RNAi silencing of brain klotho potentiates cold-induced elevation of blood pressure via the endothelin pathway

Abstract

Klotho is a recently identified antiaging gene. Brain endothelin-1 (ET1) is important in the regulation of blood pressure (BP). We hypothesized that silence of brain klotho potentiates cold-induced elevation of BP via the endothelin pathway. To silence brain klotho, we constructed adeno-associated virus (AAV) carrying rat klotho small interference hairpin RNA (KL-shRNA). AAV carrying ET1-shRNA was used to silence brain ET1. Scrambled shRNA was used as Control-shRNA. Three groups of male Sprague-Dawley rats (6 rats/group) received KL-shRNA, KL-shRNA plus ET1-shRNA, and Control-shRNA, respectively, via intracerebroventricular injection. BP was monitored daily using a telemetry system. All animals were exposed to a moderate cold environment (5°C) at 12 days after gene delivery. KL-shRNA significantly increased BP by 9 days of exposure to cold, while BP in the Control-shRNA group remained unchanged. ET1-shRNA abolished KL-shRNA-induced elevation of BP during cold exposure. Interestingly, KL-shRNA increased brain ET1 expression and plasma norepinephrine level, suggesting that silencing of brain klotho increased ET1 production and the sympathetic nervous activity. The KL-shRNA-induced increase in sympathetic nervous activity was mediated by ET1 because it could be abolished by silencing of ET1. These results demonstrated that silencing of brain klotho potentiated and expedited cold-induced elevation of BP by upregulation of ET1 and the subsequent activation of the sympathetic nervous system.

Fig. 1.

Inhibition efficiency of KL-shRNAs and ET1-shRNAs in cell cultures. Two AAV.KL-shRNAs and two AAV.ET1-shRNAs were tested for inhibition efficiency in cell cultures before packaging of the recombinant AAVs for in vivo study. A: quantitative analysis of klotho protein expression. B: representative Western blot bands of klotho protein expression (130 kDa) and β-actin (42 kDa). C: quantitative analysis of ET1 mRNA expression. D: representative bands of ET1 mRNA expression (113 bp) and β-actin (444 bp) by semiquantification RT-PCR. E: quantitative analysis of klotho mRNA expression. F: representative bands of klotho mRNA expression and β-actin. KL, klotho; ET, endothelin; shRNA, short hairpin RNA; AAV, adeno-associated virus; Data = means ± SE. *P < 0.05, **P < 0.01; n = 3 independent experiments.

Fig. 2.

Effect of silencing of brain klotho on the blood pressure (BP) in cold-exposed rats. BP was monitored using a telemetry system. A, B, C, systolic, diastolic (D-BP), and mean blood pressure (M-BP), respectively. D, E, F, net changes of systolic, D-BP, and M-BP, respectively, compared with preinjection or precold exposure levels. Data = means ± SE. *P < 0.05, n = 6.

Fig. 3.

Klotho and ET1 expression in choroid plexus (CP) of the brain. A: gel graphs represented klotho and ET1 bands by qRT-PCR. B and C: the relative level of klotho and ET1 mRNA. D: immunohistochemical analysis of klotho (top) and ET1 (bottom) protein expression in CP and ependymal cells of the cerebroventricles. Arrows indicate klotho or ET1 expression in CP and ependymal cells of cerebroventricle. E and F: relative level of klotho and ET1 protein expression, respectively. Microphotos were taken at ×60 (oil) magnification. Data = means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001; n = 4–6.

Fig. 4.

The expression of ET receptors in CP of the brain. A: ETA receptor (260 bp) and ETB receptor (208 bp) mRNA bands in agarose gels. B and C: relative level of ETA receptor and ETB receptor mRNA, respectively, in CP and ependymal cells of the brain. The data shown in the graph were normalized with β-actin. D: immunohistochemical analysis of ETA and ETB receptor protein expression in the CP of the brain. Arrows indicate ETA or ETB expression in CP and ependymal cells of cerebroventricle. E and F: relative levels of ETA and ETB receptor protein, respectively, in CP and ependyma of cerebroventricles. Microphotos were taken at ×60 (oil) magnification. Data = means ± SE. *P < 0.05, **P < 0.01, and ***P < 0.001; n = 4–6.

Fig. 5.

Effects of silencing of brain klotho on plasma level of norepinephrine. Data = means ± SE. **P < 0.01, n = 6.