The Crosstalk between Calcium Ions and Aldosterone Contributes to Inflammation, Apoptosis, and Calcification of VSMC via the AIF-1/NF- κ B Pathway in Uremia

Abstract

Vascular calcification is a major complication of maintenance hemodialysis patients. Studies have confirmed that calcification mainly occurs in the vascular smooth muscle cells (VSMC) of the vascular media. However, the exact pathogenesis of VSMC calcification is still unknown. This study shows that the crosstalk between calcium and aldosterone via the allograft inflammatory factor 1 (AIF-1) pathway contributes to calcium homeostasis and VSMC calcification, which is a novel mechanism of vascular calcification in uremia. In vivo results showed that the level of aldosterone and inflammatory factors increased in calcified arteries, whereas no significant changes were observed in peripheral blood. However, the expression of inflammatory factors markedly increased in the peripheral blood of uremic rats without aortic calcification and gradually returned to normal levels with aggravation of aortic calcification. In vitro results showed that there was an interaction between calcium ions and aldosterone in macrophages or VSMC. Calcium induced aldosterone synthesis, and in turn, aldosterone also triggered intracellular calcium content upregulation in macrophages or VSMC. Furthermore, activated macrophages induced inflammation, apoptosis, and calcification of VSMC. Activated VSMC also imparted a similar effect on untreated VSMC. Finally, AIF-1 enhanced aldosterone- or calcium-induced VSMC calcification, and NF-κB inhibitors inhibited the effect of AIF-1 on VSMC. These in vivo and in vitro results suggest that the crosstalk between calcium ions and aldosterone plays an important role in VSMC calcification in uremia via the AIF-1/NF-κB pathway. Local calcified VSMC induced the same pathological process in surrounding VSMC, thereby contributing to calcium homeostasis and accelerating vascular calcification.

Figure 1

Histological analysis of radial artery in MHD patients. (a, b) Calcification in radial artery was evaluated by Von Kossa staining. (c–h) Expression of CYP11B2, MR, and AIF-1 in radial arteries was evaluated by immunohistochemical staining.

Figure 2

Aortic calcification and inflammatory factors in uremic rats. (a) Calcification in aortic artery was evaluated by Von Kossa staining. (b–f) Expression of CYP11B2, MR, AIF-1, and CCR-2 was evaluated by western blot assay (n = 8). (g) NF-κB activity was evaluated by western blot assay (n = 8). Data were represented by means ± SD. Versus normal control group, ^∗P < 0.01; versus artery calcification group, #P < 0.01.

Figure 3

The effect of calcium ions on inflammation, apoptosis, and calcification in VSMCs. (a) Calcified nodules of VSMCs were detected by alizarin red staining. (b) Apoptosis of VSMCs was evaluated by flow cytometry. (c) Production of aldosterone in VSMCs was regulated by calcium ions. (d) Production of MCP-1 in VSMCs was regulated by calcium ions. (e–h) Expression of AIF-1, CCR-2, and NF-κB in VSMCs was evaluated by western blot assay. Data were represented by means ± SD from three independent experiments. Versus normal calcium ions, ^∗P < 0.01.

Figure 4

The effect of aldosterone on intracellular calcium ions and inflammation in VSMCs. (a) Intracellular calcium ions in VSMCs were affected by aldosterone. (b) Production of MCP-1 in VSMCs was regulated by aldosterone. (c–f) Expression of AIF-1, CCR-2, and NF-κB in VSMCs was evaluated by western blot assay. Data were represented by means ± SD from three independent experiments. Versus normal control group, ^∗P < 0.01; versus aldosterone group, #P < 0.01.

Figure 5

The effect of activated macrophages on inflammation and calcification in VSMCs. (a) Calcified nodules of VSMCs were detected by alizarin red staining. (b) Expression of AIF-1, CCR-2, and NF-κB in VSMCs was affected by activated macrophages. (c) Intracellular calcium ions, aldosterone, and MCP-1 in VSMCs was affected by activated macrophages. (d) The effect of activated VSMCs on the expression of AIF-1, CCR-2, and NF-κB in untreated VSMCs. (e) The effect of activated VSMCs on intracellular calcium ions, aldosterone, and MCP-1 in untreated VSMCs. Data were represented by means ± SD from three independent experiments. Versus normal group, ^∗P < 0.01.

Figure 6

The effect of AIF-1/NF-κB on inflammation, apoptosis, and calcification in VSMCs induced by aldosterone. (a) Calcified nodules of VSMCs were detected by alizarin red staining. (b) Apoptosis of VSMCs was evaluated by flow cytometry. (c) The effect of AIF-1/NF-κB on intracellular calcium ions, MCP-1, and ALP activity in VSMCs exposed to aldosterone. (d) The effect of AIF-1/NF-κB on inflammatory factors in VSMCs exposed to aldosterone. Data were represented by means ± SD from three independent experiments. Versus control group, ^∗P < 0.01; versus AIF-1 overexpression group, #P < 0.01.