Rat Adrenal Gland Research Articles

Extracellular vesicles derived from bone marrow mesenchymal stem cells repair functional and structural rat adrenal gland damage induced by fluoride

The adrenal glands have striking morpho-biochemical features that render them vulnerable to the effects of toxins. Aims: This study was conducted to explore the therapeutic utility of extracellular vesicles derived from bone marrow mesenchymal stem cells (BMSC-EVs) against fluoride-induced adrenal toxicity. Materials and methods: The work included isolation and further identification of BMSC-EVs by transmission electron microscopy and flow cytometric analysis. Adrenal toxicity in rats was induced by oral administration of 300 ppm of sodium fluoride (NaF) in drinking water for 60 days followed by a single dose injection of BMSC-EVs. The effects of BMSC-EVs against NaF was evaluated by adrenal oxidant/antioxidant biomarkers, hormonal assay of plasma adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) and mRNA gene expression quantitation for adrenal cortical steroidogenic pathway-encoding genes. Histopathological examination of the adrenal tissue was performed. Key findings: BMSC-EVs were effectively isolated and characterized. NaF exposure decreased adrenal superoxide dismutase and catalase activities, increased adrenal malondialdehyde levels, elevated plasma ACTH, diminished CORT concentrations and downregulated the adrenal cortical steroidogenic pathway-encoding genes. In addition, NaF-induced marked adrenal histopathological lesions. Significance: BMSC-EVs treatment repaired damaged adrenal tissue and recovered its function greatly following NaF consumption. BMSC-EVs reversed the toxic effects of NaF and reprogramed injured adrenal cells by activating regenerative processes.

Fabrication of Tunable 3D Cellular Structures in High Volume Using Magnetic Levitation Guided Assembly.

Tunable and reproducible size with high circularity is an important limitation to obtain three-dimensional (3D) cellular structures and spheroids in scaffold free tissue engineering approaches. Here, we present a facile methodology based on magnetic levitation (MagLev) to fabricate 3D cellular structures rapidly and easily in high-volume and low magnetic field. In this study, 3D cellular structures were fabricated using magnetic levitation directed assembly where cells are suspended and self-assembled by contactless magnetic manipulation in the presence of a paramagnetic agent. The effect of cell seeding density, culture time, and paramagnetic agent concentration on the formation of 3D cellular structures was evaluated for NIH/3T3 mouse fibroblast cells. In addition, magnetic levitation guided cellular assembly and 3D tumor spheroid formation was examined for five different cancer cell lines: MCF7 (human epithelial breast adenocarcinoma), MDA-MB-231 (human epithelial breast adenocarcinoma), SH-SY5Y (human bone-marrow neuroblastoma), PC-12 (rat adrenal gland pheochromocytoma), and HeLa (human epithelial cervix adenocarcinoma). Moreover, formation of a 3D coculture model was successfully observed by using MDA-MB-231 dsRED and MDA-MB-231 GFP cells. Taken together, these results indicate that the developed MagLev setup provides an easy and efficient way to fabricate 3D cellular structures and may be a feasible alternative to conventional methodologies for cellular/multicellular studies.

Selection and verification of the combination of reference genes for RT-qPCR analysis in rat adrenal gland development

Quantitative reverse transcription polymerase chain reaction (RT-qPCR) is commonly used for gene expression analysis, and the accuracy of its results depends greatly on chosen reference genes. Adrenal gland is the core of the occurrence and development of fetal-originated adult diseases. Its dysplasia or dysfunction may increase susceptibility to adult disease, which has apparent sex differences. To explore the optimal combination of reference genes for RT-qPCR in female and male rats adrenal development, we selected seven reference genes (GAPDH, β-actin, etc.), and use RT-qPCR to detect genes expression during different stages of rats adrenal development under physiological conditions. Then we analysed data using GeNorm, NormFinder and BestKeeper to select the optimal combination of reference genes. Further, we used the intrauterine growth retardation (IUGR) model of rat caused by prenatal caffeine exposure (PCE) to verify the stability and accuracy of the selected combination of reference genes under physiological conditions. The results showed that TBP + β-actin could be the optimal combination of reference genes for fetal rat adrenals under physiological conditions, without obvious sex differences. In infancy and adolescence, the optimal combination of reference genes for adrenals had sex differences, and females were GAPDH + β-actin, while males were GAPDH + SDHA. In PCE model, the optimal combination of reference genes was consistent with physiological conditions. Using combination of reference genes to analyze target genes can improve the accuracy of the results. In summary, this study provided reliable combination of reference genes for RT-qPCR and experimental supports for researches on adrenal development.

