Ethanol consumption increases the expression of endothelial nitric oxide synthase, inducible nitric oxide synthase and metalloproteinases in the rat kidney

Objectives The effects of longterm ethanol consumption on the levels of nitric oxide (NO) and the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and metalloproteinase-2 (MMP-2) were studied in rat kidney. Methods Male Wistar rats were treated with 20% ethanol (v/v) for 6 weeks. Nitrite and nitrate generation was measured by chemiluminescence. Protein and mRNA levels of eNOS and iNOS were assessed by immunohistochemistry and quantitative real-time polymerase chain reaction, respectively. MMP-2 activity was determined by gelatin zymography. Histopathological changes in kidneys and indices of renal function (creatinine and urea) and tissue injury (mitochondrial respiration) were also investigated. Results Chronic ethanol consumption did not alter malondialdehyde levels in the kidney. Ethanol consumption induced a significant increase in renal nitrite and nitrate levels. Treatment with ethanol increased mRNA expression of both eNOS and iNOS. Immunohistochemical assays showed increased immunostaining for eNOS and iNOS after treatment with ethanol. Kidneys from ethanol-treated rats showed increased activity of MMP-2. Histopathological investigation of kidneys from ethanol-treated animals revealed tubular necrosis. Indices of renal function and tissue injury were not altered in ethanol-treated rats. Conclusions Ethanol consumption increased renal metalloproteinase expression/activity, which was accompanied by histopathological changes in the kidney and elevated NO generation. Since iNOS-derived NO and MMPs contribute to progressive renal injury, the increased levels of NO and MMPs observed in ethanol-treated rats might contribute to progressive renal damage.

PTTG expression in different experimental and human prolactinomas in relation to dopaminergic control of lactotropes

Abstract Background Pituitary tumor transforming gene (pttg) is a novel oncogene that is expressed at higher level in most of the tumors analyzed to date compared to normal tissues. Nevertheless, its expression in prolactinomas and its relation with the pituitary dopamine receptor 2 (D2R) are not well defined. We sought to determine the pituitary level of pttg in three different experimental models of prolactinomas with altered dopaminergic control of the pituitary: the dopaminergic D2R knockout female mouse, the estrogen-treated rat, and the senescent female rat. These three models shared the characteristics of increased pituitary weight, hyperprolactinemia, lactotrope hyperplasia and reduced or absent dopaminergic action at the pituitary level. We also studied samples from human macroprolactinomas, which were characterized as responsive or resistant to dopamine agonist therapy. Results When compared to female wild-type mice, pituitaries from female D2R knockout mice had decreased PTTG concentration, while no difference in pttg mRNA level was found. In senescent rats no difference in pituitary PTTG protein expression was found when compared to young rats. But, in young female rats treated with a synthetic estrogen (Diethylstylbestrol, 20 mg) PTTG protein expression was enhanced (P = 0.029). Therefore, in the three experimental models of prolactinomas, pituitary size was increased and there was hyperprolactinemia, but PTTG levels followed different patterns. Patients with macroprolactinomas were divided in those in which dopaminergic therapy normalized or failed to normalize prolactin levels (responsive and resistant, respectively). When pituitary pttg mRNA level was analyzed in these macroprolactinomas, no differences were found. We next analyzed estrogen action at the pituitary by measuring pituitary estrogen receptor α levels. The D2R knockout female mice have low estrogen levels and in accordance, pituitary estrogen receptors were increased (P = 0.047). On the other hand, in senescent rats estrogen levels were slightly though not significantly higher, and estrogen receptors were similar between groups. The estrogen-treated rats had high pharmacological levels of the synthetic estrogen, and estrogen receptors were markedly lower than in controls (P < 0.0001). Finally, in patients with dopamine resistant or responsive prolactinomas no significant differences in estrogen receptor α levels were found. Therefore, pituitary PTTG was increased only if estrogen action was increased, which correlated with a decrease in pituitary estrogen receptor level. Conclusion We conclude that PTTG does not correlate with prolactin levels or tumor size in animal models of prolactinoma, and its pituitary content is not related to a decrease in dopaminergic control of the lactotrope, but may be influenced by estrogen action at the pituitary level. Therefore it is increased only in prolactinomas generated by estrogen treatment, and not in prolactinomas arising from deficient dopamine control, or in dopamine resistant compared with dopamine responsive human prolactinomas. These results are important in the search for reliable prognostic indicators for patients with pituitary adenomas which will make tumor-specific therapy possible, and help to elucidate the poorly understood phenomenon of pituitary tumorigenesis.

