| BackgroundChronic renal disease has become a global public health problem with the increasing incidence of chronic kidney disease. Hypertension is the most common secondary disease of renal parenchymal disease. Hypervolemia and renin-angiotensin system (RAS) activation is always considered to be the major determinant of hypertension in chronic renal failure (CRF). For most patients with CRF, controlling these two factors can reduce blood pressure. Recent evidence strongly suggests that increased sympathetic nervous system (SNS) activity is also involved in hypertension. More and more studies suggest that the continued increasing of sympathetic nerve activity (SNA) is due to dysfunction of central regulation mechanism of blood pressure, which lead to chronic hypertension. Key nuclei that involve in this disorder include the organum subfornicale (SFO), the paraventricular nucleus (PVN) of the hypothalamus, the rostral ventrolateral medulla (RVLM) and the nucleus of solitary tract (NTS) of the brainstem. Among these nuclei, PVN plays a pivotal role in regulating blood pressure in both the normotensive and hypertensive states.Angiotensin II (Ang II) is a member of the RAS system, which is a powerful ingredient to increase blood pressure. Ang II increase blood pressure through a variety of mechanisms, including vasoconstriction, sodium retention, vascular remodeling and increasing sympathetic nerve activity. Pressurization of Ang II is mediated by the AT1receptor, including direct vasoconstriction and stimulating the release of another hormone. Ang II also participates in other mechanisms which lead to increased cardiovascular tension. However, Ang II is a kind of neuropeptide, can through the stimulation of AT1receptors, increased neuronal excitability of central cardiovascular regulation in the hypothalamus and brain stem. Ang II binding to the AT1-R robustly activates the mitogen-activated protein kinase (MAPK) intracellular signaling pathways. Three major terminal effector kinases of the MAPK family are the ERK1/2MAPK (also called extracellular signal regulated protein kinases, ERK1/2), the stress-activated protein kinase/c-Jun NH2-terminal kinases (JNKs), and the p38MAPK. MAPK signaling pathways regulates a variety of cellular biological processes such as gene expression, proliferation, differentiation, survival and death in normal developmental processes of the brain. Numerous studies have shown MAPK activation in Ang II related hypertension model.Norepinephrine induces MAPK activation and increases the activity of Ras in spontaneously hypertensive rats. Ras-related GTP-binding protein superfamily is a class of low molecular weight proteins, the molecular weight is about20to25kD, can be very tightly bonded to the guanylate. They have two forms contain the inactive GDP-bound form and an active GTP-bound form. Ras proteins are expressed in almost all types of cells, including fibroblasts and muscle cells, and activate signal transduction pathways that regulate cellular proliferation, differentiation, and survival. In addition to its involvement in cell proliferation and differentiation, however, p21ras has also been implicated in the regulation of programmed cell death recently. Apoptosis is a cell death increasingly recognized in recent years. It is a basic biological phenomenon and plays a necessary role in multicellular organisms to remove unwanted or abnormal cells. Recent studies show that high blood pressure is an important factor in strengthening the brain oxidative stress, and the circulation between high blood pressure and oxidative stress promote their interaction. Then, high oxidative stress activates proapoptosis mechanism. Apoptosis has been identified as a critical component of the ventricular remodeling associated with long-standing hypertension. Moreover, recent investigations indicate that an upregulation of the local RAS is a critical factor in the activation of myocyte apoptosis during pressure overload and heart failure. Hypertensive rats under stress would activate pro-apoptotic mechanism and resulte in neuronal loss. Now generally believed that caspase-3is the most important terminal cut enzyme in aprocess of apoptosis. Bcl-2family is involved in the regulation of apoptosis. Bc1-2and Bax proteins belong Bcl-2family. Bcl-2is an anti-apoptotic protein while Bax is a pro-apoptotic protein, the level of both protein is directly related to the apoptosis regulation.In view of the important position and role of central nervous system occurs in the development and blood pressure regulation in hypertension, study about the central nervous system of chronic renal failure state, especially whether the