低出生体重对大鼠肾脏和血压的影响及机制研究
摘要
研究背景
胎儿生长受限(fetal growth restriction,FGR)包括胎儿宫内发育迟缓(intrauterine growth retardation,IUGR)和新生儿低出生体重(low birth weight,LBW)。我国低出生体重儿(low birth weightinfant,LBWI)的出生率约为4.75%～8.5%,LBW是围产期婴儿发病和死亡的主要危险因素之一。联合国1995年的报告估计,发生心血管疾病和中风的成年患者至少10%为LBWI,大约50%的高血压患者为LBWI。自1992年Barker首次发现心血管疾病与出生体重有关,进而提出“胚胎编程”假说以来,越来越多的流行病学和临床研究发现,LBW是心血管不良事件相对独立的危险因素之一,同时对成人期的肾脏和高血压产生不同程度的影响。现已有80项有关研究(其中涉及44.4万个病例,年龄跨度0～80岁,包括多种种族人群)证实了LBW与高血压的相关性。大量流行病学研究发现,LBW导致肾单位数目的减少,同时肾单位的减少是成人期发展为全身性高血压的一个独立的危险因素。LBW对肾脏发育和高血压的影响,引起了众多学者的关注。研究LBW引起成年后发生代谢综合征的机制,以及实施成年期疾病的早期防治已经成为一个研究热点。
人类流行病学研究和动物实验均证实LBW与肾单位数目减少有明显相关性,但是其发生机制尚不清楚。肾脏的发育过程包括快速的结构重建,尤其是后肾阶段,这个过程需要大量的细胞增殖和凋亡,并且受到多种相关基因的严格调控及互相作用,如Wt1、Bcl-2、Pax2等。同时,研究表明肾素-血管紧张素系统(RAS)不仅是维持肾脏正常发育的关键系统,而且是机体调控内环境稳定的重要体液调节系统之一。而血管紧张素Ⅱ(AngⅡ)是RAS中的主要活性肽,首先作为一种血管活性物质,可优先收缩肾小球出球小动脉,引起肾小球内高滤过损伤,亦可收缩系膜细胞,引起肾小球超滤系数下降。更为重要的是,近年来一致认为AngⅡ不仅是血管活性物质,更是一种促肾生长因子,除了可直接促使肾小球系膜细胞增生和肥大外,还能刺激其他血管活性物质和细胞因子产生,导致细胞外基质进行性积聚,促进炎症细胞浸润。AngⅡ通过组织局部的特异性受体发挥作用,如AngⅡ2型受体(AT2R)。研究发现AT2R在胎儿和围生期婴儿体内表达水平较高,表明它在细胞分化和器官发育过程中有重要作用。研究发现,AT2R的激活则对抗平滑肌细胞增殖,促进细胞凋亡。因此本实验拟观察LBW是否通过影响RAS,调节肾脏发生中关键基因的表达以及细胞的增殖和凋亡,从而引起肾单位数目减少的发生。
营养素是基因表达的重要调节因子,它可以通过控制代谢产物或代谢状态导致信使RNA、蛋白质合成与功能改变。“营养程序化”的理论指出发育关键时期的营养状况可以对机体脏器的结构和功能产生长期乃至终身的影响。早期营养环境刺激机体产生适应性的克隆选择或者分化母细胞的增殖,可以使组织细胞数量或比例永久性地得到改变。理论上在生命早期给予营养干预、改变机体营养环境,可以改善脏器的结构和功能,有助于对疾病的早期预防。因此,补充营养素来改变发育关键时期LBW人群或动物的营养状况,从而改变肾脏发育基因、信号传导通路,以及最终改变肾脏的结构和功能,达到减轻肾脏的病理损害、减轻高血压症状甚至减少高血压等发生的目的。所以选择一种合适的营养素干预是十分必要的。精氨酸是一种条件必需氨基酸,由于人体合成量较少,在婴幼儿的生长和发育时期是一种重要氨基酸,其在生物体内起生理作用的是左旋精氨酸(L-Arg),发挥着广泛而重要的生理和药理作用:促进FGR胎儿生长发育、减少AngⅡ含量、降低血压、改善肾功能等等。给LBW个体补充L-Arg,是否可以通过减少肾脏细胞凋亡、生成NO增多、减少AngⅡ生成等作用,来改善肾脏的结构和功能、减轻或杜绝高血压的发生,达到成年期疾病早期防治的目的?解决这一问题,将为L-Arg治疗LBWI提供有力的理论依据。本实验分为以下三部分:
第一部分LBW对大鼠肾脏发育和血压的影响
目的:用孕期全程低蛋白(10%蛋白)饮食法制造LBW仔鼠模型,观察LBW对仔鼠肾脏发育和血压的影响,并且探讨LBW是否是肾功能减低和高血压发生的危险因素之一。
方法:选择健康三月龄SD健康大鼠,按照雌雄1:1的比例置于同一笼中交配。受孕成功的孕鼠随机分为正常组和LBW组。正常组母鼠自受孕后第1d开始给予21%正常蛋白饮食,而LBW组母鼠自受孕后第1d开始给予10%蛋白饮食,建立LBW仔鼠模型。两组仔鼠出生后至3m龄,均给予母鼠及仔鼠21%正常蛋白饲料喂养、自由饮水。选择7d、21d、2m和3m时作为观察的4个时间点,用尾套法测量2m和3m时大鼠血压;留取7d、21d、2m和3m血标本和21d、2m和3m尿标本,检测肾功能各指标和24h尿蛋白量;2m时用酸消化法计算肾小球数目。
结果:1、LBW组仔鼠平均出生体重为(6.09±0.50)g,正常组为(6.98±0.44)g。LBW组仔鼠平均出生体重明显低于正常组(P＜0.01)。LBW组仔鼠呈现出阶段性追赶生长:7d时与正常组比较,差异有统计学意义(P＜0.05),LBW组体重显著降低;此后出现追赶生长,到21d时,两组体重比较差异无统计学意义;至2m时,LBW组大鼠体重生长再次落后,与正常组比较差异有统计学意义(P＜0.01);到3m时两组比较差异无统计学意义,LBW组达到追赶生长。
2、2m时,利用酸消化法检测两组仔鼠单侧肾脏总的肾小球数目。LBW组仔鼠肾小球数目明显低于正常组(22720±639)个/单侧vs.(28520±526)个/侧,P＜0.01。
3、2m时,LBW组和正常组大鼠的血压分别为(120.03±8.31)mmHg和(96.28±5.82)mmHg,两组比较差异有统计学意义(P＜0.01);至3m时,两组仔鼠的血压均有增高,分别为(127.03±7.13)mmHg和(109.75±9.26)mmHg,两组比较差异有统计学意义(P＜0.05)。
4、从21d～3m,LBW组大鼠Ccr与正常组比较差异有统计学意义,LBW组显著降低;3m时LBW组24h尿蛋白量与正常组比较,差异有统计学意义(117.17±10.40)mg/(kg·d)vs.(79.28±14.26)mg/(kg·d),P＜0.01,LBW组显著增加。
5、相关性分析显示:2m时,大鼠血压和肾小球数目有显著负相关性(r_1=-0.919,P_1=0.008);肾脏重量和血压有显著负相关性(r_2=-0.680,P_2=0.030);Ccr与肾脏重量成显著正相关(r_3=0.642,P_3=0.024);Ccr与肾小球数目成显著正相关(r_4=0.619,P_4=0.032)。
6、两组大鼠肾小球体积在7d时比较差异无统计学意义(μm~3×10~5)(正常组:0.41±0.05 vs.LBW组:0.50±0.08);在3m时,LBW组比正常组显著增大(μm~3×10~5)(1.19±0.09 vs.0.95±0.13,P＜0.01)。
结论:1、孕期用低蛋白饮食法成功建立了LBW大鼠模型,制模可靠,效果比较理想。
2、孕鼠低蛋白饮食可致LBW仔鼠成年期血压增高。
3、LBW大鼠肾脏发育延迟,并且肾单位数目减少了约20.34%。
4、LBW大鼠血压和肾单位数目有显著负相关,肾单位数目减少可能是LBW大鼠发生高血压的重要原因之一。
5、LBW大鼠肾功能减退可能参与了LBW大鼠高血压的发生。
第二部分LBW大鼠发生肾单位减少和高血压的机制研究
目的:研究LBW大鼠发育过程中,1、肾脏细胞的增生和凋亡及相关调控基因的表达;2、血管紧张素Ⅱ、环磷酸鸟苷(cGMP)及NO浓度的变化;3、Pax2基因的表达变化,探讨LBW大鼠发生肾脏功能减低和高血压的机制。
方法:选取LBW大鼠为研究对象,采用TUNEL法和Ki-67免疫组化法检测肾组织细胞的增殖和凋亡;RT-PCR法测定AT2R、Wt1和Bcl-2mRNA表达水平;用Western blot和免疫组化法分别检测肾组织中基因Pax2的表达。
结果:1、7d时与正常组比较,LBW组肾脏细胞凋亡指数明显增高(19.27±1.05%vs.13.24±0.89%,P＜0.05)。LBW组肾脏组织WT1mRNA表达在7d时显著减低,与正常组比较差异有统计学意义(0.83±0.11 vs.0.97±0.10,P＜0.05);LBW组肾脏组织Bcl-2 mRNA在7d时也明显降低,与正常组比较差异有统计学意义(0.88±0.06 vs.1.08±0.10,P＜0.05)。
