巨细胞病毒感染对细胞肾素基因表达的影响及其生物学意义

目的 探讨巨细胞病毒(cytomegalovirus,CMV)感染肾球旁细胞肾素基因表达的变化及其意义.方法 用病毒感染复数(MOI)为10、0.1和0的鼠CMV分别与鼠肾球旁细胞模型As4.1细胞共育5 d作为实验组;用紫外线灭活病毒的假感染(mock感染)对照组.RT-PCR检测感染细胞中CMV即刻早期基因1(IE1)的表达;免疫荧光观察肾素阳性细胞和肾素阳性颗粒在细胞的分布;双色免疫荧光染色观察肾素阳性颗粒是否出现在CMV阳性细胞;RT-PCR和Western blot检测肾素基因在感染细胞内的表达.结果 CMV感染As4.1细胞后出现典型的病毒空斑;病毒感染细胞CMV IE1 RT-PCR产物阳性;肾素阳性细胞集中在病毒空斑周围CMV新感染细胞区,肾素阳性荧光颗粒主要以块状和环状存在于病毒感染细胞质中;双色免疫荧光染色显示肾素阳性颗粒和CMV阳性颗粒出现在同一细胞;CMV感染细胞肾素基因的表达随病毒感染量增加而增加.结论 CMV感染并导致其宿主细胞肾素基因表达,可能涉及CMV加速心血管疾病发生发展的新机制.     

人巨细胞病毒感染对宿主细胞凋亡影响的研究进展

人巨细胞病毒（HCMV）属B疱疹病毒亚科，在人群中普遍感染。研究表明HCMV感染导致临床病症与细胞凋亡有密切关系，HCMV感染与细胞凋亡关系具有多样性和复杂性，在不同的阶段表现为不同的作用。研究HCMV与细胞凋亡的关系不仅有助于阐明HCMV感染细胞及细胞对其反应的分子机制和研发新的抗病毒制剂，而且能为HCMV引起临床疾病的治疗提供新的方案。     

巨细胞病毒感染时免疫功能的变化及其意义

目的　探讨巨细胞病毒感染时免疫变化及其意义。方法　以 2 0名CMV IgM(Cytomegalovirus immunoglobulinM)阳性的病儿为观察对象 ,另设对照组 30例。ELISA法检测柯萨奇B组病毒抗原 (CVB Ag)、IgM抗体 (CVB IgM)、巨细胞病毒IgM抗体 (CMV IgM)、EB病毒IgM抗体 (EBV IgM) ,单克隆抗体标记间接ABC免疫法检测外周血T细胞亚群。结果　CMV感染组的LAK细胞、NK细胞活性均明显降低 (P <0 .0 1)。合并其它感染原的CMV混合感染组CD3含量减少 ,CD8含量增加 (P <0 .0 5 ) ,CD4 CD8数值明显降低 (P <0 .0 1)。病毒混合感染并合并支原体感染组CD3含量减少 (P <0 .0 5 )、CD4 CD8数值、IgA含量明显降低 (P <0 .0 1)。结论　巨细胞病毒感染影响了T细胞亚群的平衡 ,在合并其他感染时天然免疫细胞功能下降和T细胞亚群紊乱更明显     

人巨细胞病毒感染对宿主细胞凋亡影响的研究进展

人巨细胞病毒(HCMV)属β疱疹病毒亚科,在人群中普遍感染。研究表明HCMV感染导致临床病症与细胞凋亡有密切关系,HCMV感染与细胞凋亡关系具有多样性和复杂性,在不同的阶段表现为不同的作用。研究HCMV与细胞凋亡的关系不仅有助于阐明HCMV感染细胞及细胞对其反应的分子机制和研发新的抗病毒制剂,而且能为HCMV引起临床疾病的治疗提供新的方案。     

