Abstract-We have observed recently that experimental renal failure in the rat is accompanied by increases in circulating concentrations of the cardiotonic steroid, marinobufagenin (MBG), and substantial cardiac fibrosis. We performed the following studies to examine whether MBG might directly stimulate cardiac fibroblast collagen production. In vivo studies were performed using the 5/6th nephrectomy model of experimental renal failure (PNx), MBG infusion (MBG), PNx after immunization against MBG, and concomitant PNx and adrenalectomy. Physiological measurements with a Millar catheter and immunohistochemistry were performed. In vitro studies were then pursued with cultured isolated cardiac fibroblasts. We observed that PNx and MBG increased MBG levels, blood pressure, heart size, impaired diastolic function, and caused cardiac fibrosis. PNx after immunization against MBG and concomitant PNx and adrenalectomy had similar blood pressure as PNx but less cardiac hypertrophy, diastolic dysfunction, and cardiac fibrosis. MBG induced increases in procollagen-1 expression by cultured cardiac fibroblasts at 1 nM concentration. These increases in procollagen expression were accompanied by increases in collagen translation and increases in procollagen-1 mRNA without any demonstrable increase in procollagen-1 protein stability. The stimulation of fibroblasts with MBG could be prevented by administration of inhibitors of tyrosine phosphorylation, Src activation, epidermal growth factor receptor transactivation, and N-acetyl cysteine. Based on these findings, we propose that MBG directly induces increases in collagen expression by fibroblasts, and we suggest that this may be important in the cardiac fibrosis seen with experimental renal failure. (Hypertension. 2007;49:215-224.)

The long-term effects of ouabain on transepithelial Na+ transport involve transcriptional downregulation of apical Na+/H+ exchanger isoform 3 (NHE3). The aim of this study was to determine whether ouabain could acutely regulate NHE3 via a posttranscriptional mechanism in LLC-PK1 cells. We observed that the basolateral, but not apical, application of ouabain for 1 h significantly reduced transepithelial Na+ transport. This effect was not due to changes in the integrity of tight junctions or increases in the intracellular Na+ concentration. Ouabain regulated the trafficking of NHE3 and subsequently inhibited its activity, a process independent of intracellular Na+ concentration. Ouabain-induced NHE3 trafficking was abolished by either cholesterol depletion or Src inhibition. Moreover, ouabain increased the intracellular Ca2+ concentration. Pretreatment of cells with the intracellular Ca2+ chelator BAPTA-AM blocked ouabain-induced trafficking of NHE3. Also, blockade of Na+-K+-ATPase endocytosis by a phosphatidylinositol 3-kinase inhibitor was equally effective in attenuating ouabain-induced NHE3 trafficking. These data indicate that ouabain acutely stimulates NHE3 trafficking by activating the basolateral Na+-K+-ATPase signaling complex. Taken together with our previous observations, we propose that ouabain can simultaneously regulate basolateral Na+-K+-ATPase and apical NHE3, leading to inhibition of transepithelial Na+ transport. This mechanism may be relevant to proximal tubular Na+ handling during conditions associated with increases in circulating endogenous cardiotonic steroids.

Ouabain, a cardiotonic steroid and a specific inhibitor of the Na ϩ -K ϩ -ATPase, has been shown to significantly inhibit transcellular Na ϩ transport without altering the intracellular Na ϩ concentration ([Na ϩ ]i) in the epithelial cells derived from the renal proximal tubules. We therefore studied whether ouabain affects the activity and expression of Na ϩ /H ϩ exchanger isoform 3 (NHE3) representing the major route of apical Na ϩ reabsorption in LLC-PK1 cells. Chronic basolateral, but not apical, exposure to low-concentration ouabain (50 and 100 nM) did not change [Na ϩ ]i but significantly reduced NHE3 activity, NHE3 protein, and mRNA expression. Inhibition of c-Src or phosphoinositide 3-kinase (PI3K) with PP2 or wortmannin, respectively, abolished ouabain-induced downregulation of NHE3 activity and mRNA expression. In caveolin-1 knockdown LLC-PK 1 cells, ouabain failed to downregulate NHE3 mRNA expression and NHE3 promoter activity. Ouabain response elements were mapped to a region between Ϫ450 and Ϫ1,194 nt, where decreased binding of thyroid hormone receptor (TR) and Sp1 to their cognate cis-elements was documented in vitro and in vivo by protein/DNA array analysis, EMSA, supershift, and chromatin immunoprecipitation. These data suggest that, in LLC-PK 1 cells, ouabain-induced signaling through the Na ϩ -K ϩ -ATPase-Src pathway results in decreased Sp1 and TR DNA binding activity and consequently in decreased expression and activity of NHE3. These novel findings may represent the underlying mechanism of cardiotonic steroid-mediated renal compensatory response to volume expansion and/or hypertension.

Recently, stathmin 1 has been proposed to function as an oncogene based on some relevant studies in multiple types of human cancers. However, the role of stathmin 1 in gastric cancer carcinogenesis has not been elucidated yet. The aim of this study was to investigate the expression of stathmin 1 as well as its association with overall survival of gastric cancer patients. The expression of stathmin 1 was detected by real-time quantitative reverse transcription polymerase chain reaction and Western blotting in gastric cancer and adjacent nontumor tissues. In addition, stathmin 1 expression was analyzed by immunohistochemistry in paraffin samples from 210 primary gastric cancer patients. The expression levels of stathmin 1 mRNA and protein in gastric cancer tissues were both significantly higher than those in adjacent nontumor tissues. In addition, the expression of stathmin 1 is correlated with Lauren's classification, depth of invasion, lymph node metastases, and tumor node metastasis (TNM) stage (all P < 0.05). Univariate analysis showed that high stathmin 1 expression was associated with poor prognosis in gastric cancer patients (P = 0.040). Multivariate analysis demonstrated that only lymph node metastasis and TNM stage were the independent prognostic indicators for gastric cancer. Stathmin 1 expression status is not an independent prognostic factor for patients with gastric cancer. Further subgroup analysis revealed that stathmin 1 expression was significantly correlated with prognosis in diffuse type gastric cancer. This research showed that the stathmin 1 overexpression might play an important role in the pathogenesis and subsequent progression of gastric cancer. Stathmin 1 could also be a potential therapeutic target in gastric cancer, especially for diffuse type gastric cancer.