2、2m和3m时,LBW组与正常组血浆AngⅡ浓度比较,差异有统计学意义(P＜0.01),LBW组明显高于正常组。2m时LBW组大鼠肾脏组织AT2R mRNA表达显著增高,与正常组比较差异有统计学意义(1.085±0.040 vs.0.963±0.051,P＜0.01)。在7d和21d,两组均未见Pax2的明显表达;到2m时LBW大鼠在肾小球和肾小管区可见Pax2阳性细胞的表达和蛋白的表达,3m时LBW大鼠肾脏Pax2阳性细胞和蛋白的表达均比2m更显著;而正常组仅见轻微蛋白表达。
3、LBW大鼠血清NO浓度在21d达到峰值,但在四个观察时间点均较正常组显著减低;在2m和3m时,LBW组大鼠血浆cGMP浓度显著降低。
4、相关性分析显示:2m时,AngⅡ浓度与血压成显著正相关(r=0.636,P=0.048)。
结论:1、肾发生中细胞凋亡的增加,以及Wt1、Bcl-2抗凋亡基因的表达降低可能是LBW大鼠肾单位数目减少的机制之一。
2、AngⅡ→AT2R通路可能是LBW大鼠肾脏功能减低和高血压发生的机制之一。基因Pax2的再表达可能参与了LBW大鼠肾脏功能减低和高血压的发生。
3、血cGMP和NO浓度的降低可能参与了LBW大鼠高血压发生的机制。
第三部分左旋精氨酸干预对LBW大鼠肾脏和血压的影响
目的:用左旋精氨酸(L-Arg)干预后,观察大鼠肾脏的结构和功能以及血压的变化情况,探讨L-Arg对LBW大鼠血压及肾脏影响的机制。
方法:L-Arg干预组(简称LT组)母鼠自受孕后第1天到仔鼠出生止给予10%低蛋白饮食;在仔鼠出生至21d哺乳期内,给予母鼠和仔鼠21%正常蛋白饲料喂养,另外加L-Arg水干预,按L-Arg 200mg/(kg.d)加入50ml饮水中白天给予,夜间自由饮水。正常组和LBW组造模及喂养方法同第一部分。于7d、21d、2m和3m时,用化学法测血清中NO浓度;放射免疫法测血浆中血管紧张素(AngⅡ)和环磷酸鸟苷(cGMP)浓度;尾套法测2m和3m时大鼠血压;常规生化法检测肾功能各指标和24h尿蛋白量;RT-PCR法检测肾组织Desmin mRNA表达;WesternBlot法测大鼠Pax2基因蛋白的表达;电镜观察肾小球系膜细胞和足细胞超微结构的变化。
结果:1.用L-Arg干预后,LT组大鼠体重增长较LBW多,21d时甚至超过正常组。
2.用L-Arg干预后,21d,2m和3m时LT组大鼠血清NO浓度均比LBW组显著升高。
3.用L-Arg干预后,2m时LT组血浆AngⅡ浓度比LBW组显著降低。2m和3m时,LT组cGMP浓度比LBW组显著增加,两组比较差异有统计学意义(P＜0.05)。
4.用L-Arg干预后,2m时与LBW组比较,LT组大鼠血压显著降低(P＜0.05)。
5.用L-Arg干预后,2m和3m时与LBW组比较,LT组Ccr显著增加(P＜0.05);3m时LT组24h尿蛋白量比LBW组显著降低(P＜0.01)。
6.用L-Arg干预后,21d时LT组Desmin mRNA表达与正常组比较,差异有统计学意义(P＜0.05),LT组表达增高。2m时,LT组比LBW组显著降低(P＜0.01),而与正常组比较,差异无统计学意义。
7.用L-Arg干预后,Western Blot检测发现,到2月和3m时,与LBW组比较,LT组大鼠肾组织Pax2蛋白表达显著减少(P＜0.01)。
8.用L-Arg干预后,电镜发现,2m时LBW组和LT组肾小球系膜细胞增生、滤过膜足突减少及部分溶合;LT组系膜细胞增生较LBW组减少;正常组系膜细胞无明显增生,滤过膜足突形态正常。
结论:1.L-Arg干预后,LT组大鼠体重增长较LBW多,2m时LT组血清NO水平和血浆cGMP浓度升高、AngⅡ浓度降低。
2.L-Arg干预后,LT组大鼠2m时Ccr明显增加,24h尿蛋白量3m龄时明显减少。
3.L-Arg干预后,LT组大鼠肾脏Desmin mRNA和Pax2表达减少。
4.L-Arg干预后,LT组大鼠肾脏系膜细胞增生减少、超微结构有一定改善。
综上所述,本研究结论如下:
1、用低蛋白(10%蛋白)饮食法成功地建立了LBW大鼠模型,LBW大鼠肾单位数目减少、肾脏发育落后,出现了肾功能的减低和高血压。提示:LBW可能是肾功能减退和高血压的原因之一。
2、LBW大鼠的肾小球中细胞凋亡显著增加,WT1和Bcl-2抗凋亡基因表达的下调。细胞凋亡增加可能是肾单位数目减少的机制之一。
3、LBW大鼠血浆AngⅡ浓度增加,AT2R mRNA表达的上调,基因Pax2的再表达。AngⅡ→AT2R通路可能是LBW大鼠肾脏功能减低和高血压发生的机制之一。基因Pax2的再表达可能参与了LBW大鼠肾脏功能减低和高血压的发生。
4、血浆cGMP和血清NO浓度的降低可能参与了LBW大鼠发生高血压的机制。
5、L-Arg干预后,LT组大鼠血NO和cGMP浓度升高、AngⅡ浓度一定程度降低,起一定程度降低血压的作用;LT组大鼠Ccr增加和尿蛋白水平降低,L-Arg干预起一定程度改善肾功能作用;LT组大鼠肾脏Desmin mRNA和Pax2表达减少、肾脏系膜细胞增生减少,L-Arg干预使肾脏病理损害有一定修复作用。
Background
Fetal growth restriction(FGR) has two means,one is intrauterine growth retardation(IUGR),the other is low birth weight(LBW)of newborn. In China,the rate of low birth weight infant is about 4.75%～8.5%,LBW is one of important serious reasons to increase morbidity and mortality of babies in perinatal period.In 1995,one reporter of the United Nations indicated that adults with cardiovascular disease and blood-stroke were LBWI at least 10%,those sickers with high blood pressure(HBP) were almost 50%with LBW.In 1992,the relationship of angiocardiopathy and LBW was found by Barker at first time,and then he introduced hypothesis about "fetal programming".From then more and more epidemiologies and clinical studies found that LBW was one of relative independent risk factors in angiocardiopathy,at the same time it has different effects on kidney and HBP of adults.Now,the associativity about LBW and HBP was confirmed by 80 studies.More epidemiologic studies showed the reason about reduction of nephrons is LBW,then the reduction of nephrons was one of independent risk factors in angiocardiopathy in adults.The effect of LBW on kidney and HBP provoked numerous scholars to study. To study the mechanisms of metabolism symptom which caused by LBW in adult period,and to practice prevention and cure of adults diseases in nonage had been one of investigative hot spots.
