| Chapter one BackgroundRenal parenchymal disease is the most common secondary cause of hypertension, The pathogenesis of hypertension in patients with renal disease has been classically considered to be due to the combined result of volume overload/sodium retention and inappropriate activation of the rennin-angiotensin-aldosterone system (RAAS). Recent evidence now strongly suggests that increased sympathetic nervous system (SNS) activity is also involved.1Pathogenesis of Hypertension in Renal FailurePressure natriuresis is a central component of the feedback system for the long-term control of fluid volume and arterial pressure. However, in the presence of a defect in renal sodium excretory function, blood pressure is elevated to offset the abnormal pressure natriuresis relationship. Salt retention and the subsequent volume expansion also contribute to the phenomena of autoregulation, whereby increased tissue blood flow stimulates widespread vascular constriction, further increasing blood pressure. Therefore, volume retention and sodium balance is important in the hypertension associated with renal disease and has been well documented in end-stage renal disease (ESRD) patients.There is overwhelming evidence for a role of the RAAS in the development of hypertension and progression of kidney disease in renal failure. This is most apparent when considering the success of angiotensin-converting enzyme (ACE) inhibitors or angiotensin (Ang) II receptor antagonists in controlling blood pressure in renal insufficiency and the fact that these drugs also provide blood pressure-independent renoprotection.Afferent signals from the kidney are detected by chemoreceptors, which respond to ischaemic metabolites, uraemic toxins and the overall ionic composition of the renal interstitium, and mechanoreceptors, which monitor hydrostatic pressure changes in the kidney. The associated stimulation of renal afferents evokes a reflex increase in sympathetic activity and blood pressure. Whether mechanoreceptor-mediated responses or activation of renal chemoreceptors, the kidney has both direct and indirect connections to central nuclei of the SNS that are involved in arterial pressure regulation. The hypothalamus, in particular, plays a number of important roles in the integrated renal afferent sympathetic response. Not only does it have the ability to regulate renal efferent sympathetic nerve activity directly via presympathetic PVN neurons, but it also has a neurohormonal influence, augmenting the release of arginine vasopressin and oxytocin into the systemic circulation.2Brain Control of Blood PressureThe brain plays a critical role in the regulation of blood pressure (BP) through the incorporation of a complex signaling network that processes information from the circulation regarding changes in vascular tone. In turn, this network responds by modulating sympathetic nerve activity (SNA) and by secreting hormones into the circulation help to adjust the degree of vascular resistance in order to maintain a homeostatic BP set point that is known to be governed by renal pressure-natriuresis mechanisms. However, an accumulating body of work has suggested that dysfunctions in the central nervous system (CNS) regulatory mechanisms of BP control can lead to sustained increase in SNA, which can lead to chronic hypertension.The BP regulatory brain centers are situated, for the most part, in the hypothalamus of the forebrain and in the medulla of the brainstem. These nuclei are highly networked and their major function in cardiovascular control involves the regulation of SNA to modulate BP tone. In addition, these central cardiovascular pathways mediate neurohormone secretion, including arginine-vasopressin (AVP) that stimulates vasoconstriction and modulates the renal pressure-natriuresis mechanism. Hypothalamic control centers are also important in the regulation of electrolyte and plasma fluid volume via stimulation of thirst and increasing salt appetite.Animal models of hypertension have been vital in detailing the contribution of various cardiovascular regulatory brain nuclei to neurogenic hypertension. Key nuclei that have been involved in this disorder include; the organum subfornicale (SFO), the paraventricular nucleus (PVN) of the hypothalamus, the rostral ventrolateral medulla (RVLM) and the nucleus of the solitary tract (NTS) of the brainstem. The PVN plays a pivotal role in regulating blood pressure in both the normotensive and hypertensive states. This nucleus receives neural input concerning cardiovascular status from regions such as the SFO and NTS, integrates the input and controls sympathetic outflow by sending projections directly to the intermediolateral cell column (IML) of the spinal cord, and by sending projections to other sympathetic regulatory regions including the RVLM. The PVN also regulates the production of hormones that influence cardiovascular function including glucocorticoids and vasopressin.3Brain Renin-Angiotensin System and Blood PressureThe RAS is one of the most important mechanisms for regulating blood pressure, and the major bioactive hormone of this system is Ang II. The pressor actions of Ang II are mediated by its binding to AT1R. Since then, AT1R and all the components for the RAS have been found in