Relationship Between Regional Brain Angiotensinogen a Local Blood Flow and Volume in the Adrenalectomize Rat: Application of Approach to Quantification of Brain Corticosterone Receptors

Abstract: Prior studies from this laboratory have established that angiotensinogen, the prohormone of angiotensin, is unevenly distributed in the rat brain and that adrenalectomy selectively perturbs levels of the prohormone in regions associated with cardiovascular neural pathways. However, plasma angiotensinogen levies are 10²-10³ times higher in plasma than in brain, so that the observation of a unique distribution of brain angiotensinogen may reflect variable plasma contamination. Studies were therefore undertaken to establish whether adrenalectomy selectively alters regional blood flow, blood volume, or plasma contamination of brain tissue, thereby artifactitiously altering apparent angiotensinogen levels. Radioactive 2-deoxyglucose, iodoantipyrine, and inulin were employed in these analyses. We conclude that variations in blood flow do not explain the selective effects of adrenalectomy, but that a variable extent of residual plasma contamination (remaining after transcardiac perfusion) is partially reflected in our earlier data. However, after correcting for plasma contamination, we still find significant changes in selected areas of the rat brain following adrenalectomy. Finally, our results demonstrate the necessity for direct quantitation of plasma contamination of brain tissue segments. This is shown to have relevance in other situations, such as corticosterone binding globulin contamination of brain corticosterone receptor binding.     

Detailed Localization of Augmented Angiotensinogen mRNA and Protein in Proximal Tubule Segments of Diabetic Kidneys in Rats and Humans

In the intrarenal renin-angiotensin system, angiotensinogen levels are well known to be increased in diabetes, and these enhanced intrarenal angiotensinogen levels may initiate the development and accelerate the progression of diabetic nephropathy. However, the specific localization of the augmented angiotensinogen in proximal tubule segments in diabetes is still unknown. We investigated the detailed localization of angiotensinogen in 3 proximal tubule segments in the diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats and the control Long-Evans Tokushima Otsuka (LETO) rats. We also prepared OLETF rats treated with angiotensin II type 1 receptor blocker, olmesartan or with a combination of vasodilator agents. Moreover, biopsied samples of human kidney cortex were used to confirm the results of animal studies. We examined the co-localization of angiotensinogen with segment-specific markers by double staining using fluorescence in situ hybridization and/or immunofluorescence. Angiotensinogen mRNA expression was barely detectable in segment 1. In segment 3, the area of angiotensinogen mRNA expression was augmented in the OLETF rats compared with the LETO rats. Angiotensinogen protein expression areas in segments 1 and 3 were also increased in the OLETF rats compared with the LETO rats. Chronic treatment with olmesartan ameliorated these areas of augmented angiotensinogen expression. Biopsied human kidney samples showed similar results. These data suggest that the augmented angiotensinogen mRNA levels in segment 3 and angiotensinogen protein levels in segments 1 and 3 may contribute to the progression of diabetic nephropathy.     

Hypoxia-Induced Endothelial Progenitor Cell Function Is Blunted in Angiotensinogen Knockout Mice

Angiotensinogen (AGT), the precursor of angiotensin I, is known to be involved in tumor angiogenesis and associated with the pathogenesis of coronary atherosclerosis. This study was undertaken to determine the role played by AGT in endothelial progenitor cells (EPCs) in tumor progression and metastasis. It was found that the number of EPC colonies formed by AGT heterozygous knockout (AGT^+/−) cells was less than that formed by wild-type (WT) cells, and that the migration and tube formation abilities of AGT^+/− EPCs were significantly lower than those of WT EPCs. In addition, the gene expressions of vascular endothelial growth factor (VEGF), Flk1, angiopoietin (Ang)-1, Ang-2, Tie-2, stromal derived factor (SDF)-1, C-X-C chemokine receptor type 4 (CXCR4), and of endothelial nitric oxide synthase (eNOS) were suppressed in AGT^+/− EPCs. Furthermore, the expressions of hypoxia-inducible factor (HIF)-1α and -2α were downregulated in AGT^+/− early EPCs under hypoxic conditions, suggesting a blunting of response to hypoxia. Moreover, the activation of Akt/eNOS signaling pathways induced by VEGF, epithelial growth factor (EGF), or SDF-1α were suppressed in AGT^+/− EPCs. In AGT^+/− mice, the incorporation of EPCs into the tumor vasculature was significantly reduced, and lung tumor growth and melanoma metastasis were attenuated. In conclusion, AGT is required for hypoxia-induced vasculogenesis.     