brain RAS system is activated, and the control mechanism of blood pressure will further deepen understanding the mechanism of hypertension of chronic renal failure and other chronic kidney disease.The present study first verify that Ang II and AT1R is activated in PVN of renal hypertension rats. Then, experiment was conducted to determine whether elevated AT1R activates caspase-3through the Ras/p38MAPK/ERK1/2pathway in PVN of renal hypertension rats, if so, to determine whether activation of this pathway is involved in the increased sympathoexcitation in renal hypertension rats. Toward this end, we examined the activity of Ras, p38MAPK, ERK1/2, proapoptotic proteins Bax, antiapoptotic protein Bcl-2, and caspase-3in the PVN of renal hypertension and Sham-operated rats. Then we performed intracerebroventricular (ICV) injections of a AT1R inhibitor, a Ras inhibitor, an ERK1/2inhibitor, a p38inhibitor and a caspase-3inhibitor, and examined the changes in blood pressure and SNA.MethodChapter Two:Study of the expresion of RAS/MAPK and apoptosis protein on brain nuclei PVN of renal hypertension rats. 1. Animal model preparationsSD male rats were maintained on a12/12h light/dark cycle at22℃and fed adlibitum a standard laboratory chow for a1week acclimation period. Subsequently, the animals were randomly assigned to two groups:sham operated (n=12) and five-sixth nephrectomy (5/6Nx)(n=12). Surgical5/6Nx and sham operations in rats were performed. Briefly, under pentobarbital anesthesia, two-thirds of the left kidney was removed in the first stage of the procedure, and a week later the right kidney was totally excised. Control animals were sham-operated with only decapsulation of the kidney.2. Blood pressure and urine collection24-hour urine samples were collected for three consecutive days before the end of the study period, and the blood pressure was determined via a pressure transducer placed in the femoral artery in anesthetized rats.3. The collection of sample3.1Blood collection and specimen collectionAt the completion of each protocol, rats were anesthetized with pentobarbital sodium (40mg/kg, i.p.), blood was collected in chilled vacuum tubes, and plasma samples were separated and stored at-80℃until assayed. To collect brain tissue for follow-up study, rats were perfused with200ml of ice-cold normal saline.The tissue of PVN was isolated from the brain and quickly placed in liquid nitrogenand then kept in the-80℃refrigerator until protein and total RNA extraction.The remaining rats were continued perfused with4%400ml paraformaldehyde, After completion, the brain was removed and cut into chunks according to the respective requirements, fixed for6hours, then dehydrated in a graded with different concentrations of alcohol, Finally, the samples were embedded in paraffin and sliced in5μm sheet. 3.2Serum creatinine was detected by clinical automatic biochemical analyzer.3.324h urinary protein was detected by Bradford method.4. Determination of AT1R, Ras, ERK1/2, P38, Bax, Bcl-2and caspase-3protein expression4.1Western blottingThe tissue of PVN was taken out from the-80℃refrigerator and transferred to1.5ml EP tubes, the lysate was added equal to the ratio of1:4to tissue weight, one5ml syringe was used to repeatedly pipetting samples on ice. After complete splitting, the lysate was incubated on ice for15min and then centrifuged at13,000g for15min at4℃. The supernatant was used to measure protein concentration by the Bradford method. Samples were diluted4:1(V/v)with5xLaemmli buffer, heated at95℃and then proteins were detected by western blotting. The actived Ras protein was first purified by a kit name "Ras Pull-Down and Detection". After this process, the actived Ras protein were detected by western blotting.4.2ImmunohistochemistryTaking three paraffin sections of PVN in each rat, after dewaxing and graded ethanol hydration, immunohistochemical staining was adopted, photographs were captured by the microscope and analyzed using Image-ProPlus software. Finally, the number of positive cells were counted.5. Determination of AT1R, Bax, Bcl-2mRNA by real-time PCRTotal RNA of the tissue of PVN was extracted with TRIZOL reagent. AT1R, Bax and Bcl-2mRNA were detected by real-time PCR according to the manufacturer protocol.6. StaticsAll data are presented as mean±SE. The independent t test is used between two groups. Significance was defined as P<0.05. Chapter three:Angiotensin Ⅱ Type1Receptor-triggered cell apoptosis through Ras/ERKl/2signaling in paraventricular nucleus mediates sympathetic excitation in renal hypertension rats.1. Preparation of Animal ModelSurgical five-sixth nephrectomy and sham operations in rats were performed. Control