Co-individuazione delle isoforme di mRNA per il recettore dei mineralcorticoidi (MR) e dell' enzima 11ß-idrossisteroide deidrogenasi in cellule e tessuti umani atipici, rispondenti all'aldosterone. Studio particolareggiato in pazienti affetti da iperaldosteronismo primitivo e ricerca di mutazioni nel gene MR in una famiglia con diagnosi di pseudo-ipoaldosteronismo.

The steroid hormone aldosterone, that normally controls the urinary Na+/K+ balance, and the osmolarity in the kidney, colon, salivary and swab glands epithelial tissues, exerts also many different effects, by binding to the mineralocorticoid receptor (MR). This receptor binds both aldosterone and glucocorticoids (mostly cortisol) with approximately the same affinity. However, cortisol has a 1000-fold higher concentration in plasma than that of aldosterone. In its classical target tissues, the mineralocorticoid selectivity mainly depends on the enzymatic activity of the type 2 11ß-hydroxysteroid dehydrogenase (11ß -HSD2), which converts cortisol into inactive cortisone, a corticosteroid that doesn't bind to the MR. The human MR gene (hMR) maps on chromosome 4 and contains 8 coding exons, with the start of the open reading frame (ORF) in the exon 2, which gives a 984 aminoacid product. The untranslated exon 1, which exists in the two isoforms 1? and 1ß, may give rise, by alternative splicing, to the hMR? and hMRß mRNA variants. The hMRß seems to be expressed only in some tissues and cell types. In 2001, two other hMR mRNA variants were described and they were called hMR?5 and hMR?5,6, because they lack the exon 5 or both exons 5 and 6, respectively. The hMR? transcript isoforms, first detected in the kidney, encode for a receptor which lack a fragment or the whole LBD domain, and they are both widely expressed among tissues, where they act as transcription factors that do not depend upon the ligand aldosterone. In this work, I first demonstrated, by RT-PCR assays, the expression of hMR gene in spermatozoa, even though, in these cells, it was less detectable than in human mononuclear leukocytes (LMN). These important data were confirmed by immunofluorescence, in which the hMR protein was detectable in the middle piece and in the tail of the spermatozoa. These results may provide useful insight into mechanisms that can modify sperm motility in the presence of aldosterone in the milieu. It is well known, from the scientific literature, that the activation of a number of genes, consequently to the specific MR-aldosterone interaction, may lead to the synthesis of some pro-inflammatory substances, in the heart, kidney, and in the vascular endothelium. In primary hyperaldosteronism, the presence of high aldosterone plasma levels may finally cause fibrosis in the same districts. The first steps of the inflammatory processes are characterized by a massive invasion of LMN, so, in the present research, I analyzed, by RT-PCR reactions, the expression of hMR gene variants in LMN from patients with Conn syndrome, and in both the normal adrenal cortex tissue, and in the adjacent adenoma producing aldosterone (APA) samples. I demonstrated a variable expression of the hMRß isoform, in comparison with the hMR? variant, which is homogeneously detectable in all the analyzed samples, and it was found that the hMRß mRNA was less expressed in the APA tissues, than in the normal adrenal tissue and in the LMN of the same patients. In the same samples, I also verified the co-expression of mRNA for both the type 1 and 2 11ß-hydroxysteroid dehydrogenase (11ß-HSD 1 and 2). A very interesting outcome from this step of my study was the demonstration of the 11ß -HSD type 2 expression in LMN of most of the patients with primary aldosteronism, while the same mRNA was not detectable in LMN from healthy volunteers. These important results, if they are confirmed in a significantly greater number of patients, would provide more information in order to predict the presence of a primary aldosteronism itself. In this work, I also analyzed, by RT-PCR, the expression of the above mentioned genes in five normal tissue samples from human larynx. Even in this case, the hMR?, hMRß and the 11ß-HSD type 2 mRNAs were all detectable in the larynx tissue, and so this can be considered as a novel tissue specifically responsive, in a genomic manner, to aldosterone by its interaction with MR. The meaning of this result will be investigate in the future. Many human diseases depend upon a number of important mutations in almost all the hMR exons, causing type I pseudo-hypoaldosteronism (PHA I), which mimics the absence of aldosterone, even if it is synthesized in a great amount. In the last period of my work, I investigated the hMR gene for mutations in each exon, in a family with PHA I, composed by a newborn, that was identified as the case-control, and their parents. Since no important nucleotide substitutions was revealed in the analyzed coding regions, this family is probably affected by the renal variant of the above mentioned diseases, where the mutations involve the gene encoding for the epithelial sodium channels subunits in the kidney.