Hybridization in situ studies of angiotensinogen gene expression in rat adrenal and lung

Both rat adrenal and lung contain low levels of angiotensinogen mRNA, as shown by Northern blot and nuclease S1 analyses of RNA extracted from these tissues. We sought to identify the cellular localization of angiotensinogen mRNA in these two tissues using hybridization in situ of tissues obtained from both control rats and rats administered a combination of dexamethasone, ethynylestradiol, and T3. For the adrenal of hormone-treated rats, angiotensinogen mRNA was identified in periadrenal fibroblast-like cells and brown adipose tissue. For control rats, positive hybridization was obtained for fibroblast-like cells immediately adjacent to the adrenal capsule, but not for periadrenal brown adipose tissue. No hybridization was obtained for cells of the adrenal cortex, medulla, capsule or vessels. For the lung of hormone treated, but not control rats, angiotensinogen mRNA was identified in perivascular and peribronchial fibroblast-like cells and brown adipose tissue in the lung hilum. No hybridization was obtained for pulmonary parenchyma, bronchi, or vessels. These results confirm the widespread tissue distribution of angiotensinogen mRNA, and provide further evidence for the formation of angiotensin within tissues by mechanisms independent of the circulating renin-angiotensin system.     

Chronic salt loading upregulates expression of adrenomedullin and its receptors in adrenal glands and kidneys of the rat

The vasodilator peptide adrenomedullin (AM) elicits diuresis and natriuresis and inhibits aldosterone secretion. The aim of this study was to better understand the role of AM in maintaining water and electrolyte balance during chronic salt loading. Male Wistar rats were divided into a high salt (HS) group that received a diet containing 8% sodium chloride (NaCl) and a normal salt group that received a diet containing 0.4% NaCl. Plasma AM concentrations as well as expression of AM mRNA in the adrenal gland and kidney were then measured after 3, 7, 14, and 28 days. After 28 days, sodium and water excretion were significantly higher in HS rats than in control, although blood pressure and fluid volume were not significantly affected. Moreover, although plasma AM remained unchanged for up to 14 days, it was increased 2.5-fold in HS rats after 28 days on a high salt diet, and there were corresponding 3-fold and 1.5-fold increases in the levels of AM mRNA in the adrenal gland and kidney, respectively. At the same time, expression of calcitonin receptor-like receptor mRNA was significantly upregulated in both kidney and adrenal gland, as was expression of receptor activity-modify protein 1 (RAMP1) and RAMP2 mRNA in the adrenals and expression of RAMP3 in kidneys. Taken together, these results suggest that AM plays a role in the regulation of water and electrolyte balance in animals chronically ingesting high levels of salt.     

老年大鼠心脏及肾脏血管紧张素AT1受体mRNA水平与受体密度的变化

目的探讨老年大鼠心脏及肾脏中血管紧张素Ⅱ受体第１亚型（ＡＴ１）ｍＲＮＡ水平与受体密度的变化。方法采用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）法测定３、１２、２４月龄Ｗｉｓｔａｒ大鼠心脏及肾脏ＡＴ１受体ｍＲＮＡ水平，用免疫组织化学法检测ＡＴ１受体密度的改变。结果正常大鼠肾脏中ＡＴ１受体ｍＲＮＡ的表达高于其在心脏中的表达。随年龄增长心脏ＡＴ１受体ｍＲＮＡ水平呈下降趋势，与３月龄组比较，１２及２４月龄组分别下降至３月龄组的８７．０％±１１．８％及７０．８％±１８．８％；而肾脏中未发现明显改变。免疫组织化学方法观察结果表明：２４月龄大鼠阳性反应物面积下降，灰度无明显改变；在不同月龄大鼠肾脏中均未发现明显改变。结论随年龄增长心脏ＡＴ１受体ｍＲＮＡ水平下降、受体密度降低，肾脏ＡＴ１受体未发生明显改变，提示心脏功能更易受到老龄化的影响     

Renin gene expression in the adrenal and kidney of patients with primary aldosteronism.

