Altered Expression of AQP1 and AQP4 in Brain Barriers and Cerebrospinal Fluid May Affect Cerebral Water Balance during Chronic Hypertension

Abstract

Hypertension is the leading cause of cardiovascular affection and premature death worldwide. The spontaneously hypertensive rat (SHR) is the most common animal model of hypertension, which is characterized by secondary ventricular dilation and hydrocephalus. Aquaporin (AQP) 1 and 4 are the main water channels responsible for the brain’s water balance. The present study focuses on defining the expression of AQPs through the time course of the development of spontaneous chronic hypertension. We performed immunofluorescence and ELISA to examine brain AQPs from 10 SHR, and 10 Wistar−Kyoto (WKY) rats studied at 6 and 12 months old. There was a significant decrease in AQP1 in the choroid plexus of the SHR-12-months group compared with the age-matched control (p < 0.05). In the ependyma, AQP4 was significantly decreased only in the SHR-12-months group compared with the control or SHR-6-months groups (p < 0.05). Per contra, AQP4 increased in astrocytes end-feet of 6 months and 12 months SHR rats (p < 0.05). CSF AQP detection was higher in the SHR-12-months group than in the age-matched control group. CSF findings were confirmed by Western blot. In SHR, ependymal and choroidal AQPs decreased over time, while CSF AQPs levels increased. In turn, astrocytes AQP4 increased in SHR rats. These AQP alterations may underlie hypertensive-dependent ventriculomegaly.

Keywords: SHR; aquaporins; cerebrospinal fluid; choroid plexus; ependyma; hydrocephalus; hypertension.

Figure 1

Confocal and optical microscopy images of the immunostaining of AQP1 in the ChP of WKY and SHR rats. An increase of AQP1 was observed in the ChP of SHR in the 6 months group when compared to WKY (a,c,e,g), while in the 12 months group, a decrease of AQP1 was observed in the SHR (b,d,f,h) rats. At the bottom, stain intensities values in relative units (gray units) for AQP1 (i,j) are represented as the means ± SD (n = five animals per group). The differences between WKY and SHR were significant. One-way ANOVA test with post hoc analysis using the Tukey post hoc test (* p < 0.05) was applied. LV: lateral ventricle; ChP: choroid plexus; GU: grey units. Scale bars 40 μm.

Figure 2

Confocal microscopy images of AQP4 in the ependymal cells of the lateral ventricle of WKY and SHR rats. A slight decrease of AQP4 was detected in the ependyma of SHR in the 6 months group when compared to WKY (a–d), while in the 12 months group, a significant decrease of AQP4 was observed in the SHR (e–h) rats. White arrows point to AQP4 immunoreactive material (b). At the bottom, stain intensities in relative units (gray units) for AQP4 (i,j) are represented as the means ± SD (n = five animals per group). The differences between WKY and SHR were significant when applied to a One-way ANOVA test with post hoc analysis using the Tukey post hoc test (* p < 0.05). LW: lateral wall; SVZ: subventricular zone; EP: ependyma; LV: lateral ventricle; GU: grey units. Scale bars 20 μm.

Figure 3

Confocal and optical microscopy images of AQP4 in the blood vessels of brain parenchyma of WKY and SHR rats. A significant increase of AQP4 was observed in SHR in the 6 months group when compared to WKY (a,b,e,f), in the 12 months group was found a more significant presence of AQP4 in the SHR (c,d,g,h) rats. Stain intensities in relative units for AQP4 (i,j) are represented as the means ± SD (n = five animals per group). The differences between WKY and SHR were significant when applied to a One-way ANOVA test with post hoc analysis using the Tukey post hoc test (* p < 0.05). BV: blood vessel; GU: grey units. Scale bars 10 μm.

Figure 4

Variation in the timeline of AQP values in CSF. Representation of age-related mean values of AQP1 and AQP4 in CSF of WKY and SHR are shown in (a) and (b), respectively. Expression of AQP1 and AQP4 in CSF (c). Graphic representation of values of AQP1 and AQP4 in CSF by Western blot (d,e).

Figure 5

Schematic representation of the expression of AQP1 and AQP4 in 6 months and 12 months WKY and SHR rats. In 6 months and 12 months WKY (a,c), rat expression of AQP 1 is detected in the apical pole of the ChP epithelium associated with active production of CSF. It is also found in the CSF, most likely associated with EVs. Per contra, AQP4 is expressed in the astrocyte end-feet and the basolateral membrane of the ependymal cells associated with brain CSF homeostasis. In addition, it is found in the CSF. In 6 months SHR rats (b), AQP1 expression is significantly increased in the apical pole of the ChP epithelium, and AQP4 expression increases in the astrocytes end-feet but not in the ependymal cells. This AQP disbalance may contribute to the mechanisms that trigger ventriculomegaly. In the CSF, the expression of AQPs remains the same as in 6 months WKY. In the 12 months SHR rats (d), AQP1 expression significantly decreased in the ChP epithelium while proportionally increasing in the CSF compared to 12 months WKY (c). Thus, it could reduce CSF production as a possible compensatory mechanism for ventriculomegaly. In turn, AQP4 decreased in the ependymal layer while it increased in the CSF and in the astrocytes’ end-feet, which could also contribute to reducing the production of CSF (ependymal layer) and the increase of the CSF absorption (astrocyte end-feet).