The obvious correlation about LBW and nephron reduction had been cofirmed by epidemiologic study and animal experiment,but its mechanism is still unknown.We all know the procession about renal development,especially the period of metanephros,needs more cell proliferation,apoptosis and moreover related gene control each other,such as Wt1,Bcl-2,Pax2,et al.At the same time,it is well know that the renin-angiotensin system(RAS) is not only the maintenance of normal kidney development in critical systems,but also is one of the importance regulation systems which can control internal environment humoral regulation systems.AngⅡis the main active peptide in RAS.First of all as a vasoactive substance,it had a priority to let small arteries out of the ball glomerular to contract,caused high filtration injury by glomerular, could also cause glomerular ultrafiltration coefficient decreased by contraction of mesangial cells.More importantly,in recent years,AngⅡis not only a vasoactive substances,also is a renal growth factor for promotion.In addition to directly promote glomerular mesangial cell hyperplasia and hypertrophy,but also it could stimulate other vasoactive substances and cell factor to generate,leading to extracellular matrix progressive accumulation,and promoting infiltration of inflammatory cells. AngⅡplayed a role through partial specific receptor of the organization, such as AngⅡ2 receptor(AT2R).Studies found that AT2R had higher expression levels in vivo at fetal and perinatal infants,indicating that it had an important role in the process of cell differentiation and organ development.The study found that activation of AT2R was against smooth muscle cell proliferation and promoted cell apoptosis.So our purpose on this experiment is to observe if LBW can regulate the expression of key genes and cell apoptosis through RAS in the kidney, thus leading to the diminished nephron number and HBP.
Nutrients are important regulatory factors of gene expression,it can lead to mRNA,protein synthesis and function change by controlling the metabolites or metabolic status."Nutritional programming" theory points out that in the key period of development the nutrition will influence the organ's function for a long time or lifetime.Early nutritional environment can timulate the body produce adaptive clonal selection or differentiation of the cell proliferation,and can make permanent change of tissue cells quantity or proportion.So during early life,if given nutritional intervention and changing nutritional environment,can improve the organ's structure and function,which can contribute to the early prevention of disease in theory.Therefore,adding nutrients will change the nutritional status in the critical period of development of LBW body in order to change the nutritional status of renal developmental genes,signal transduction pathways and ultimately change the structure and function of the kidney to alleviate the pathology of kidney damage,to alleviate symptoms of HBP,even eliminate the occurrence of HBP.Arginine is a conditional essential amino acid,because of low synthesis amount in body,it is an important amino acid in infants and young children during the growth and development.Its physiological effects in vivo are L-arginine(L-Arg), leading a broad effects of physiological and pharmacological,it can promote growth and development of the FGR fetal,to degrade blood pressure,to improve kidney function,to reduce AngⅡconcentration,and so on.If we feed LBW rat with L-Arg,it can increase NO level,reduce renal cell apoptosis and AngⅡgeneration,improve kidney structure and function,reduce or eliminate the occurrence rate of HBP? At last,in order to play a role to prevent and cure adult hypertension and kidney diseases.But it is still no theory to confirm.If we can solve this problem,it will provide a strong theoretical basis on treatment LBWI with L-Arg.
This experiment is divided into the following three parts:
PartⅠThe effect of LBW on blood pressure and kidney in rat
Objective
To make low birth weight neonatal rat model by feeding pregnant rats with 10%low protein diet.To observe the effect of LBW on renal growth and blood pressure,to observe if LBW is one of risk facors when HBP happened.
Methods
1.The building of low birth weight neonatal rat model and subgroups:twelve male and twelve female healthy three-month SD rats were bought respectivly.Male and female rats were put into a cage at the proportion of 1:1.The pregnant rats were divided into two groups:1) the model of low birth weight neonatal rats was made by feeding 10%low protein diet to the pregnant rats of LBW group during gestation and feeding 21%normal protein diet after dilivery;2) feeding 21%normal protein diet to the pregnant rats in normal group;3) All femal rats were given running warter.To observe the newly born member per nest,body weight of neonatal rat,IUGR incidence rate and perinatal mortality in each group.
2.Rat's blood pressure was measured by tail cuff at 2m and 3m,at 7d,21d,2m and 3m,to collect blood and urine for detecting biochemical indicators of renal function and 24h-urine protein,the glomeruli number was calculated by acid digestion method at 2m.
Results
1.At 7d,the body weight of offspring in model group was lower than that in normal group.LBW group rats showed a stagewise catching up with body weight.At 21d,the body weight in LBW group reached normal level.While at 2m the body weight in LBW group was under the normal level obviously.There were no significant differences between two groups. But at 3m,the body weight reached normal level in LBW group.
2.At 2m,the glomeruli number was calculated with acid digestion method,the number of glomeruli in LBW group was reduced obviously than that in normal group.
3.At 2m,the blood pressure in LBW group was higher bviously than that in normal group.But at 3m,there was no difference between two groups.
4.From 21d to 3m,the level of Ccr in LBW group was degraded significantly than that in normal group.At 3m,the level of 24h-Urine protein was notably increased in LBW group than that in normal group.
5.At 7d,the volume of glomeruli had no difference in 2 groups.But at 3m,the volume of glomerulu in LBW group was swelled more than that in normal group.