mRNA levels for renin in the adrenal gland and kidney were measured by ribonuclease protection assay (RPA). Renin mRNA was not detected by RPA in aldosteronoma and kidney tissues obtained from two patients with primary aldosteronism (PA). In these patients, the PRA values, plasma concentrations of active renin (ARC), and total renin (TRC = ARC + prorenin) were below the assay limit (less than 0.03 ng/L.s, 2.5 ng/L, and 10 ng/L, respectively). On the other hand, renin mRNA was recognized by RPA in aldosteronoma and kidney tissues obtained from two other patients with PA treated with 50 mg/day spironolactone for more than 2 months. Their TRC values were 49.8 and 16.6 ng/L, but their PRA and ARC were undetectable. Renin mRNA content was greater in normal adrenocortical tissue and in the normal kidneys obtained from three hypertensive patients with renal cell carcinoma. In these patients, the mean values of PRA, ARC, and TRC were 0.28 +/- 0.03 (mean +/- SD) ng/L.s, 18.4 +/- 7.8 ng/L, and 110 +/- 15 ng/L, respectively. This is the first report of the lack of renin gene expression in aldosteronoma and kidney tissues obtained from untreated patients with PA. Furthermore, treatment with spironolactone resulted in an increase in the levels of renin mRNA in the aldosteronoma and kidney tissues of patients with PA.     

Increased expression of adrenomedullin 2/intermedin in rat hearts with congestive heart failure

Adrenomedullin 2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide family. To investigate the pathophysiological role of AM2/IMD in heart failure, we examined the expression of AM2/IMD, adrenomedullin (AM) and receptor complex components (calcitonin receptor-like receptor, three types of receptor activity-modifying proteins) by quantitative RT-PCR and immunohistochemistry in the hearts and kidneys of rats with congestive heart failure (CHF). Significantly increased levels of AM2/IMD mRNA were found in the atrium, right ventricle, non-infarcted part of the left ventricle and the infarcted part of the left ventricle of CHF rats, compared with sham operated rats (about 2.8-fold, 1.7-fold, 1.7-fold and 2.5-fold, respectively). Expression levels of mRNA encoding AM and the receptor complex components were also increased in the hearts of CHF rats. In a separate experiment, AM2/IMD mRNA levels in the heart did not differ between Wistar-Kyoto and spontaneously hypertensive rats. In both sham operated and CHF rats, the myocardium was diffusely immunostained with AM2/IMD. The fibrotic infarcted layer was not immunostained with AM2/IMD but was surrounded by positively immunostained myocardial layers. These findings suggest that the expression of AM2/IMD is enhanced in the failing heart, and AM2/IMD has a certain pathophysiological role in heart failure.     

Estrogen and phytoestrogen regulate the mRNA expression of adrenomedullin and adrenomedullin receptor components in the rat uterus

The serum estrogen surge in the uterus triggers precisely-timed physiological and biochemical responses required establishing and maintaining pregnancy. Previous reports have shown that consumption of phytoestrogen-containing plants may disrupt the precise control of pregnancy. To evaluate the effects of phytoestrogens in the uterus, we screened for estradiol (E2)-inducible genes in immature rat uteri. We identified the gene for receptor-activity-modifying protein 2 (Ramp2), known to be a component of the adrenomedullin (ADM) receptor, as responsive to both E2 and the phytoestrogen coumestrol (Cou). We further examined the expression of ADM and ADM signaling components Ramp2, Ramp3, and CRLR in the immature rat uterus and found that both E2 and Cou regulated these genes expression. In addition, treatment with ADM increased uterine weight and edema similar to that observed after Cou treatment. Our findings indicated that the phytoestrogen caused the abnormal induction of vasoactive factors in the uterus.     

The production of congenital heart defects with the use of antisera to rat kidney,placenta, heart,and lung homogenates

Pregnant Long-Evans rats received antisera to adult rat kidney, lung, and heart, rat placenta, and purified glomerular basement membrane on the ninth to eleventh days of gestation in doses ranging from 1 to 3 ml. per 100 Gm. The teratogenic antisera produced increased resorption rates, reductions in fetal weight, and significant malformations—with the exception of heart antiserum, which was not teratogenic in this experiment. Kidney and placenta antisera had a malformation rate of 76 and 80 per cent, with nearly three fourths of these being cardiovascular defects. Glomerular basement membrane and lung antisera were also teratogenic but less so. The cardiovascular malformations included situs inversus associated with truncus (4), as well as single ventricle (2), truncus arteriosus (8), various transpositions (7), interventricular septal defects (6), and several aortic outflow tract or proximal aorta abnormalities. These abnormalities were compared to those produced by trypan blue, which produces mostly transposition complexes, as well as to those produced by other teratogenic agents. The results suggest that antiserum produces its own unique spectrum of malformations, somewhat different from those produced by trypan blue, and therefore that the two agents may have different modes of action.     

Olfactory receptors, Golf alpha and adenylyl cyclase mRNA expressions in the rat heart during ontogenic development.