Activation and expression of endogenous CREB-regulated transcription coactivators (CRTC) 1, 2 and 3 in the rat adrenal gland.

The activation and nuclear translocation of cAMP‐response element binding protein (CREB)‐regulated transcription coactivator (CRTC)2 occurs in the rat adrenal gland, in response to adrenocorticotrophic hormone (ACTH) and stressors, and has been implicated in the transcriptional regulation of steroidogenic acute regulatory protein (StAR). We have recently demonstrated the activation of CRTC isoforms, CRTC1 and CRTC3, in adrenocortical cell lines. In the present study, we aimed to determine the activation and expression of the three CRTC isoforms in vivo in relation to Star transcription, under basal conditions and following a robust endotoxic stress challenge. Rat adrenal glands and blood plasma were collected following i.v. administration of either an ultradian‐sized pulse of ACTH or administration of lipopolysaccharide, as well as under unstressed conditions across the 24‐hour period. Plasma ACTH and corticosterone (CORT) were measured and the adrenal glands were processed for measurement of protein by western immunoblotting, RNA by a quantitative reverse transcriptase‐polymerase chain reaction and association of CRTC2 and CRTC3 with the Star promoter by chromatin immunoprecipitation. An increase in nuclear localisation of CRTC2 and CRTC3 followed increases in both ultradian and endotoxic stress‐induced plasma ACTH, and this was associated with increased CREB phosphorylation and corresponding increases in Star transcription. Both CRTC2 and CRTC3 were shown to associate with the Star promoter, with the dynamics of CRTC3 binding corresponding to that of nuclear changes in protein levels. CRTC isoforms show little variation in ultradian expression or variation across 24 hours, although evidence of long‐term down‐regulation following endotoxic stress was found. We conclude that co‐transcription factors CRTC2 and, more clearly, CRTC3 appear to act alongside phosphorylated CREB in the generation of ultradian pulses of Star transcription, essential for the maintenance of basal StAR expression. Similarly, our findings suggest CRTC2 and CRTC3 mediate Star transcriptional initiation following an endotoxic stressor; however, other transcription factors are likely to be responsible for the long‐term up‐regulation of adrenal Star transcription.

Background data of 2-year-old male and female F344 gpt delta rats.

Although gpt delta rats, as reporter gene-transgenic rats, were originally developed for in vivo mutation assays, they have also been used to evaluate chemical carcinogenesis and comprehensive toxicity. Therefore, it is necessary to accumulate background data on carcinogenicity and general toxicity in gpt delta rats. Here, we investigated the background data of 110-week-old male and female F344 gpt delta rats and wild-type rats. There was no effect of reporter gene transfection on animal survival rates and body weights during the experiment. The relative weight of male gpt delta rat adrenals was significantly higher than that of wild-type rats, possibly due to the higher incidence of pheochromocytoma. There were no intergenotype differences in the incidence of nonneoplastic lesions in both sexes, including chronic progressive nephropathy and focus of cellular alteration in the liver, which had a higher incidence in both genotypes. Additionally, the significantly higher incidence of adrenal pheochromocytoma in male gpt delta rats than that in wild-type rats was likely incidental because of the lack of differences in the incidences of preneoplastic (male and female) and neoplastic (female) adrenal lesions in both genotypes. Other neoplastic lesions in both sexes showed no intergenotype differences in incidence rates, although large granular lymphocytic leukemia in the spleen and Leydig cell tumors in the testes of males showed higher incidence rates. Overall, there were no effects of reporter gene transfection on the spectrum of spontaneous lesions in F344 gpt delta rats, thus supporting their applicability in evaluating chemical toxicity and carcinogenicity.

Elucidation of the Mechanisms for the Underlying Depolarization and Reversibility by Photoactive Molecule.

Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.

