Unsupervised Network Analysis of the Plastic Supraoptic Nucleus Transcriptome Predicts Caprin2 Regulatory Interactions.

The supraoptic nucleus (SON) is a group of neurons in the hypothalamus responsible for the synthesis and secretion of the peptide hormones vasopressin and oxytocin. Following physiological cues, such as dehydration, salt-loading and lactation, the SON undergoes a function related plasticity that we have previously described in the rat at the transcriptome level. Using the unsupervised graphical lasso (Glasso) algorithm, we reconstructed a putative network from 500 plastic SON genes in which genes are the nodes and the edges are the inferred interactions. The most active nodal gene identified within the network was Caprin2. Caprin2 encodes an RNA-binding protein that we have previously shown to be vital for the functioning of osmoregulatory neuroendocrine neurons in the SON of the rat hypothalamus. To test the validity of the Glasso network, we either overexpressed or knocked down Caprin2 transcripts in differentiated rat pheochromocytoma PC12 cells and showed that these manipulations had significant opposite effects on the levels of putative target mRNAs. These studies suggest that the predicative power of the Glasso algorithm within an in vivo system is accurate, and identifies biological targets that may be important to the functional plasticity of the SON.

Changes in plasma aldosterone and electrolytes levels, kidney epithelial sodium channel (ENaC) and blood pressure in normotensive WKY and hypertensive SHR rats following gonadectomy and chronic testosterone treatment.

We hypothesized that testosterone-induced increase in blood pressure involve changes in aldosterone levels and expression of epithelial sodium channel (ENaC) in the kidneys. METHODS: Ovariectomized female normotensive Wistar Kyoto (WKY) and Spontaneous hypertensive (SHR) rats were given six weeks treatment with testosterone via subcutaneous silastic implant. The rats were anesthetized and mean arterial pressure (MAP) was measured via direct cannulation of the carotid artery. Animals were sacrificed and kidneys were removed and subjected for α, ß and γ-ENaC protein and mRNA expression analyses by Western blotting and Real-time polymerase chain reaction (qPCR), respectively. Distributions of α, ß and γ-ENaC proteins in kidneys were observed by immunofluorescence. Plasma testosterone, aldosterone, electrolytes, osmolality, urea and creatinine levels were determined by biochemical assays. Analysis were also performed in non-testosterone treated orchidectomized and sham-operated male WKY and SHR rats. RESULTS: Treatment of ovariectomized female WKY and SHR rats with testosterone causes increased in MAP but decreased in plasma aldosterone, sodium (Na+), osmolality and expression and distribution of α, ß and γ-ENaC subunits in the kidneys. Orchidectomy decreased the MAP but increased plasma aldosterone, Na+, osmolality and α, ß and γ-ENaC expression and distribution in the kidneys of male WKY and SHR rats. CONCLUSIONS: Decreased in plasma aldosterone, Na+ and ENaC levels in kidneys under testosterone influence indicated that testosterone-induced increased in MAP were not due to increased plasma aldosterone and ENaC levels in kidneys, and thus the testosterone effect on MAP likely involve other mechanisms.

Sex-specific differences in cardiovascular and metabolic hormones with integrated signalling in the paraventricular nucleus of the hypothalamus.

NEW FINDINGS: What is the topic of this review? We describe roles of crucial signalling molecules in the paraventricular nucleus of the hypothalamus and highlight recent data suggesting sex-specific changes in the expression of crucial signalling molecules and their receptors, which may underlie sex differences in both cardiovascular and metabolic function. What advances does it highlight? This review highlights the integrative capacity of the paraventricular nucleus in mediating cardiovascular and metabolic effects by integrating information from multiple signalling molecules. It also proposes that these signalling molecules have sex-specific differential gene expression, indicating the importance of considering these differences in our ongoing search to understand the female-male differences in the regulation of crucial autonomic systems. Many traditional cardiovascular hormones have been implicated in metabolic function. Conversely, many hormones traditionally involved in metabolic regulation have an effect on cardiovascular function. Many of these signalling molecules exert such effects through specific actions in the paraventricular nucleus, an integrative autonomic control centre located in the hypothalamus. Here, we focus on four cardiovascular/metabolic peptide hormones that signal within the paraventricular nucleus, namely angiotensin II, orexin, adiponectin and nesfatin-1. Each of these hormones has specific electrophysiological effects on paraventricular nucleus neurons that can be related to its physiological actions. In addition, we introduce preliminary transcriptomic data indicating that the genes for some of these hormones and their receptors have sex-specific differential expression.

