Chronic Sarpogrelate Treatment Reveals 5-HT7 Receptor in the Serotonergic Inhibition of the Rat Vagal Bradycardia.

5-Hydroxytryptamine (5-HT) modulates the cardiac parasympathetic neurotransmission, inhibiting the bradyarrhythmia by 5-HT2 receptor activation. We aimed to determine whether the chronic selective 5-HT2 blockade (sarpogrelate) could modify the serotonergic modulation on vagal cardiac outflow in pithed rat. Bradycardic responses in rats treated with sarpogrelate (30 mg·kg·d; orally) were obtained by electrical stimulation of the vagal fibers (3, 6, and 9 Hz) or intravenous (IV) injections of acetylcholine (1, 5, and 10 µg/kg). 5-HT7 receptor expression was quantified by Western blot in vagus nerve and right atrium. The IV administration of 5-HT (10-200 µg/kg) dose dependently decreased the vagally induced bradycardia, and agonists 5-CT (5-HT1/7), 8-OH-DPAT (5-HT1A), or AS-19 (5-HT7) (50 µg/kg each) mimicked the 5-HT-induced inhibitory effect. Neither agonists CGS-12066B (5-HT1B), L-694,247 (5-HT1D), nor 1-phenylbiguanide (5-HT3) modified the electrically-induced bradycardic responses. Moreover, SB-258719 (5-HT7 antagonist) abolished the 5-HT-, 5-CT-, 8-OH-DPAT-, and AS-19-induced bradycardia inhibition; 5-HT or AS-19 did not modify the bradycardia induced by IV acetylcholine; and 5-HT7 receptor was expressed in both the vagus nerve and the right atrium. Our outcomes suggest that blocking chronically 5-HT2 receptors modifies the serotonergic influence on cardiac vagal neurotransmission exhibiting 5-HT as an exclusively inhibitory agent via prejunctional 5-HT7 receptor.

Absence of K-Ras Reduces Proliferation and Migration But Increases Extracellular Matrix Synthesis in Fibroblasts.

The involvement of Ras-GTPases in the development of renal fibrosis has been addressed in the last decade. We have previously shown that H- and N-Ras isoforms participate in the regulation of fibrosis. Herein, we assessed the role of K-Ras in cellular processes involved in the development of fibrosis: proliferation, migration, and extracellular matrix (ECM) proteins synthesis. K-Ras knockout (KO) mouse embryonic fibroblasts (K-ras(-/-) ) stimulated with transforming growth factor-ß1 (TGF-ß1) exhibited reduced proliferation and impaired mobility than wild-type fibroblasts. Moreover, an increase on ECM production was observed in K-Ras KO fibroblasts in basal conditions. The absence of K-Ras was accompanied by reduced Ras activation and ERK phosphorylation, and increased AKT phosphorylation, but no differences were observed in TGF-ß1-induced Smad signaling. The MEK inhibitor U0126 decreased cell proliferation independently of the presence of K-ras but reduced migration and ECM proteins expression only in wild-type fibroblasts, while the PI3K-AKT inhibitor LY294002 decreased cell proliferation, migration, and ECM synthesis in both types of fibroblasts. Thus, our data unveil that K-Ras and its downstream effector pathways distinctively regulate key biological processes in the development of fibrosis. Moreover, we show that K-Ras may be a crucial mediator in TGF-ß1-mediated effects in this cell type. J. Cell. Physiol. 231: 2224-2235, 2016. © 2016 Wiley Periodicals, Inc.

ALK1 heterozygosity increases extracellular matrix protein expression, proliferation and migration in fibroblasts.

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-ß1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-ß1 with an important role in angiogenesis whose function in cellular biology and TGF-ß signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1(+/+) and ALK1(+/-) mouse embryonic fibroblasts (MEFs) after TGF-ß1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-ß/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.

Endothelial endoglin is involved in inflammation: role in leukocyte adhesion and transmigration.

Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng(+/-)) and their wild-type siblings Eng(+/+) treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng(+/-) than in Eng(+/+) mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglin-coated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5ß1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5ß1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking.

