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The cardiotonic steroid marinobufagenin (MBG) has been implicated in the pathogenesis of experimental uremic cardiomyopathy, which is characterized by progressive cardiac fibrosis. We examined whether the transcription factor Friend leukemia integration-1 (Fli-1) might be involved in this process. Fli-1-knockdown mice demonstrated greater cardiac collagen-1 expression and fibrosis compared with wild-type mice; both developed increased cardiac collagen expression and fibrosis after 5/6 nephrectomy. There was a strong inverse relationship between the expressions of Fli-1 and procollagen in primary culture of rat cardiac and human dermal fibroblasts as well as a cell line derived from renal fibroblasts and MBG-induced decreases in nuclear Fli-1 as well as increases in procollagen-1 expression in these cells. Transfection of a Fli-1 expression vector prevented increased procollagen- 1 expression from MBG. MBG exposure induced a rapid translocation of the [delta]-isoform of protein kinase C (PKC[delta]) to the nucleus. This translocation was prevented by pharmacological inhibition of phospholipase C, and MBG-induced increases in procollagen-1 expression were prevented with a PKC[delta]- but not a PKC[alpha]-specific inhibitor. Finally, immunoprecipitation studies strongly suggest that MBG induced phosphorylation of Fli-1. We feel these data support a causal relationship with MBG-induced translocation of PKC[delta], which results in phosphorylation of as well as decreases in nuclear Fli-1 expression, which, in turn, leads to increases in collagen production. Should these findings be confirmed, we speculate that this pathway may represent a therapeutic target for uremic cardiomyopathy as well as other conditions associated with excessive fibrosis. renal failure; cardiotonic steroids; fibrosis

We recently demonstrated that the cardiotonic steroid marinobufagenin (MBG) induced fibrosis in rat hearts through direct stimulation of collagen I secretion by cardiac fibroblasts. This stimulation was also responsible for the cardiac fibrosis seen in experimental renal failure. In this study, the effect of MBG on the development of renal fibrosis in rats was investigated. Four weeks of MBG infusion triggered mild periglomerular and peritubular fibrosis in the cortex and the appearance of fibrotic scars in the corticomedullary junction of the kidney. MBG also significantly increased the protein levels and nuclear localization of the transcription factor Snail in the tubular epithelia. It is known that activation of Snail is associated with epithelial-to-mesenchymal transition (EMT) during renal fibrosis. To examine whether MBG alone can trigger EMT, we used the porcine proximal tubular cell line LLC-PK1. MBG (100 nM) caused LLC-PK1 cells grown to confluence to acquire a fibroblast-like shape and have an invasive motility. The expressions of the mesenchymal proteins collagen I, fibronectin, and vimentin were increased twofold. However, the total level of E-cadherin remained unchanged. These alterations in LLC-PK1 cells in the presence of MBG were accompanied by elevated expression and nuclear translocation of Snail. During the time course of EMT, MBG did not have measurable inhibitory effects on the ion pumping activity of its natural ligand, [Na.sup.+]-[K.sup.+]-ATPase. Our data suggest that the MBG may be an important factor in inducing EMT and, through this mechanism, elevated levels of MBG in chronic renal failure may play a role in the progressive fibrosis. cardiotonic steroid hormone; ouabain; [Na.sup.+]-[K.sup.+]-ATPase; transcription factor Snail

The effect of cardiac glycosides to increase cardiac inotropy by altering [Ca.sup.2+] cycling is well known but still poorly understood. The studies described in this report focus on defining the effects of ouabain signaling on sarcoplasmic reticulum [Ca.sup.2+]-ATPase function. Rat cardiac myocytes treated with 50 [micro]M ouabain demonstrated substantial increases in systolic and diastolic [Ca.sup.2+] concentrations. The recovery time constant for the [Ca.sup.2+] transient, [[[tau].sub.[Ca.sup.2+]],was significantly prolonged by ouabain. Exposure to 10 [micro]M [H.sub.2][O.sub.2], which causes an increase in intracellular reactive oxygen species similar to that of 50 [micro]M ouabain, caused a similar increase in [[tau].sub.Ca.sup.2+]. Concurrent exposure to 10 mM N-acetylcysteine or an aqueous extract from green tea (50 mg/ml) both prevented the increases in [[tau]].sup.Ca.sup.2+] as well as the changes in systolic or diastolic [Ca.sup.2+] concentrations. We also observed that 50 [micro]M ouabain induced increases in developed pressure in addition to diastolic dysfunction in the isolated perfused rat heart. Coadministration of ouabain with N-acetylcysteine prevented these increases. Analysis of sarcoplasmic reticulum [Ca.sup.2+]-ATPase protein revealed increases in both the oxidation and nitrotyrosine content in the ouabain-treated hearts. Liquid chromatography-mass spectrometric analysis confirmed that the sarcoplasmic reticulum [Ca.sup.2+]-ATPase protein from ouabain-treated hearts had modifications consistent with oxidative and nitrosative stress. These data suggest that ouabain induces oxidative changes of the sarcoplasmic reticulum [Ca.sup.2+]-ATPase structure and function that may, in turn, produce some of the associated changes in [Ca.sup.2+] cycling and physiological function. reactive oxygen species; cardiac signaling