Intralymphocyte magnesium decrease in patients with primary aldosteronism. Possible links with cardiac remodelling

ARTICLE

Auteur(s) : Pietro Delva, Alessandro Lechi

Department Biomedical and Surgical Sciences, University of Verona, Verona, Italy.

Introduction

Hyperaldosteronism is characterised by magnesium deficiency, yet the data which could explain this phenomenon are extremely limited and refer mainly to plasma and urinary magnesium.

Aldosterone influences renal magnesium handling, causing magnesium wasting [1]. The acute administration of mineralocorticoids fails to modify magnesium and calcium excretion in humans [2] though clinical and experimental data suggest that the excretion of magnesium may be affected by long-term action of mineralocorticoids [1]. Very few data are available on plasma magnesium in subjects with primary aldosteronism. In 1954, Mader and Iseri reported one case with hypomagnesemia [3]. In 1962, Horton and Biglieri described five cases with an increased renal magnesium clearance despite normal plasma magnesium levels [1]. Recently, Resnick and Laragh described ten cases of patients with primary aldosteronism with normal plasma magnesium values [4].

As far as the role of aldosterone on cellular magnesium homeostasis are concerned, very few data are available so far. Our group have described a group of 16 patients with primary aldosteronism which is characterized by significantly decreased intralymphocyte ionized magnesium concentration [5]. Furthermore, in vitro, the effect of aldosterone appears to be mediated by the specific receptor of the hormone in that the decrease in intracellular ionized magnesium is not observed when canrenoic acid, a specific antagonist of the receptor is present. Moreover, the effect of the hormone seems to involve the classic genomic pathway of steroid action since it is completely eliminated by actinomycin D and cycloeximide, inhibitors of transcription and protein synthesis respectively. The effects of aldosterone on the intralymphocyte contents of ionized magnesium appear to be dose-dependent with a half-maximal effect (EC₅₀) of approximately 0.5-1 nmol/l of aldosterone [5].

One of the main causes of myocardial fibrosis is aldosterone. An increase in the concentration of plasma aldosterone induces biventricular fibrosis in rats and can be prevented by spironolactone [6]. The administration of DOCA in uninephrectomized rats leads to an increase in fibrous tissue [7]. Aldosterone induces collagen synthesis in fibroblasts in culture [8]. Finally, ACE inhibitors, even at non-hypotensive doses, prevent myocardial fibrosis in pressure-overloaded hearts [9]. From a clinical point of view, patients with suprarenal adenoma present a large perivascular fibrosis of the coronary and systemic arteries [10].

What is lacking in the hypothetical chain linking a negative magnesium balance and fibrosis is the demonstration that a decrease of extra or intracellular magnesium may induce an augmented synthesis of collagen. Consequently, to shed some light on the relationships between aldosterone and tissue fibrosis we have tested the in vitro effects of incubating human fibroblasts in low magnesium medium on mRNA collagen I and III gene expression.

Methods

Cell Cultures. We incubated cells from a human lung-derived fibroblast cell line (SVG1) in the presence (MgSO₄ 0.8 mmol/l) and in the absence of extracellular magnesium for 24 hours and then we measured collagen I and III mRNA gene expression. Cells were cultured in Ham F12 containing 10% fetal calf serum (FCS) and, when needed, were made quiescent through incubation with FCS 0.4% for 24 hours. The FCS utilized was previously dialysed overnight.

Measurement of collagen I and III mRNA expression. The method has already been described in detail [11]. Briefly, for studying mRNA collagen expression and histone H3 expression, the latter as an index of proliferation rate, cells at confluence were harvested and homogenized in 4Hguanidine thiocyanate and the homogenate was stratified over CsCl and centrifuged at 130,000 g for 17 hours. Five m g of total RNA was fractionated by electrophoresis on a 1% agarose gel containing formaldehyde and then blotted on Hybond N membrane. UV-irradiated filters were then hybridized with a random-primed [32g] collagen I, collagen III or histone H3 cDNAs probe. The intensity of the bonds was quantified using Phosphor Imager (Molecular Dinamics).

Results

We measured the effect of incubating human fibroblasts in two different state of proliferation. In cells made quiescent with a low percentage of FCS (0.4%) and in proliferating cells i.e. cells incubated in culture medium with 10% of FCS added. The two different conditions of proliferation were checked by measuring the istone H3 gene expression which was clearly decreased in cells incubated with low FCS, percentage as compared to cells incubated in 10% FCS, as shown in figure 1. We incubated human fibroblasts in both a state of proliferation and in a condition of magnesium deprivation for 24 hours and then we measured the mRNA collagen I and III gene expression. Both collagen I and III mRNA gene expression were increased by magnesium deprivation as shown in figure 1. The increase in collagen expression was similar for both collagen I and III and for the two states of proliferation considered.

Discussion

Our results show that by depriving the cell culture medium of magnesium there is an increase in the expression of the two genes assigned to the synthesis of collagen. The effect of extracellular magnesium deprivation does not seem to be linked to the actual state of cell proliferation as demonstrated by the similar results obtained in cells incubated in medium with either high FCS (10%) or low (0.4%) FCS concentration. These data are in favour of a potential link between magnesium and collagen synthesis. An obvious limitation of the present study relies on the in vitro model which is far from the physiological condition in that the cells underwent 24 hours of complete magnesium deprivation. Despite this limitation we believe that these preliminary data confirm the anecdotal reports of increased fibrosis in animal models of magnesium deficiency. Shivakumar et al. describes in rats that the aortas of rats on a magnesium-deficient diet demonstrate an increase in the rate of collagen synthesis [12]. The same authors reported a similar increase in collagen synthesis in the heart of rats on a magnesium-deficient diet [13].

A physiopathologic mechanism linking magnesium homeostasis to the state of collagen turnover may have important clinical correlates. In fact, it may provide partial explanations for the effects of spironolactone in congestive heart failure. A group of 1,633 patients with moderate-severe heart failure was studied and an improvement in mortality of 30% was shown when a low dose of spironolactone (25 mg daily) was added to the usual therapy of a combination of diuretics, ACE-inhibitors and digoxin. The decrease in mortality in the group randomized to treatment with spironolactone applied to cases of both progressive heart failure and of sudden death [14]. We might hypothesize that spironolactone produces some of its beneficial effects by restoring a normal magnesium homeostasis in these patients who are characterized by a negative magnesium balance (for review see 15). This latter may produce a cardiac fibrosis and remodelling resulting in an increased mortality. A normal magnesium balance may thus restore a normal collagen turnover.

Acknowledgement

The authors wish to thank Prof. Marta Menegazzi for technical assistance in northern blot analysis.

References

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