Chronic restraint stress induces serotonin transporter expression in the rat adrenal glands

Chronic restraint stress (CRS) magnifies restraint-induced corticosterone secretion through a mechanism involving increased adrenocortical 5-HT content and turnover. We analysed the impact of CRS on serotonin transporter (SERT) expression and distribution in rat adrenal glands. Male Wistar rats were submitted to CRS (20 min/day) or undisturbed control conditions for 14 days. Exposure to CRS induced a remarkable increase in SERT-like immunoreactivity in the adrenal cortex, which closely matched that of chromogranin A immunostaining, along with a significant increase in SERT protein and mRNA levels in whole adrenals as determined by immunohistochemistry, Western blot and RT-PCR assays, respectively; all these CRS-induced changes occurred almost exclusively in left adrenals. Closely similar results were obtained in animals that received a 14-day chronic corticosterone treatment. These results unravel an interesting association between chronic stress exposure and SERT expression in adrenocortical chromogranin A-positive cells, which seems to be a glucocorticoid-dependent phenomenon.

Effects of β-HgS on cell viability and intracellular oxidative stress in PC-12 cells.

Traditional Tibetan medicines containing β-HgS have been used to treat chronic ailments for thousands of years. However, there has recently been speculation regarding the safety of these medicines due to their high mercury content. Although the toxic effect of β-HgS has been previously investigated in vivo, the mechanism underlying the toxicity of this compound remains unclear. In this study, we investigate the mechanism of β-HgS cytotoxicity via experiments performed on rat adrenal gland tumor cells (PC-12). Specifically, we analyze the viability and intracellular oxidative stress state of PC-12 cells treated with varying concentrations of β-HgS. For comparison purposes, the effects of MeHgCl and HgCl2, two Hg-based compounds, on ROS generation and MDA, GSH/GSSG, Nrf2, NQO-1, and HO-1 levels are also determined. It should be noted that we used the small-molecule thiols of cell culture medium, such as cysteine, to increase the solubility of β-HgS and prepare a β-HgS solution to treat PC-12 cells. The obtained results show that β-HgS inhibits cell viability at concentrations of 200-1000 ng Hg mL-1 (48 h treatment). In the concentration range of 200-600 ng Hg mL-1 (24 h treatment), the inhibitory effect of β-HgS is stronger than that of MeHgCl; however, this trend is reversed at higher concentrations (800-1000 ng mL-1) and longer exposure times (48 h). Moreover, β-HgS significantly promotes MDA, but has no appreciable influence on cell apoptosis and ROS generation in PC-12 cells, which suggests that its inhibitory effect on cell viability might be related to the stimulation of ROS-independent oxidative stress. Notably, β-HgS and HgCl2 significantly increase the GSH content, GSH/GSSG ratio, NQO-1 mRNA expression, and HO-1 protein expression in PC-12 cells, indicating that the antioxidant protection against these compounds is triggered by Nrf2 activation. HPLC-AFS analysis shows that in β-HgS and HgCl2 solutions, mercury exists in the same form of Hg2+, but the cytotoxicity of the former is greater. This is probably due to the additional oxidative damage induced by the S2- ion in β-HgS. In conclusion, β-HgS induces ROS-independent oxidative stress in PC-12 cells, and thus, is obviously cytotoxic. At the same time, it promotes the antioxidant capacity of cells by activating the Nrf2 pathway.

A new model for the HPA axis explains dysregulation of stress hormones on the timescale of weeks.

Stress activates a complex network of hormones known as the hypothalamic–pituitary–adrenal (HPA) axis. The HPA axis is dysregulated in chronic stress and psychiatric disorders, but the origin of this dysregulation is unclear and cannot be explained by current HPA models. To address this, we developed a mathematical model for the HPA axis that incorporates changes in the total functional mass of the HPA hormone‐secreting glands. The mass changes are caused by HPA hormones which act as growth factors for the glands in the axis. We find that the HPA axis shows the property of dynamical compensation, where gland masses adjust over weeks to buffer variation in physiological parameters. These mass changes explain the experimental findings on dysregulation of cortisol and ACTH dynamics in alcoholism, anorexia, and postpartum. Dysregulation occurs for a wide range of parameters and is exacerbated by impaired glucocorticoid receptor (GR) feedback, providing an explanation for the implication of GR in mood disorders. These findings suggest that gland‐mass dynamics may play an important role in the pathophysiology of stress‐related disorders.

