| Objective:1. To build up cystic fibrosis transmembrane conductance regulator (CFTR) gene knockout model;2. To observe the effect of CFTR on blood pressure and vascular function;3. To explore the novel role of CFTR in high fructose and salt-induced hypertension and investigate mechanism pathway WNKs and oxidative stress.Methods:1. Breed CFTR knockout mice. Female and male heterozygous Stock Cftrtm1Unc Tg (FABPCFTR)1Jaw/J (CFTR+/-) were purchased from the Jackson Laboratory (Bar Harbor, Maine) and then housed in our animal facility. After breeding, genotyping was performed in those littermates and8-12week old homozygous CFTR+/+and CFTR-/-were used in this study. Mice were surgically implanted with radiotelemetry transmitters (HD-X11, Data Science International). After2week recovery period from surgery, blood pressure and ECG were measured and obtained in conscious, unrestrained mice (Dataquest ART4.3, Data Science International).2. Build up hypertension model. High fructose diet (64.7%fructose,17.7%protein,7.2%fat, cellulose, mineral mix, vitamin mix, choline bitartrate and TBHQ) and starch control diet (58.9%corn starch,17.7%protein and7.2%fat, maltodextrin, cellulose, mineral mix, vitamin mix, choline bitartrate and TBHQ) were obtained from Harlan Laboratory (Madison, Wisconsin). The high salt (2%sodium chloride) was given in drinking water by adding5g sodium chloride into250ml tap water. CFTR KO and CFTR WT mice were respectively divided into two groups:1) High fructose+high salt group (FS),65%fructose diet and2% salt drinking water for8weeks;2) Control group (Con), starch diet and regular drinking water for8weeks.3. Evaluate the effects of CFTR knockout on blood pressure, vascular function, protein expression and oxidative stress. Echocardiography and Doppler were performed using a Vevo2100Imaging System (VisualSonics, Toronto, Canada). Images were captured on cine loops at the time of the study and afterwards measured by the Vevo2100standard measurement package. The aortic arch was imaged from a suprasternal view to assess the descending aorta peak velocity (DAoPV) using pulsed-waved (PW) Doppler-mode assisted by Color Doppler-mode at angle of60degree. Peak systolic and end-diastolic renal artery blood velocities were measured using Color and PW Doppler-mode at angle of60degree in a supine position. Renal resistive index (RI) was automatically calculated by the Vevo2100standard measurement package. Real-time RCP and Western Blot were performed to detect CFTR and WNKs levels. Specific fluorescent probes were used to determine the ROS and GSH levels. In order to explore the further mechanisms, we isolated mitochondria and analyzed the mitochondria purity and function to make mitochondrial GSH intake assay.Results:1. CFTR knockout-induced changes in blood pressure and vascular functionWe monitored blood pressure by telemetry to assess whether CFTR knockout had effect on basal blood pressure. CFTR knockout significantly elevated24-hour blood pressure (△SBP=12mmHg and△DBP=10mmHg, all P<0.05). CFTR KO mice displayed not only higher daytime blood pressure but also higher nighttime blood pressure. CFTR disruption decreased aortic distensibility (12.7±2.1%versus7.4±3.0%, P<0.05).2. CFTR knockout impacts changes in blood pressure induced by high fructose and salt dietHigh fructose and salt diet significantly increased the BP of CFTR WT mice. SBP was increased by12mmHg and DBP was elevated by 9mmHg after high fructose and salt feeding (P<0.05). Hypertension development was ameliorated in CFTR KO mice. Only increased DBP in CFTR KO mice was obtained since week5of high fructose and salt diet.3. WNK1and WNK4regulate CFTR expressionAortic CFTR mRNA level was90%lower in WT FS group than WT Con group (1±0.06versus0.1±0.02, P<0.05) and mesenteric arterial CFTR protein expression was decreased by80%in WT FS group compared with WT Con group (1±0.02versus0.2±0.04, P<0.05). We found that high fructose and slat induced reduction in WNK1expression in aortas and mesenteric arteries. Aortic WNK1mRNA level was decreased by50%in WT mice (1±0.02versus0.5±0.02, P<0.05) and decreased by17%in KO mice (0.6±0.02versus0.5±0.07, P<0.05) after8-week high fructose and salt feeding. Mesenteric arterial WNK1protein expression was decreased by50%in WT mice (1±0.05versus0.5±0.01, P<0.05) and decreased by36%in KO mice (1.4±0.04versus0.9±0.1, P<0.05) after8-week high fructose and salt feeding. WNK4expression in vasculature of both WT and CFTR KO mice was also decreased after the administration of high fructose and salt. Aortic WNK4mRNA level was decreased by90%in WT mice (1±0.05versus0.1±0.006, P<0.05) and decreased by85%in KO mice (0.2±0.03versus0.03±0.00, P<0.05) after8-week high fructose and salt feeding. Mesenteric arterial WNK4protein expression was decreased by70%in WT mice (1±0.08versus0.3±0.01, P<0.05) and decreased by60%in KO mice (0.5±0.009versus0.2±0.01, P<0.05) after8-week high fructose and salt feeding.4. CFTR disruption mediates oxidative stressWe found that aortic CFTR mRNA level was90%lower in WT FS group than WT Con group and mesenteric arterial CFTR protein expression was decreased by80%in WT FS group compared with WT Con group. High fructose and salt diet induced an increase in ROS level of aorta from WT mice (1.8±0.05versus1±0.01, P<0.05). The aortic ROS level in CFTR KO mice was3.4-fold higher than that in WT mice (3.4±0.3versus1±0.01, P<0.05). We detected the mitochondrial ROS level and found the similar increase which suggests mitochondrial oxidative stress contributes to tissue elevated ROS. However the mitochondrial GSH intake assay showed mitochondrial GSH intake from CFTR KO mice was faster than that from WT mice while mitochondrial ROS decline from CFTR KO mice was slower than that from WT mice which supports decreased GSH is the result of elevated ROS. Conclusion:1. CFTR regulates basal blood pressure and vascular function;2. CFTR knockout resists to the elevation of blood pressure induced by high fructose and salt diet;3. The regulation pathway of CFTR involves WNKs expression and oxidative stress. |
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