various brain regions that are involved in the regulation of SNA as well as the regulation of fluid and electrolyte balance. Furthermore, the physiological significance of a brain RAS is underscored by observations of increased sensitivity to Ang II, and of increased ATIR expression in the cardiovascular regulatory brain areas of animal models of neurogenic hypertension. In addition, genetic or pharmacological interventions of brain RAS components have been shown to attenuate neurogenic hypertension.In addition, increased RAS activity in the brain increases AVP secretion, which is found to exacerbate hypertension in rats. At the other extreme, a deficit of RAS function in key regions in the brain, such as the magnocellular region of the PVN or the SON, results in impaired production and secretion of AVP, the main cause of centrally mediated diabetes insipidus.Each component of the RAS has been identified in the brain. Evidence of their role in central BP control and function in hypertension has been determined, both pharmacologically and by genetic manipulation. Data from multiple laboratories show that brain RAS components are compartmentalized within specific brain nuclei and even within specific cells, which would result in increased efficiency of RAS function. These findings help assert that a functional and competent RAS is active in the brain.4Dietary salt and hypertension There is broad agreement that excess dietary salt (NaCl) is the single most important controllable factor responsible for the rise in BP with advancing age in our culture and, thus, for the high incidence of essential hypertension. According to widely accepted dogma, salt and, consequently, water retention by the kidneys is a major factor in the pathogenesis of salt-induced hypertension. Slightly elevated plasma [Na+](by about1-3mM) with apparent normovolemia is often observed in hypertensive humans. The high-salt intake in salt-sensitive subjects elevates not only plasma [Na+] but cerebrospinal fluid (CSF)[Na+] as well.A variety of lesion and stimulation studies have indicated the importance of areas of the anteroventral region of the third ventricle (AV3V), specifically the MnPO and SFO, in cardiovascular and fluid-balance regulation. Additionally, there is substantial neuroanatomical and electrophysiological evidence indicating that the circumventricular organs, such as the organum vasculosum of lamina terminalis and SFO project to the MnPO and PVN. The organum vasculosum of lamina terminalis, SFO, AV3V, and MnPO were identified by the viral neuronal label, and they were found to be a part of the descending sympathetic pathway linked to cardiovascular control. In addition to the anatomical evidence, the stimulation studies showed that electrical stimulation of the SFO produces action potentials in efferent neurons of the PVN. These circumventricular organs may play an important role in the sympathoexcitatory pathway by providing information regarding the circulating humoral milieu, since their neuronal cell bodies are positioned at the site of weak blood-brain barrier and can thereby interact with molecules in the circulation, such as ANG II. There is evidence that ANG II acts as a neurotransmitter from the SFO to the PVN.A brain region crucial for the regulation of body fluid homeostasis is the paraventricular nucleus of the hypothalamus (PVN). Located lateral to the third ventricle, it is anatomically and functionally connected to neurons residing in forebrain sensory circumventricular organs, parts of the brain lacking a functional blood-brain barrier known for their ability to sense small changes in sodium concentration/osmolality. It includes large vasopressin-producing magnocellular neurons and smaller parvocellular neurons that project to spinal preganglionic sympathetic neurons and premotor sympathetic neurons in the brainstem. The PVN is thus a plausible site for coordination of neurogenic and hormonal actions on the cardiovascular system and the kidney in response to changes in sodium concentration/osmolality.Hypertension is a common presentation of CRF. The present study was conducted to test the hypothesis that dysregulation of RAS occur in brain of CRF rats, which contribute to the sympathetic overactivity and development of hypertension in CRF, and then we will clarify the relationship between brain RAS and sympathetic activity in CRF rats.Materials and methodsPreparation of Animal ModelAll animal procedures were approved by the Animal Experiment and Care Committee of the Sourthern Medical University. Sixty adult male Sprague-Dawley rats (initial weight,150-180g, Sourthern Medical University Animal Experiment Center) were maintained under shandardized conditions. The animals were subjected to two-step,5/6nephrectomy (5/6Nx, by performing a right nephrectomy with surgical resection of the two-thords of the left kidney) or to sham operation (controls). Ten weeks after the operation, the5/6Nx rats were randomized into subgroups.Experimental data and specimen collection1. Blood pressure and urine collection Three 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. Systolic blood pressure before and14days after for rats on different stimulate were monitored via a pressure transducer placed in the femoral artery, the change in