Transforming growth factors (TGF) are potent multifunctional polypeptides that are involved in renal function and glomerular sclerosis. We postulated that dietary salt modified renal production of TGF-β. An increase in dietary salt produced sustained increases in steady-state levels of mRNA for TGF-β1, -β2, and -β3 in the rat kidney. While serum concentration of TGF-β1 did not change, the 8.0% NaCl diet increased urinary excretion of TGF-β1, indicating enhanced renal production was the source of TGF-β1. Increasing urinary flow rates with diuretics did not further increase synthesis of TGF-β1 in animals receiving the 8.0% NaCl diet. The 8.0% NaCl diet increased production of TGF-β1 in both glomeruli and tubules, although active TGF-β1 was secreted in greater amounts only from glomeruli. Enhanced glomerular production of both inactive and active TGF-β1 induced by the 8.0% NaCl diet was inhibited by tetraethylammonium (TEA) and not glybenclamide. Cardiac production of TGF-β1 also increased on the 8.0% NaCl diet but was not affected by TEA. The results demonstrated that increased dietary salt augmented glomerular TGF-β production by a mechanism that included a TEA-sensitive potassium channel. Dietary salt, by facilitating glomerular expression of TGF-β, may directly promote development of glomerulosclerosis.

The amount of NaCl in the diet plays an important role in modulating nitric oxide (NO) synthesis in vivo. In the glomerulus, dietary NaCl also regulates transforming growth factor-beta1 (TGF-beta1) production. We hypothesized that dietary NaCl intake regulated expression of the endothelial isoform of nitric oxide synthase (NOS3) and TGF-beta1 in the aorta. Administration of 8.0% NaCl diet to rats for 7 days did not affect blood pressure but increased steady-state mRNA and protein levels of NOS3 in the arterial wall compared with animals on 0.3% NaCl diet. Northern analysis demonstrated increased steady-state amounts of mRNA of TGF-beta1 in aortas of rats on 8.0% NaCl diet. By ELISA, both total and active TGF-beta1 were increased in these vessel segments. Endothelial denudation of aortic rings reduced active TGF-beta1 secretion to undetectable levels. Addition of a neutralizing antibody to TGF-beta to aortic ring segments attenuated NO production but not to that observed in animals on the 0.3% NaCl diet. The data showed that dietary NaCl intake modulated NOS3 and TGF-beta1 expression in the arterial wall; NOS3 expression was at least partially regulated by endothelial cell production of TGF-beta1.

We have previously demonstrated that tamoxifen inhibits the growth of human cholangiocarcinoma cells in culture and inhibits tumor growth when cells are injected into nude mice. However, the mechanism of action of tamoxifen remains unknown. Here we demonstrate that tamoxifen and trifluoperazine, both potent calmodulin antagonists, induce apoptosis in vitro, probably acting via the Fas system, in human cholangiocarcinoma cells. Human cholangiocarcinoma cell lines heterogeneously express Fas antigen on their surface. Fas-negative and Fas-positive surface-expressing cells were isolated, cloned, and cultured. Fas antibody, tamoxifen, and trifluoperazine induced dose-dependent apoptosis only in Fas-positive cells; Fas-negative cells were unaffected. Furthermore, apoptosis induced by tamoxifen in Fas-positive cells was blocked by an inhibitory Fas antibody. Tamoxifen was not acting through an anti-estrogenic mechanism, because neither Fas-negative nor Fas-positive cells expressed estrogen receptors and the pure anti-estrogen compound, ICI 182780, did not induce apoptosis in either cell line. Fas-negative cells, but not Fas-positive cells, were able to produce tumors when subcutaneously injected into nude mice. These findings suggest Fas may be a candidate oncogene involved in the pathogenesis of cholangiocarcinoma. Furthermore, the similarity between the pro-apoptotic effects of tamoxifen and trifluoperazine support an underlying molecular mechanism for Fas-mediated apoptosis that involves calmodulin.