Mineralocorticoid receptor is involved in rat and human ocular chorioretinopathy

Abstract

Central serous chorioretinopathy (CSCR) is a vision-threatening eye disease with no validated treatment and unknown pathogeny. In CSCR, dilation and leakage of choroid vessels underneath the retina cause subretinal fluid accumulation and retinal detachment. Because glucocorticoids induce and aggravate CSCR and are known to bind to the mineralocorticoid receptor (MR), CSCR may be related to inappropriate MR activation. Our aim was to assess the effect of MR activation on rat choroidal vasculature and translate the results to CSCR patients. Intravitreous injection of the glucocorticoid corticosterone in rat eyes induced choroidal enlargement. Aldosterone, a specific MR activator, elicited the same effect, producing choroid vessel dilation -and leakage. We identified an underlying mechanism of this effect: aldosterone upregulated the endothelial vasodilatory K channel KCa2.3. Its blockade prevented aldosterone-induced thickening. To translate these findings, we treated 2 patients with chronic nonresolved CSCR with oral eplerenone, a specific MR antagonist, for 5 weeks, and observed impressive and rapid resolution of retinal detachment and choroidal vasodilation as well as improved visual acuity. The benefit was maintained 5 months after eplerenone withdrawal. Our results identify MR signaling as a pathway controlling choroidal vascular bed relaxation and provide a pathogenic link with human CSCR, which suggests that blockade of MR could be used therapeutically to reverse choroid vasculopathy.

Figures

Figure 1. Induction of choroidal thickening by MR activation in rat eyes.

(A) In vivo OCT scans taken in the same region of the retina before and 24 hours after IVT of high-dose corticosterone (10 μM). Thickness of the choroid (arrows) was increased by corticosterone and associated with choroid vessel dilation (stars). Aldosterone (20 nM) also enhanced choroidal thickness and vasodilation. Scale bars: 200 μm. (B) Historesine sections illustrating increased choroidal thickness 24 hours after 10 μM corticosterone or 20 nM aldosterone compared with vehicle-injected eyes (NaCl; 0.9% added with 0.01% ethanol). Choriocapillaries (Cc) and choroid veins (CV) were dilated in corticosterone- and aldosterone-injected eyes. Arrowheads delineate the limit of the choroid and the sclera. ONL, outer nuclear layer; Chor, choroid. Scale bar: 25 μm. (C) Aldosterone-induced choroidal thickening occurred all along the retina (measured on serial photographs), from the optic nerve to the periphery. Values from aldosterone-treated (Aldo) eyes were higher than those of vehicle-injected (NaCl) eyes (n = 12–14 per condition). P < 0.001, vehicle vs. aldosterone, 2-way ANOVA. (D) Quantification of choroidal thickness measured on historesine sections. Aldosterone (20 nM) induced a significant increase in choroidal thickness compared with control vehicle-injected eyes; this was significantly inhibited by coinjection of aldosterone with 500-fold excess of a specific MR antagonist, but not a specific GR antagonist (n = 4–5 per condition). *P < 0.05, ***P < 0.001.

Figure 2. Expression and aldosterone regulation of KCa2.3 in rat eyes.

(A) Immunofluorescence localization of KCa2.3 and CD31 in the retina. KCa2.3 labeling was restricted to the choroid vessels, CD31 labeled both choroid and retinal vessels (arrowhead). Higher magnification of KCa2.3 labeling of choroid vessels (arrowheads) and nuclei (blue) is shown below. Scale bars: 100 μm (top); 25 μm (bottom). (B) KCa2.3 and CD31 were colocalized (arrowhead) in choroid vessel endothelial cells. Scale bar: 25 μm. (C) Colocalization of KCa2.3 and CD31 (arrowhead) in a choroid vessel. Scale bar: 25 μm. See Supplemental Figure 4 for lower magnification and separate signals of individual markers. (D) KCa2.3 expression was increased in aldosterone-injected eyes compared with vehicle injection, an effect prevented by coinjection with the MR antagonist canreonate (Canre) in 500-fold excess. Shown are Western blot on choroid samples and quantification of KCa2.3/β-actin, relative to vehicle-injected eyes (n = 8 per condition). **P < 0.01, ***P < 0.001.

