细胞外信号调节蛋白激酶介导醛固酮诱导的肾小球系膜细胞增殖

Objective To determine the role of extracellular signal-regulated kinases (ERK1/2) in aldosterone-induced rat mesangial cells (RMCs) proliferation. Methods RMCs were obtained from intact glomeruli of 4- to 6-week-old Sprague-Dawley rats and characterized according to published methods. RMCs between passages 5 and passages 10 were used. Protein levels of mineralocorticoid receptor(MR) in RMCs were analyzed by Western blotting. The cells were divided into the following groups: control group, PD98059(10 ?滋mol/L) group, eplerenone (1 ?滋mol/L) group, aldosterone (100 nmol/L) group, aldosterone (100 nmol/L) ＋PD98059 (10 ?滋mol/L) group, aldosterone(100 nmol/L)＋eplerenone (1 ?滋mol/L) group. ERK1/2 activity was measured by Western blotting. Cell proliferation of RMCs was evaluated by [3H]-thymidine uptake measurements. Results MR protein expression in RMCs was confirmed by Western blotting. Aldosterone activated ERK1/2, and the maximal ERK1/2 activation induced by aldosterone was at a concentration of 100 nmol/L. Aldosterone (100 nmol/L)-induced activation of ERK1/2 peaked at 10 minutes (P<0.05). Pretreatment with a selective MR antagonist eplerenone (1 ?滋mol/L) significantly attenuated aldosterone-induced ERK1/2 phosphorylation. Aldosterone (100 nmol/L) treatment for 30 hours increased [3H]-thymidine incorporation of RMCs (135%±8% of controls, P<0.05). Cellular proliferation induced by aldosterone could be prevented by pretreatment with eplerenone or an ERK (MEK) inhibitor PD988059. Conclusion Aldosterone induces RMCs proliferation through MR and ERK1/2 activation, which may contribute to the pathogenesis of glomerular mesangial injury.     

Aldosterone induces CTGF in mesangial cells by activation of the glucocorticoid receptor.

BACKGROUND: Aldosterone contributes substantially to cardiac and renal injury by acting on target cells not involved in the regulation of salt and water balance. The profibrotic protein connective tissue growth factor (CTGF) has been identified as one of the target proteins of aldosterone. However, the molecular mechanisms of aldosterone-mediated CTGF induction have not been characterized. METHODS: Mesangial cells were treated with aldosterone or dexamethasone. CTGF expression was characterized at the mRNA and protein level. Translocation of the glucocorticoid receptor (GR) was detected by immunocytochemistry and by Western blotting. RESULTS: Aldosterone and dexamethasone induced CTGF at the mRNA and protein level in a time- and concentration-dependent manner. Specific antagonists of the mineralocorticoid receptor, spironolactone, canrenoate or eplerenone, did not inhibit CTGF induction. However, inhibition of the GR by RU486 prevented dexamethasone-as well as aldosterone-induced CTGF expression, indicating the importance of the GR in aldosterone-mediated regulation of CTGF. This notion was confirmed by translocation of the GR to the nucleus upon stimulation with aldosterone. CONCLUSIONS: CTGF is a functional target of aldosterone in mesangial cells, but aldosterone-induced CTGF gene expression is not directly mediated by the mineralocorticoid receptor.     

姜黄素联合安体舒通改善醛固酮诱导大鼠肾小管上皮细胞转分化

目的：观察姜黄素联合安体舒通对大鼠肾小管上皮细胞（TECs）转分化影响。方法：将体外培养的醛固酮处理组大鼠TEC分别给予姜黄素、安体舒通和姜黄素联合安体舒通共培养,利用倒置显微镜观察细胞形态,采用RT-PCR及Western blot观察TEC细胞TGF-β1、α-SMA和SGK1表达。结果：醛固酮（10-6M）处理后,大鼠TEC细胞明显发生转分化,TGF-β1、α-SMA和SGK1表达明显升高,姜黄素（10-3M）和安体舒通（10-5M）处理则明显抑制醛固酮诱导TEC细胞转分化效应,二者具有明显的协调效应。结论：醛固酮能显著促进大鼠肾小管上皮细胞TGF-β1、α-SMA和SGK1的表达和细胞转分化,姜黄素联合安体舒通可通过抑制SGK1表达拮抗醛固酮的促炎促纤维化效应。     

Aldosterone-induced kidney injury is mediated by NFκB activation

Background  

Aldosterone induces inflammation and fibrosis in the kidney, while nuclear factor κB (NFκB) plays key roles in inflammation mediated by various cytokines. Here, we determined the roles of NFκB activation in aldosterone-induced kidney injury.     