H-Ras isoform modulates extracellular matrix synthesis, proliferation, and migration in fibroblasts.

Ras GTPases are ubiquitous plasma membrane transducers of extracellular stimuli. In addition to their role as oncogenes, Ras GTPases are key regulators of cell function. Each of the Ras isoforms exhibits specific modulatory activity on different cellular pathways. This has prompted researchers to determine the pathophysiological roles of each isoform. There is a proven relationship between the signaling pathways of transforming growth factor-ß1 (TGF-ß1) and Ras GTPases. To assess the individual role of H-Ras oncogene in basal and TGF-ß1-mediated extracellular matrix (ECM) synthesis, proliferation, and migration in fibroblasts, we analyzed these processes in embryonic fibroblasts obtained from H-Ras knockout mice (H-ras(-/-)). We found that H-ras(-/-) fibroblasts exhibited a higher basal phosphatidylinositol-3-kinase (PI3K)/Akt activation than wild-type (WT) fibroblasts, whereas MEK/ERK 1/2 activation was similar in both types of cells. Fibronectin and collagen synthesis were higher in H-ras(-/-) fibroblasts and proliferation was lower in H-ras(-/-) than in WT fibroblasts. Moreover, H-Ras appeared indispensable to maintain normal fibroblast motility, which was highly restricted in H-ras(-/-) cells. These results suggest that H-Ras (through downregulation of PI3K/Akt activation) could modulate fibroblast activity by reducing ECM synthesis and upregulating both proliferation and migration. TGF-ß1 strongly increased ERK and Akt activation in WT but not in H-ras(-/-) fibroblasts, suggesting that H-Ras is necessary to increase ERK 1/2 activation and to maintain PI3K downregulation in TGF-ß1-stimulated fibroblasts. TGF-ß1 stimulated ECM synthesis and proliferation, although ECM synthesis was higher and proliferation lower in H-ras(-/-) than in WT fibroblasts. Hence, H-Ras activation seems to play a key role in the regulation of these effects.

The furosemide stress test and computational modeling identify renal damage sites associated with predisposition to acute kidney injury in rats.

Acute kidney injury (AKI) diagnosis relies on plasma creatinine concentration (Crpl), a relatively insensitive, surrogate biomarker of glomerular filtration rate that increases only after significant damage befalls. However, damage in different renal structures may occur without increments in Crpl, a condition known as subclinical AKI. Thus, detection of alterations in other aspects of renal function different from glomerular filtration rate must be included in an integral diagnosis of AKI. With this aim, we adapted to and validated in rats (for preclinical research) the furosemide stress test (FST), a tubular function test hitherto performed only in humans. We also tested its sensitivity in detecting subclinical tubular alterations. In particular, we predisposed rats to AKI with 3 mg/kg cisplatin and subsequently subjected them to a triggering insult (ie, 50 mg/kg/d gentamicin for 6 days) that had no effect on nonpredisposed animals but caused an overt AKI in predisposed rats. The FST was performed immediately before adding the triggering insult. Predisposed animals showed a reduced response to the FST (namely, reduced furosemide-induced diuresis and K+ excretion), whereas nonpredisposed animals showed no alteration, compared to the controls. Computational modeling of epithelial transport of solutes and water along the nephrons applied to experimental data suggested that proximal tubule transport was only minimally reduced, the sodium-chloride symporter was upregulated by 50%, and the renal outer medullary potassium channel was downregulated by 85% in predisposed animals. In conclusion, serial coupling of the FST and computational modeling may be used to detect and localize subclinical tubular alterations.

Activation of Erk1/2 and Akt following unilateral ureteral obstruction.

Chronic unilateral ureteral obstruction is a well characterized model of renal injury leading to tubulointerstitial fibrosis and distinct patterns of cell proliferation and apoptosis in the obstructed kidney. In this study we assessed the contribution of the mitogen activated protein kinase (MAPK)-ERK1/2 and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways to early renal changes following unilateral obstruction. Increased activation of small Ras GTPase and its downstream effectors ERK1/2 and Akt was detected in ligated kidneys. The use of specific pharmacological inhibitors to either ERK1/2 or Akt activation led to decreased levels of fibroblast-myofibroblast markers in the interstitium while inhibition of PI3K reduced the number of proliferating cells and the amount of interstitial extracellular matrix deposition. Treatment with an ERK1/2 inhibitor diminished the number of apoptotic tubule and interstitial cells. Our results suggest a role for the MAPK-ERK1/2 and PI3K-Akt systems in early changes induced by ureteral obstruction and that inhibition of these signaling pathways may provide a novel approach to prevent progression of renal fibrosis.