Conclusions
1.Feeding 10%low protein diet to the pregnant rat was an effective method to make low birth weight neonatal rat model.
2、The number of glomeruli per kidney in the model was reduced about 20.34%,at the same time,HBP was elevated during adult period.
3、The reduced number of glomeruli may be one of important reasons to elevate blood pressure.
4、The decreased renal function was probably taken part in HBP in LBW group.
PartⅡThe Study of the mechanism on decreasing the nephron number and HBP in LBW rat
Objective
To study the mechanisms of nephron numbers reduction and HBP by next measures:1.renal cell proliferation,apoptosis and related genes expression in LBW rat;2.the levels of AngⅡ,cGMP and NO;3.the expression of Pax2 gene in LBW group.
Methods
To establish a rat model of LBW with low protein(10%protein) diet. To select LBW rats as objects to study,to observe the cells proliferation and apoptosis by using the methods of TUNEL and Ki-67 immunohisto-chemistry in renal tissues,to detect the levels of AT2R,WT land Bcl-2 mRNA by the method of RT-PCR,to check the levels of AngⅡ,cGMP and NO in blood by chemical methods,to examine the expression of Pax2 protein by Western blot analysis in renal tissues at 4 time points.
Results
1.At 7d,the cells apoptotic index in LBW group raised up clearly than that in normal group(13.24±0.89%vs.19.27±1.05%,P＜0.05 ).And the expressions of WT1and Bcl-2 mRNA were decreased at 7d(0.83±0.11 vs.0.97±0.10,0.88±0.06 vs.1.08±0.10,P＜0.05 )
2.From 7d to 2m,the levels of AngⅡin two groups were raised gradually.Till 2m,the levels of AngⅡreached peak values.After that time, they showed a downtrend.At 2m and 3m,they were higher in LBW group than those in normal group.At 2m,the expression of AT2R mRNA in LBW group was higher obviously than that in normal group(1.085±0.040 vs.0.963±0.051,P＜0.01 ).At 7d and 21d,the expression of Pax2 was out of sight in two groups,but at 2m and 3m,the expression of Pax2 was increased highly in LBW group.The expression of Pax2 in LBW group at 3m was increased clearly than that at 2m.But it's expression in normal group was very lower than that in LBW group.
3.At 21d,the levels of NO reached peak values in 2 groups,but the level of NO was depressed notably in LBW group.The levels of cGMP had no differences in two groups at 7d and 21d,but it was reduced significently in LBW group at 2m and 3m.
Conclusions
1.The increasing cells apoptosis during kidney development and the decreasing expressions of WT1and Bcl-2 mRNA may be one of the mechanisms on decreased nephron number in LBW rat.
2.The passageway of AngⅡ→AT2R and the expression of Pax2 may be one of the important molecular mechanisms to reduce the numbers of nephrons and HBP in LBW rat.
3.The lower levels of cGMP and NO possibly participated in the mechanisms of nephrons reduction and HBP.
PartⅢThe Effect of Intervention on Kidney and Blood Pressure in LBW Rat wih L-Arg
Objective
After treating by L-Arg,to observe the dynamic change of rat kidney structure and function,as well as blood pressure,and to carry out related researchs,in order to investigate the mechanism on blood pressure and kidney in LBW rat with L-Arg.
Methods
The building of low birth weight neonatal rat model and subgroups: eightteen male and eighteen female healthy three-month SD rats were bought.Male and female rats were put into a cage at the proportion of 1:1. The pregnant rats were divided into three groups:1) the model of low birth weight neonatal rats was made by feeding 10%low protein diet to the pregnant rats of LBW group during gestation and feeding normal protein diet during lactation;2) feeding 21%normal protein diet to the pregnant rats in normal group;3) a part of LBW group were given a supplementation with L-Arg(200mg/Kg) drinking water(,LT group) only during 21d lactation;other femal rats were given running warter.To observe the newly born member per nest,body weight of neonatal rat, IUGR incidence rate and perinatal mortality in each group.
At 7d,21d,2m and 3m,to measure the level of serum NO by chemical method,to detecte the levels of AngⅡand cGMP in blood plasma,to measure blood pressure with tail cuff,to test renal function and 24h urine protein by biochemistry method,to use the method of RT-PCR to determinate the level of desmin mRNA,to use the method of western blot to detecte the expression of Pax2 protein in renal organizations,to observe ultrastructural change in mesangial cell and podocyte by electron microscope.
Results
1.With the intervention of L-Arg,the body weight in LT group increased more than that in LBW group.Even surpassed more than that in normal group at 21d.
2.With the intervention of L-Arg,the levels of NO and cGMP were increased in LT group than those in LBW group at 2m.
3.With the intervention of L-Arg,at 2m,the level of AngⅡwas decreased clearly in LT group than that in LBW group,but at 3m,the level of AngⅡwas raised clearly in LT group than that in normal group.
4.With the intervention of L-Arg,at 2m,the blood pressure in LT group was lower obviously than that in LBW group.
5.With the intervention of L-Arg,the level of Ccr in LT group was increased significantly than that in LBW group at 21d,2m and 3m.The level of 24h urine protein was significant decreased in LT group than that in LBW group at 3m.
6.With the intervention of L-Arg,the expression of desmin mRNA was decreased significently in LT group than that in LBW group at 2m.
7.With the intervention of L-Arg,at 7d and 21d,the expression of Pax2 protein was out of sight in LT group.But at 2m and 3m,the expressions of Pax2 protein were decreased highly in LT group than those in LBW group by western blot.
8.It showed that glomerulus mesangial cell proliferation,filtration membranes impairment and colliquefaction were serious in LBW group and LT group by electron microscope,and the level of lesion was a little improved in LT group than that in LBW group.But in normal group,they were not found any hurt.
Conclusions
1) L-Arg can increase body weight,the levels of NO and cGMP,at the same ime,it can decrease the levels of blood pressure.
2) L-Arg can decrease the expression of desmin mRNA and Pax2 protein in LT group,increase the level of Ccr and reduce 24h urine protein.
3) L-Arg can improve renal ultramicrostructure and function. The main contents and conclusions of the research were summarized as follows:
1.Feeding 10%low protein diet to the pregnant rat was an effective method to make LBW neonatal rat model.The numbers of glomerulus in the models were reduced,the development of kidney was delayed,the renal function was damaged and HBP was raised in later time.This hinted that LBW was perhaps one of reasons on renal injury and HBP in LBW rat.
2.The reduced numbers of nephrons in LBW rat had relationship with cell apoptosis,WT1and Bcl-2 gene expression both were downward adjustment during nephrogenesis.The increased cell apoptosis was probably one of the mechanisms on decreased nephron in LBW rat.
3.The level of AngⅡand the expression of AT2R mRNA increased and Pax2 gene appeard again.The road of AngⅡ→AT2R was possibly one of the important mechanisms to reduce nephrons and HBP in LBW rat.
4.The decreased levels of cGMP in blood plasma and NO in serum were probable one of the important mechanisms on HBP in LBW rat.
5.With L-Arg intervention,the levels of NO and Ccr were raised and the quantity of 24h-proteinufia was decreased.The lesser NO levels probably one of important reasons to decrease Ccr and increase the quantity of 24h-proteinuria in LBW rats.The expression of Desmin mRNA and Pax2 protein were decreased in intervention group.It showed L-Arg can lessen renal pathological lesion in a certain extent.