Aldosterone induces collagen synthesis via activation of extracellular signal-regulated kinase 1 and 2 in renal proximal tubules

Aim: Aldosterone plays a crucial role in renal fibrosis by inducing mesangial cell proliferation and promoting collagen synthesis in renal fibroblasts. However, renal proximal tubule involvement in aldosterone-induced collagen synthesis has not yet been identified. The aim of this study was to examine the potential role of aldosterone in collagen expression and its possible mineralocorticoid receptor (MR)-dependent pathway, mediated by activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in cultured human renal proximal tubular epithelial (HKC) cells. Methods: After HKC cells were stimulated by aldosterone with different concentrations for various time and periods, the gene expression and protein synthesis of collagen I, II, III and IV were measured by real-time polymerase chain reaction and western blot, respectively. ERK1/2 activation, α-smooth muscle actin (α-SMA), and E-cadherin were also detected by western blot. Results: Aldosterone can increase ERK1/2 phosphorylation of human renal proximal tubular epithelial cells in a time- and dose-dependent manner. Although aldosterone had no effect on collagen I and II expression, it increased expression of α-SMA and collagen III and IV and decreased that of E-cadherin in HKC cells after 48 h. These effects could be prevented by a ERK pathway inhibitor, U0126, or by a selective MR antagonist, spironolactone. Conclusion: The results suggest that aldosterone plays a pivotal role in tubulointerstitial fibrosis by promoting tubular epithelial–mesenchymal transition and collagen synthesis in proximal tubular cells. The process is MR-dependent, and mediated by ERK1/2 mitogen-activated protein kinase pathway.     

Combined Prognostic Value of Both RelA and IκB-α Expression in Human Non–Small Cell Lung Cancer

Background We sought to investigate the prognostic significance of nuclear factor (NF)-κB activity, especially nuclear RelA and IκB-α expression patterns, in non–small cell lung cancer (NSCLC). Methods A total of 116 patients with pathologically confirmed stage I to II NSCLC were included. Immunohistochemical analysis and electrophoretic mobility shift assays of NF-κB were performed to determine RelA and phosphorylated IκB-α staining, and DNA binding activity of NF-κB in human NSCLC. Downstream genes, including VEGF and IL-8, were also assessed. The prognostic significance of a single expression of RelA, phosphorylated IκB-α, and b-composite expressions was evaluated by Cox proportional hazard regression models and by Kaplan-Meier survival analyses. Correlation between RelA/IκB-α expression status and clinicopathological features of NSCLC was also analyzed. Results NF-κB DNA binding activity, VEGF, and IL-8 showed correlation with nuclear RelA and cytoplasmic pIκB-α expression. Expression of nuclear RelA/NF-κB showed an increase in NSCLC tissue compared with adjacent normal tissue and normal lung tissue. There was a positive correlation between NF-κB activation (nuclear translocation of RelA) and tumor clinicopathological features such as tumor grade, including T stages, N stages, and tumor, node, metastasis system stages, smoking status, and age. Positive correlation was observed between nuclear RelA and cytoplasmic pIκB-α. Both nuclear RelA and cytoplasmic pIκB-α were associated with poor prognosis by univariate and multivariate analyses. Conclusions Nuclear RelA and cytoplasmic pIκB-α expression are associated with a poorer prognosis in NSCLC patients. In particular, composite application of these two biomarkers might be of greater value than application of a single marker to identify patients at high risk, even at an early clinical stage. D.Z. and X.J. contributed equally to this work.     