Triphenyltin chloride reduces the development of rat adrenal cortex during puberty

Triphenyltin has been classified as an endocrine disruptor. However, whether triphenyltin interferes with the adrenal glands during puberty remains unknown. Here, we reported the effects of triphenyltin on the adrenal glands in rats. Male Sprague Dawley rats (age of 35 days) were orally administered with 0, 0.5, 1, or 2 mg/kg/day triphenyltin for 18 days. Triphenyltin significantly lowered corticosterone levels at 1 and 2 mg/kg and adrenocorticotropic hormone at 2 mg/kg. The RNA-Seq analysis detected multiple differentially expressed genes. Four down-regulated genes were transcription factor genes (Nr4a1, Nr4a2, Nr4a3, and Ppard), which might be associated with the suppression of the adrenal cortex function. RNA-seq and qPCR showed that triphenyltin dose-dependently down-regulated the expression of the genes for cholesterol transport and biosynthesis, including Scarb1, Ldlr, Hmgcs1, Hmgcr, and Hsd17b7. Further Western blotting revealed that it lowered NR4A1, PPRAD, LDLR, and HMGCS1 protein levels. We treated H295R adrenal cells with 1–100 nM triphenyltin for 72 h. Triphenyltin induced significant higher ROS production at 100 nM and did not induce apoptosis at 10 and 100 nM. In conclusion, triphenyltin inhibits production of corticosterone via blocking the expression of cholesterol uptake transporters and cholesterol biosynthesis.

Characterization of Group I Metabotropic Glutamate Receptors in Rat and Human Adrenal Glands.

Glutamate and its receptors have been demonstrated to promote both basal and nicotine-evoked catecholamine release in bovine chromaffin cells. Multiple glutamate receptors, including metabotropic glutamate receptors (mGluRs), are found in the adrenal glands of several species, as well as in chromaffin cells. However, there is limited information available regarding the expression of glutamate metabotropic receptor (GRM)1-8 mRNAs and the detailed localization of group I mGluRs (mGluR1 and mGluR5) in the rat and human adrenal cortex and medulla. Therefore, we examined mRNA expression of GRM1-8 subunits using reverse transcription-polymerase chain reaction (RT-PCR) and the distribution of mGluR1 and mGluR5 by immunostaining. The results showed that the GRM1-8 mRNAs were expressed in both the cortex and medulla of rat and human adrenal glands with the exception of GRM1, which was not detectable in the rat adrenal cortex. Immunostaining of mGluR1 revealed that it was localized only in the adrenal medulla of rats but was present in both the adrenal cortex and medulla in humans. In the adrenal medulla, the central part of the adrenal glands, mGluR1 was detected in chromaffin cells but not in nerve fibers and ganglion cells. Immunoactivity of mGluR5 was visible in the capillary wall throughout the adrenal cortex and medulla in rat and human samples. Its immunoactivity was also observed in ganglion cells in the rat adrenal medulla. There was no mGluR5 immunoactivity detected in chromaffin cells and nerve fibers in the rat and human adrenal medulla. Using dissected rat adrenal medulla as a model, we found that treatment with a mGluR1 agonist activated extracellular signal-regulated kinase (ERK) 1/2 and increased the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis. Moreover, these results showed that mGluR1 signaling was involved in hypoxia-induced upregulation of TH in the rat adrenal medulla. This study shows the expression of GRM1-8 mRNAs in rat and human adrenal glands and indicates that glutamate, through the activation of mGluRs, may play various physiological roles in the adrenal gland. Furthermore, mGluR1 may be involved in catecholamine biosynthesis by regulating TH, and mGluR5 may affect cortical and medullar hormone levels by regulating microvascular function.