巨细胞病毒感染对造血功能影响的研究进展

人巨细胞病毒(HCMV)是人群中广泛存在的感染因子，随着免疫力低下人群(器官移植、恶性肿瘤、艾滋病等)的增加，近10年来，HCMV感染引起严重疾病的机制再次成为医学领域的研究热点，HCMV几乎可感染人体所有组织，整个造血系统尤其是造血祖细胞是HCMV的主要潜伏部位。通过抑制细胞免疫、诱导细胞凋亡，逃避吞噬细胞溶酶体的降解而早潜伏性感染，经过转移活化刺激、细胞分化和细胞因子等作用再激活，造血系统经常受累，其中骨髓抑制、血小板减少和免疫功能紊乱较常见。     

人巨细胞病毒感染对小鼠脑组织同源框基因表达的影响

目的　研究小鼠脑组织同源框基因 (homeoboxgene ,Hox)的表达状态及人巨细胞病毒(humancytomegalovirus ,HCMV)感染对小鼠脑组织Hox基因表达的影响 ,以探讨HCMV致畸的分子机制。方法　昆明系小鼠 4 8只 ,随机分为 :实验对照组 (16只 )和病毒感染组 (32只 ) ,病毒感染为脑内注射。在感染后 7、15、30、6 0d分别以病理学方法观察病理损伤 ,以免疫组化方法检查HCMV LA(latean tigen) ,PCR检测小鼠脑组织中HCMV DNA。在建立HCMV脑部感染的小鼠模型基础上 ,用RT PCR测定Hox基因在病毒感染组和实验对照组小鼠脑组织中的表达 ,并用同位素标记的Hox寡核苷酸探针进行Northernblot检测相应的表达状况。结果　 (1)接种HCMVAD16 9株的小鼠脑组织发生了病理改变 ,免疫组化和PCR方法在神经细胞内查到了HCMV LA和DNA ;(2 )RT PCR和Northernblot发现 :对照组的小鼠脑组织表达Hoxa2、Hoxb1和Hoxb2 ,但不表达Hoxb5、Hoxb8;HCMV感染后 ,小鼠脑组织被诱导表达Hoxa1,与对照组比较差异有显著性意义 (P <0 .0 1) ,且于感染后的 15d达到高峰 ,而Hoxb1的表达下调 (P <0 .0 5 ) ;Hoxa2和Hoxb2无明显变化 ;Hoxb5和Hoxb8仍旧不表达。结论　HCMVAD16 9株可感染小鼠神经系统。HCMV感染可诱导小鼠神经系统发育基因Hox基因表达改变 ,这对     

人巨细胞病毒感染对小鼠脑组织同源盒基因表达的影响

目的研究小鼠脑组织同源盒基因（homeobox gene，Hox）的表达状态及人类巨细胞病毒（human cytomegalovims，HCMV）感染对小鼠脑组织Hox基因表达的影响，以探讨HCMV致畸的分子机制。方法昆明系小鼠48只，随机分为实验对照组（16只）和病毒感染组（32只），病毒感染为脑内注射。在感染后7、15、30、60d分别以病理学方法观察病理损伤，以免疫组化方法检查HCMV—LA（1ateantigen），PCR检测小鼠脑组织中HCMV—DNA。在建立HCMV脑部感染的小鼠模型基础上，用RT-PCR测定Hox基因在病毒感染组和实验对照组小鼠脑组织中的表达,并用同位素标记的cDNA探针进行Northern-blot检测相应的表达状况。结果（1）接种HCMVAD169株的小鼠脑组织发生了病理改变，免疫组化和PCR方法在神经细胞内查到了HCMV-LA和DNA；（2）RT-PCR和Northern-blot发现：对照组的小鼠脑组织表达Hox-A9、Hox-A11、Hox-A12和Hox-A13，但不表达Hox-B13；HCMV感染后，小鼠脑组织被诱导表达Hox-B13，与对照组比较差异有统计学意义（P〈0.01），且于感染后的30d达到高峰，而Hox—A9、Hox—A11的表达下调（P〈0.05）；Hox—A10和Hox—A13的表达增强。结论HCMVAD169株可感染小鼠神经系统。HCMV感染可诱导小鼠神经系统发育基因Hox表达改变，这对研究HCMV感染致畸的分子机制提供了有价值的理论依据。     