Characterization of murine S-endoglin isoform and its effects on tumor development.

Endoglin is a transmembrane glycoprotein that acts as an auxiliary receptor for transforming growth factor-beta (TGF-beta) and modulates cellular responses to this pleiotropic cytokine. Endoglin is strongly expressed in endothelial cells, where it appears to exert a crucial role in vascular development and angiogenesis. Two endoglin isoforms (L and S), differing in their cytoplasmic domains, have been previously characterized in human tissues. We now demonstrate the existence of similar L- and S-endoglin variants in murine tissues with 47 and 35 amino acids, respectively, in their cytoplasmic tail. RT-PCR analysis showed that L is the predominant endoglin isoform expressed in mouse tissues, although S-endoglin mRNA is significantly expressed in liver and lung, as well as in endothelial cell lines. Furthermore, a protein of size equivalent to recombinant S-endoglin expressed in mammalian cells was detected in mouse endothelial cells by Western blot analysis. L- and S-endoglin isoforms can form disulfide-linked heterodimers, as demonstrated by cotransfection of L- and S-endoglin constructs. To address the role of S-endoglin in vivo, an S-Eng(+) transgenic mouse model that targets S-endoglin expression to the endothelium was generated. The lethal phenotype of endoglin-null (Eng(-/-)) mice was not rescued by breeding S-Eng(+) transgenic mice into the endoglin-null background. S-Eng(+) mice exhibited reduced tumor growth and neovascularization after transplantation of Lewis lung carcinoma cells. In addition, S-Eng(+) mice showed a drastic inhibition of benign papilloma formation when subjected to two-stage chemical skin carcinogenesis. These results point to S-endoglin as an antiangiogenic molecule, in contrast to L-endoglin which is proangiogenic. Oncogene (2005) 24, 4450-4461. doi:10.1038/sj.onc.1208644 Published online 4 April 2005.

Gentamicin induces Jun-AP1 expression and JNK activation in renal glomeruli and cultured mesangial cells.

Reactive oxygen species (ROS) mediate MC contraction, proliferation and apoptosis induced by gentamicin (G) in vitro and in vivo. Sustained increases in cytosolic free calcium, increased iNOS expression and elevated nitric oxide (NO) production are associated with MC apoptosis in vitro. As NO strongly activated c-Jun N-terminal kinase (JNK) and increased AP1 expression, and these two factors are involved in MC proliferation in vitro, we have measured Jun-AP1 expression in rat glomeruli from G-treated rats, and the effect of G on Jun-AP1 expression and JNK activity in cultured MC. Moreover, we studied the expression of inducible (iNOS) and constitutive (cNOS) NO synthases in rat glomeruli. Glomeruli were obtained from rats treated with G (100 mg/kg body weight/day) along 6 days, and MC primary cultures were evaluated after 24, 48 and 72 h incubation with 10(-5) M G. G induced an increase in the expression of iNOS, cNOS and Jun-AP1 in rat glomeruli and in MC cultures. Moreover, G activated JNK; JNK activation was reduced by co-incubation with the calcium channel blocker verapamil and with the ROS scavengers superoxide dismutase and catalase. These results strongly suggest a role for reactive oxygen/nitrogen species produced by increased NOS activity in G-induced MC activation. These reactive oxygen molecules and increased intracellular free calcium may mediate the increase in Jun-AP1 expression and JNK activation induced by G treatment in MC.

Effect of angiotensin II and small GTPase Ras signaling pathway inhibition on early renal changes in a murine model of obstructive nephropathy.