胎儿生长受限(fetal growth restriction,FGR)包括胎儿宫内发育迟缓(intrauterine growth retardation,IUGR)和新生儿低出生体重(low birth weight,LBW)。我国低出生体重儿(low birth weightinfant,LBWI)的出生率约为4.75%～8.5%,LBW是围产期婴儿发病和死亡的主要危险因素之一。联合国1995年的报告估计,发生心血管疾病和中风的成年患者至少10%为LBWI,大约50%的高血压患者为LBWI。自1992年Barker首次发现心血管疾病与出生体重有关,进而提出“胚胎编程”假说以来,越来越多的流行病学和临床研究发现,LBW是心血管不良事件相对独立的危险因素之一,同时对成人期的肾脏和高血压产生不同程度的影响。现已有80项有关研究(其中涉及44.4万个病例,年龄跨度0～80岁,包括多种种族人群)证实了LBW与高血压的相关性。大量流行病学研究发现,LBW导致肾单位数目的减少,同时肾单位的减少是成人期发展为全身性高血压的一个独立的危险因素。LBW对肾脏发育和高血压的影响,引起了众多学者的关注。研究LBW引起成年后发生代谢综合征的机制,以及实施成年期疾病的早期防治已经成为一个研究热点。
人类流行病学研究和动物实验均证实LBW与肾单位数目减少有明显相关性,但是其发生机制尚不清楚。肾脏的发育过程包括快速的结构重建,尤其是后肾阶段,这个过程需要大量的细胞增殖和凋亡,并且受到多种相关基因的严格调控及互相作用,如Wt1、Bcl-2、Pax2等。同时,研究表明肾素-血管紧张素系统(RAS)不仅是维持肾脏正常发育的关键系统,而且是机体调控内环境稳定的重要体液调节系统之一。而血管紧张素Ⅱ(AngⅡ)是RAS中的主要活性肽,首先作为一种血管活性物质,可优先收缩肾小球出球小动脉,引起肾小球内高滤过损伤,亦可收缩系膜细胞,引起肾小球超滤系数下降。更为重要的是,近年来一致认为AngⅡ不仅是血管活性物质,更是一种促肾生长因子,除了可直接促使肾小球系膜细胞增生和肥大外,还能刺激其他血管活性物质和细胞因子产生,导致细胞外基质进行性积聚,促进炎症细胞浸润。AngⅡ通过组织局部的特异性受体发挥作用,如AngⅡ2型受体(AT2R)。研究发现AT2R在胎儿和围生期婴儿体内表达水平较高,表明它在细胞分化和器官发育过程中有重要作用。研究发现,AT2R的激活则对抗平滑肌细胞增殖,促进细胞凋亡。因此本实验拟观察LBW是否通过影响RAS,调节肾脏发生中关键基因的表达以及细胞的增殖和凋亡,从而引起肾单位数目减少的发生。
营养素是基因表达的重要调节因子,它可以通过控制代谢产物或代谢状态导致信使RNA、蛋白质合成与功能改变。“营养程序化”的理论指出发育关键时期的营养状况可以对机体脏器的结构和功能产生长期乃至终身的影响。早期营养环境刺激机体产生适应性的克隆选择或者分化母细胞的增殖,可以使组织细胞数量或比例永久性地得到改变。理论上在生命早期给予营养干预、改变机体营养环境,可以改善脏器的结构和功能,有助于对疾病的早期预防。因此,补充营养素来改变发育关键时期LBW人群或动物的营养状况,从而改变肾脏发育基因、信号传导通路,以及最终改变肾脏的结构和功能,达到减轻肾脏的病理损害、减轻高血压症状甚至减少高血压等发生的目的。所以选择一种合适的营养素干预是十分必要的。精氨酸是一种条件必需氨基酸,由于人体合成量较少,在婴幼儿的生长和发育时期是一种重要氨基酸,其在生物体内起生理作用的是左旋精氨酸(L-Arg),发挥着广泛而重要的生理和药理作用:促进FGR胎儿生长发育、减少AngⅡ含量、降低血压、改善肾功能等等。给LBW个体补充L-Arg,是否可以通过减少肾脏细胞凋亡、生成NO增多、减少AngⅡ生成等作用,来改善肾脏的结构和功能、减轻或杜绝高血压的发生,达到成年期疾病早期防治的目的?解决这一问题,将为L-Arg治疗LBWI提供有力的理论依据。本实验分为以下三部分:
第一部分LBW对大鼠肾脏发育和血压的影响
目的:用孕期全程低蛋白(10%蛋白)饮食法制造LBW仔鼠模型,观察LBW对仔鼠肾脏发育和血压的影响,并且探讨LBW是否是肾功能减低和高血压发生的危险因素之一。
方法:选择健康三月龄SD健康大鼠,按照雌雄1:1的比例置于同一笼中交配。受孕成功的孕鼠随机分为正常组和LBW组。正常组母鼠自受孕后第1d开始给予21%正常蛋白饮食,而LBW组母鼠自受孕后第1d开始给予10%蛋白饮食,建立LBW仔鼠模型。两组仔鼠出生后至3m龄,均给予母鼠及仔鼠21%正常蛋白饲料喂养、自由饮水。选择7d、21d、2m和3m时作为观察的4个时间点,用尾套法测量2m和3m时大鼠血压;留取7d、21d、2m和3m血标本和21d、2m和3m尿标本,检测肾功能各指标和24h尿蛋白量;2m时用酸消化法计算肾小球数目。
结果:1、LBW组仔鼠平均出生体重为(6.09±0.50)g,正常组为(6.98±0.44)g。LBW组仔鼠平均出生体重明显低于正常组(P＜0.01)。LBW组仔鼠呈现出阶段性追赶生长:7d时与正常组比较,差异有统计学意义(P＜0.05),LBW组体重显著降低;此后出现追赶生长,到21d时,两组体重比较差异无统计学意义;至2m时,LBW组大鼠体重生长再次落后,与正常组比较差异有统计学意义(P＜0.01);到3m时两组比较差异无统计学意义,LBW组达到追赶生长。
2、2m时,利用酸消化法检测两组仔鼠单侧肾脏总的肾小球数目。LBW组仔鼠肾小球数目明显低于正常组(22720±639)个/单侧vs.(28520±526)个/侧,P＜0.01。
3、2m时,LBW组和正常组大鼠的血压分别为(120.03±8.31)mmHg和(96.28±5.82)mmHg,两组比较差异有统计学意义(P＜0.01);至3m时,两组仔鼠的血压均有增高,分别为(127.03±7.13)mmHg和(109.75±9.26)mmHg,两组比较差异有统计学意义(P＜0.05)。
4、从21d～3m,LBW组大鼠Ccr与正常组比较差异有统计学意义,LBW组显著降低;3m时LBW组24h尿蛋白量与正常组比较,差异有统计学意义(117.17±10.40)mg/(kg·d)vs.(79.28±14.26)mg/(kg·d),P＜0.01,LBW组显著增加。
5、相关性分析显示:2m时,大鼠血压和肾小球数目有显著负相关性(r_1=-0.919,P_1=0.008);肾脏重量和血压有显著负相关性(r_2=-0.680,P_2=0.030);Ccr与肾脏重量成显著正相关(r_3=0.642,P_3=0.024);Ccr与肾小球数目成显著正相关(r_4=0.619,P_4=0.032)。
6、两组大鼠肾小球体积在7d时比较差异无统计学意义(μm~3×10~5)(正常组:0.41±0.05 vs.LBW组:0.50±0.08);在3m时,LBW组比正常组显著增大(μm~3×10~5)(1.19±0.09 vs.0.95±0.13,P＜0.01)。
结论:1、孕期用低蛋白饮食法成功建立了LBW大鼠模型,制模可靠,效果比较理想。
2、孕鼠低蛋白饮食可致LBW仔鼠成年期血压增高。
3、LBW大鼠肾脏发育延迟,并且肾单位数目减少了约20.34%。
4、LBW大鼠血压和肾单位数目有显著负相关,肾单位数目减少可能是LBW大鼠发生高血压的重要原因之一。
5、LBW大鼠肾功能减退可能参与了LBW大鼠高血压的发生。
第二部分LBW大鼠发生肾单位减少和高血压的机制研究
目的:研究LBW大鼠发育过程中,1、肾脏细胞的增生和凋亡及相关调控基因的表达;2、血管紧张素Ⅱ、环磷酸鸟苷(cGMP)及NO浓度的变化;3、Pax2基因的表达变化,探讨LBW大鼠发生肾脏功能减低和高血压的机制。
方法:选取LBW大鼠为研究对象,采用TUNEL法和Ki-67免疫组化法检测肾组织细胞的增殖和凋亡;RT-PCR法测定AT2R、Wt1和Bcl-2mRNA表达水平;用Western blot和免疫组化法分别检测肾组织中基因Pax2的表达。
结果:1、7d时与正常组比较,LBW组肾脏细胞凋亡指数明显增高(19.27±1.05%vs.13.24±0.89%,P＜0.05)。LBW组肾脏组织WT1mRNA表达在7d时显著减低,与正常组比较差异有统计学意义(0.83±0.11 vs.0.97±0.10,P＜0.05);LBW组肾脏组织Bcl-2 mRNA在7d时也明显降低,与正常组比较差异有统计学意义(0.88±0.06 vs.1.08±0.10,P＜0.05)。