MicroRNA expression profile in head and neck cancer: HOX-cluster embedded microRNA-196a and microRNA-10b dysregulation implicated in cell proliferation

Abstract Background Current evidence implicates aberrant microRNA expression patterns in human malignancies; measurement of microRNA expression may have diagnostic and prognostic applications. Roles for microRNAs in head and neck squamous cell carcinomas (HNSCC) are largely unknown. HNSCC, a smoking-related cancer, is one of the most common malignancies worldwide but reliable diagnostic and prognostic markers have not been discovered so far. Some studies have evaluated the potential use of microRNA as biomarkers with clinical application in HNSCC. Methods MicroRNA expression profile of oral squamous cell carcinoma samples was determined by means of DNA microarrays. We also performed gain-of-function assays for two differentially expressed microRNA using two squamous cell carcinoma cell lines and normal oral keratinocytes. The effect of the over-expression of these molecules was evaluated by means of global gene expression profiling and cell proliferation assessment. Results Altered microRNA expression was detected for a total of 72 microRNAs. Among these we found well studied molecules, such as the miR-17-92 cluster, comprising potent oncogenic microRNA, and miR-34, recently found to interact with p53. HOX-cluster embedded miR-196a/b and miR-10b were up- and down-regulated, respectively, in tumor samples. Since validated HOX gene targets for these microRNAs are not consistently deregulated in HNSCC, we performed gain-of-function experiments, in an attempt to outline their possible role. Our results suggest that both molecules interfere in cell proliferation through distinct processes, possibly targeting a small set of genes involved in cell cycle progression. Conclusions Functional data on miRNAs in HNSCC is still scarce. Our data corroborate current literature and brings new insights into the role of microRNAs in HNSCC. We also show that miR-196a and miR-10b, not previously associated with HNSCC, may play an oncogenic role in this disease through the deregulation of cell proliferation. The study of microRNA alterations in HNSCC is an essential step to the mechanistic understanding of tumor formation and could lead to the discovery of clinically relevant biomarkers.

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations

Abstract Background Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC Methods Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. Results A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) <5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR <5%, p-value ≤0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p <0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes’ as the main enriched category. A module map analysis of the protein-coding genes significantly (p <0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p <0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p <0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. Conclusion Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats

Abstract Background: Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. Findings: By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. Conclusions: SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects.

Hepatocyte nuclear factors 1α/4α and forkhead box A2 regulate the solute carrier 2A2 (Slc2a2) gene expression in the liver and kidney of diabetic rats

AIMS: Solute carrier 2a2 (Slc2a2) gene codifies the glucose transporter GLUT2, a key protein for glucose flux in hepatocytes and renal epithelial cells of proximal tubule. In diabetes mellitus, hepatic and tubular glucose output has been related to Slc2a2/GLUT2 overexpression; and controlling the expression of this gene may be an important adjuvant way to improve glycemic homeostasis. Thus, the present study investigated transcriptional mechanisms involved in the diabetes-induced overexpression of the Slc2a2 gene. MAIN METHODS: Hepatocyte nuclear factors 1α and 4α (HNF-1α and HNF-4α), forkhead box A2 (FOXA2), sterol regulatory element binding protein-1c (SREBP-1c) and the CCAAT-enhancer-binding protein (C/EBPβ) mRNA expression (RT-PCR) and binding activity into the Slc2a2 promoter (electrophoretic mobility assay) were analyzed in the liver and kidney of diabetic and 6-day insulin-treated diabetic rats. KEY FINDINGS: Slc2a2/GLUT2 expression increased by more than 50% (P<0.001) in the liver and kidney of diabetic rats, and 6-day insulin treatment restores these values to those observed in non-diabetic animals. Similarly, the mRNA expression and the binding activity of HNF-1α, HNF-4α and FOXA2 increased by 50 to 100% (P<0.05 to P<0.001), also returning to values of non-diabetic rats after insulin treatment. Neither the Srebf1 and Cebpb mRNA expression, nor the SREBP-1c and C/EBP-β binding activity was altered in diabetic rats. SIGNIFICANCE: HNF-1α, HNF-4α and FOXA2 transcriptional factors are involved in diabetes-induced overexpression of Slc2a2 gene in the liver and kidney. These data point out that these transcriptional factors are important targets to control GLUT2 expression in these tissues, which can contribute to glycemic homeostasis in diabetes.

