【作者】 龙泉；

【导师】 顾勇； 陈靖； 郝传明；

【作者基本信息】 复旦大学 ， 内科学， 2009， 博士

【摘要】 第一部分短期低蛋白合并α-酮酸饮食对维持性血液透析患者难治性高磷血症的影响目的：高磷血症是维持性血液透析患者常见并发症之一，与患者心血管并发症和肾性骨病的发生密切相关，然而临床上治疗却很棘手，血磷控制达标率很低。限制蛋白质摄入必然会减少磷的摄入，但是出于对营养状况的担忧，临床上缺乏在血液透析患者中应用低蛋白饮食的经验。开同^?是复方α-酮酸制剂，合并低蛋白饮食应用，能补充慢性肾脏病患者体内缺乏的必需氨基酸，维持正氮平衡；由于含钙，还能在一定程度上结合磷。因此，本研究旨在观察短期低蛋白合并α-酮酸饮食对维持性血液透析患者难治性高磷血症的影响。方法：在专业营养师的指导下按照患者体重指数（BMI）及饮食习惯制定个体化的低蛋白食谱，热卡为30-35kcal／kg／d，蛋白质摄入为0.8g／kg／d，同时予以复方α-酮酸制剂12片／天口服，治疗8周。通过3日饮食日记、营养状况评分、人体测量学指标（上臂肌围、肱三头肌皮褶厚度、干体重、体重指数等）和生化指标（血清肌酐、校正的蛋白质分解率、血清总蛋白、血清白蛋白、血浆氨基酸谱）对患者进行营养状态评估。测定治疗前后的高敏C反应蛋白（hs CRP）、尿素清除率（Kt／V）和CO₂结合力（CO₂-CP），分别观察患者的炎症状态、透析充分性和代谢性酸中毒情况。测定治疗前后的血钙、磷（计算钙磷乘积）和全段甲状旁腺激素（iPTH）的水平，评价该疗法对钙磷代谢，营养状况等的影响。结果：所有患者平均摄入的总热卡为：30.10±3.70kcal／kg／d，蛋白质为：0.86±0.15g／kg／d，磷为：669.55±134.51mg／d。营养状况评分、人体测量学指标和生化指标在试验前后的差异均无统计学意义（p值均＞0.05）。hs CRP和Kt／V水平在治疗前后的差异均无统计学意义（p值均＞0.05），CO₂-CP水平在试验后显著升高[（25.34±2.81vs18.49±1.77）mmol／L，p＜0.001]。血磷及钙磷乘积水平在试验后较试验前显著下降[P：（5.59±1.20 vs7.26±1.42）mg／dl，p＜0.001；Ca×P：（52.94±12.80 Vs 70.60±12.39）mg²／dl²，p＜0.001]。血Ca和iPTH水平在治疗前后的差异均无统计学意义[Ca：（9.44±1.04 vs 9.80±1.00）mg／dl；iPTH：（454.23±36.51 vs 531.28±48.00）pg／ml]（p值均＞0.05）。结论：短期低蛋白饮食合并α-酮酸疗法是一种安全有效治疗维持性血液透析患者难治性高磷血症的方法。长期使用该方法的疗效和安全性有待于更多大规模、多中心、随机对照的临床研究加以证实。第二部分低蛋白合并α-酮酸饮食对肾脏保护作用机制的动物研究（一）低蛋白合并α-酮酸饮食对5／6肾大部切除大鼠的肾脏保护作用和机制探讨目的：低蛋白合并α-酮酸饮食的营养疗法在临床上常用于中重度肾功能损害的慢性肾脏病患者以延缓病程进展，但是目前对其治疗安全性的认识仍存在争议，而且其肾脏保护作用的具体机制也不清楚。因此本研究旨在观察低蛋白合并α-酮酸饮食对5／6肾大部切除大鼠肾功能的影响，并探讨相关可能的机制。方法：30只雄性SD大鼠行5／6肾大部切除术建立慢性肾功能衰竭模型，1周后给予不同蛋白含量饲料喂养，根据喂养分组如下：（1）正常蛋白组（NPD组）：予18％酪蛋白；（2）低蛋白组（LPD组）：予6％酪蛋白；（3）低蛋白+α-酮酸组（LK组）：予5％酪蛋白+1％α-酮酸，每组10只大鼠。另取10只雄性SD大鼠行假手术后予以正常蛋白（18％酪蛋白）含量饲料作对照组（Sham组）。12周后麻醉处死大鼠，留取血、尿和肾脏组织标本。常规生化法检测血清白蛋白（Alb）、总蛋白（TP）、血尿素氮（BUN）、血肌酐（Scr）、甘油三酯（TG）、胆固醇（CHO）、高密度脂蛋白（HDL）、低密度脂蛋白（LDL）和空腹血糖（FBG）；放免法检测大鼠空腹血清胰岛素；考马斯亮蓝结合法测定24h尿蛋白排泄量（24UPro）；扫描电镜观察肾小球滤过膜的超微结构。蛋白组学法检测血清中差异蛋白质的表达；比色法测定血清中的丙二醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽髓过氧化物酶（GSH-Px）的水平。免疫组织化学和免疫印迹法检测肾脏中转化生长因子（TGF-β₁）的表达。糖原染色法（PAS）观察肾小球硬化指数（GSI）和细胞外基质（ECM）增生病变。结果：1．一般情况：建模3个月后4组大鼠的体重、血清白蛋白和血清总蛋白水平的差异均无统计学意义（p值均＞0.05）。NPD、LPD、LK组大鼠的Scr较Sham组显著升高[（NPD：58.67±4.80 vs．LPD：59.40±3.65vs．LK：58.60±4.56vs．Sham：34.67±5.47）umol／L]（p值均＜0.05），但前3组间的差异均无统计学意义（p值均＞0.05）；LPD组（7.26±1.29）和LK组大鼠（6.63±2.20）的BUN水平较NPD组（11.03±2.45）显著降低（p值均＜0.05）。