systolic blood pressure is used as the statistic.2. Blood collect and brain perfusionAt 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. Part of the rat brain tissue were collected for western blot studies, and the others were used for immunohistochemical studies.Chapter two Affects of salt diet on central renin-angiotensin system of5/6nephrectomy rat modelAfter14days stimulation, urine and Blood were collected for biochemical indicators test; the brain tissue were used for brain RAS detect by immunohistochemistry or western blot.Chapter three Cellular localization of brain RAS in5/6Nx rats and the relationship between the RAS and the neuron activationThe rat brain paraffin section from Chapter two were used for double lable brain RAS with NSE or GFAP by immunofluorescence, or for c-fos quantitative by immunohistochemistry. Chapter four The relationship between brain RAS and blood pressure or SNA in5/6Nx ratsTen week after5/6Nx, the rats were fed a high-salt (4%) diet, randomized into9groups (n=12in each group) and treated as follows for14days, respectively:①Losartan Omg/kg/d by intragastric gavage (IG):Rats were administrated with daily intragastric injection of endotoxin-free phosphate-buffered saline (PBS)(pH7.4);②Losartan lmg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (lmg/kg per day, Sigma Chemical, St Louis, MO, USA);③Losartan50mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (50mg/kg per day);④Losartan500mg/kg/d IG:Rats were administrated with daily intragastric injection of losartan (500mg/kg per day);⑤Losartan Omg/kg/d by intracerebroventricular injection (ICV):Rats were administrated with daily intracerebroventricular injection of artificial cerebrospinal fluid (aCSF);⑥Losartan1mg/kg/d ICV:Rats were administrated with daily intracerebroventricular injection of losartan(1mg/kg per day);⑦Clonidine5.76g/kg/d ICV:Rats were administrated with daily intracerebroventricular injection of clonidine (5.76g/kg per day, Sigma Chemical, St Louis, MO, USA);⑨Renal denervation (RDX);⑨Tempol30mg/kg/d IG:Rats were administrated with daily intragastric injection of tempol (30mg/kg per day, Sigma Chemical, St Louis, MO, USA).After14days stimulation, urine and Blood were collected for biochemical indicators test; the brain tissue were used for brain RAS and TH and c-fos detect by immunohistochemistry or western blot.Statistical AnalysesAll 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.Results1. Characteristics of rats post-5/6nephrectomy versus sham-operated rats on10weeksThe base line blood pressure (systolic blood pressure) was determined in conscious rats by the indirect tail-cuff method. Systolic pressure and serum creatinine and24-hour urinary protein excretion were higher in5/6Nx rats versus sham rats, prove5/6Nx rats modeling success.2. Changes in general characteristics after14days different salt diet stimulation in sham-operated and5/6Nx ratsBlood pressure, serum sodium levels and24h urine protein had no significant difference in sham-operated rats with14days different salt diet (P>0.05). Compared with sham-operated rats,5/6Nx rats had a significantly higher blood pressure with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the blood pressure and serum sodium levels and24h urine protein in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the blood pressure of the rats with5/6Nx (P<0.05).The24h urinary volume and24h urinary sodium excretion were increased with increasing dietary salt (P<0.05), but the blood Ang II has been suppressed with increasing dietary salt in both sham and5/6Nx rats (P<0.05).3. Changes in systolic blood pressure before and14days after for rats on low, normal and high salt dietsSBP of femoral blood pressure had no significant difference in sham-operated rats with14days different salt diet (P>0.05). Compared with normal salt5/6Nx group, the SBP of femoral blood pressure in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the blood pressure of the rats with5/6Nx (P<0.05).4. Levels of Ang Ⅱ in SFO and PVN after14days different salt diet stimulation in sham-operated and5/6Nx ratsCompared with sham-operated rats,5/6Nx rats had a significantly higher Ang II in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the Ang II in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the Ang Ⅱ in SFO and PVN in sham and5/6Nx rats (P<0.05).5. Levels of AT1R in SFO and PVN after14days different salt diet stimulation in sham-operated and5/6Nx ratsCompared with sham-operated rats,5/6Nx rats had a significantly higher AT1R in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the AT1R in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the AT1R in SFO and PVN in sham and5/6Nx rats (P<0.05).6. Cellular localization of Ang Ⅱ or AT1R in SFO and PVN in5/6Nx ratsAng II or AT1R immunoreactivity is colocalized on neurons within SFO and PVN.7. Neurons activation in SFO and PVN of5/6Nx rats with different salt dietCompared with sham-operated rats,5/6Nx rats had a significantly higher c-fos in SFO and PVN with the same normal salt diet (P<0.05). Compared with normal salt5/6Nx group, the c-fos in SFO and PVN in high salt5/6Nx group was significantly increased (P<0.05); The low-salt diet can significantly lower the c-fos in SFO and PVN in sham and5/6Nx rats (P<0.05). 