树突状细胞在人巨细胞病毒感染中的作用

人巨细胞病毒(human cytomegalovirus,HCMV)在人群中感染率极高,通过多种免疫逃避机制,实现在宿主体内的长期潜伏感染。树突状细胞(dendritic cells,DC)是重要的抗原提呈细胞,在诱导和维持特异性免疫应答中发挥重要的作用。人体内的DC根据来源、表型分为两群:髓系DC(myeloid DC,mDC)和浆细胞样DC(plasmacytoid DC,pDC),大量研究证实HCMV介导的多种免疫逃避机制中,部分是通过影响DC功能实现的。HCMV不仅可以感染mDC,影响mDC表型、迁移、分泌细胞因子、激活T细胞功能,而且还可以抑制pDC分泌干扰素水平及激活T细胞能力,并且激活过度的B细胞反应,导致机体抗病毒细胞免疫反应的抑制和体液免疫紊乱,实现病毒长期潜伏感染。本文主要讨论HCMV是如何改变两种DC亚型的功能以实现免疫逃避目的。     

巨细胞病毒感染与细胞凋亡的研究进展

CMV病毒和细胞凋亡的关系相当复杂，有时表现为诱导凋亡作用，有时表现为抗凋亡作用。从病毒的角度看，病毒诱导小胶质细胞、巨噬细胞、造血干细胞、中性粒细胞及淋巴细胞等发生凋亡，可干扰机体抗病毒免疫反应，使病毒不至于被机体免疫系统消除，对于病毒感染的有些宿主细胞，病毒则利用自身抗凋亡作用以利于在感染细胞内完成病毒的复制和生活周期。从宿主的角度出发，机体会调动全身和局部抗病毒免疫反应、宿主细胞会启动凋亡机制来清除病毒。     

巨细胞病毒对骨髓基质细胞粘附功能的影响

目的:探讨巨细胞病毒(CMV)对骨髓基质细胞表面粘附分子ICAM-1 和VCAM-1的表达及骨髓基质细胞对正常造血细胞的粘附率的影响。方法:用流式细胞仪检测骨髓基质细胞表达粘附分子ICAM-1和VCAM-1阳性率,用MTT方法检测骨髓基质细胞对正常骨髓造血细胞的粘附率。结果:本实验所用的CMV可以感染骨髓基质成纤维细胞;100TCID₅₀剂量以下CMV对骨髓基质细胞无明显破坏作用;活性CMV 感染骨髓基质细胞早期(18 h)粘附分子ICAM-1表达明显较高,晚期(120 h)ICAM-1表达明显较低;灭活的CMV也能使骨髓基质细胞ICAM-1表达增高,作用与有活性的CMV相似;骨髓基质细胞在CMV感染早期,对造血细胞的粘附率增高。CMV对VCAM-1的表达无明显影响。结论:CMV 感染骨髓基质细胞早期,骨髓基质细胞对造血细胞粘附能力增高,CMV感染晚期骨髓基质细胞对造血细胞粘附能力降低。     

Role of the macula densa in the control of renal renin gene expression in two-kidney/one-clip rats

This study was designed to examine whether macula densa function is involved in the changes of renal renin gene expression upon acute hypoperfusion of one kidney. To block macula densa function, rats with free access to salt and water were subcutaneously infused with furosemide (12 mg/day) for 6 days. Then, 4 days after the start of the infusion, the left renal arteries were clipped with 0.2-mm silver clips and renin mRNA levels in ipsilateral and contralateral kidneys, as well as plasma renin activities (PRA), were determined 48 h after clipping. In non-clipped animals furosemide increased PRA from 10 to 47 ng angiotensin I · h^–1 · ml^–1 and raised renin mRNA levels in both kidneys 2.5-fold. In vehicle-infused animals, clipping of the left renal artery increased PRA to 37 ng angiotensin I · h^–1 · ml^–1 and led to a 5-fold rise of renin mRNA levels in the ipsilateral kidneys and to a suppression to 20% of the control values in the contralateral kidneys. PRA values in clipped and furosemide-infused animals were 45 ng angiotensin I · h^–1 · ml^–1. In these animals renin mRNA levels increased in the ipsilateral kidneys to similar absolute values as in vehicle-infused rats, whilst contralateral renin mRNA levels fell to about 25% of the respective controls. These findings indicate that the stimulations of renin gene expression by inhibition of macula densa salt transport and by renal artery clipping are not additive, suggesting that the macula densa mechanism may participate in the stimulation of renin gene expression upon hypoperfusion. The macula densa mechanism, however, appears to be not essentially involved in the suppression of renin gene expression in the contralaterals to stenosed kidneys.     