高血压患者血管紧张素原基因调控及与细胞因子关系的研究

在高血压患者中研究血管紧张素原(AGT)基因M235T多态性、等位基因分离及与细胞因子的关系。采用聚合酶链反应结合内切酶消化进行AGT基因型分析。用双抗体夹心ELISA酶联法测定细胞因子(IL-1、IL-6、TNF)。分析高血压病组和对照组的血管紧张素原(AGT)基因235位氨基酸残基的基因型及等位基因频率,并分析细胞因子对AGT基因M235T变异的影响。结果表明：高血压组中AGT 235为TT型38例(55．88％),TM型24例(35．29％),MM型6例(8．82％),其T型和M型等位基因频率分别为73．53％和26．47％。对照组中开型28例(47．46％),TM型25例(42．37％),MM型6例(10．17％),T型和MM型等位基因频率分别为68．65％和31．35％。在AGT 235T等位基因型组中,高血压病组IL-1、IL-6、TNF浓度显著高于对照组。在AGT 235M等位基因型组中,TNF浓度高于对照组。无论大于60岁或小于60岁的高血压病与相应年龄对照组比较,IL-1、IL-6、TNF浓度均显著升高。而在高血压病或对照组内,大于60岁组与小于60岁组之间比较,IL-1、IL-6、TNF浓度均无显著性差异。说明IL-1、IL-6、TNF升高与疾病有关而与年龄无关。研究结论：高血压病AGT 235TT型和T等位基因频率高于健康对照。AGT 235TT型和T等位基因频率增高可能与高血压发生和发展有关。在AGT 235T型等位基因频率增高的人群中,细胞因子IL-1、IL-6、TNF增高可能促进高血压发病。细胞因子对高血压病患者AGT 235TT型转录及表达可能有一定的调控作用。     

Association of the T174M Polymorphism of the Angiotensinogen Gene with Essential Hypertension in Russians and Tatars from Bashkortostan

The alleles and genotypes of the T174M polymorphism of the angiotensinogen gene were PCR-analyzed in Russians and Tatars from Bashkortostan. The genotype frequency distribution observed in either ethnic group did not differ from that reported for other populations. The T174M polymorphism was tested for association with essential hypertension (EH). Genotypes TT, TM, and MM were found respectively in 82.56, 13.95, and 3.49% normotensive Russians and in 83.81, 16.19, and 0% normotensive Tatars. The frequency of genotype TM in patients with EH onset beyond 45 years of age was significantly higher than in controls of the same age without signs of cardiovascular disorders (51.72 vs. 11.11% in Russians and 45.45 vs. 16% in Tatars). Patients with EH onset under 45 did not differ in genotype frequency distribution from normotensive subjects of the same age. Genotype TM was associated with higher risk of EH in people over 45.     

Effects of glucocorticoids and antiglucocorticoid on angiotensinogen production by hepatoma cells in culture

Summary Angiotensinogen is synthesized in large amounts by Fao cells derived from the Reuber H35 rat hepatoma in a medium enriched with 5% fetal bovine serum (FBS). Treatment of FBS with dextran-coated charcoal removed endogenous steroids without modifying angiotensinogen production. This treatment allowed the study of the effects of steroids on angiotensinogen production. Hydrocortisone increased the angiotensinogen synthesis in a dosedependent manner. The antiglucocorticoid RU 38486 did not change the basal rate of angiotensinogen production but inhibited the stimulation by hydrocortisone. Similar results were obtained with dexamethasone. Angiotensinogen biosynthesis seems to be regulated by two distinct mechanisms: (a) glucocorticoid independent, controlling the basal rate of angiotensinogen production and (b) glucocorticoid dependent, mediating the increased rate of angiotensinogen production upon glucocorticoid treatment. This work was supported in part by a grnat from Inserm (CRL 824022).