| BackgroundCardiovascular dieases are the most common complication of the ESRD patients. Arterial stiffness is now considered an independent risk factor for the progression of cardiovascular and chronic kidney disease (CKD). Results of animal and human experiments suggest that central arteries stiffnes is associated with adverse cardiovascular outcomes in many different patient groups and in general population. The representative arterial changes in CKD patients are arteriosclerosis and atheroscleros. In fact, there are some connections between arteriosclerosis and atherosclero. Arteriosclerosis is prone to progress into atherosclero. Media thickness, medial elastin fatigue and a consequent increased loading on stiffer collagen fibres are pathological characteristics of arteriosclerosis.For million years the ancestors of humans ate a diet containing less than 0.25 g of salt per day. We Chinese discovered that salt could be used to preserve food 5000 years ago.Then salt became of great economic importance as it was possible to preserve food during the winter and allowed the development of settled communities. Salt was the most taxed and traded commodity in the world, with intake reaching a peak around the 1870s. Salt consumption had been declining with the invention of refrigerators because salt was no longer required as a preservative. However, with the high prevailing of highly salted processed food, salt intake is now increasing towards levels similar to those of the 1870s, and is approximately 9-12 g/day,50 times more than our evolutionary salt intake. These changes of salt intake may raise a question that whether high salt intake will bring a health problem. Evidence show that high salt intake causes a rise in blood pressure (BP), thereby increasing the risk of cardiovascular disease (CVD-strokes,heart attacks and heart failure) and renal disease. Furthermore, high salt intake has direct harmful effects (increasing the risk of stroke, left ventricular hypertrophy, progression of renal disease and proteinuria) independent of but additive to the effect of salt on blood pressure. There is also increasing evidence that salt intake is indirectly related to obesity through soft drink consumption,associated with an increasing risk of renal stones and osteoporosis,and is probably a major cause of stomach cancer. Therefore, the harful effects of salt intake can not be neglected.It has been well studied that the renin-angiotensin system (RAS) or the renin-angiotensin-aldosterone system (RAAS) is a hormone system that regulates blood pressure and water (fluid) balance. More attentions have been paid on the effects of local RAS in diseases. Locally expressed renin-angiotensin systems have been found in a number of tissues, including the kidneys, adrenal glands, the heart, vasculature andnervous system, and have a variety of functions, including local cardiovascular regulation, in association or independently of the systemic renin-angiotensin system, as well as non-cardiovascular functions. However, in the process of some disease such as atherosclerosis, myocardial fibrosis and chronic kidney disease, local RAS activates in those target organs.Our previous work suggested that high salt intake caused local RAS activation in kidney, fat tissue and brain. Local RAS activation was thought to be related with the pathological changes in those organs.However, the exact effects of high salt intake on aorta in patients of chronic kidney disease (CKD) remain unknown.In this study, we used rat model with 5/6 nephrectomy to observe the effects of different salt intake on the aorta as well as the potential mechanism of local rennin-angiotensin system. We also detected the effects of renin-angiotensin system inhibitor on aorta in 5/6Nx rats with high salt diet.Materials and methodsPreparation of Animal ModelAll animal procedures were approved by the Animal Experiment and Care Committee of the Sourthern Medical University. Male SD rats (Provide by Sourthern Medical University Animal Experiment Center, maintained under standardized conditions) initial weighting 150 to180g were chosed. After a week of adaptive feeding,5/6 nephrectomy surgery was done in two-step method as follows:Rats were in the prone position. Firstly,make sure the position of left kidney by feeling the right costovertebral angle(to find the kidney at accurate position), then the left hand holding the forceps to lift the corresponding part of the skin, right hand cut a 2cm incision with scissors,and then cut the muscle until the abdominal cavity. Pull out the perirenal fat of the kidney using tweezers. Strip the renal capsule from the lower pole to up (so as not to damage the adrenal gland).Then surgical resected of the two-thords of the left kidney then put the gelatin sponge on the two ends of the remanent kidney and with some press to stop bleeding. At the end of the operation, Suture the muscle, skin respectively. The sham operation procedure should be ended when the renal capsule was removed. A week later, the right kidney was removed by ligating the renal pedicle then carefully cutting the kidney from the renal pedicle.Grouping of animals1.The effects of different salt intake on the aorta in 5/6Nx rats:Ten weeks after the 5/6 nephrectomy or sham operation, rats were randomized into subgroups as follow. The period of salt intake is 2 weeks.(n=6)① Low salt diet (0.02%Nacl)+Sham;②Normal salt diet(0.4%Nacl)+Sham;③High salt diet(4%Nacl)+Sham;④Low salt diet(0.02%Nacl)+5/6Nx;⑤Normal salt diet(0.4%Nacl)+5/6Nx;⑥High salt diet(4%Nacl)+5/6Nx.2.Effects of blockade with various doses of losartan on aorta in 5/6Nx rats with high salt dietTen weeks after the 5/6 nephrectomy, rats were randomized into subgroups with 2 weeks of high salt diet and inhibitors respectively.