Typical and atypical aspects of renin secretion from juxtaglomerular epithelioid cells

Summary A survey is given about features of renin synthesis and secretion from juxtaglomerular epithelioid cells that are largely atypical as compared to those of other secretory systems. Renin-producing cells have the capability of reversible metaplastic transformation into vascular smooth muscle cells, their secretory granules are very closely related to lysosomes, and they react paradoxically, i.e. with an inhibition instead of a stimulation of renin secretion, to a rise in intracellular free Ca⁺⁺. The modes of renin secretion and activation of the enzyme as well as possible mechanisms involved in adjusting the ratio of secreted active to inactive renin to the current needs of the renin-angiotensin system are discussed.These studies were supported by the German Research Foundation within the Forschergruppe Niere/Heidelberg     

Furosemide stimulates renin expression in the kidneys of salt-supplemented rats

This study was conducted to obtain information about a possible influence of salt transport by the thick ascending limb of Henle (TALH) and the macula densa on the expression of renin in the kidney. To this end, adult male rats were subcutaneously infused with furosemide (12 mg/24 h), an established inhibitor of TALH and macula densa salt transport, or with vehicle for 6 days. The animals had free access to chow, water and salt water (0.8% NaCl, 0.1% KCl) to maintain salt and water balance. Chronic furosemide treatment led to a 20-fold increase in urine flow rates and 50% increase in kidney weights, while urine osmolality decreased by 60% and body weight gain decreased by 40% in the furosemide-treated animals. Plasma renin activities increased from 2.9±0.5 ng angiotensin I h^–1 ml^–1 in controls to 10.6±2.2 ng angiotensin I h^–1 ml^–1 in furosemide-treated rats. In parallel, kidney areas immunoreactive for renin increased by 80% and the renal content of renin mRNA increased by 120% in the animals receiving furosemide. Under the assumption that the effects seen on renal renin expression were primarily due to the inhibition of TALH and macula densa function by furosemide, our findings suggest that salt transport across the TALH and macula densa exerts a negative control function not only on the secretion but also on the expression of renin in the kidney.     

Amiloride enhances the secretion but not the synthesis of renin in renal juxtaglomerular cells

In this study we have examined a potential role of the sodium/proton exchange system in the regulation of renin secretion. We found that the inhibitors of the Na⁺/H⁺ antiport, amiloride (1 mM) and ethylisopro-pylamiloride (EIPA, 50 M), led to a 125% increase of renin secretion from cultured mouse juxtaglomerular cells. The stimulatory effect of EIPA on renin secretion was dependent on the extracellular concentrations of sodium and hydrogen ions. While lowering the extracellular pH from 7.3 to 7.0, and lowering [Na⁺]_e from 130 mM to 5 mM had no effect on basal renin release, it markedly attenuated or even blunted the effect of EIPA on renin secretion. The stimulatory effect of forskolin on renin secretion, however, was not altered by decreases of extracellular pH and of sodium. Inhibition of basal renin release was achieved with angiotensin II (1 M). In the presence of EIPA the inhibitory effect angiotensin II was markedly attenuated. Although effective on renin secretion, neither amiloride nor EIPA exerted a significant effect on the de novo synthesis of renin in cultured mouse JG cells. These findings are compatible with the idea that an amiloride-sensitive transport process, presumably the Na⁺/H⁺ exchanger, acts indirectly as an inhibitory signal transduction system for renin secretion from renal juxtaglomerular cells.     