Selection of suitable endogenous reference genes for qPCR in kidney and hypothalamus of rats under testosterone influence.

Real-time quantitative PCR (qPCR) is the most reliable and accurate technique for analyses of gene expression. Endogenous reference genes are being used to normalize qPCR data even though their expression may vary under different conditions and in different tissues. Nonetheless, verification of expression of reference genes in selected studied tissue is essential in order to accurately assess the level of expression of target genes of interest. Therefore, in this study, we attempted to examine six commonly used reference genes in order to identify the gene being expressed most constantly under the influence of testosterone in the kidneys and hypothalamus. The reference genes include glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin beta (ACTB), beta-2 microglobulin (B2m), hypoxanthine phosphoribosyltransferase 1 (HPRT), peptidylprolylisomerase A (Ppia) and hydroxymethylbilane synthase (Hmbs). The cycle threshold (Ct) value for each gene was determined and data obtained were analyzed using the software programs NormFinder, geNorm, BestKeeper, and rank aggregation. Results showed that Hmbs and Ppia genes were the most stably expressed in the hypothalamus. Meanwhile, in kidneys, Hmbs and GAPDH appeared to be the most constant genes. In conclusion, variations in expression levels of reference genes occur in kidneys and hypothalamus under similar conditions; thus, it is important to verify reference gene levels in these tissues prior to commencing any studies.

Effects of gonadectomy and testosterone treatment on aquaporin expression in the kidney of normotensive and hypertensive rats.

We tested the hypothesis that testosterone-induced increase in blood pressure was due to changes in aquaporin (AQP) expression in kidneys. In this study, expression level of kidney AQPs was investigated under testosterone influence. Adult normotensive Wistar Kyoto (WKY) and hypertensive SHR male and female rats underwent gonadectomy. For female rats, testosterone was given for six weeks duration, two weeks following ovariectomy via subcutaneous silastic implant. Mean arterial pressure (MAP) was measured in all the rats after eight weeks via carotid artery cannulation and the rats were then sacrificed and kidneys were harvested for analyses of AQP-1, 2, 3, 4, 6, and 7 mRNA and protein expressions by quantitative real-time PCR and Western blotting, respectively. Distribution of AQP subunits' protein in kidneys was observed by immunofluorescence. In male WKY rats, MAP, AQP-1, 2, 4, and 7 protein; and mRNA expression decreased however AQP-3 protein and mRNA expression increased following orchidectomy. The vice versa effects were observed in testosterone-treated ovariectomized female WKY rats. However, no changes in AQP-6 expression were observed. Meanwhile, in adult male SHR rats, MAP and expression level of all AQP subunits decreased following orchidectomy. The opposite effects were seen in ovariectomized female SHR rats following testosterone treatment. Immunofluorescence study showed AQP-1 and AQP-7 were distributed in the proximal convoluted tubules (PCT) while AQP-2, AQP-4, and AQP-6 were distributed in the collecting ducts (CDs). AQP-3 was distributed in the PCT and CD. In conclusion, changes in AQP subunit expression in kidneys could explain changes in blood pressure under testosterone influence. Impact statement This study provides fundamental understanding on the mechanisms underlying testosterone-induced increase in blood pressure which involve regulation of aquaporin channel subunits in the kidneys. A better understanding of this issue can help to explain the reason for higher blood pressure in males as compared to females and may explain the reason for higher blood pressure in females after menopause than females before menopause, the former most probably related to the changes in female androgen.

Effects of testosterone on mean arterial pressure and aquaporin (AQP)-1, 2, 3, 4, 6 and 7 expressions in the kidney of orchidectomized, adult male Sprague-Dawley rats.