Increased SGLT1 expression in salivary gland ductal cells correlates with hyposalivation in diabetic and hypertensive rats

Background Oral health complications in diabetes and hypertension include decreased salivary secretion. The sodium-glucose cotransporter 1 (SGLT1) protein, which transports 1 glucose/2 Na+/264 H2O molecules, is described in salivary glands. We hypothesized that changes in SGLT1 expression in the luminal membrane of ductal cell may be related to an altered salivary flow. Findings By immunohistochemistry, we investigated SGLT1 expression in ductal cells of parotid and submandibular glands from Wistar Kyoto rats (WKY), diabetic WKY (WKY-D), spontaneously hypertensive rats (SHR) and diabetic SHR (SHR-D), as well as in parotid glands from WKY subjected to sympathetic stimulation, with or without previous propranolol blockade. Diabetes and hypertension decreased the salivary secretion and increased SGLT1 expression in the luminal membrane of ductal cells, and their association exacerbated the regulations observed. After 30 min of sympathetic stimulation, SGLT1 increased in the luminal membrane of ductal cells, and that was blocked by previous injection of propranolol. Conclusions SGLT1 expression increases in the luminal membrane of salivary gland ductal cells and the salivary flow decreases in diabetic and hypertensive rats, which may be related to sympathetic activity. This study highlights the water transporter role of SGLT1 in salivary glands, which, by increasing ductal water reabsorption, may explain the hyposalivation of diabetic and hypertensive subjects

Altered phenotype and function of dendritic cells in individuals with chronic periodontitis

OBJECTIVE: To investigate the effects of periodontal bacterial lysates on maturation and function of mature monocyte-derived dendritic cells (m-MDDCs) derived from individuals with chronic periodontitis (CP) or healthy periodontal tissue (HP). DESIGN: m-MDDCs derived from peripheral blood monocytes, cultured for 7 days in presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF), were stimulated with lysates of Streptococcus sanguinis, Prevotella intermedia, Porphyromonas gingivalis, or Treponema denticola on day 4, and were then phenotyped. IL-10, IL-12 and IFN-gamma concentration in the supernatant of cultures were measured. RESULTS: Expression of HLA-DR was lower in bacterial-unstimulated mature m-MDDC from CP compared to HP (p=0.04), while expression of CD1a and CD123 were higher in CP. The expression pattern of HLA-DR, CD11c, CD123, and CD1a did not change on bacterial stimulation, regardless of the bacteria. Stimulation with P. intermedia upregulated CD80 and CD86 in CP cells (p≤0.05). Production of IL-12p70 by bacterial-unstimulated m-MDDCs was 5.8-fold greater in CP compared to HP. Bacterial stimulation further increased IL-12p70 production while decreasing IL-10. Significantly more IFN-gamma was produced in co-cultures of CP m-MDDCs than with HP m-MDDCs when cells were stimulated with P. intermedia (p=0.009). CONCLUSIONS: Bacterial-unstimulated m-MDDC from CP exhibited a more immature phenotype but a cytokine profile biased towards proinflammatory response; this pattern was maintained/exacerbated after bacterial stimulation. P. intermedia upregulated co-stimulatory molecules and IFN-gamma expression in CP m-MDDC. These events might contribute to periodontitis pathogenesis