Influence of dietary NaCl intake on renin gene expression in the kidneys and adrenal glands of rats

The aim of this study was to examine the influence of dietary NaCl intake on renin gene expression in the kidneys and adrenal glands of adult rats. Rats were kept on low (0.02%, w/w), normal (0.6%) or high (4%) NaCl diets and plasma renin activity (PRA) and the relative abundance of renin messenger ribonucleic acid (mRNA) in renal and adrenal tissue were followed for 20 days. In animals on a normal-salt diet PRA and renal renin mRNA levels did not change with time. PRA values in animals on the low-salt diet increased transiently (about threefold) and then declined again during the third week of treatment. Renal renin mRNA levels in these animals paralleled the changes of PRA. Conversely, in the animals kept on a high-salt diet PRA values decreased transiently and renal renin mRNA decreased continuously to about 50% of control values. Arterial blood pressure measured in conscious animals was not significantly influenced by the different salt diets. To establish whether the changes in renin mRNA levels are mediated by renal nerve input, animals on the different diets were also studied after unilateral renal denervation. Renal nerve section led to a 50% decrease of renin mRNA levels in the denervated kidneys in animals kept on the normal-salt diet. In the animals on the low-salt diet renin mRNA rose to similar levels in the denervated to those in the innervated kidney, while in animals receiving a high-salt diet renin mRNA was further decreased in the denervated kidneys. The abundance of renin mRNA in adrenal tissue was low and was estimated to be around 1% of that found in the kidneys. Adrenal renin mRNA levels also increased in animals kept on a low-salt diet and decreased in animals on high-salt diet. Taken together, our findings suggest that renin secretion and renin gene expression are inversely related to salt intake and that the influence of salt diet on these parameters has both transient and constant temporal components. Changes of blood pressure or nerve activity are not likely mediators of the effect of salt intake on renin expression. Since renal and adrenal renin mRNA levels change in parallel in response to alterations of salt intake we hypothesize the existence of a humoral factor that links renin expression to the rate of salt intake.     

Endothelium derived relaxing factor is involved in the pressure control of renin gene expression in the kidney

To study the influence of endothelium derived relaxing factor/nitric oxide (EDNO) on renin gene expression, the effects of a 2-day treatment with the NO-synthase inhibitor nitro-L-arginine-methylester (L-NAME, 40 mg/kg twice a day) on plasma renin activity (PRA) and renal and adrenal renin m-RNA levels were examined in conscious rats with and without unilateral renal clips (0.2 mm). In sham-clipped animalsL-NAME led to a decrease of PRA from 7.5 to 2.5 ng angiotensin I (ANGI) · h^–1 · ml^–1 and to a 35% decrease of renal renin m-RNA levels. Unilateral renal artery clipping increased PRA to 35 and to 13 ng ANGI · h^–1 · ml^–1 in vehicle and inL-NAME-treated rats, respectively. In the clipped kidneys renin m-RNA levels increased to 450% of control values in vehicle-treated animals and to 220% of control values inL-NAME-treated animals. In the contralaterals as opposed to clipped kidneys, renin m-RNA levels decreased to 16% and 50% of the control values in vehicle- and inL-NAME-treated animals, respectively. In the adrenal glands renin m-RNA levels were not significantly changed either by clipping of one renal artery or by treatment of animals withL-NAME. The NO-donor sodium nitroprusside (100 M) was found to increase renin secretion and renin m-RNA levels in primary cultures of renal juxtaglomerular cells. These findings suggest that EDNO is involved in the control of the renin gene by the renal perfusion pressure.     

A sensitive method for determination of renin activity in the single juxtaglomerular apparatus of the rat kidney

Summary We have established a method for the determination of renin activity in minute amounts of tissue. This method was used for single juxtaglomerular apparatuses (JGA's) of rats. Enzyme kinetic studies were performed to establish the optimal conditions for microdissected JGA's, using purified rat renin and its substrate.Maximal formation of angiotensin occurs at pH 6.5. The effect of incubation and homogenization time, temperature, substrate and renin concentration on the angiotensin formation rate was studied.For the studies reported here purified rat renin substrate was used. The purification methods revealed a substrate content of appr. 1000 ng/mg protein. This highly purified rat renin substrate improved the sensitivity of renin determination in micro amounts of tissue. Hence renin activity of single microdissected JGA's could be quantitatively determined within 1 hour of incubation in a volume of 0.1 ml. Mean renin activity in 18 JGA's from rats kept on standard Altromin diet was 15.2±S.D. 7.0 ng/0.1 ml·hour.Supported by the Deutsche Forschungsgemeinschaft.     

