is dialysed against Chelex to model the possible reaction of the resin with Ehrlich-cell Zn-MT. However, Chelex does compete slowly for MT-bound zinc when resin and MT are directly mixed. Analysis of the known and possible pathways of zinc metabolism in cells in relationship to these rate constants shows that biodegradation of MT protein cannot account for the rate of loss of zinc from Zn-MT.