SUN-LB43 DLK1 Expressing Cells Contribute to the Zonation of the Adrenal Gland

The adrenal cortex is a dynamic organ that undergoes self-renewal and remodeling in response to the demand for steroids. In the mouse it is divided into two concentric layers, the outer zona glomerulosa and the inner zona fasciculata (ZF), that secrete aldosterone and corticosterone, respectively. Cell fate mapping studies have shown that the maintenance of the cortex relies on a pool of stem/progenitor cells located in the capsular and subcapsular compartments. Two interconnected cell populations have been identified, subcapsular undifferentiated cells secreting the morphogen Sonic Hedgehog (Shh) and capsular Gli1+ cells, which can transduce the Shh signal (1); both populations are precursors of steroidogenic cells and newly formed cells migrate in a centripetal fashion to repopulate the gland until they reach the juxtamedullary region where they undergo senescence and apoptosis. Moreover, our lab has shown that the Notch atypical ligand Delta-Like homologue 1 (Dlk1) is expressed in partially undifferentiated cells of the subcapsular region in rat (2)and human (3)adrenals, whilst it is mostly expressed in capsular cells in mice (4,5). To investigate whether Dlk1 expressing cells contribute to the zonation of the adrenal cortex we conducted lineage tracing analyses using a tamoxifen inducible Dlk1CreERT2mouse model carrying the R26tdTom reporter. Pregnant dames were injected with tamoxifen at embryonic day (e) 12.5 and pups were culled at postnatal day (p) 10 and p38. Analysis of tdTomato expression showed that 35% (p10) and 24% (p38) of Steroidogenic Factor 1(Sf1)+cortical cells were tdTomato+, revealing that capsular Dlk1+cells are steroidogenic precursors. On the other hand, postnatal tamoxifen injections (p0) showed tdTomato+/Sf1+ cells only in 1-2% in cortical cells after 24-months chase, suggesting that the contribution of Dlk1+cells to adrenocortical self-renewal is limited postnatally.However, the Dlk1+population could be reactivated in the adult mouse treated with dexamethasone and was shown to contribute to the regeneration of the ZF once dexamethasone treatment was ceased. 1. King P, et al. Shh signaling regulates adrenocortical development and identifies progenitors of steroidogenic lineages. Proc Natl Acad Sci(2009) 106:21185-211902. Guasti L, et al. Dlk1 Up-Regulates Gli1 Expression in Male Rat Adrenal Capsule Cells Through the Activation of β1 Integrin and ERK1/2. Endocrinology(2013) 154:4675-46843. Hadjidemetriou I, et al. DLK1/PREF1 marks a novel cell population in the human adrenal cortex. J Steroid Biochem Mol Biol(2019) 193:1054224. Guasti L, Candy Sze WC, McKay T, Grose R, King PJ. FGF signalling through Fgfr2 isoform IIIb regulates adrenal cortex development. Mol Cell Endocrinol(2013) 371:182-1885. Heikkilä M, et al. Wnt-4 Deficiency Alters Mouse Adrenal Cortex Function, Reducing Aldosterone Production. Endocrinology(2002) 143:4358-4365

OR19-03 Effects of Nonpeptide Orally Bioavailable ACTH Antagonists on Adrenal Gland Size and Function in Rats

Cushing’s disease (CD) and Ectopic ACTH syndrome (EAS) stem from excess circulating adrenocorticotropic hormone (ACTH) and resulting hypercortisolemia. In CD, excess ACTH is secreted from pituitary tumors, whereas excess ACTH in EAS arises from nonpituitary tumors. ACTH acts on the adrenal melanocortin type 2 (MC2) receptor to control the synthesis and secretion of adrenal hormones, including the stress hormone cortisol (corticosterone in rats) which accounts for the comorbidities of CD and EAS. Availability of a potent ACTH antagonist that can normalize cortisol in patients with diseases of excess ACTH will be a major advance in endocrinology. Additionally, an ACTH antagonist will have utility in congenital adrenal hyperplasia (CAH) because of its ability to block production of excess adrenal androgens.Crinetics is evaluating and developing ACTH antagonists for the treatment of diseases of excess ACTH. To our knowledge, these compounds represent the first potent nonpeptide ACTH antagonists to demonstrate in vitro potency and in vivo efficacy. As a result, the direct effects of sustained MC2 receptor blockade on the structure and function of the adrenal gland have never been able to be assessed. We examined the effects of several orally bioavailable ACTH antagonists across a range of doses on Sprague-Dawley rat adrenal gland weight, histology, and hormone levels in repeat dosing (7-14 days) studies. Sustained MC2 receptor antagonism dose dependently blocked activity of ACTH at the level of the adrenal gland and reduced plasma corticosterone levels. In the normal rat, this resulted in dose-dependent atrophy of the adrenal gland as assessed by organ weights and microscopically. The atrophy was primarily observed in the cortisol producing zona fasciculata, as well as in the zona reticularis, with smaller reductions noted in the aldosterone producing zona glomerulosa. Additionally, hypertrophy of the adrenal glands caused by continuous subcutaneous administration of exogenous ACTH was reversed by treatment with an ACTH antagonist. The adrenal effects were accompanied by expected changes in corticosterone levels. These preclinical findings demonstrate the therapeutic potential of ACTH antagonism and provide a strong rationale for development of an orally bioavailable drug that can be used to combat CD, EAS, and CAH.

