Project description:Primary aldosteronism (PA) is a common form of endocrine hypertension suspected in the presence of arterial hypertension (HT), hypokalemia and low plasma renin levels. While for a long time the genetic causes of PA have remained mysterious, recent findings have revealed an important role for an altered function of potassium channels in the pathogenesis of this disease. The basis for the unique sensitivity of adrenal zona glomerulosa (ZG) cells for plasma potassium concentration is a very high background potassium conductance, which is dependent on expression at high levels of two 2-pore domain (K2P) potassium channels, Task1 (KCNK3) and Task3 (KCNK9. Recent studies have shown that mice lacking Kcnk3 (Task1) or both Kcnk3 and Kcnk9 have PA. In Kcnk3 null mice, hyperaldosteronism is present in young animals of either sex, but is corrected in males after puberty. Strikingly, in these animals hyperaldosteronism associates with abnormal adrenal cortex functional zonation, since Cyp11b2, the rate-limiting enzyme for aldosterone production, is expressed in the inner region of the adrenal cortex and not in the glomerulosa. Additionally, this phenotype is under the control of sex hormones, as shown by the fact that castration of male Kcnk3 -/- animals prevents normal Cyp11b2 distribution, while testosterone injection in female Kcnk3 null mice restores expression of Cyp11b2 in ZG cells. We took advantage of the unique characteristics of the Kcnk3 null mouse model to search for genes that can modify their phenotype of PA and adrenocortical functional zonation. Using gene expression profiling in the adrenal glands of Kcnk3 null mice and exploiting the possibility of modulation of their phenotype by sexual hormones, we identified a cluster of genes closely associated with hyperaldosteronism in a sex- and hormone-dependent dynamic fashion. Among these genes, we focused our attention on Dkk3 (dickkopf3), encoding a peculiar member of the dickkopf family of Wnt signalling modulators because of its close association with aldosterone-producing cells in humans (11). Inactivation of Dkk3 in the Kcnk3 null background causes extension of the hyperaldosteronemic phenotype and increased expression of Cyp11b2 in the adrenal gland to the male sex, without affecting functional zonation. These data indicate that Dkk3 is a component of the genetic circuitry regulating expression of Cyp11b2 and suggest that it may be implicated in the pathogenesis of low-renin hyperaldosteronism in humans. One color -experiment with 2 strains of mice: C57BL/6 WT or Kcnk3 null (KO): male and female in basal conditions or following hormonal treatment (castration for males and testosterone injection for females), corresponding to 8 groups of animals. Total of 24 samples (n=3).

Project description:Primary aldosteronism (PA) is a common form of endocrine hypertension suspected in the presence of arterial hypertension (HT), hypokalemia and low plasma renin levels. While for a long time the genetic causes of PA have remained mysterious, recent findings have revealed an important role for an altered function of potassium channels in the pathogenesis of this disease. The basis for the unique sensitivity of adrenal zona glomerulosa (ZG) cells for plasma potassium concentration is a very high background potassium conductance, which is dependent on expression at high levels of two 2-pore domain (K2P) potassium channels, Task1 (KCNK3) and Task3 (KCNK9. Recent studies have shown that mice lacking Kcnk3 (Task1) or both Kcnk3 and Kcnk9 have PA. In Kcnk3 null mice, hyperaldosteronism is present in young animals of either sex, but is corrected in males after puberty. Strikingly, in these animals hyperaldosteronism associates with abnormal adrenal cortex functional zonation, since Cyp11b2, the rate-limiting enzyme for aldosterone production, is expressed in the inner region of the adrenal cortex and not in the glomerulosa. Additionally, this phenotype is under the control of sex hormones, as shown by the fact that castration of male Kcnk3 -/- animals prevents normal Cyp11b2 distribution, while testosterone injection in female Kcnk3 null mice restores expression of Cyp11b2 in ZG cells. We took advantage of the unique characteristics of the Kcnk3 null mouse model to search for genes that can modify their phenotype of PA and adrenocortical functional zonation. Using gene expression profiling in the adrenal glands of Kcnk3 null mice and exploiting the possibility of modulation of their phenotype by sexual hormones, we identified a cluster of genes closely associated with hyperaldosteronism in a sex- and hormone-dependent dynamic fashion. Among these genes, we focused our attention on Dkk3 (dickkopf3), encoding a peculiar member of the dickkopf family of Wnt signalling modulators because of its close association with aldosterone-producing cells in humans (11). Inactivation of Dkk3 in the Kcnk3 null background causes extension of the hyperaldosteronemic phenotype and increased expression of Cyp11b2 in the adrenal gland to the male sex, without affecting functional zonation. These data indicate that Dkk3 is a component of the genetic circuitry regulating expression of Cyp11b2 and suggest that it may be implicated in the pathogenesis of low-renin hyperaldosteronism in humans.

Project description:The mineralocorticoid aldosterone mainly produced by the adrenal gland is essential for life but an abnormal excessive secretion causes severe pathological effects including hypertension and target organ injury in the heart and kidney. The aim of this study was to determine the gene regulatory network triggered by aldosterone secretagogues in a non transformed cell system. Freshly isolated rat adrenal zona glomerulosa cells were stimulated with the two main aldosterone secretagogues, angiotensin II and potassium, for two hours and subjected to whole genome expression studies using multiple biological and bioinformatics tools. Several genes were differentially expressed by Ang II (n=133) or potassium (n=216). Genes belonging to the nucleic acid binding and transcription factor activity categories were significantly enriched. A subset of the most regulated genes were confirmed by real-time RT-PCR and then their expression analyzed in time curve studies. Differentially expressed genes were grouped according to their time-response expression pattern and their promoter regions analyzed for common regulatory transcription factors binding sites. Finally, data mining with gene promoters, transcription factors and literature databases were performed to generate gene interaction networks for either Ang II or potassium. This study provides for the first time a complete study of the genes that are regulated, and the interaction between them, by aldosterone secretagogues in rat adrenal cells. Increasing our knowledge of adrenal physiology and gene regulation in non transformed cell systems would lead us to a better approach for discovery of candidate genes involved pathological conditions of the adrenal cortex. Experiment Overall Design: Freshly isolated rat adrenal cells were treated with Ang II (100 nM) or potassium (16 mM) for 2 h at 37C. Experiment Overall Design: Total RNA from three independent experiments were isolated, labeled and hybridized to whole genome high-density oligonucleotide microarrays (Affymetrix Rat Genome 230 2.0). Experiment Overall Design: 3 independent cell preparations. Experiment Overall Design: In each experiement there is one tube control, one Ang II (100 nM) and potassium (16 mM).

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The E3 ubiquitin ligase Siah1 regulates adrenal gland organization and hyperaldosteronism

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