Fluoride effects on cell viability and ENaC expression in kidney epithelial cells.

Fluoride (F) at micromolar (µM) concentrations induces apoptosis in several cell lines. Moreover, proteomic studies have shown major changes in the profile of proteins involved in signal transduction. These effects may negatively affect ion transport in the kidneys. The activity of epithelial sodium channels (ENaCs) is a limiting factor for sodium and water resorption in the kidneys, which is essential for the maintenance of the electrolyte balance and homeostasis of the body. Here we investigated the effects of F, at different concentrations (10, 40, 100, 200, and 400 µM), on the viability of renal epithelial cells (M-1), and ENaC expression. We showed that sodium fluoride (NaF) reduces cell viability in a concentration-dependent manner (p < 0.05) up to a 96-h time-point when compared to control. Sodium fluoride at moderate concentrations (100 and 200 µM), upregulated the ENaC subunit genes Scnn1a and Scnn1g, but not Scnn1b. Sodium fluoride downregulated all three ENaC subunit genes at a higher concentration of 400 µM (p < 0.05). Immunofluorescence analysis showed that Scnn1a and Scnn1g expression was decreased within 24 h of NaF treatment. After 48 h, NaF (400 µM) increased the expression of Scnn1a but not Scnn1g. However, NaF decreased the expression of Scnn1g at all studied concentrations. We conclude that F, at µM concentrations, modulates the expression of ENaC subunit genes, which is likely to significantly affect molecular signaling in kidney epithelial cells.

The effect of fluoride on the structure, function, and proteome of intestinal epithelia.

Fluoride exposure is widespread, with drinking water commonly containing natural and artificially added sources of the ion. Ingested fluoride undergoes absorption across the gastric and intestinal epithelia. Previous studies have reported adverse gastrointestinal effects with high levels of fluoride exposure. Here, we examined the effects of fluoride on the transepithelial ion transport and resistance of three intestinal epithelia. We used the Caco-2 cell line as a model of human intestinal epithelium, and rat and mouse colonic epithelia for purposes of comparison. Fluoride caused a concentration-dependent decline in forskolin-induced Cl- secretion and transepithelial resistance of Caco-2 cell monolayers, with an IC50 for fluoride of about 3 mM for both parameters. In the presence of 5 mM fluoride, transepithelial resistance fell exponentially with time, with a t1/2 of about 7 hours. Subsequent imaging by immunofluorescence and scanning electron microscopy showed structural abnormalities in Caco-2 cell monolayers exposed to fluoride. The Young's modulus of the epithelium was not affected by fluoride, although proteomic analysis revealed changes in expression of a number of proteins, particularly those involved in cell-cell adhesion. In line with its effects on Caco-2 cell monolayers, fluoride, at 5 mM, also had profound effects on Cl- secretion and transepithelial resistance of both rat and mouse colonic epithelia. Our results show that treatment with fluoride has major effects on the structure, function, and proteome of intestinal epithelia, but only at concentrations considerably higher than those likely to be encountered in vivo, when much lower fluoride doses are normally ingested on a chronic basis.

The effect of fluoride on the structure, function, and proteome of a renal epithelial cell monolayer.

High concentrations of fluoride in the body may cause toxic effects. Here, we investigated the effects of fluoride on the structure, function, and proteome of a cortical collecting duct epithelium in vitro. Kidney tubule cells (M-1) were chosen because the concentration of fluoride in the kidney is 4-5-fold higher than that in plasma. Mouse M-1 cell monolayers were incubated in fluoride-containing media, and the amiloride-sensitive short-circuit current and transepithelial resistance were measured. The Young's modulus of the epithelium was determined using atomic force microscopy, and the effect of fluoride on epithelial structure was assessed using scanning and transmission electron microscopy, and immunofluorescence. Differences in the expression of membrane proteins were evaluated using proteomics and bioinformatics. Fluoride exposure reduced both transepithelial Na+ transport and resistance. The IC50 for fluoride was ∼300 µM for both effects, and the half-times for the decays of ion transport and resistance were 8.4 h and 3.6 days, respectively. Fluoride treatment did not affect the sensitivity of Na+ transport to amiloride. The Young's modulus of the epithelium was also unaffected by fluoride; however, the functional effects of fluoride were accompanied by marked structural effects. Proteomic analysis revealed changes in expression of a number of proteins, and particularly mitochondrial proteins. Treatment with fluoride had profound effects on the structure, function and proteome of a model cortical collecting duct epithelium. Significantly, however, these effects were produced only at concentrations considerably higher than those likely to be encountered in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1455-1467, 2017.