| BackgroundSince Goldblatt adopted the method of clamping the dog bilateral renal arteries to induce a risein the blood pressure in 1934, using clamping renal arteries to obtain hypertension model hasbeen more and more widely applied in studying the cardiovascular diseases, the most commonone being the Two-Kidney, One Clip method. It has been demonstrated that angiotensinII(AngII) plays an important role in developing and maintaining the pressure of hypertensionmodels by the method of Two-Kidney, One Clip. There are two main receptor subtypes ofangiotensinII, type 1 (AT1) and type 2 (AT2) receptors, and AngII primarily acts on AT1,exerting effects on the constriction of blood vessels as well as the proliferation of cells.Furthermore, there exist two subtypes of AT1, AT1a and AT1b. Studies reported that AT1aplayed a major role in regulating the blood pressure, whereas AT1b was also involved in such aprocess. With the progress of transgenesis and gene knockout technologies, the mouse has beenused on a growing scale in investigating the cardiovascular diseases. Consequently, obtainingrenovascular hypertension models of mouse successfully appears necessary. However, rats aremainly chosen to obtain this model interiorly, and there is little information about using themouse to get the renovascular hypertension models by the method of Two-Kidney, One Clip. Inaddition, it is still unclear how the condition of hypertension influences the expression of AT1receptor subtype.ObjectiveHere, we established the mouse renovascular hypertension model by the Two-Kidney, One Clipmethod, and used it to investigate the effects of hypertension on the AT1 receptor subtypeexpression levels, aiming to provide some theoretical basis for the fundamental research as wellas prevention and treatment of hypertension diseases. MethodMethodsHealthy male C57BL/6 mice (Class: SPF; Age: 8-12 weeks old; weight: 24-28 grams) weresupplied by Experimental Animal Center, Shantou University Medical College. Seventeen micewere randomly divided into two groups: the sham operation group (Sham, eight) and the Two-Kidney, One Clip group (2K1C, nine). In the 2K1C group, right-side renal artery was clampedwith a U-shaped silver clip, the width of whose opening was 0.12 mm. The operation on mice inthe Sham group was similar to that of the 2K1C except that silver clip wasn’t settled in the rightrenal artery. The time was defined as 0 day on the day of operation, and systolic blood pressureof the conscious mice was observed by tail blood pressure. Mice whose systolic blood pressurewas higher than 140mmHg on the 27th day were chosen as the hypertensive ones. Mice wereexecuted on the 28th day after operation. Then the body weight along with the weight of heartand bilaterally kidneys was obtained, before the ratio of heart weight to body weight and that ofbilaterally kidneys to body weight were analyzed (mg/g); HE staining was applied to observethe pathological changes in the heart and kidney of mouse; Reverse transcription polymerasechain reaction (RT-PCR) and restriction enzymes(EcoRI and HapII)were used to analyze theexpression levels of AT1 receptor in the mouse heart, kidney, adrenal gland, hypothalamus,pituitary, brain stem and abdominal aorta; Examination system of vessel ring tension wasadopted to test contraction responses induced by AngII in the isolated abdominal aorta frommice; Statistical soft system of SPSS 16.0 was chosen to analyze the data.Results1. In the experiment, the systolic blood pressure in the Sham group was between 101.9±14.4mmHg and 114.1±17.1mmHg, no rise being observed, whereas the systolic blood pressure ofmouse in the 2K1C group showed a clear upward trend, with the highest level being156.0±17.7mmHg (the 23th day). On the 27th day, the systolic blood pressure of the Sham groupand 2K1C group was 114.1±17.1mmHg and 150.9±11.9mmHg respectively. There wassignificant difference between the two groups (P <0.01).2. On the 28th day after operation, there was no significant difference in the body weightbetween the two groups. Compared with that in the Sham group, the levels of the heart weight, the left-side kidney weight as well as the ratio of heart weight to body weight and ratio of theleft-side kidneys to body weight were higher, whereas the right-side kidney weight and the ratioof the right-side kidneys to body weight were remarkably decreased. There was significantdifference between the two groups (P <0.01).3. The volume in right-side kidneys of 2K1C group was obviously decreased in contrast withthat in the Sham group. Results by HE staining show that the diameters of myocardium cellswere thickened and the glomerulus of kidney were compensatory hypertrophic in the left-sidekidney, whereas part of the glomerulus and renal tubules were atrophied in the right-sidekidneys of the 2K1C group. Additionally, the disarrangement of smooth muscle cells and theincrease of extracellular matrix in the left-side kidney arterioles were observed in the 2K1Cgroup.4. It was found that the AT1 subtype was dominantly expressed in the heart, kidney, adrenalgland, hypothalamus, pituitary and brain stem of the Sham group along with 2K1C group bymeans of RT-PCR and restriction enzymes(EcoRI and HapII), without obvious difference beingobserved between the two groups. However, in the Sham group, AT1b subtype was dominantlyexpressed in the abdominal aorta, whereas AT1a subtype was dominantly expressed in theabdominal aorta in the 2K1C group.5. The contraction responses induced by 10nM AngII in the isolated abdominal aorta from micewere 49.0±13.8% and 25.6±11.2%, respectively. The difference between the two groups wasstatistically different (P <0.05).ConclusionConclusions1. The increase of mouse systolic blood pressure and the morphological changes of the heart aswell as the kidney indicate that we have successfully established the 2K1C renovascularhypertension model with the C57BL/6 mice.2. In 2K1C hypertension model, AT1a becomes the predominant subtype in abdominal aorta,and this is accompanied by a decrease in vasoconstrictor response to AngII, suggesting thatAT1a may plays a role that is distinct from 1b in the vascular wall during the development ofhypertension. |
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