MON-LB129 A Pilot Genome Wide Association Study (GWAS) on Primary Aldosteronism Patients in a Multi-Ethnic Malaysian Cohort

Studies on excised aldosterone-producing lesions have found somatic mutations in five genes (KCNJ5, CACNA1D, ATP1A1, ATP2B3, and CTNNB1) commonly causes the excess aldosterone production. Interestingly, Oriental cohorts had the highest frequency of KCNJ5 mutations whereas CACNA1D mutations were most common in Black African Caribbean patients, suggesting that genetic background affects the prevalence and distribution of aldosterone-driving somatic mutation. We therefore aimed to identify the common germline variants that associates with excess aldosterone production through performing a pilot genome wide association study (GWAS) on primary aldosteronism (PA) patients. GWAS was performed using the Human Infinium OmniExpressExome-8 v1.4 BeadChip containing 960,919 markers to compare gDNA of 154 PA patients with 78 healthy controls. Samples were checked for sex discordance, heterozygosity rate, missing rate and the degree of recent shared ancestry for each pair of individuals using the PLINK program and Genome Studio (Illumina). In total, 150 patients and 75 controls (112 males and 113 females) were included in the downstream analysis. 630,749 markers that passed quality control steps (missing call rate <95% and minor allele frequency in controls >1%) were used to perform association analysis using the Chi-square Test which was then subjected to multiple testing corrections (Bonferroni correction). As expected with a pilot sample size, no variants passed the suggestive significant threshold of Bonferroni corrected P-value < 5 x 10-6 (-log10 P = 5.3). However, 27 SNPs had the uncorrected P-value<0.0002, odds ratio >2, and differences of frequencies in cases compared to control >0.1 or <-0.2, of which 3 genes (SRGAP3, AUTS2, and RORA) associated with these SNPs were also highlighted in the UK Biobank database of 72 patients with primary aldosteronism (https://biobankengine.stanford. edu/coding/HC189). Of these, RORA has recently been found to be down-regulated in adrenals from PA patients and spontaneously hypertensive rat adrenals compared to control adrenalsa,b. RORA encodes for the protein retinoic acid receptor (RAR)-related orphan receptor alpha, a member of the NR1 subfamily of nuclear hormone receptors (NR1F1). Interestingly, adrenal is the second organ to skin with the highest expression of RORA and treatment of angiotensin II in the adrenocortical cell line H295R increases RORA expressionc,d. Taken together, this pilot GWAS highlights RORA as a potential nuclear hormone receptor that regulates aldosterone production.References aChu et al., Int J Clin Exp Pathol 2017;10(9):10009-10018. bTanaka et al., Hypertens Res 2019;42(2):165-173. cNogueira et al., Mol Cell Endocrinol 2009; 302(2): 230–236. dGTEx Analysis Release V7 (dbGaP Accession phs000424.v7.p2)AcknowledgementsThis research was supported by the Malaysian Ministry of Higher Education Grant (FRGS/1/2015/SKK08/UKM/02/3), The National University of Malaysia (UKM) University Grant (GUP-2016-083), and The UKM Medical Center Fundamental Grant (FF-2016-302).

Regulatory effect of Jiaotai Pills on central and peripheral neurotransmitters in rats with heart-kidney imbalance insomnia

To explore the pathogenesis of heart and kidney imbalance insomnia and the regulatory effect of Jiaotai Pills, in order to study the changes of central and peripheral neurotransmitters in rat. Insomnia rats with heart and kidney imbalance were induced through intraperitoneal injection with p-chlorophenylalanine(PCPA, 400 mg·kg~(-1)·d~(-1)), and the model rats were intragastrically administrated with Jiaotai Pills(3.3 g·kg~(-1)·d~(-1)) for 7 days. Nine neurotransmitters were determined by UPLC-MS/MS and principal component analysis(PCA) method in serum, urine, brain, heart, liver, kidney and adrenal gland of rats. The results showed that the ratio of 5-HT and 5-HIAA in platelet of insomnia rats was significantly lower than that in the normal group, and Jiaotai Pills had a significant up-regulatory or down-regulatory effect. Compared with the normal group, the changed neurotransmitters in blood of insomnia rats were 5-HIAA, E, NE, DA, Glu and ACH, and except ACH, the changes of 7 kinds of neurotransmitters in urine were more significant, Jiaotai Pills had a significant up-regulatory or down-regulatory effect. Compared with the normal group, all of the 8 neurotransmitters in insomnia rats except HVA were changed. Jiaotai Pills could regulate the neurotransmitters in each tissue of insomnia rats, especially in brain, liver and adrenal gland. In conclusion, insomnia is caused by not only a change of neurotransmitters in brain, but also a series of changes in peripheral tissues. It indicates that insomnia is a systematic imbalance of neurotransmitters. Jiaotai Pills not only regulates the central nervous system, but also has a certain protective effect on other organs, reflecting the multi-target and systematic effect of Jiaotai Pills in the treatment of insomnia.