2、2m和3m时,LBW组与正常组血浆AngⅡ浓度比较,差异有统计学意义(P＜0.01),LBW组明显高于正常组。2m时LBW组大鼠肾脏组织AT2R mRNA表达显著增高,与正常组比较差异有统计学意义(1.085±0.040 vs.0.963±0.051,P＜0.01)。在7d和21d,两组均未见Pax2的明显表达;到2m时LBW大鼠在肾小球和肾小管区可见Pax2阳性细胞的表达和蛋白的表达,3m时LBW大鼠肾脏Pax2阳性细胞和蛋白的表达均比2m更显著;而正常组仅见轻微蛋白表达。
3、LBW大鼠血清NO浓度在21d达到峰值,但在四个观察时间点均较正常组显著减低;在2m和3m时,LBW组大鼠血浆cGMP浓度显著降低。
4、相关性分析显示:2m时,AngⅡ浓度与血压成显著正相关(r=0.636,P=0.048)。
结论:1、肾发生中细胞凋亡的增加,以及Wt1、Bcl-2抗凋亡基因的表达降低可能是LBW大鼠肾单位数目减少的机制之一。
2、AngⅡ→AT2R通路可能是LBW大鼠肾脏功能减低和高血压发生的机制之一。基因Pax2的再表达可能参与了LBW大鼠肾脏功能减低和高血压的发生。
3、血cGMP和NO浓度的降低可能参与了LBW大鼠高血压发生的机制。
第三部分左旋精氨酸干预对LBW大鼠肾脏和血压的影响
目的:用左旋精氨酸(L-Arg)干预后,观察大鼠肾脏的结构和功能以及血压的变化情况,探讨L-Arg对LBW大鼠血压及肾脏影响的机制。
方法:L-Arg干预组(简称LT组)母鼠自受孕后第1天到仔鼠出生止给予10%低蛋白饮食;在仔鼠出生至21d哺乳期内,给予母鼠和仔鼠21%正常蛋白饲料喂养,另外加L-Arg水干预,按L-Arg 200mg/(kg.d)加入50ml饮水中白天给予,夜间自由饮水。正常组和LBW组造模及喂养方法同第一部分。于7d、21d、2m和3m时,用化学法测血清中NO浓度;放射免疫法测血浆中血管紧张素(AngⅡ)和环磷酸鸟苷(cGMP)浓度;尾套法测2m和3m时大鼠血压;常规生化法检测肾功能各指标和24h尿蛋白量;RT-PCR法检测肾组织Desmin mRNA表达;WesternBlot法测大鼠Pax2基因蛋白的表达;电镜观察肾小球系膜细胞和足细胞超微结构的变化。
结果:1.用L-Arg干预后,LT组大鼠体重增长较LBW多,21d时甚至超过正常组。
2.用L-Arg干预后,21d,2m和3m时LT组大鼠血清NO浓度均比LBW组显著升高。
3.用L-Arg干预后,2m时LT组血浆AngⅡ浓度比LBW组显著降低。2m和3m时,LT组cGMP浓度比LBW组显著增加,两组比较差异有统计学意义(P＜0.05)。
4.用L-Arg干预后,2m时与LBW组比较,LT组大鼠血压显著降低(P＜0.05)。
5.用L-Arg干预后,2m和3m时与LBW组比较,LT组Ccr显著增加(P＜0.05);3m时LT组24h尿蛋白量比LBW组显著降低(P＜0.01)。
6.用L-Arg干预后,21d时LT组Desmin mRNA表达与正常组比较,差异有统计学意义(P＜0.05),LT组表达增高。2m时,LT组比LBW组显著降低(P＜0.01),而与正常组比较,差异无统计学意义。
7.用L-Arg干预后,Western Blot检测发现,到2月和3m时,与LBW组比较,LT组大鼠肾组织Pax2蛋白表达显著减少(P＜0.01)。
8.用L-Arg干预后,电镜发现,2m时LBW组和LT组肾小球系膜细胞增生、滤过膜足突减少及部分溶合;LT组系膜细胞增生较LBW组减少;正常组系膜细胞无明显增生,滤过膜足突形态正常。
结论:1.L-Arg干预后,LT组大鼠体重增长较LBW多,2m时LT组血清NO水平和血浆cGMP浓度升高、AngⅡ浓度降低。
2.L-Arg干预后,LT组大鼠2m时Ccr明显增加,24h尿蛋白量3m龄时明显减少。
3.L-Arg干预后,LT组大鼠肾脏Desmin mRNA和Pax2表达减少。
4.L-Arg干预后,LT组大鼠肾脏系膜细胞增生减少、超微结构有一定改善。
综上所述,本研究结论如下:
1、用低蛋白(10%蛋白)饮食法成功地建立了LBW大鼠模型,LBW大鼠肾单位数目减少、肾脏发育落后,出现了肾功能的减低和高血压。提示:LBW可能是肾功能减退和高血压的原因之一。
2、LBW大鼠的肾小球中细胞凋亡显著增加,WT1和Bcl-2抗凋亡基因表达的下调。细胞凋亡增加可能是肾单位数目减少的机制之一。
3、LBW大鼠血浆AngⅡ浓度增加,AT2R mRNA表达的上调,基因Pax2的再表达。AngⅡ→AT2R通路可能是LBW大鼠肾脏功能减低和高血压发生的机制之一。基因Pax2的再表达可能参与了LBW大鼠肾脏功能减低和高血压的发生。
4、血浆cGMP和血清NO浓度的降低可能参与了LBW大鼠发生高血压的机制。
5、L-Arg干预后,LT组大鼠血NO和cGMP浓度升高、AngⅡ浓度一定程度降低,起一定程度降低血压的作用;LT组大鼠Ccr增加和尿蛋白水平降低,L-Arg干预起一定程度改善肾功能作用;LT组大鼠肾脏Desmin mRNA和Pax2表达减少、肾脏系膜细胞增生减少,L-Arg干预使肾脏病理损害有一定修复作用。
Background
Fetal growth restriction(FGR) has two means,one is intrauterine growth retardation(IUGR),the other is low birth weight(LBW)of newborn. In China,the rate of low birth weight infant is about 4.75%～8.5%,LBW is one of important serious reasons to increase morbidity and mortality of babies in perinatal period.In 1995,one reporter of the United Nations indicated that adults with cardiovascular disease and blood-stroke were LBWI at least 10%,those sickers with high blood pressure(HBP) were almost 50%with LBW.In 1992,the relationship of angiocardiopathy and LBW was found by Barker at first time,and then he introduced hypothesis about "fetal programming".From then more and more epidemiologies and clinical studies found that LBW was one of relative independent risk factors in angiocardiopathy,at the same time it has different effects on kidney and HBP of adults.Now,the associativity about LBW and HBP was confirmed by 80 studies.More epidemiologic studies showed the reason about reduction of nephrons is LBW,then the reduction of nephrons was one of independent risk factors in angiocardiopathy in adults.The effect of LBW on kidney and HBP provoked numerous scholars to study. To study the mechanisms of metabolism symptom which caused by LBW in adult period,and to practice prevention and cure of adults diseases in nonage had been one of investigative hot spots.