Golf alpha (a G heterotrimeric protein which shares a high homology with Gs alpha) expression was studied in the rat heart before birth and until weaning. Since Golf alpha in the neuro-olfactory epithelium is coupled to olfactory receptors and type III adenylyl cyclase, we looked for the presence of such molecules in the heart. Golf alpha mRNA was detected in the rat heart, highest levels being found in 21-day old fetuses until 3 days post partum. The protein amounts measured by Western blots paralleled the Golf alpha mRNA levels. Immunohistochemical studies revealed the presence of Golf alpha in atrial and ventricular cardiomyocytes. OL1 and latrophilin mRNAs, G protein-coupled olfactory receptors, were expressed at early postnatal stages. Adenylyl cyclase mRNAs for type II, type III, type V and type VI were expressed before birth and until weaning. Elements for an unexpected signaling pathway involving odorant receptors like OL1 and latrophilin, Golf alpha and type III adenylyl cyclase were expressed in rat heart, and appeared developmentally regulated.     

Regulation of renin gene expression in rat adrenal zona glomerulosa cells.

Our previous studies indicated that the amount of renin present in cultured adrenal zona glomerulosa cells increased after stimulation with adrenocorticotropic hormone or potassium. In the present study, we investigated the effects of adrenocorticotropic hormone or potassium on renin gene expression in cultured rat adrenal zona glomerulosa cells. The amount of rat renin messenger RNA (mRNA) was measured by complementary DNA synthesis and the competitive polymerase chain reaction method. The effects of adrenocorticotropic hormone or potassium on adrenal zona glomerulosa cell renin activity and renin mRNA content were compared with the activity and content of control cells. After 1 and 4 hours of stimulation by adrenocorticotropic hormone or potassium, total renin in the medium increased slightly; at the same time, the percent change in the amount of renin mRNA was 281% and 291%, respectively, in the adrenocorticotropic hormone-stimulated group and 218% and 348%, respectively, in the potassium-stimulated group. Twenty-four hours after adrenocorticotropic hormone or potassium stimulation, total renin in the medium increased significantly, by 689% and 220%, respectively; percent change in the renin mRNA content was 754% and 278%, respectively. These results demonstrate that adrenocorticotropic hormone and potassium increased the activity of adrenal renin through an increase in the level of renin mRNA.     

人促肾上腺髓质素基因导入对高盐饮食诱导的高血压大鼠血压及心肾功能的影响

目的 :研究人促肾上腺髓质素 (AM)基因导入对高盐饮食诱导的高血压大鼠的降压作用和对心、肾功能的保护作用。方法 :8周龄雄性Wistar大鼠以高盐 (8%氯化钠 )饲料及 0 .85 %氯化钠喂养 8周后形成高血压动物模型。再经尾静脉注射含人AMcDNA的重组质粒。然后监测血压 ;收集尿液以测定尿微量清蛋白 ;处死动物前插管测定心功能 ,处死动物后测定人AM基因在各主要器官的表达。结果 :一次静脉注射人AM基因的重组质粒后的第 4天即出现降低高盐饮食诱导的高血压大鼠的血压 ,最大降压效应出现在导入AM基因后的第 12天 ;实验组动物血压降低的同时 ,尿微量清蛋白的值亦出现显著下降 ;实验组右侧颈总动脉插管测定的心功能较对照组显著改善。结论 :人AM基因的导入可能有助于降低血压 ,并预防高血压所导致的心、肾功能损害。为临床应用AM基因治疗并发心、肾功能损害的原发性高血压提供了可能。     

肾上腺髓质素在肾上腺疾病中的免疫组化研究

目的：应用免疫组织化学技术观察免疫反应性肾上腺髓质素（ir-ADM)在人正常肾上腺、良恶性肾上腺肿瘤及肾上腺皮质增生组织中的分布特点。方法：对人正常肾上腺皮质（n＝10)和髓质（n=10)、嗜铬细胞瘤（n=31)、原发性醛固酮增多症的肾上腺皮质腺瘤（n=13)和增生（n=9)、皮质醇增多症的肾上腺皮质腺瘤（n=12)、增生（n=10)和腺癌（n=4)、无功能肾上腺皮质腺癌组织（n=7)进行ir-ADM的免疫组化染色。结果：10例人正常肾上腺髓质组织均显示ir-ADM染色阳性;31例嗜铬细胞瘤组织中16例染色阳性,大部分嗜铬细胞瘤组织中的ir-ADM免疫组化染色强度低于正常肾上腺髓质组织（P＜0．01）。在嗜铬细胞瘤中阵发性高血压组阳性染色例数多于持续性高血压组（P＜0．05）;人正常肾上腺皮质及各组皮质病变组织中,ir-ADM免疫组化染色均为阴性。结论：ir-ADM在嗜铬细胞瘤组织中分布呈不均一性,可能与嗜铬细胞瘤患者血压表现的多样化有关,应进一步探讨ADM在嗜铬细胞瘤发病中的病理生理作用。     