①Losartan Omg/kg/d by intragastric gavage (IG):Rats were administrated with daily intragastric injection of endotoxin-free phosphate-buffered saline (PBS) (pH 7.4);② Losartan lmg/kg/d IG:Rats were administrated with daily intragastric injection of losartan(lmg/kg per day, Sigma Chemical, St Louis, MO, USA);③Losartan 50mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (50mg/kg per day);④Losartan 500mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (500mg/kg per day);⑤hydralazine 15mg/kg/d by intragastric gavage (IG):Rats were administrated with daily intragastric injection of endotoxin-free phosphate-buffered saline (PBS) (pH 7.4).3.Effects of central RAS blockade with losartan on aorta in 5/6Nx rats with high salt diet① Losartan Omg/kg/d by intracerebroventricular injection (ICV):Rats were administrated with daily intracerebroventricular injection of artificial cerebrospinal fluid (aCSF);②Losartan lmg/kg/d ICV:Rats were administrated with daily intracerebroventricular injection oflosartan(1mg/kg per day);Experimental data and specimen collection1. Blood pressure taken and urine sample collectionThree days before the end of the study period,24-hour urine samples were collected for three consecutive days, and the blood pressure was determined in conscious rats by the indirect tail-cuff method also.2. Blood sample, perfusion and tissue collectionAt the completion of each protocol, rats were anesthetized with pentobarbital sodium (40mg/kg, i.p.), Trunk blood was collected in chilled vacuum tubes, and plasma samples were separated and stored at -80℃ until assayed. After the slowly organ perfusion with cold normal saline, fresh aorta was taken out carefully. Part of the rat tissue were collected and stored at -80℃ for western blot and PCR analyses, while the others were fixed in 4%paraformaldehyde for 24 hours for HE stainig and immunohistochemical studies. Blood samples were collected for the dectection of renal function, Serum electrolytes and AngⅡ.3. Statistical AnalysesAll data represent more than three repeated experimental results, presented as mean±standard deviation, Continuous variables between groups werecompared using one-way ANOVA, followed by LSD method when P< 0.05.Nonparametric test is used when heterogeneity of variance. Statistical analyses wereconducted with SPSS 13.0 for Windows (SPSS, Chicago, IL). P<0.05 was considered statistically significant.Results1.Characteristics of basic parametersNo difference had been found between groups in body weight and food intake of rats. In 5/6Nx rats, high salt diet caused a significantly higher blood pressure, higher serum sodium level and heavier urine protein (P<0.05 vs normal salt diet). The expression of rennin-angiotensin system’s compound was suppressed with the increasing of dietary salt in both 5/6Nx group and sham group.2. Effects of different salt intake on aorta in ratsAfter the stimulation of different concentrations of salt intake for 2 weeks, aorta was fixed and made into pathological sections for HE staining as well as masson staining. The media thickness of aorta in 5/6Nx rats was significantly increased when comparing with the sham rats with the same normal salt diet(P<0.05).Media thickness of aorta in 5/6Nx rats increased significantly with high salt intake (P< 0.05), while no significant difference was seen in the sham rats(P>0.05).The result of masson staining reflected the collagen deposition in the aorta. 5/6Nx rats with high salt diet had the severest collagen deposition with the increase of Nacl concentration (P<0.05), however, there was no difference between the sham groups.3. Effects of different salt intake on the expression of local RAS compounds in aortaWe used methods of Western blot and immunochemistry to observe the protein expressions of local rennin-angiotensin system compounds, and Real time PCR for gene (mRNA) expressions. After the stimulation of different levels of salt intake for 2 weeks, fixed aorta was made into pathological sections for immunochemistry studies and fresh tissues were for western blot and real time PCR analyses. RAS compounds included AGT、ACE、Ang Ⅱ and AT1, however, Ang Ⅱ could not be detected by western bolt or PCR method. Results of immunochemistry showed that local RAS compounds of aorta in 5/6Nx rats increased significantly with the increasing dietary salt(P<0.05 vs normal salt diet), while no difference had been found in the sham groups.The results of western blot and real time PCR corresponds to that of the immunochemistry.4. Effects of blockade with various doses of losartan on aorta in 5/6Nx rats with high salt dietWe used methods of Western blot and immunochemistry to observe the protein expressions of local rennin-angiotensin system compounds, and real time PCR for gene (mRNA) expressions after the blockade of losartan. Expressions of local RAS compounds decreased in 50mg/kg/d losartan group. What’s more, the 500 mg/kg/d of Losartan suppressed the RAS expression to the lowest level (P<0.05 vs 50mg/kg/d Losartan IG). No obvious RAS suppressant effect was found in the hydralazine group though it decreased blood pressure significantly.After blocking the local RAS of aorta, we detected the effects of ameliorateing aorta stiffness. No difference was found between 1mg/kg/d losartan IG group and control group. However, when the dose of Losartan increased to 50 mg/kg/d, benefits of ameliorating aorta stiffness showed.50 mg/kg/d of Losartan IG significantly decresed the media thickness of aorta and lessen the collagen deposition (P<0.05 vs control group).500mg/kg/d losartan group had obvious therapeutic effect (P<0.05 vs 50mg/kg/d Losartan IG). No effect on aorta was found in the hydralazine group.5. Effects of central RAS blockade with losartan on aorta in 5/6Nx rats with high salt dietLosartan intracerebroventricular injection with dose of lmg/kg/d decreased the expression of local RAS significantly (P<0.05 vs Omg/kg/d losatan ICV) which had the same effect of as Losartan IG with dose of 500mg/kg/d (P>0.05 compared with Losartan 500mg/kg/d IG). After inhibiting the central RAS activation, Losartan ICV group had a pleased effect of thinning the aorta wall and lessening collagen deposition.ConclusionsIn this artical, we used rat model with 5/6 nephrectomy to observe the effects of different concentration of salt in diet on structural changes of the aorta as well as the potential mechanism of local rennin-angiotensin system. We also detected the effects of renin-angiotensin system inhibitor in ameliorateing aorta stiffness.Our results suggested that a high salt diet caused aorta stiffness by thickening the media thickeness and increasing collagen deposition via the local RAS activation. Losartan administration via both intragastric gavage and intracerebroventricular injection ameliorated the process of arterial stiffness. The effects were dose-dependent. No therapeutic effect was found in the hydralazine group. It suggested that the harmful effect of high salt diet on aorta was blood pressure independent. |
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