Tm Seccia

The Medical and Endovascular Treatment of Atherosclerotic Renal Artery Stenosis (METRAS) study: rationale and study design.

It is unclear whether revascularization of renal artery stenosis (RAS) by means of percutaneous renal angioplasty and stenting (PTRAS) is advantageous over optimal medical therapy. Hence, we designed a randomized clinical trial based on an optimized patient selection strategy and hard experimental endpoints. Primary objective of this study is to determine whether PTRAS is superior or equivalent to optimal medical treatment for preserving glomerular filtration rate (GFR) in the ischemic kidney as assessed by 99mTcDTPA sequential renal scintiscan. Secondary objectives of this study are to establish whether the two treatments are equivalent in lowering blood pressure, preserving overall renal function and regressing target organ damage, preventing cardiovascular events and improving quality of life. The study is designed as a prospective multicentre randomized, un-blinded two-arm study. Eligible patients will have clinical and angio–CT evidence of RAS. Inclusion criteria is RAS affecting the main renal artery or its major branches either >70% or, if <70, with post-stenotic dilatation. Renal function will be assessed with 99mTc-DTPA renal scintigraphy. Patients will be randomized to either arms considering both resistance index value in the ischemic kidney and the presence of unilateral/bilateral stenosis. Primary experimental endpoint will be the GFR of the ischemic kidney, assessed as quantitative variable by 99TcDTPA, and the loss of ischemic kidney defined as a categorical variable.

HIGH EXPRESSION OF THE PRO-RENIN RECEPTOR IN ALDOSTERONE PRODUCING ADENOMA CAUSING HUMAN PRIMARY ALDOSTERONISM: 4D.02

Objective: Primary Aldosteronism (PA) is the most prevalent form of endocrine hypertension but its underlying mechanisms are unknown. The detection of prorenin, despite the suppression of renin, in plasma of PA patients suggests that prorenin, by acting via the Pro-Renin Receptor (PRR), could play a pathophysiologic role in PA by causing adrenocortical cell growth and hyperaldosteronism. Hence, we hypothesized that the PRR is expressed in the human zona glomerulosa (ZG) and in aldosterone producing adenoma (APA). Design and Method: To test this hypothesis we investigated the presence of the PRR by using beforehand a whole transcriptome analysis approach (in 24 APA). We then used Real time RT-PCR to quantify more precisely the PRR transcript in APA (n = 12), in two adrenocortical carcinoma cell lines (H295 and HAC15) and in immunomagnetic bead separated CD56+ human adrenocortical ZG cells (Caroccia, Endocrinology 2010). To confirm the expression of the PRR at the protein level immunohistochemistry and immunoblotting were also performed. Results and Conclusions: Microarray analysis evidenced the expression of PRR in all APAs. Quantitative gene expression studies demonstrated a level of expression of the PRR gene in APA which was on average much higher than that of the established adrenocortical house-keeping gene PBGD (PRR Ct = 22,69 ± 1,11; PBGD Ct = 27,49 ± 1,69 p < 0.0001). The expression of the PRR in the human adrenal cortical tissue was confirmed in CD56+ ZG cells, and in H295 and HAC15 cells. Immunohistochemistry confirmed the expression of the PRR at the protein level in all these cells. It also allowed to localize it more precisely to the adrenocortical ZG. These results are consistent with the hypothesis that circulating prorenin can activate the PRR in PA patients. Therefore, experiments are ongoing to investigate the functional relevance of these findings with the ultimate goal of demonstrating the role of the PRR in human PA.

[PP.22.18] UROTENSIN II EXERTS PRESSOR EFFECTS BY STIMULATING RENIN AND ALDOSTERONE SYNTHASE GENE EXPRESSION

Objective: The pressor effects of the potent vasoconstrictor Urotensin II (UII) remain uncertain. We investigated the effects of UII on blood pressure (BP) and the renin-angiotensin-aldosterone system. Design and method: We randomized normotensive Sprague-Dawley rats into 4 groups that received a 7-days UII infusion (cases, 600 pmol/kg/h) or a vehicle (controls). Group 1 received a normal sodium intake; Group 2 underwent unilateral nephrectomy and salt loading; Group 3 received spironolactone, besides unilateral nephrectomy and salt loading; Group 4 only received spironolactone. BP was monitored by radio-telemetry; changes of gene and protein level of renin and Cyp11b2 expression were measured. Results: UII raised BP transiently after a lag phase of 24–48 hours in Group 1, and progressively over the week in Group 2. Spironolactone abolished both pressor effects of UII. UII increased by 7-fold the renal expression of renin in Group 2 rats (p < .01 vs control); it also increased aldosterone synthase expression (p < .01, both gene and protein level) in the adrenocortical zona glomerulosa, and prevented the blunting of renin expression induced by high salt. Double immunohistochemistry showed localization of renin and UII receptor in the human renal cortex. Conclusions: UII raises BP transiently when sodium intake and renal function are normal, but progressive in salt-loaded uni-nephrectomized rats. Moreover, UII counteracts the suppression of renin induced by salt loading and stimulation of aldosterone secretion. This novel action of UII in the regulation of renin and aldosterone synthesis can play a role in several clinical conditions where UII levels are up-regulated.