PI-3’ kinase and NF-ΚB cross-signaling in human pancreatic cancer cells

Because tumor necrosis factor-alpha (TNF-α) and some chemotherapeutic agents activate both apoptosis andNF-κB-dependent antiapoptotic genes, they may neutralize their own antitnmor effects. The cell-signaling mechanisms for such chemoresistance are not clear but may involve phosphotidylinositol 3′ kinase (PI3K). To clarify this we examined whether cross-signaling between PI3K and NF-κ B enhances the antitnmor effect of TNF-α in human pancreatic cancer cells. Quiescent pancreatic cancer cells (Pant-1, MiaPaCa-2) with TNF-α, Ly294002 (PI3K inhibitor), alone or combined, were restimulated with mitogen (10% fetal calf serum [FCS] to induce cell cycle entry). Proliferation (monotetrazolium), cell cycle progression (ApoBrDU and fluorescence-activated cell sorter analysis), and apoptosis (PARP cleavage; caspase-3 activation) were measured. Akt activation (Akt kinase assay) and IKB~ degradation were determined by Western blot analysis. Translocation of NF-κ B into the nucleus was examined by EMSA, whereas an NF-κ B/hiciferase reporter gene was used to quantify NF-κ B-dependent gene expression. Statistical analysis was carried out by means of two-tailed t test (P <0.05). PI3K inhibition significantly enhanced the antiproliferative and proapoptotic effects of TNF-α in both cell lines. Ly294002 also blocked TNF-α -induced Akt activation but failed to alter cytoplasmic IκBα degradation or subsequent NF-κ B nuclear translocation. NF-κ B-dependent gene expression, however, was ultimately suppressed by Ly294002, suggesting that PI3k-dependent activation of NF-κ B is Iκ Bα independent. PI3K inhibition can block NF-κ B-dependent gene expression regardless of cytoplasmic IKBNF-κ B activation. Because it also regulates the antitumor effects of TNF-α PI3K may in part determine NF-κ B-induced chemoresistance in human pancreatic cancer. Presented at the Forty-Second Annual Meeting of The Society for Surgery of the Alimentary Tract, Atlanta, Ga., May 20–23, 200l.     

Aldosterone stimulates proliferation of mesangial cells by activating mitogen-activated protein kinase 1/2, cyclin D1, and cyclin A

Recently, attention has been focused on the role of aldosterone in the pathophysiology of hypertension and cardiovascular disease. Several clinical and experimental data support the hypothesis that aldosterone contributes to the progression of renal injury. However, the molecular mechanisms of the effects of aldosterone in signal transduction and the cell-cycle progression of mesangial cells are not well known. For determining the signaling pathway of aldosterone in cultured mesangial cells, the effects of aldosterone on the mitogen-activated protein kinase 1/2 (MAPK1/2) pathway and the promoter activities of cyclin D1, cyclin A, and cyclin E were investigated. First, it was shown that the mineralocorticoid receptor (MR) was expressed in rat mesangial cells and glomeruli and that aldosterone stimulated the proliferation of mesangial cells via the MR and MAPK1/2 pathway. Next, it was demonstrated that aldosterone stimulated Ki-RasA, c-Raf kinase, MEK1/2, and MAPK1/2 in rat mesangial cells. Aldosterone induced cyclin D1 and cyclin A promoter activities and protein expressions, as well as the increments of CDK2 and CDK4 kinase activities. The presence of CYP11B2 and 11beta-HSD2 mRNA in rat mesangial cells also was shown. In conclusion, aldosterone seems to exert mainly MR-induced effects that stimulate c-Raf, MEK1/2, MAPK1/2, the activities of CDK2 and CDK4, and the cell-cycle progression in mesangial cells. MR antagonists may serve as a potential therapeutic approach to mesangial proliferative disease.     

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.     

Aldosterone stimulates reactive oxygen species production through activation of NADPH oxidase in rat mesangial cells

It has recently been shown that glomerular mesangial injury is associated with increases in renal cortical reactive oxygen species (ROS) levels in rats treated chronically with aldosterone and salt. This study was conducted to determine the mechanisms responsible for aldosterone-induced ROS production in cultured rat mesangial cells (RMC). Oxidative fluorescent dihydroethidium was used to evaluate intracellular production of superoxide anion (O(2)(-)) in intact cells. The lucigenin-derived chemiluminescence assay was used to determine NADPH oxidase activity. The staining of dihydroethidium was increased in a dose-dependent manner by aldosterone (1 to 100 nmol/L) with a peak at 3 h in RMC. Aldosterone (100 nmol/L for 3 h) also significantly increased NADPH oxidase activity from 232 +/- 18 to 346 +/- 30 cpm/5 x 10(4) cells. Immunoblotting data showed that aldosterone (100 nmol/L for 3 h) increased p47phox and p67phox protein levels in the membrane fraction by approximately 2.1- and 2.3-fold, respectively. On the other hand, mRNA expression of NADPH oxidase membrane components, p22phox, Nox-1, and Nox-4, were not altered by aldosterone (for 3 to 12 h) in RMC. Pre-incubation with the selective mineralocorticoid receptor (MR) antagonist, eplerenone (10 micromol/L), significantly attenuated aldosterone-induced O(2)(-) production, NADPH oxidase activation and membranous translocation of p47phox and p67phox. These results suggest that aldosterone-induced ROS generation is associated with NAPDH oxidase activation through MR-mediated membranous translocation of p47phox and p67phox in RMC. These cellular actions of aldosterone may play a role in the pathogenesis of glomerular mesangial injury.     