Myocardial expression of survivin, an apoptosis inhibitor, in aging and heart failure. An experimental study in the spontaneously hypertensive rat

BACKGROUND: Apoptosis plays a major role in the transition to heart failure (HF) in systemic hypertension although the underlying mechanisms are still unclear. The aim of this study was to determine the relationship between apoptosis, left ventricular remodeling, heart failure and the myocyte expression of survivin, an inhibitor of apoptosis. METHODS: Spontaneously hypertensive rats (SHR) were used as a model of hypertensive cardiopathy, and Wistar Kyoto Stars rats (WKY) were used as controls. Animals were allowed to survive up to 18 months of age. The animals underwent echocardiography (EDD, ESD and FS were measured). The median section of the heart was processed for in situ end-labeling of DNA fragmentation (TUNEL) and for survivin expression by immunohistochemistry. RESULTS: All SHR presented features of adverse cardiac remodeling. Apoptotic cells were increased in SHR compared with WKY, measured as apoptotic cells per high power field (1.08+/-0.43 vs. 0.27+/-0.15, P<0.001), and as apoptotic rate (0.16+/-0.06% vs. 0.04+/-0.02%, P<0.001). The incidence of apoptosis showed a positive correlation with unfavorable ventricular remodeling, assessed by echocardiogram. Survivin expression was found in all cases, but the survivin expression index was significantly lower in SHR vs. WKY (43+/-40% vs. 86+/-18%, respectively, P=0.014). Moreover the survivin expression index was inversely correlated with features of adverse remodeling (i.e., Heart Weight, R=-0.79, P<0.001) and with apoptosis (i.e., apoptotic rate, R=-0.52, P=0.050). CONCLUSION: Survivin myocardial expression in aging SHR is associated with reduced apoptosis and more favorable cardiac remodeling. Modulation of this pathway may prove beneficial in preventing pressure overload cardiac remodeling and heart failure.     

Spironolactone increases integrin beta3 gene expression in kidney and heart muscle cells

In clinical trials of heart failure, spironolactone, an antagonist of the mineralocorticoid receptor (MR), reduced mortality rates by unknown mechanisms. We hypothesized that spironolactone functions by upregulating expression of certain cardiovascular genes. An RNA differential display technique was used to identify genes whose expression was increased by spironolactone in an Xenopus kidney epithelial cell line (A6), a known target of aldosterone. We found that integrin beta3 gene expression was increased by spironolactone, and reversed by aldosterone or dexamethasone in a dose dependent manner. Competition binding studies and RT-PCR indicate the presence of MR in A6 cells, suggesting that regulation of expression occurred primarily through MR. Spironolactone also increased integrin beta3 expression in rat neonatal cardiomyocytes. In summary, spironolactone increases integrin beta3 gene expression in kidney epithelial cells and cardiomyocytes. The findings suggest new mechanisms for spironolactone actions with possible relevance to treatment of heart disease.     

Vascular remodeling and growth factor gene expression in the rat lung during hypoxia

Recent studies suggest that the vasoactive peptides endothelin-1 and -3 and the mitogens VEGF and PDGF-A and -B could be involved in the pathogenesis of hypoxic pulmonary hypertension. We were interested to investigate whether these peptides could also be involved in the vascular remodeling occurring during chronic hypoxia (10% oxygen; 1 and 3 weeks) in the rat. Hypoxia increased significantly systolic right ventricular pressure and typical morphological signs of vascular remodeling were found. This was accompanied by increased ET-1 and the ET-3 mRNA expression after acute (6 h; P<0.05) and chronic hypoxia of 1 (P<0.05) and 3 weeks (P<0.05). In contrast, we found no effects of hypoxia on the gene expression of VEGF and PDGF-A and -B in the lung. Our findings indicate that ET-3 in addition to ET-1 could be involved in the process of hypoxia-induced vascular remodeling, whereas it appears less likely that the mitogens VEGF and PDGF-A and -B are essentially involved in the pathogenesis of hypoxic pulmonary hypertension.