A Moderate Increase in Ambient Temperature Influences The Structure and Hormonal Secretion of Adrenal Glands in Rats.

Objective As a consequence of global warming, the increase in the average annual temperature is observed, while the living organisms actively adapt to these changes. High environmental temperature initiates numerous physiological, autonomic, and behavioral responses, and activates the stress response. Thus, the aim of the study was to investigate effect of a moderate increase in ambient temperature on the activity of the hypothalamic-pituitary-adrenocortical (HPA) axis by determining histological changes in adrenal glands and hormonal levels in adult male rats. Material and Methods: In this experimental study, the morpho-functional state of adrenal glands was estimated by stereological evaluation of parameters, including the adrenal volume, adrenocortical cell/nuclear size and number, and the volume density of vascular tissues after four days of exposure to a moderate increase in ambient temperature of 35 ± 1˚C. Novelli histochemical and vascular endothelial growth factor (VEGF) immunohistochemical staining provided insight into the adrenal gland vascular network. Additionally, the adrenal levels of aldosterone, corticosterone, and pituitary adrenocorticotropic hormone (ACTH) were determined as crucial indicators of the hypothalamic-pituitary- adrenocortical (HPA) axis activity. Results Prolonged exposure to a moderate increase in ambient temperature for four days resulted in a significant increase in ACTH level up to 24%, which altered adrenal glands both structurally and functionally. The adrenocortical volume and number of cells in all cortical zones were markedly increased (P<0.05). A statistically significant increase was shown in the level of aldosterone (16%) and corticosterone (25%) in serum levels of individuals. ConclusionIncreased activity of the HPA axis reflects the response to a moderate increase in ambient temperature during four days, showing the capacity of the HPA axis to adapt the organism to daily temperature changes.

Morphological and Cytophysiological Changes in the Adult Rat Adrenal Medulla after Prenatal and Postnatal Exposure to Endocrine-Disrupting DDT.

The aim of the investigation was to study morphological and cytophysiological changes in the adult rat adrenal medulla after prenatal and postnatal exposure to endocrine-disrupting chemical dichlorodiphenyltrichloroethane (DDT).Materials and MethodsThe experiment was carried out on male Wistar rats (n=30). The rats were exposed to low doses of DDT during prenatal and postnatal development (n=10) and during postnatal development (n=10) only. The average daily intake of DDT by rats was 3.30±0.14 μg/kg. The control (n=10) and experimental animals were sacrificed at the age of 10 weeks (post-puberty), when rat adrenal glands reached their maximum development. Histological samples of the equatorial sections of the adrenal glands were prepared. The adrenal medulla was studied using light microscopy and computer morphometry. Immunohistochemistry was performed to evaluate tyrosine hydroxylase expression in chromaffin cells. Plasma epinephrine content was measured using enzyme-linked immunosorbent assay.ResultsPostnatal exposure of rats to low doses of the endocrine disruptor was found to interfere with adrenal medulla development and cytophysiology of chromaffin cells. Prenatal and postnatal exposure resulted in more profound delay in adrenal medulla development of adult rats as compared to the values of rats exposed postnatally, which was manifested by reduced area of the adrenal medulla and reduced total area of chromaffin cells in its section. Postnatal exposure is manifested by more significant functional disorders including decreased synthesis of tyrosine hydroxylase enzyme in the cytoplasm of chromaffin cells and elevated number of tyrosine-hydroxylase-negative cells in the medulla section, leading to a decrease in plasma epinephrine concentration.ConclusionExposure to low doses of DDT in the early stages of development leads to morphological and cytophysiological changes in the adult rat adrenal medulla and, therefore, reduced functional activity of chromaffin cells, which allows considering the endocrine disruptor DDT a potential risk factor disrupting the function of the neuroendocrine system.