We hypothesized that higher blood pressure in males than females could be due to testosterone effects on aquaporin (AQP) expression in kidneys. METHODS: Orchidectomized adult male Sprague-Dawley (SD) rats received seven days subcutaneous testosterone treatment (125 µg/kg/day or 250 µg/kg/day), with or without flutamide or finasteride. Following completion of treatment, MAP was determined in rats under anaesthesia via carotid artery cannulation. In another cohort of rats, kidneys were removed following sacrifice and AQP-1, 2, 3, 4, 6 and 7 protein and mRNA levels were determined by Western blotting and Real-time PCR respectively. Distribution of AQP subunits' protein in the nephrons were visualized by immunofluorescence. RESULTS: Testosterone caused MAP, AQP-1, 2, 4, 6 and 7 protein and mRNA levels in kidneys to increase while AQP-3 protein and mRNA levels in kidneys to decrease (p < 0.05). AQP-1 and 7 were found to be distributed in the proximal convoluted tubule (PCT) while AQP-2, 3, 4 and 6 were found to be distributed in the collecting ducts (CD). Effects of testosterone were antagonized by flutamide and finasteride. CONCLUSIONS: Elevated expression of AQP-1, 2, 4, 6 and 7 under testosterone influence in kidneys could lead to increase H2O reabsorption which eventually lead to increase in blood pressure.

Sub-chronic testosterone treatment increases the levels of epithelial sodium channel (ENaC)-α, ß and γ in the kidney of orchidectomized adult male Sprague-Dawley rats.

Testosterone has been reported to cause blood pressure to increase. However mechanisms that underlie the effect of this hormone on this physiological parameter are currently not well understood. The aims of this study were to investigate effects of testosterone on expression of α, ß and γ-epithelial sodium channel (ENaC) proteins and messenger RNAs (mRNAs) in kidneys, the channel known to be involved in Na(+) reabsorption, which subsequently can affect the blood pressure. Methods. Adult male Sprague-Dawley (SD) rats were orchidectomized fourteen days prior to receiving seven days treatment with testosterone propionate (125 µg/kg/day or 250 µg/kg/day) with or without flutamide (androgen receptor blocker) or finasteride (5α-reductase inhibitor). Following sacrifice, the kidneys were removed and were subjected for α, ß and γ-ENaC protein and mRNA expression analyses by Western blotting and Real-time PCR (qPCR) respectively. The distribution of α, ß and γ-ENaC proteins in kidneys were observed by immunofluorescence. Results. The α, ß and γ-ENaC proteins and mRNA levels in kidneys were enhanced in rats which received testosterone-only treatment. In these rats, α, ß and γ-ENaC proteins were distributed in the distal tubules and collecting ducts of the nephrons. Co-treatment with flutamide or finasteride resulted in the levels of α, ß and γ-ENaC proteins and mRNAs in kidneys to decrease. In conclusions, increases in α, ß and γ-ENaC protein and mRNA levels in kidneys mainly in the distal tubules and collecting ducts under testosterone influence might lead to enhance Na(+) reabsorption which subsequently might cause an increase in blood pressure.

RNA binding protein Caprin-2 is a pivotal regulator of the central osmotic defense response.

In response to an osmotic challenge, the synthesis of the antidiuretic hormone arginine vasopressin (AVP) increases in the hypothalamus, and this is accompanied by extension of the 3' poly(A) tail of the AVP mRNA, and the up-regulation of the expression of RNA binding protein Caprin-2. Here we show that Caprin-2 binds to AVP mRNAs, and that lentiviral mediated shRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail at the same time as reducing transcript abundance. In a recapitulated in vitro system, we confirm that Caprin-2 over-expression enhances AVP mRNA abundance and poly(A) tail length. Importantly, we show that Caprin-2 knockdown in the hypothalamus decreases urine output and fluid intake, and increases urine osmolality, urine sodium concentration, and plasma AVP levels. Thus Caprin-2 controls physiological mechanisms that are essential for the body's response to osmotic stress.

Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus.

BACKGROUND: Arginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression. RESULTS: The effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress. CONCLUSION: Here we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.