The role of angiotensin II in the feedback control of renin gene expression

 This study aimed to characterize the influence of endogenous angiotensin II on renal renin gene expression during different states of a stimulated and of a suppressed renin system. To this end the renin system in male Sprague Dawley rats was stimulated by unilateral renal artery clipping (0.2 mm clip), by furosemide (60 mg/kg per diem) or isoproterenol (160 μg/kg per diem), and by ingestion of a low-salt diet (0.02%), or was suppressed by setting a contralateral renal artery clip (0.2-mm clip) or by ingestion of a high-salt diet (4%). During the last 2 days of these different treatment regimens, the animals were treated with the angiotensin II AT1 receptor antagonist losartan (40 mg/kg per diem) and renal renin mRNA levels were assayed. Renin gene expression was stimulated four- to fivefold by renal artery clipping and isoproterenol infusion, two- to threefold by furosemide and a low-salt diet, and about fourfold by losartan. Additional treatment with losartan potentiated the stimulatory effects of a low-salt diet, of furosemide and of isoproterenol infusion on renin gene expression, whilst there was no significant additional effect of losartan on renin gene expression in clipped kidneys. Both contralateral renal artery clipping and a high-salt diet decreased renin mRNA levels to about 50% of the control value. In rats with a unilateral clip, additional losartan treatment caused renin mRNA to increase to about 350% of the control value in the contralateral kidney but to only 110% of the control value in animals on a high-salt diet. These findings suggest that the enhanced formation of angiotensin II during a low-salt intake, during tubular inhibition of salt reabsorption or during β-adrenoreceptor activation plays a relevant negative feedback role in the activation of the renin gene. Moreover, in rats with one hypoperfused kidney, angiotensin II could be involved in the inhibition of renin gene expression in the contralateral kidney. In hypoperfused kidneys, however, and in animals on a high-salt diet, angiotensin II appears to play only a minor feedback role in the regulation of the renin gene. Received: 10 September 1996 / Received after revision: 17 February 1997 / Accepted: 25 February 1997     

Factors controlling the release of renin

Summary Blood was collected by micropuncture from the efferent arteriole and superficial venules of the cat's kidney. Blood samples were also collected from the femoral artery and the renal vein. The blood renin concentrations (ng Al ml^–1 2 h^–1) in a basal state were 37.2±3.5 (S.E.M.) (n=60) (artery), 32.5±5.2 [7] (efferent arteriole), 53.5±4 (116) (superficial venule), 54.2±5.4 (43) (renal vein). The corresponding values after haemorrhage were 389±98 (21), 345±115(6), 963±208 (37), 907±290 (17), ng Al ml^–1 2 h^–1. The efferent arteriolar renin did not differ from that in the artery but the concentrations in superficial venular blood and renal vein were higher than arterial concentration. Thus renin entered the circulation between the efferent arteriole and the superficial venule. The blood renin concentration of different superficial venules at a steady arterial renin concentration varied markedly. Into certain venules there appeared to be little or no renin secretion, into others a marked renin secretion, suggesting a heterogeneity of renin secretion by the different nephrons.When the flow of tubular fluid to the macula densa of a group of nephrons was blocked, the renin concentration fell and was significantly less than the renin concentration in venules draining non blocked nephrons and less than in the renal vein.These results suggest that the juxtaglomerular apparatus of the nephrons do not release renin in a synchronous fashion. The release appears to be episodic and is inhibited when flow to the macula densa is ceased. This implies that a high sodium concentration at the macula densa stimulates renin release.Supported by the National Heart Foundation of Australia