The obvious correlation about LBW and nephron reduction had been cofirmed by epidemiologic study and animal experiment,but its mechanism is still unknown.We all know the procession about renal development,especially the period of metanephros,needs more cell proliferation,apoptosis and moreover related gene control each other,such as Wt1,Bcl-2,Pax2,et al.At the same time,it is well know that the renin-angiotensin system(RAS) is not only the maintenance of normal kidney development in critical systems,but also is one of the importance regulation systems which can control internal environment humoral regulation systems.AngⅡis the main active peptide in RAS.First of all as a vasoactive substance,it had a priority to let small arteries out of the ball glomerular to contract,caused high filtration injury by glomerular, could also cause glomerular ultrafiltration coefficient decreased by contraction of mesangial cells.More importantly,in recent years,AngⅡis not only a vasoactive substances,also is a renal growth factor for promotion.In addition to directly promote glomerular mesangial cell hyperplasia and hypertrophy,but also it could stimulate other vasoactive substances and cell factor to generate,leading to extracellular matrix progressive accumulation,and promoting infiltration of inflammatory cells. AngⅡplayed a role through partial specific receptor of the organization, such as AngⅡ2 receptor(AT2R).Studies found that AT2R had higher expression levels in vivo at fetal and perinatal infants,indicating that it had an important role in the process of cell differentiation and organ development.The study found that activation of AT2R was against smooth muscle cell proliferation and promoted cell apoptosis.So our purpose on this experiment is to observe if LBW can regulate the expression of key genes and cell apoptosis through RAS in the kidney, thus leading to the diminished nephron number and HBP.
Nutrients are important regulatory factors of gene expression,it can lead to mRNA,protein synthesis and function change by controlling the metabolites or metabolic status."Nutritional programming" theory points out that in the key period of development the nutrition will influence the organ's function for a long time or lifetime.Early nutritional environment can timulate the body produce adaptive clonal selection or differentiation of the cell proliferation,and can make permanent change of tissue cells quantity or proportion.So during early life,if given nutritional intervention and changing nutritional environment,can improve the organ's structure and function,which can contribute to the early prevention of disease in theory.Therefore,adding nutrients will change the nutritional status in the critical period of development of LBW body in order to change the nutritional status of renal developmental genes,signal transduction pathways and ultimately change the structure and function of the kidney to alleviate the pathology of kidney damage,to alleviate symptoms of HBP,even eliminate the occurrence of HBP.Arginine is a conditional essential amino acid,because of low synthesis amount in body,it is an important amino acid in infants and young children during the growth and development.Its physiological effects in vivo are L-arginine(L-Arg), leading a broad effects of physiological and pharmacological,it can promote growth and development of the FGR fetal,to degrade blood pressure,to improve kidney function,to reduce AngⅡconcentration,and so on.If we feed LBW rat with L-Arg,it can increase NO level,reduce renal cell apoptosis and AngⅡgeneration,improve kidney structure and function,reduce or eliminate the occurrence rate of HBP? At last,in order to play a role to prevent and cure adult hypertension and kidney diseases.But it is still no theory to confirm.If we can solve this problem,it will provide a strong theoretical basis on treatment LBWI with L-Arg.
This experiment is divided into the following three parts:
PartⅠThe effect of LBW on blood pressure and kidney in rat
Objective
To make low birth weight neonatal rat model by feeding pregnant rats with 10%low protein diet.To observe the effect of LBW on renal growth and blood pressure,to observe if LBW is one of risk facors when HBP happened.
Methods
1.The building of low birth weight neonatal rat model and subgroups:twelve male and twelve female healthy three-month SD rats were bought respectivly.Male and female rats were put into a cage at the proportion of 1:1.The pregnant rats were divided into two groups:1) the model of low birth weight neonatal rats was made by feeding 10%low protein diet to the pregnant rats of LBW group during gestation and feeding 21%normal protein diet after dilivery;2) feeding 21%normal protein diet to the pregnant rats in normal group;3) All femal rats were given running warter.To observe the newly born member per nest,body weight of neonatal rat,IUGR incidence rate and perinatal mortality in each group.
2.Rat's blood pressure was measured by tail cuff at 2m and 3m,at 7d,21d,2m and 3m,to collect blood and urine for detecting biochemical indicators of renal function and 24h-urine protein,the glomeruli number was calculated by acid digestion method at 2m.
Results
1.At 7d,the body weight of offspring in model group was lower than that in normal group.LBW group rats showed a stagewise catching up with body weight.At 21d,the body weight in LBW group reached normal level.While at 2m the body weight in LBW group was under the normal level obviously.There were no significant differences between two groups. But at 3m,the body weight reached normal level in LBW group.
2.At 2m,the glomeruli number was calculated with acid digestion method,the number of glomeruli in LBW group was reduced obviously than that in normal group.
3.At 2m,the blood pressure in LBW group was higher bviously than that in normal group.But at 3m,there was no difference between two groups.
4.From 21d to 3m,the level of Ccr in LBW group was degraded significantly than that in normal group.At 3m,the level of 24h-Urine protein was notably increased in LBW group than that in normal group.
5.At 7d,the volume of glomeruli had no difference in 2 groups.But at 3m,the volume of glomerulu in LBW group was swelled more than that in normal group.
Conclusions
1.Feeding 10%low protein diet to the pregnant rat was an effective method to make low birth weight neonatal rat model.