[PP.25.04] UROTENSIN II EXERTS PRESSOR EFFECTS BY STIMULATING RENIN AND ALDOSTERONE SECRETION

Objective: As Urotensin II (UII) induced a transient pressor response in normotensive rats similar to the ‘escape phenomenon’ to aldosterone, we investigated if it activated the synthesis of renin and aldosterone. Design and method: Sprague-Dawley normotensive rats were randomly divided into 4 groups. Group 1 received either a 7-days infusion of UII (600 pmol/kg/h) (cases) or a vehicle (controls). Group 2 underwent unilateral nephrectomy and a high sodium intake to antagonize the ‘escape phenomenon’. Group 3, besides unilateral nephrectomy and high sodium intake, received spironolactone, while Group 4 only received spironolactone. Blood pressure (BP) was continuously monitored by telemetry. Changes of genes and proteins expression of renin and Cyp11b2 were measured with Q-RT-PCR and immunohistochemistry, respectively. Results: In Group 1, after a lag phase of 24–48 hours, UII transiently raised BP. In Group 2 it induced a progressive increase of BP over the week. In Group 3 and 4 spironolactone abolished both the early and the late pressor effects of UII. UII increased by 7-fold the expression of renin in the kidney of uninephrectomized rats (p < .01) in spite of salt-loading; it also increased Cyp11b2 gene and aldosterone synthase protein expression (p < .01) in the adrenocortical zona glomerulosa and prevented the salt-induced blunting of Cyp11b2 expression. Conclusions: Thus, with normal sodium intake and excretory capabilities UII raised BP transiently; in salt-loaded uni-nephrectomized rats it raised BP progressively by counteracting the suppression of renin induced by salt loading. Overall these results suggest a role of UII enhanced renin and aldosterone synthesis in hypertension forms with impaired renal function.

[PP.25.06] ANGIOTENSIN-(1–7) PEPTIDE AND MAS RECEPTOR AND ANGIOTENSIN II- TYPE 2 RECEPTOR ARE INVOLVED IN ALDOSTERONE AND CORTISOL PRODUCTION IN HUMAN ADRENOCORTICAL CELL LINE (NCI-H295)

Objective: Aldosterone and cortisol secretion are tightly regulated by angiotensin II (Ang II) acting via angiotensin type 1 receptor (AT1R) in adrenal tissue. In several tissues, AT2R and the MasR, target of Ang-(1–7), can counteregulate AT1R effects. Nevertheless, whether AT2R and MasR play any role in the adrenal cortex is still unknown. Aim: To investigate: 1) the presence of AT2R and MasR in human adrenocortical tissue 2) if Ang-(1–7) could play a role in modulation of aldosterone and cortisol production in a human adrenocortical cell line (NCI-H295) in vitro. Design and method: RT-PCR, immunoblotting, and immunohistochemistry were used to detect AT2R and MasR Moreover, in NCI-H295 cells, we used Ang-(1–7) to stimulate MasR, Ang II to stimulate AT1R and AT2R, irbesartan and A779 as blockers for AT1R and MasR respectively. RT-PCR was used to quantify CYP11B1/CYP11B2 genes expression. Results: AT2R and MasR are heterogeneously expressed in human adrenal cortex and in APA. Ang-(1–7) had no effect at low concentrations after 12 hours, although at high concentrations it significantly increased CYP11B1 and CYP11B2 transcriptional expression compared to control, and affected AngII effects. A779 did not significantly blunt the effects of Ang-(1–7) at high doses whereas irbesartan abolished these effects. Conclusions: These results showed the expression of AT2R and MasR in human adrenal cortex, albeit at much lower levels than the AT1R. They suggested a role of high local concentrations of Ang-(1–7) in the modulation of aldosterone and cortisol production.

EXPRESSION OF THE ADIPOKINE CTRP-1 IN THE HUMAN ADRENAL GLAND: A LINK BETWEEN HYPERALDOSTERONISM AND HYPERTENSION IN OVERWEIGHT-OBESE PATIENTS?: PP.18.171

Objective: CTRP1 (Complement-C1q TNFα-related protein), a recently identified adipokine, was found to stimulate aldosterone production in the adrenocortical carcinoma cell line H295R, suggesting that it represents a pathophysiologic link between hyperaldosteronism and hypertension in overweight-obese patients. Thus, we investigated whether CTRP-1 is expressed in the human adrenal gland and aldosterone producing adenomas (APAs) and sought to identify the cell types that synthesize CTRP-1. Methods: In the periadrenal fat (n = 4), the normal adrenal tissue (n = 6), APA (n = 7), pheochromocytoma (n = 5) and myelolipoma (n = 2) CTRP-1 gene expression was quantified with real-time RT-PCR and its protein expression with immunohistochemistry (IHC) and immunoblot. Results: CTRP-1 gene was expressed in the periadrenal fat, the normal adrenal tissue, and all examined pathologic adrenal tissues. CTRP-1 protein in the adrenal gland was confirmed with immunoblot and IHC. Noteworthy in 2 APA with concurrent myelolipoma the latter showed a marked immunostaining for CTRP-1 not only in the adipose cells but also in the hematopoietic tissue. IHC and cytofluorimetry experiments revealed that cells expressing CTRP-1 are of myeloid lineage. Conclusions: The finding that CTRP-1 expression is not confined to adipocytes, but involves also normal and pathologic adrenocortical tissues supports the contention that CTRP-1 modulates aldosterone synthesis in both the normal zona glomerulosa and in APA, albeit through unknown receptors and signalling mechanisms. The expression of CTRP-1 in myelolipomas concurrent with APA suggests that CTRP-1 might trigger aldosterone excess and ZG cell growth.