SIÇAN BÖBREKÜSTÜ BEZİNİN HİSTOLOJİK YAPISI ÜZERİNE ELEKTROMANYETİK ALANIN ETKİSİNİN STEREOLOJİK YÖNTEMLE ARAŞTIRILMASI

Objective: Cell phone emitting electromagnetic field (EMF) are routinely used in our daily life. Long-term use of these Cell phones has caused public health concerns in recent years. Also, reports regarding the effect of EMF exposure on the biosystem are inconclusive. In this study, we aimed to investigate the possible effects of 900 MHz EMA on the adrenal glands of adult male rats. Method: Twelve adult rats were randomly selected and divided into two groups as&nbsp;follow: control and EMF. Finally, we&nbsp;analysed&nbsp;the suprarenal gland tissues using&nbsp;stereological&nbsp;method. Results: We found no significant difference in the volume of the adrenal zona glomerulosa d in the EMF group compared to the control group. The volume of the adrenal zona fasciculate was significantly increased In the EMF group compared to the control group (p&lt;0.05). There was also&nbsp;significant&nbsp;increase in the mean volume of the zona reticularis (p&lt; 0.05). Conclusion: Our finding showed that exposure to 900 MHz EMF caused&nbsp;morphometrical&nbsp;change in the rat adrenal gland.

Иммуногистохимическое исследование иннервации надпочечника крысы

The aim of the study was to study the innervation of the cortical part and medulla of the rat adrenal gland using neuroimmunohistochemical markers. Original data on the innervation of the rat Wistar (n = 8) adrenal cortex and medulla were obtained using immunohistochemical detection of the PGP 9.5 protein, synaptophysin, tyrosine hydroxylase, peripherin and serotonin. It was established that all zones of the cortex (glomerular, bundle, and reticular) are innervated by nerve fibers of various neurotransmitter identity. In the cortex, the innervation of the glomerular zone is most pronounced. A thick terminal plexus of varicose axons around islets of endocrinocytes, arterioles and nerve cells was detected in this zone. Cholinergic nerve structures (plexus of preterminal nerve fibers and the terminal synaptic network, which consists of varicose parasympathetic axons located around groups of neuroendocrine cells and sinusoidal capillaries) prevail in the medulla. Parasympathetic and sympathetic intramural ganglia were found in the medulla. Chromaffin cells immunoreactive to various catecholamines and serotonin were found in the adrenal medulla. It was assumed that different sources of sympathetic innervation of the organ are present. These are the medulla's own neurons and sympathetic trunk neurons. Such innervation is important for the regulation of local blood circulation.The results obtained in this study can be used both in scientific research and in the diagnosis of adrenal diseases.

Long-term triphenyltin exposure disrupts adrenal function in adult male rats.

Triphenyltin is an organotin, which is widely used as a fungicide in agriculture. Here, we reported the effects of triphenyltin on adrenal function in adult male rats. Adult male Sprague Dawley rats were daily gavaged with triphenyltin (0, 0.5, 1, and 2 mg/kg body weight) from postnatal day 56-86. Triphenyltin significantly decreased serum corticosterone levels at 1 and 2 mg/kg without affecting serum levels of aldosterone and adrenocorticotropic hormone. Triphenyltin increased thickness of zona glomerulosa without affecting that of zona fasciculata. Triphenyltin did not affect cell number in zona fasciculata and zona glomerulosa. Triphenyltin down-regulated the expression of Scarb1, Star, Cyp11a1, Hsd3b1, Cyp21, Cyp11b1, and Hsd11b1 at 1 and/or 2 mg/kg while it up-regulated the expression of At1, Nr4a2, and Hsd11b2 at 2 mg/kg. Triphenyltin activated the phosphorylation of AMPKα while suppressed the phosphorylation of AKT1 and SIRT1/PGC-1α in rat adrenals invivo and H295R cells invitro. Invitro, triphenyltin also induced ROS production in H295R cells at 100 nM, a concentration at which no apoptosis was induced. In conclusion, triphenyltin disrupts glucocorticoid synthesis in rat adrenal cortex via several mechanisms: 1) lowering AKT1 phosphorylation and SIRT1/PGC-1α levels; 2) activating AMPKα; and 3) possibly inducing ROS production.