Tubulointerstitial fibrosis is a major feature of chronic kidney disease. Unilateral ureteral obstruction (UUO) in rodents leads to the development of renal tubulointerstitial fibrosis consistent with histopathological changes observed in advanced chronic kidney disease in humans. The purpose of this study was to assess the effect of inhibiting angiotensin II receptors or Ras activation on early renal fibrotic changes induced by UUO. Animals either received angiotensin II or underwent UUO. UUO animals received either losartan, atorvastatin, and farnesyl transferase inhibitor (FTI) L-744,832, or chaetomellic acid A (ChA). Levels of activated Ras, phospho-ERK1/2, phospho-Akt, fibronectin, and α-smooth muscle actin were subsequently quantified in renal tissue by ELISA, Western blot, and/or immunohistochemistry. Our results demonstrate that administration of angiotensin II induces activation of the small GTPase Ras/Erk/Akt signaling system, suggesting an involvement of angiotensin II in the early obstruction-induced activation of renal Ras. Furthermore, upstream inhibition of Ras signalling by blocking either angiotensin AT1 type receptor or by inhibiting Ras prenylation (atorvastatin, FTI o ChA) reduced the activation of the Ras/Erk/Akt signaling system and decreased the early fibrotic response in the obstructed kidney. This study points out that pharmacological inhibition of Ras activation may hold promise as a future strategy in the prevention of renal fibrosis.

Functional expression of angiotensinogen depends on splicing enhancers in exon 2.

Angiotensinogen belongs to the family of serpins and is the only precursor of the potent cardiovascular peptide, angiotensin II, the main effector of the renin-angiotensin system. The gene coding for this protein carries an internal exon (exon 2), the length of which (859 bp) by far exceeds the mean length of internal exons in vertebrates (<300 bp). Here, we show that this essential exon is skipped in about 20% of all transcripts in liver, brain, and kidney of rats and mice. Deletion mutants of exon 2 revealed a 62 bp region located at its 5'-end which is important for its inclusion in the mature angiotensinogen mRNA in transfected COS7 cells. Using an artificial minigene, we defined sequences inside this region as exonic splicing enhancers. These data reveal a novel molecular mechanism important for the renin-angiotensin system with implications in the basic understanding and the therapeutical assessment of cardiovascular diseases.

Endoglin as a marker in cervical paragangliomas.

BACKGROUND: Endoglin is expressed on endothelium and is implicated in the control of angiogenesis. This study compares the expression of endoglin with vascular endothelial growth factor (VEGF), commonly used as a marker for neoangiogenesis in cervical paragangliomas (CPG). METHODS: The CPG were surgically obtained from 5 patients and compared with nontumoral lung obtained from patients subjected to pulmonary resection. Detection with specific antibodies was used to determine the expression of the proteins VEGF and endoglin. The expressions of hypoxia-inducible factor (HIF) and vascular cell adhesion molecule-1 (VCAM-1) were used to determine the degree of hypoxia and capillarization, respectively. RESULTS: Endoglin is located at the plasma membrane of endothelial cells. The relative expression of endoglin is significantly higher in CPG respect to lung (p < .02), whereas that of VEGF is similar. CONCLUSION: Endoglin expression in CPG is significantly superior to that of VEGF and correlates with tumor vascularization.

Reduced tumor growth and angiogenesis in endoglin-haploinsufficient mice.

Endoglin is a transforming growth factor-beta(1) (TGF-beta(1)) accessory receptor which is highly expressed in tumor vessels. To study the role of endoglin in tumor growth and angiogenesis we induced a highly vascularized tumor in mice heterozygous for endoglin (Eng+/-) and in their control littermates (Eng+/+) by injecting 10(6) Lewis lung carcinoma (3LL) cells subcutaneously. Nine days after injection, the tumor was removed and weighed. Capillary density (CD31 immunohistochemistry), hemoglobin content and vascular cell adhesion molecule-1 (VCAM-1) expression were used to assess tumor vascularization. Tumor perfusion rate was measured by laser-Doppler technique. Expression of the hypoxia-inducible factor (HIF), endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) were determined by Western blot analysis. The aerobic metabolism and oxygen dependency were inferred from the measurement of ATP in tumoral tissue. Tumor weight, capillary density, hemoglobin and VCAM-1 were reduced by about 30% in Eng+/- compared to Eng+/+ littermates. The protein levels of eNOS and phosphorylated eNOS were significantly reduced in Eng+/- compared to Eng+/+ mice. HIF expression was slightly reduced whereas VEGF level was slightly increased in Eng+/- compared to Eng+/+. Tumor tissue levels of ATP and ADP were similar in both types of mice. These data demonstrate that endoglin plays a major role in tumor neoangiogenesis.