animals were sham-operated with only decapsulation of the kidney. Ten week after5/6Nx, the rats were randomized into8groups (n=5in each group) and treated as follows for14days, respectively:①Sham-operated rats with no treatment;②5/6Nx group with no treatment;③5/6Nx group with artificial cerebrospinal fluid (aCSF) ICV, rats were administrated with daily intracerebroventricular injection of artificial cerebrospinal fluid (aCSF);④5/6Nx group with Losartan ICV, rats were administrated with daily intracerebroventricular injection of losartan (1mg/kg per day, Sigma Chemical, USA);⑤5/6Nx group with farnesyl thiosalicylic acid (FTS) ICV, rats were administrated with daily intracerebroventricular injection of FTS (1mmol/L, Cayman Chemical, USA);⑥5/6Nx group with PD98059ICV, rats were administrated with daily intracerebroventricular injection of PD98059(20μmol/L, Sigma Chemical, USA);⑦5/6Nx group with SB203580ICV, rats were administrated with daily intracerebroventricular injection of SB203580(100μmol/L, Sigma Chemical, USA);⑧5/6Nx group with N-Benzyloxycarbonyl-Asp (OMe)-Glu (OMe)-Val-Asp-(OMe)-fluoro-methylketone (Z-DEVD-FMK) ICV, rats were administrated with daily intracerebroventricular injection of Z-DEVD-FMK (750μmol/L, calbiochem, USA).After14days stimulation, urine and Blood were collected for biochemical indicators test; the brain tissues were used for western blot.2. Blood pressure and urine collection24-hour urine samples were collected for three consecutive days before the end of the study period, and the blood pressure was determined via a pressure transducer placed in the femoral artery in anesthetized rats.3. The collection of sample3.1Blood collection and specimen collectionAt the completion of each protocol, rats were anesthetized with pentobarbital sodium (40mg/kg, i.p.), blood was collected in chilled vacuum tubes, and plasma samples were separated and stored at-80℃until assayed. To collect brain tissue for follow-up study, rats were perfused with200ml of ice-cold normal saline. The tissue of PVN was isolated from the brain and quickly placed in liquid nitrogenand then kept in the-80℃refrigerator.3.2Serum creatinine was detected by clinical automatic biochemical analyzer.3.324h urinary protein was detected by Bradford method.4. Western blotting detect the expression of AT1R, Ras, ERK1/2, P38, Bax, Bcl-2and caspase-3proteinThe tissue of PVN was taken out from the-80℃refrigerator and transferred to1.5ml EP tubes, the lysate was added equal to the ratio of1:4to tissue weight, one5ml syringe was used to repeatedly pipetting samples on ice. After complete splitting, the lysate was incubated on ice for15min and then centrifuged at13,000xg for15min at4℃. The supematant was used to measure protein concentration by the Bradford method. Samples were diluted4;1(V/v)with5xLaemmli buffer, heated at95℃and then proteins were detected by western blotting. The actived Ras protein was first purified by a kit name "Ras Pull-Down and Detection". After this process, the actived Ras protein were detected by western blotting.5. StaticsAll data are presented as mean±SE. Continuous variables between groups were compared using one-way ANOVA, followed by LSD method when P<0.05. Nonparametric test is used when heterogeneity of variance. Statistical analyses were conducted with SPSS13.0for Windows (SPSS, Chicago, IL). Significance was defined as P<0.05.Result1. Characteristics of rats with post-5/6nephrectomy versus sham-operation on10weeksThe blood pressure was determined via a pressure transducer placed in the femoral artery in anesthetized rats. Systolic pressure, serum creatinine and24-hour urinary protein excretion were higher in5/6Nx rats versus sham rats (P<0.05), proving that the model of renal hypertension is successful.2. Levels of AGT, Ang II and AT1R in PVN of renal hypertension rats and sham-operated ratsImmunohistochemistry was used to detect the expression of AGT, Ang II, AT1R, compared with sham-operated rats, levels of AGT, Ang II and AT1R in PVN of renal hypertension rats were significantly higher (P<0.05). Western blot and Real-time PCR was also used to detect the expression of AT1R, compared with sham-operated rats, levels of AT1R in PVN of renal hypertension rats were significantly higher (P<0.05).3. Levels of Ras-GTP in PVN of renal hypertension rats and sham-operated ratsWestern blot was used to detect the expression of Ras-GTP, compared with sham-operated rats, levels of Ras-GTP in PVN of renal hypertension rats were significantly higher (P<0.05).4. Levels of p-ERKl/2and p-p38in PVN of renal hypertension rats and sham-operated rats Immunohistochemistry and western blot was used to detect the expression