Exploiting Our Knowledge of NF-κB Signaling for the Treatment of Mammary Cancer

Nuclear Factor-kappaB (NF-κB) has been implicated in the lobuloalveolar development of the mammary gland. In breast cancer its activation has been linked to tumor progression via stimulation of cell proliferation, pro-survival, and angiogenesis pathways and metastasis. Whether NF-κB activation in the immune system influences mammary cancer remains unclear. In addition to the constitutive activation frequently found in mammary carcinoma tissue, radio- and chemotherapeutic agents used in the treatment of mammary cancer can lead to activation of NF-κB. This effect has been postulated to contribute to the development of resistance to these agents and suggests the use of NF-κB inhibitors as sensitizers for therapy. The review describes principle targets and drugs used to inhibit NF-κB function and discusses future perspectives in the use of these inhibitors for the treatment of mammary cancer.     

Inhibition of the classical NF-κB pathway prevents osteoclast bone-resorbing activity

The classical NF-κB pathway plays an important role in osteoclast formation and differentiation; however, the role of NF-κB in osteoclast bone-resorbing activity is not well understood. To elucidate whether NF-κB is important for osteoclast bone-resorbing activity, we used a selective peptide inhibitor of the classical NF-κB pathway named the NBD peptide. Osteoclasts were generated using bone marrow macrophages in the presence of M-CSF and RANKL. The NBD peptide dose-dependently blocked the bone-resorbing activity of osteoclasts by reducing area, volume (p < 0.001) and depths (p < 0.05) of pits. The reduced resorption by the peptide was due to reduced osteoclast bone-resorbing activity, but not reduced differentiation as the number of osteoclasts was similar in all groups. The peptide inhibited bone resorption by reducing TRAP activity, disrupting actin rings and preventing osteoclast migration. Gene expressions of a panel of bone resorption markers were significantly reduced. The NBD peptide dose-dependently reduced the RANKL-induced c-Src kinase activity, which is important for actin ring formation and osteoclast bone resorption. Therefore, these data suggest that the classical NF-κB pathway plays a pivotal role in osteoclast bone-resorbing activity.     

Water solution of onion crude powder inhibits RANKL-induced osteoclastogenesis through ERK, p38 and NF-κB pathways

Summary  Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-κB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. Introduction  A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-κB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. Methods  The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-κB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-κB activity. Results  Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-κB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. Conclusions  Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-κB activation. R.-S. Yang, and W.-M. Fu contributed equally to this study.     

Aldosterone and glomerular podocyte injury

Aldosterone is traditionally viewed as a hormone regulating electrolyte and blood pressure homeostasis by acting on the distal nephron. Accumulating evidence suggests that aldosterone also plays pathogenetic roles in cardiovascular and renal injury. For example, aldosterone is a potent inducer of proteinuria. We demonstrated that podocyte injury underlies the pathogenesis of proteinuria in aldosterone-infused rats on a high salt diet. Mineralocorticoid receptor was detected in the podocytes in vivo and in vitro, and aldosterone caused induction of its effector kinase Sgk1, activation of NADPH oxidase and generation of reactive oxygen species. Selective aldosterone blocker eplerenone, as well as antioxidant tempol, ameliorated aldosterone-induced podocyte injury and proteinuria. Aldosterone was also involved in the podocyte damage and proteinuria of metabolic syndrome model SHR/NDmcr-cp. Adipocyte-derived aldosterone releasing factors were suggested to contribute to the aldosterone excess of this model. Furthermore, high salt diet markedly worsened the renal injury of SHR/NDmcr-cp. Although salt lowered serum aldosterone levels, it caused MR activation in the kidney. Accordingly, eplerenone dramatically improved the salt-evoked nephropathy. Taken together, aldosterone blockers can be an excellent therapeutic strategy for the treatment of podocyte injury, proteinuria, and cardiovascular and renal complications, not only in high aldosterone states but also in patients with activated MR signaling in the target tissue, whose circulating aldosterone level is not necessarily high. Addition of aldosterone blockers in patients treated with ACEIs or ARBs are also promising, because of "aldosterone breakthrough" phenomenon. Careful monitoring of hyperkalemia is necessary, especially in patients with impaired renal function.     