Gene expression fingerprinting for human hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) or Osler-Weber-Rendu syndrome is an autosomal dominant vascular disorder characterized by telangiectases and internal arteriovenous malformations. It is caused by mutations in elements of the transforming growth factor-beta (TGF-beta) receptor complex: endoglin, a co-receptor, responsible for HHT1, or ALK1 (activin receptor-like kinase 1), a type I receptor leading to HHT2. Recently, we have established cultures of HHT endothelial cells, primary targets of the disease. These cells showed deficient TGF-beta signaling and angiogenesis, representing a useful human model to study the molecular mechanism of this disease. To understand the pathogenic mechanism underlying HHT, we have used total RNA probes to compare HHT versus non-HHT cells by expression microarrays. This work represents a systematic study to identify target genes affected in HHT cells. Given the similarity of symptoms in HHT1 and HHT2, special interest has been put on the identification of common targets for both HHT types. As a result, 277 downregulated and 63 upregulated genes were identified in HHT versus control cells. These genes are involved in biological processes relevant to the HHT pathology, such as angiogenesis, cytoskeleton, cell migration, proliferation and NO synthesis. The type of misregulated genes found in HHT endothelial cells lead us to propose a model of HHT pathogenesis, opening new perspectives to understand this disorder. Moreover, as the disease is originated by mutations in proteins of the TGF-beta receptor complex, these results may be useful to find out targets of the TGF-beta pathway in endothelium.

Endoglin regulates renal ischaemia-reperfusion injury.

BACKGROUND: Renal ischaemia-reperfusion (I-R) can cause acute tubular necrosis and chronic renal deterioration. Endoglin, an accessory receptor for Transforming Growth Factor-beta1 (TGF-beta1), is expressed on activated endothelium during macrophage maturation and implicated in the control of fibrosis, angiogenesis and inflammation. METHODS: Endoglin expression was monitored over 14 days after renal I-R in rats. As endoglin-null mice are not viable, the role of endoglin in I-R was studied by comparing renal I-R injury in haploinsufficient mice (Eng(+/-)) and their wild-type littermates (Eng(+/+)). Renal function, morphology and molecular markers of acute renal injury and inflammation were compared. RESULTS: Endoglin mRNA up-regulation in the post-ischaemic kidneys of rats occurred at 12 h after I-R; endoglin protein levels were elevated throughout the study period. Expression was initially localized to the vascular endothelium, then extended to fibrotic and inflamed areas of the interstitium. Two days after I-R, plasma creatinine elevation and acute tubular necrosis were less marked in Eng(+/-) than in Eng(+/+) mice. Significant up-regulation of endoglin protein was found only in the post-ischaemic kidneys of Eng(+/+) mice and coincided with an increased mRNA expression of the TGF-beta1 and collagen IV (alpha1) chain genes. Significant increases in vascular cell adhesion molecule-1 (VCAM-1) and inducible nitric oxide synthase (iNOS) expression, nitrosative stress, myeloperoxidase activity and CD68 staining for macrophages were evident in post-ischaemic kidneys of Eng(+/+), but not Eng(+/-) mice, suggesting that impaired endothelial activation and macrophage maturation may account for the reduced injury in post-ischaemic kidneys of Eng(+/-) mice. CONCLUSIONS: Endoglin is up-regulated in the post-ischaemic kidney and endoglin-haploinsufficient mice are protected from renal I-R injury. Endoglin may play a primary role in promoting inflammatory responses following renal I-R.

Effect of the long-term treatment with trandolapril on endoglin expression in rats with experimental renal fibrosis induced by renal mass reduction.