of p-ERKl/2, levels of p-ERKl/2in PVN of renal hypertension rats were significantly higher (P<0.05) in renal hypertension rats than sham-operated rats. Western blot was used to detect the expression of p-p38, compared with sham-operated rats, levels of p-p38in PVN of renal hypertension rats were significantly higher (P<0.05).5. Levels of Bax, Bcl-2and cleaved caspase-3in PVN of renal hypertension rats and sham-operated ratsThe results of three technique including immunohistochemistry, Western blot and RT-PCR showed that the expression of Bax in PVN of renal hypertension rats were significantly higher in renal hypertension rats than that of sham rats (P<0.05). And the expression of the antiapoptosis protein Bcl-2were significantly lower in PVN of renal hypertension rats than that of sham rats testing by Western blot and RT-PCR (P <0.05). Western blot was used to detect the expression of cleaved caspase-3, compared with sham-operated rats, levels of cleaved caspase-3in PVN of renal hypertension rats were significantly higher (P<0.05).6. General characteristics respond to14days of blockade with various agents in renal hypertension ratsThere was no difference between renal hypertension rats with intracerebroventricular injection (I.C.V) CSF and various agents on body weight and serum creatinine. Compared with renal hypertension rats with aCSF, intracerebroventricular injection with Losartan, FTS, PD98059and Z-DEVD-FMKICV can significantly attenuated systolic blood pressure (P<0.05), urinary protein (P<0.05) and level of plasma norepinephrine (P<0.05), which prove that intraventricular injection of these interventions is through acting on the central nervous system to produce antihypertensive effect. However, the p38inhibitor SB203580did not reduce blood pressure and the level of plasma NE. 7. Levels of AT1R expression in PVN after14days of blockade with various agents in renal hypertension ratsIn order to determine the possible regulation mechanism of AT1R on blood pressure and sympathetic nerve activity, the protein expression of ATIR in PVN of renal hypertension rats with intracerebroventricular injection aCSF, Losartan, FTS, PD98059and Z-DEVD-FMKICV in PVN of renal hypertension rats was detected by western blot. The results show that the high expression of AT1R in PVN of renal hypertension rats was suppressed only by ICV losartan (P<0.05). Whereas the FTS, PD98059, SB203580or Z-DEVD-FMK by ICV had no effect on the protein expression of AT1R.8. The activity of Ras in PVN after14days of blockade with various agents in renal hypertension ratsThe activity of Ras was significantly lower in PVN of renal hypertension rats with intracerebroventricular injection Losartan and FTS (P<0.05). But the PD98059, SB203580and Z-DEVD-FMK ICV given had no effect on the activity of Ras. This shows that ATIR can regulate the activation of Ras, while the other blocking agents can not regulate the activity of Ras in the PVN of renal hypertension rats.9. The level of ERK1/2phosphorylation in PVN after14days of blockade with various agents in renal hypertension ratsThe level of ERK1/2phosphorylation was significantly lower in PVN of renal hypertension rats with intracerebroventricular injection Losartan, FTS and PD98059(P<0.05). While the SB203580and Z-DEVD-FMK ICV given had no effect on the activity of ERK1/2. This means that, both ATIR and Ras can regulate the phosphorylation of ERK1/2, however, the other blocking agents can not regulate the activity of ERK1/2in the PVN of renal hypertension rats.10. Levels of Bax, Bcl-2and cleaved caspase-3expression in PVN after14days of blockade with various agents in renal hypertension ratsBax and cleaved caspase-3were significantly higher in PVN of renal hypertension rats and rats with intracerebroventricular injection aCSF than Sham-operated rats(P<0.05),meanwhile, Bcl-2was significantly lower(P<0.05). Whereas intracerebroventricular injection Losartan, FTS and PD98059in renal hypertension rats significantly reduce the expression of Bax and significantly increase the expression of Bcl-2(P<0.05).And intracerebroventricular injection Losartan, FTS, PD98059and Z-DEVD-FMK in renal hypertension rats significantly reduce the expression of cleaved caspase-3.Summary1. RAS was activated in PVN of renal hypertension rats.2. Ras and MAPK signal pathway was activated in PVN of renal hypertension rats.3. There exists neurons apoptosis in PVN of renal hypertension rats.4. The activation of AT1R can induce apoptosis, then regulate the sympathetic activity and increased blood pressure, through the pathway of Ras/ERK1/2/caspase-3in PVN of renal hypertension rats. |
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