Study On The Mechanism Of Kidney And Blood Pressure In Low Birth Weight Rat

BackgroundFetal 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 ratObjectiveTo 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.Methods1.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.Results1.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.Conclusions1.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 ratObjectiveTo 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.MethodsTo 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.Results1.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. Conclusions1.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-ArgObjectiveAfter 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.MethodsThe 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.Results1.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.Conclusions1) 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.