BACKGROUND: Endoglin is a membrane glycoprotein that regulates TGF-beta1 signaling. Previous studies have revealed that endoglin is upregulated in several models of experimental fibrosis, and that endoglin expression can counteract the fibrogenic effects of TGF-beta1. As treatment with angiotensin converting enzyme (ACE) inhibitors reduces renal fibrosis by mechanisms that are, in part, not dependent on angiotensin II blockade, we have assessed the hypothesis that this effect could be mediated by endoglin upregulation. METHODS: We have used the 5/6-nephrectomy renal mass reduction (RMR) model of renal fibrosis in rats treated (RMR+T) or not treated with the ACE inhibitor trandolapril (0.7 mg/kg/day). One, 3 and 5 months after RMR, mean arterial pressure and renal function were measured. In addition, renal fibrosis was evaluated quantitatively and endoglin, TGF-beta1, collagen type I and collagen type IV expression was assessed by Northern blot and immunohistochemistry. RESULTS: RMR induced a progressive increase in mean arterial pressure, urinary protein excretion and glomerular and tubulointerstitial fibrosis, which is accompanied by an increased expression of TGF-beta1, endoglin and collagen types I and IV. Trandolapril treatment reduced systemic blood pressure and lessened proteinuria after RMR, as well as expression of TGF-beta1, endoglin and collagens. CONCLUSION: The present study demonstrates an increased TGF-beta1, endoglin, collagen type I and collagen type IV expression in rats with severe hypertension and renal damage. The effect of trandolapril to decrease renal fibrosis seems to be based in a reduced TGF-beta1 expression but not in an increased expression of endoglin.

Gentamicin treatment induces simultaneous mesangial proliferation and apoptosis in rats.

BACKGROUND: Gentamicin (G)-induced acute renal failure is characterized by an impairment of glomerular function without apparent changes in glomerular structure. However, G stimulates reactive oxygen species (ROS)-mediated mesangial cell proliferation in vitro. We studied whether G promotes mesangial cell apoptosis in vitro, and if apoptosis and proliferation in parallel may occur in glomerular cells in vivo after a renal damage induced by G treatment. METHODS: For in vivo studies, rats were treated with G (100 mg/kg body weight/day) for 6 days, and functional and histologic studies were performed. For in vitro studies, mesangial cell proliferation and apoptosis were evaluated after 24, 48, and 72 hours of 10(-5) mol/L G incubation. RESULTS: After G injections, the number of nuclei per glomerulus did not change, whereas proliferating and apoptotic cell numbers increased. G increases DNA synthesis and cell number in cultured mesangial cells, and increases markedly the apoptotic cell number. ROS scavengers superoxide dismutase and catalase reduce G-induced mesangial cell apoptosis, whereas the incubation with the ROS donor system xanthine plus xanthine oxidase increases apoptosis to levels similar to G. G-induced cellular proliferation and apoptosis either in vitro or in vivo is associated to an early increase in the pro-apoptotic protein Bax and a delayed increase in the survival protein Bcl-2. CONCLUSION: G simultaneously induces proliferation and apoptosis of mesangial cells in vitro and glomerular mesangial cells in vivo. ROS may mediate G-induced mesangial apoptosis in vitro. The equilibrium proliferation/apoptosis may maintain mesangial cell number within normal limits after a G-induced glomerular insult.

Intrarenal administration of molsidomine, a molecule releasing nitric oxide, reduces renal ischemia-reperfusion injury in rats.

Ischemia reperfusion (I-R)-induced renal damage is reduced by systemic administration of the NO-dependent vasodilator molsidomine. The aim of this study was to estimate the effect of direct intrarenal molsidomine administration on renal dysfunction and inflammatory reaction after experimental I-R in rats, in order to assess only renal NO effects and to obviate its systemic hemodynamic action. Ischemia was induced by renal pedicle ligation (60 min) followed by reperfusion and contralateral nephrectomy. Molsidomine (4 mg/kg) was infused into the renal artery 15 min before reperfusion and its effects were compared with those of the NO-independent vasodilator hydralazine (2 mg/kg). Survival rates after 7 days were 100% in the sham-operated group and 75% in the I-R rats. Molsidomine treatment almost completely prevented the I-R-induced renal dysfunction, and survival reached 100%. Molsidomine prevented an I-R-induced increase in superoxide anion and reduced plasma levels of pro-inflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma), whereas it enhanced anti-inflammatory cytokines (IL-6 and IL-10). Inflammatory cell infiltration and cell-adhesion molecules (ICAM-1, PECAM-1, VCAM-1 and P-selectin) were lower in the molsidomine-treated kidneys than in the untreated animals. All these protective effects were not observed after hydralazine administration. In conclusion, intrarenal administration of molsidomine before reperfusion improved renal function and decreased inflammatory responses after I-R.