8. General characteristics respond to14days of blockade with various approaches in5/6Nx ratsSystolic blood pressure and urinary albumin excretion markedly decreased in salt-loaded5/6Nx rats with losartan ICV at lmg/kg/d (P<0.05). Losartan IG at higher doses (50-500mg/kg/d), clonidine ICV, renal denervation, or tempol IG also significantly attenuated systolic blood pressure and urinary albumin excretion in high-salt-fed5/6Nx animals (P<0.05). NE level markedly decreased in salt-loaded5/6Nx rats combined with losartan ICV at lmg/kg/d, clonidine ICV or renal denervation (P<0.05). Losartan IG at higher doses (50-500mg/kg/d) and tempol IG also significantly attenuated renal NE levels in high-salt-fed5/6Nx animals (P<0.05).9. Changes in systolic blood pressure before and14days after losartan or clonidine blockade in sham-operated and5/6Nx ratsSystolic blood pressure markedly decreased in salt-loaded5/6Nx rats with losartan ICV at lmg/kg/d (P<0.05). Losartan IG at higher doses (50-500mg/kg/d), clonidine ICV, renal denervation, or tempol IG also significantly attenuated systolic blood pressure in high-salt-fed5/6Nx animals (P<0.05).10. Levels of RAS expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx ratsWe previously showed that high-salt diet enhance RAS expression in SFO and PVN nucleus in5/6Nx rats. To verify the role of central RAS in the development of hypertension and the relationship between central RAS and SNS, the5/6Nx rats treated with high-salt diet were blockaded by different approaches to central or periphery RAS and SNS. Immunohistochemistry was used to map the expression of Ang II. The operation of renal denervation eliminate the increase of Ang II expression induced by high-salt diet (P<0.05vs5/6Nx rats with IG Omg of losartan), conversely, lmg of losartan given by ICV or500mg losartan given by IG further increased Ang II expression in both SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (lmg and50mg) or tempol given by IG and clonidine given by ICV had no effect.Immunohistochemistry was also used to map the expression of ATIR. Changes of AT1R expression response to various blockades are different to Ang II. Low dose of losartan (lmg) given by ICV prominently suppressed the expression of ATIR in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), while the same dose or even much larger dose of losartan (50mg) given by IG didn’t have any effect on expression of ATIR, the effect of500mg of losartan given by IG was comparable with that of1mg of losartan given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan). The operation of renal denervation had restrained the high expression of AT1R in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan). Clonidine or tempol had no effect on AT1R expression.Western blot was used to further determine the ATIR changes, and obtained similar results to immunohistochemistry.The high expression of AT1R in SFO and PVN nucleus induced by high-salt diet were suppressed by ICV1mg of losartan or IG500mg of losartan or renal denervation (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (1mg and50mg) or tempol given by IG and clonidine given by ICV had no effect.11. Levels of tyrosine hydroxylase (TH) expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx ratsTo verify the relationship between central RAS and SNS, tyrosine TH levels in SFO and PVN nucleus in5/6Nx rats were detected by western blot. ICV lmg of losartan significantly decreased the expression of TH in SFO and PVN nucleus (P<0.05vs5/6Nx rats with IG Omg of losartan), high dose of losartan (500mg) given by IG or renal denervation had similar effect on TH (P<0.05vs5/6Nx rats with IG Omg of losartan), whereas the losartan (lmg and50mg) or tempol given by IG and clonidine given by ICV had no effect.12. Levels of c-fos expression in SFO and PVN nucleus after14days of blockade with various approaches in5/6Nx ratsTo clarify the effect of various blockades on neurons activation in SFO and PVN of5/6Nx rats treated with high-salt diet, the c-fos was quantified by immunohistochemistry. We found that high level of c-fos was restored by Low dose of losartan (1mg) given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan), in contrast, the same dose of losartan and even more large of losartan (50mg) given by IG didn’t change the c-fos expression in SFO and PVN nucleus, while the large dose of losartan (500mg) had comparable effect with that of lmg of losartan given by ICV (P<0.05vs5/6Nx rats with IG Omg of losartan). Moreover, we found that c-fos was significantly restored by renal denervation (P<0.05vs5/6Nx rats with IG Omg of losartan), but not by clonidine or tempol. Paper summarizes1. RAS was activated in brain cardiovascular centre (sympathetic integration site) in5/6Nx rats;2. Overactive brain RAS may raises blood pressure by stimulating the central sympathetic nervous system in5/6Nx rats;3. High-salt diet can aggravate kidney injury and further activation brain RAS by afferent nerves from the kidneys, thereby activating central sympathetic nervous system and worsen the hypertension;4. There exists a positive feedback loop between the kidney and brain cardiovascular centre (sympathetic integration site), which can accelerated the progress of kidney injury. |
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