2、The number of glomeruli per kidney in the model was reduced about 20.34%,at the same time,HBP was elevated during adult period.
3、The reduced number of glomeruli may be one of important reasons to elevate blood pressure.
4、The decreased renal function was probably taken part in HBP in LBW group.
PartⅡThe Study of the mechanism on decreasing the nephron number and HBP in LBW rat
Objective
To study the mechanisms of nephron numbers reduction and HBP by next measures:1.renal cell proliferation,apoptosis and related genes expression in LBW rat;2.the levels of AngⅡ,cGMP and NO;3.the expression of Pax2 gene in LBW group.
Methods
To establish a rat model of LBW with low protein(10%protein) diet. To select LBW rats as objects to study,to observe the cells proliferation and apoptosis by using the methods of TUNEL and Ki-67 immunohisto-chemistry in renal tissues,to detect the levels of AT2R,WT land Bcl-2 mRNA by the method of RT-PCR,to check the levels of AngⅡ,cGMP and NO in blood by chemical methods,to examine the expression of Pax2 protein by Western blot analysis in renal tissues at 4 time points.
Results
1.At 7d,the cells apoptotic index in LBW group raised up clearly than that in normal group(13.24±0.89%vs.19.27±1.05%,P＜0.05 ).And the expressions of WT1and Bcl-2 mRNA were decreased at 7d(0.83±0.11 vs.0.97±0.10,0.88±0.06 vs.1.08±0.10,P＜0.05 )
2.From 7d to 2m,the levels of AngⅡin two groups were raised gradually.Till 2m,the levels of AngⅡreached peak values.After that time, they showed a downtrend.At 2m and 3m,they were higher in LBW group than those in normal group.At 2m,the expression of AT2R mRNA in LBW group was higher obviously than that in normal group(1.085±0.040 vs.0.963±0.051,P＜0.01 ).At 7d and 21d,the expression of Pax2 was out of sight in two groups,but at 2m and 3m,the expression of Pax2 was increased highly in LBW group.The expression of Pax2 in LBW group at 3m was increased clearly than that at 2m.But it's expression in normal group was very lower than that in LBW group.
3.At 21d,the levels of NO reached peak values in 2 groups,but the level of NO was depressed notably in LBW group.The levels of cGMP had no differences in two groups at 7d and 21d,but it was reduced significently in LBW group at 2m and 3m.
Conclusions
1.The increasing cells apoptosis during kidney development and the decreasing expressions of WT1and Bcl-2 mRNA may be one of the mechanisms on decreased nephron number in LBW rat.
2.The passageway of AngⅡ→AT2R and the expression of Pax2 may be one of the important molecular mechanisms to reduce the numbers of nephrons and HBP in LBW rat.
3.The lower levels of cGMP and NO possibly participated in the mechanisms of nephrons reduction and HBP.
PartⅢThe Effect of Intervention on Kidney and Blood Pressure in LBW Rat wih L-Arg
Objective
After treating by L-Arg,to observe the dynamic change of rat kidney structure and function,as well as blood pressure,and to carry out related researchs,in order to investigate the mechanism on blood pressure and kidney in LBW rat with L-Arg.
Methods
The building of low birth weight neonatal rat model and subgroups: eightteen male and eighteen female healthy three-month SD rats were bought.Male and female rats were put into a cage at the proportion of 1:1. The pregnant rats were divided into three groups:1) the model of low birth weight neonatal rats was made by feeding 10%low protein diet to the pregnant rats of LBW group during gestation and feeding normal protein diet during lactation;2) feeding 21%normal protein diet to the pregnant rats in normal group;3) a part of LBW group were given a supplementation with L-Arg(200mg/Kg) drinking water(,LT group) only during 21d lactation;other femal rats were given running warter.To observe the newly born member per nest,body weight of neonatal rat, IUGR incidence rate and perinatal mortality in each group.
At 7d,21d,2m and 3m,to measure the level of serum NO by chemical method,to detecte the levels of AngⅡand cGMP in blood plasma,to measure blood pressure with tail cuff,to test renal function and 24h urine protein by biochemistry method,to use the method of RT-PCR to determinate the level of desmin mRNA,to use the method of western blot to detecte the expression of Pax2 protein in renal organizations,to observe ultrastructural change in mesangial cell and podocyte by electron microscope.
Results
1.With the intervention of L-Arg,the body weight in LT group increased more than that in LBW group.Even surpassed more than that in normal group at 21d.
2.With the intervention of L-Arg,the levels of NO and cGMP were increased in LT group than those in LBW group at 2m.
3.With the intervention of L-Arg,at 2m,the level of AngⅡwas decreased clearly in LT group than that in LBW group,but at 3m,the level of AngⅡwas raised clearly in LT group than that in normal group.
4.With the intervention of L-Arg,at 2m,the blood pressure in LT group was lower obviously than that in LBW group.
5.With the intervention of L-Arg,the level of Ccr in LT group was increased significantly than that in LBW group at 21d,2m and 3m.The level of 24h urine protein was significant decreased in LT group than that in LBW group at 3m.
6.With the intervention of L-Arg,the expression of desmin mRNA was decreased significently in LT group than that in LBW group at 2m.
7.With the intervention of L-Arg,at 7d and 21d,the expression of Pax2 protein was out of sight in LT group.But at 2m and 3m,the expressions of Pax2 protein were decreased highly in LT group than those in LBW group by western blot.
8.It showed that glomerulus mesangial cell proliferation,filtration membranes impairment and colliquefaction were serious in LBW group and LT group by electron microscope,and the level of lesion was a little improved in LT group than that in LBW group.But in normal group,they were not found any hurt.
Conclusions
1) L-Arg can increase body weight,the levels of NO and cGMP,at the same ime,it can decrease the levels of blood pressure.
2) L-Arg can decrease the expression of desmin mRNA and Pax2 protein in LT group,increase the level of Ccr and reduce 24h urine protein.
3) L-Arg can improve renal ultramicrostructure and function. The main contents and conclusions of the research were summarized as follows:
1.Feeding 10%low protein diet to the pregnant rat was an effective method to make LBW neonatal rat model.The numbers of glomerulus in the models were reduced,the development of kidney was delayed,the renal function was damaged and HBP was raised in later time.This hinted that LBW was perhaps one of reasons on renal injury and HBP in LBW rat.
2.The reduced numbers of nephrons in LBW rat had relationship with cell apoptosis,WT1and Bcl-2 gene expression both were downward adjustment during nephrogenesis.The increased cell apoptosis was probably one of the mechanisms on decreased nephron in LBW rat.
3.The level of AngⅡand the expression of AT2R mRNA increased and Pax2 gene appeard again.The road of AngⅡ→AT2R was possibly one of the important mechanisms to reduce nephrons and HBP in LBW rat.
4.The decreased levels of cGMP in blood plasma and NO in serum were probable one of the important mechanisms on HBP in LBW rat.
5.With L-Arg intervention,the levels of NO and Ccr were raised and the quantity of 24h-proteinufia was decreased.The lesser NO levels probably one of important reasons to decrease Ccr and increase the quantity of 24h-proteinuria in LBW rats.The expression of Desmin mRNA and Pax2 protein were decreased in intervention group.It showed L-Arg can lessen renal pathological lesion in a certain extent.
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