Figure 3. Aldosterone-induced choroidal thickening depends on KCa2.3 activity in rat eyes.

(A) Historesine sections and quantification showed vasodilation of choriocapillaries and choroid veins 24 hours after aldosterone injection compared with vehicle-injected eyes (NaCl, 0.9% added with acetic acid 0.01%), as well as its reduction by coinjection with apamin (Apa), a specific KCa channel blocker (n = 5–6 per condition). Arrowheads delineate the limit of the choroid. Scale bar: 25 μm. (B) Quantification of vessel, extravascular, and total choroid area in tissue sections. Aldosterone injection led to a coordinate increase in area; apamin reversed this swelling, although the difference with respect to vascular area was not statistically significant (n = 5–11 per condition). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Choroid vasodilation and vascular leak after IVT aldosterone in rat eyes.

After ICG injection in the tail vein, successive confocal in vivo angiography images were taken at early time points (1 and 2.5 minutes) and at a late phase (24 minutes) of the angiographic sequence (n = 5 per group). Most superficial vessels (red arrowheads) are retinal vessels; the choroid is located in the deep regions (yellow arrowheads). Aldosterone-induced dilation of choroid, not retinal, vessels was observed at 1–2.5 minutes; at 24 minutes, the dye was washed out from vessels in the control eye, but stained focal vascular areas (arrows), attesting to the vascular leakage in aldosterone-treated eyes. Scale bar: 200 μm.

Figure 5. OCT and angiographic findings of CSCR Patient 1 before and after eplerenone treatment.

(A) OCT oblique scans of the right eye. At presentation, the patient exhibited a bubble of serous retinal detachment in the macular area, whose surface increased 4 months later. Initial treatment with 25 mg/d eplerenone resulted in a rapid decrease of subretinal fluid accumulation 1 week later. Eplerenone was then increased to 50 mg/d; a further reduction of the serous detachment was observed 1 week later. Near-complete resolution was observed 3 weeks after the beginning of the treatment. Eplerenone was withdrawn after 5 weeks of administration, and retinal morphology remained normal and stable 5 months after treatment interruption. Scale bar: 200 μm. (B) Fluorescein angiography of the right eye at presentation. The early phase of the angiogram (left) showed multiple RPE disruption white dots (arrows), and leakage at the late phase of the angiogram (right) inducing filling of the subretinal bubble with fluorescein (arrows). Scale bar: 2 mm. (C) Choroidal thickness before and after eplerenone treatment. Other OCT oblique scans focused on deep zones of the eye (choroid vessels): at presentation, choroid vessels were dilated, which was reduced on the same oblique sections of the same zone after 1 and 2 weeks of eplerenone treatment (50 mg/d). Scale bar: 200 μm. Red arrows indicate the orientation and location of the captured image; small arrows delineate the lower limit of the choroid.

Figure 6. Kinetics of OCT vertical scans (red arrows) of CSCR Patient 2 before and after eplerenone treatment.

In the right eye, OCT at presentation showed a bubble of serous retinal detachment (white arrows) close to the macula. Color mapping representation of the retinal surface showed red areas illustrating the extent of retinal detachment. Improvement was observed 15 days after beginning eplerenone treatment that remained stable at 3 weeks and 5 months after treatment was discontinued. In the left eye, OCT before treatment showed submacular fluid accumulation and intraretinal cysts (white arrows). Color mapping representation of the retinal surface showed red elevation at the center of the macula resulting from the fluid accumulation below and inside the retina. The retina recovered its normal morphology and structure with total disappearance of fluid 15 days after eplerenone treatment and 3 weeks and 5 months after eplerenone withdrawal. Scale bars: 2 mm (color mapping); 400 μm (OCT).