Endoglin expression regulates basal and TGF-beta1-induced extracellular matrix synthesis in cultured L6E9 myoblasts.

BACKGROUND/AIMS: TGF-beta1 plays a major role in extracellular matrix (ECM) accumulation in tissue fibrosis. Connective tissue growth factor appears to play a critical role in this effect. Endoglin is a component of the transforming growth factor b (TGF-beta) receptor complex. Endoglin is upregulated by TGF-beta1, but its functional role in ECM regulation is unknown. Using rat myoblasts as a model system, we have assessed the role of endoglin on regulating CTGF expression and ECM synthesis and accumulation in the presence or absence of TGF-beta1. METHODS: L6E9 myoblast cell line was transfected with human endoglin, and collagen, fibronectin and CTGF production was assessed by Western blot and by proline incorporation to collagen proteins. RESULTS: Northern blot analysis revealed that parental rat myoblasts L6E9 do not express endogenous endoglin. Upon endoglin transfection, endoglin-expressing cells displayed a decreased CTGF expression and decreased collagen and fibronectin accumulation respect to mock transfectants. Northern blot analysis also revealed a decreased alpha2 (I) procollagen mRNA expression in endoglin transfectants. TGF-beta1 treatment induced an increase in CTGF expression and collagen synthesis and accumulation in L6E9 myoblasts. This effect was significantly lower in endoglin-transfected than in mock-transfected cells. CONCLUSION: These results demonstrate that endoglin expression negatively regulates basal and TGF-beta1-induced CTGF and collagen expression and synthesis.

Involvement of reactive oxygen species on gentamicin-induced mesangial cell activation.

BACKGROUND: Reactive oxygen species (ROS) have been shown to be involved in the reduction of glomerular filtration rate observed after gentamicin (Genta) treatment in vivo, a phenomenon directly related with mesangial cell (MC) contraction. Our previous study reported that Genta induces concentration-dependent MC contraction and proliferation in vitro. METHODS: To study the possible mediation of ROS in the effect of Genta, ROS production was measured in primary cultures of rat MC stimulated with Genta (10-5 mol/L). In addition, the MC response to Genta in the presence of the ROS scavengers superoxide dismutase (SOD) and catalase (CAT) was studied. MC activation and O2- production were studied in the presence of an inhibitor of the NADP(H) oxidase, diphenylene iodinium (DPI), and in the presence of L-NAME, an inhibitor of nitric oxide synthases (NOS). Finally, the effects of Genta on SOD activity and mRNA expression were examined. RESULTS: Genta (10-5 mol/L) induced an increase in O2- production and SOD activity that was neither accompanied by an elevation in cytosolic Cu/Zn-SOD mRNA expression nor by H2O2 accumulation. Genta induced MC contraction and proliferation that were inhibited by SOD plus CAT. Both the extracellular and intracellular ROS donor systems, xantine+xantine oxidase (X+XO) and dimethoxinaphtoquinone (DMNQ), respectively, also stimulated MC contraction and proliferation. Genta-induced MC activation and O2- production were inhibited by DPI. Genta-induced O2- production was inhibited by L-NAME. Furthermore, Genta did not induce detectable changes in membrane fluidity and lipid peroxidation. CONCLUSIONS: These results strongly suggest that an oxidative-mediated pathway exists in Genta-induced MC activation. A portion of the production of O2- may be due to NADP(H) oxidase and NOS activation. The amount of ROS produced, rather than having a toxic effect, might play a role as a mediator of Genta-induced MC activation