In Vivo Reduction of the Nuclear Factor-κB Activity Using Synthetic <Emphasis Type="Italic">cis</Emphasis>-Element Decoy Oligonucleotides Suppresses Intimal Hyperplasia in the Injured Carotid Arteries in Rabbits

Purpose Nuclear factor-κB (NF-κB) plays a critical role in inflammation-related reactions, and is also found in the injured arterial wall. The purpose of this study was to introduce synthetic double-stranded cis-element “decoy” oligonucleotides (ODNs) into the arterial wall using the hemagglutinating virus of Japan (HVJ) liposome, and to investigate the inhibitory potential of decoy ODN against balloon injury-induced intimal hyperplasia by reducing NF-κB activity. Methods Fluorescein isothiocyanate (FITC)-labeled decoy ODNs using the HVJ-liposome method were tranfected in balloon-injured rabbit carotid arteries. We then performed electrophoretic mobility shift assay to examine NF-κB activity using balloon-injured arteries, and we introduced NF-κB decoy into balloon-injured arteries. Results Transfection of FITC-labeled decoy ODNs by using the HVJ-liposome method demonstrated highly efficient protein expression with diffuse, frequent, and widespread nuclear signals over the entire medial layer, while the same amount of naked ODNs showed much less efficiency with scattered distribution of fluorescence in balloon-injured carotid arteries. Electrophoretic mobility shift assay showed activation of NF-κB in balloon-injured arteries. In vivo transfection of decoy ODNs mediated by HVJ liposome abolished the NF-κB activity in injured arteries with specific binding affinity to NF-κB protein. Intimal hyperplasia of carotid artery after balloon injury was reduced by approximately 50% by NF-κB decoy transfection compared with buffer treatment or scrambled decoy transfection. Conclusion Our results demonstrated involvement of NF-κB in intimal formation after arterial injury, and indicated that NF-κB can be an appropriate molecular target for anti-restenosis therapy.     

Cox-2 inhibition results in alterations in nuclear factor (nf)-κb activation but not cytokine production in acute pancreatitis

Acute pancreatitis is characterized by local inflammation and cytokine production, and release is thought to contribute to this process. Nuclear factor (NF)-κB activation and cytokine production are linked and inhibition of NF-κB has been shown to decrease the severity of pancreatitis. We have shown that inhibition of COX-2 ameliorates pancreatitis; however, the mechanism by which this effect occurs is unclear. Swiss Webster mice were injected intraperitoneally with either saline (control) or caerulein (CAE; 50 mg/kg) hourly for 8 hours; mice receiving CAE were further subdivided to receive saline or the cyclooxygenase-2 (COX-2) selective inhibitor (SC-58125; 10 mg, intraperitoneally) at the time of the first injection of CAE. Pancreata were harvested, histologic sections were scored, and protein was extracted to determine cytokine (interleukin [IL]-6, IL-1β) levels and NF-κB subunits by ELISA and NF-κB activation by gel shift. In addition, serum was collected for measurement of cytokines. COX-2 inhibition resulted in decreased inflammation and a decrease in NF-κB activation. IL-6 and IL-1β levels after COX-2 inhibition, however, remained elevated to levels equivalent to those of mice with histologic inflammation after CAE alone. COX-2 inhibition decreases inflammation as well as late-phase NF-κB activation but does not diminish levels of inflammatory cytokines, thus suggesting a two-phase activator of NF-κB. The attenuation of inflammation, despite unaltered cytokine levels, suggests that cytokines may not be critical for the inflammatory phase of pancreatitis. This paper was presented, in part, at the Annual Meeting of the Society for Surgery of the Alimentary Tract, May 18–22, 2003, Orlando, Florida, and published in abstract form (Gastroenterology 2003;124:A501, A804). This work was supported by grants from the National Institutes of Health (RO1 DK48498, PO1 DK35608, and T32 DK07639).