NPD、LPD、LK组大鼠的24UPro水平也较Sham组明显增多（p值均＜0.05），但其中LK组24UPro水平较NPD组和LPD组显著降低[（NPD：94.23±20.15 vs LPD：58.70±8.18vs LK：38.30±5.50 vs Sham：17.70±8.10）mg／24h]（p值均＜0.05）。NPD组大鼠的肾小球滤过膜存在轻度足突融合和内皮细胞屏障结构紊乱，而LPD组和LK组大鼠的肾小球滤过膜结构基本正常。2．应用蛋白组学分析了4组大鼠血清中共328种蛋白质。NPD、LPD和LK组与Sham组相比：5种蛋白质表达增高，8种蛋白质（如：抗氧化作用的谷胱甘肽过氧化物酶GSH-Px等）表达降低；LPD组与NPD组相比：5种蛋白质（如：抗氧化酶GSH-Px等）表达增高，10种蛋白质（如：急性反应期蛋白：胰蛋白酶抑制剂重链4-ITIH4等）表达降低；LK组和NPD组相比：14种蛋白质（如：营养相关的转铁蛋白serotransferrin和抗氧化酶GSH-Px等）表达增高，7种蛋白质（如ITIH4和载脂蛋白ApoE等）表达降低；LK组和LPD组相比：9种蛋白质（如：抗氧化酶GSH-Px等）表达增高，6种蛋白质（如：载脂蛋白ApoE和纤维化相关的层粘连蛋白laminin）表达降低。3．糖脂代谢：FBG在4组大鼠间的差异均无统计学意义（p值均＞0.05），但NPD、LPD和LK组大鼠的空腹血清胰岛素（NeD：41.86±6.13；LPD：27.49±4.15；LK：18.71±3.92）、HOMA-IR（NPD：19.93±1.52；LPD：14.68±0.10；LK：10.23±2.02）均较Sham组[Insulin：（12.48±2.24）；HOMA-IR：（4.51±0.61）]显著升高（p值均＜0.05），而在LK组中两项指标均较LPD组和NPD组显著降低（p值均＜0.05）。HDL在4组大鼠间的差异亦均无统计学意义（p值均＞0.05），但NPD、LPD和LK组大鼠的TG：（NPD：1.25±0.46；LPD：1.20±0.36；LK：0.92±0.33）、CHO：（NPD：2.89±0.65；LPD：2.56±0.51；LK：2.47±0.52）和LDL：（NPD：0.33±0.12；LPD：0.28±0.10；LK：0.25±0.08）水平均较Sham组[TG：（0.71±0.16）；CHO：（1.53±0.11）；LDL：（0.16±0.03）]显著升高（p值均＜0.05），而仅仅CHO和TG水平在LK组中较NPD组中显著降低（p值均＜0.05）。4．循环中氧化应激：NPD、LPD和LK组大鼠的血清中MDA含量均显著高于Sham组（3.1992±0.1781）（p值均＜0.05），但其中LK组（4.6056±0.1217）和LPD组（5.2752±0.1572）血清中MDA含量均较NPD组（6.4898±0.2621）显著减少，LK组血清中MDA含量又较LPD组进一步减少（p值均＜0.05）；而NPD、LPD和LK组大鼠的血清中SOD和GSH-Px水平均显著低于Sham组[SOD：（46.5054±2.4142）；GSH-Px：（332.2500±33.8189）]（p值均＜0.05），但其在LK组[SOD：（28.0876±1.0904）；GSH-Px：（173.7770±4.0926）]和LPD组[SOD：（22.4170±1.2948）；GSH-Px：（117.5703±5.4685）]血清中的水平均较NPD组[SOD：（15.2142±1.9010）；GSH-Px：（63.0785±4.8479）]显著升高，在LK组血清中的水平又较LPD组进一步升高（p值均＜0.05）。5．肾脏纤维化：纤维化因子TGF-β₁蛋白在NPD组中的表达（0.6459±0.0442）显著高于在LPD组（0.3763±0.0314）和LK组（0.3128±0.0296）中的表达（p值均＜0.05）。NPD、LPD和LK组大鼠的肾小球硬化指数（GSI）和细胞外基质（ECM）增生评分均较Sham组显著增高（p值均＜0.05），但在LK组和LPD组中均较NPD组显著降低[GSI：（NPD：37.50±6.31vs LPD：18.32±3.22 vs LK：14.91±2.33 vs Sham：2.71±1.02）；ECM增生评分：（NPD：0.176±0.05 vs LPD：0.078±0.02 vs LK：0.056±0.02 vs Sham：0.032±0.01）；p值均＜0.05]。结论：低蛋白合并α-酮酸饮食能在保证5／6肾大部切除大鼠稳定营养状态的同时，改善血脂代谢紊乱和胰岛素抵抗，并具有减轻5／6肾大部切除大鼠氮质血症、减少足突融合、降低蛋白尿排泄等肾脏保护作用，可能与改善肾脏纤维化病变和减轻循环中增强的氧化应激有关。（二）低蛋白合并α-酮酸饮食对5／6肾大部切除大鼠肾素-血管紧张素系统的影响目的：肾脏局部肾素-血管紧张素系统的激活，能导致肾脏局部氧化应激增强和肾脏纤维化病变加重，是慢性肾脏病进展的关键因素之一。已有研究证实高蛋白饮食会导致肾脏局部肾素产生增多，因此本研究旨在观察低蛋白合并α-酮酸饮食能否通过影响5／6肾大部切除大鼠肾素-血管紧张素系统的兴奋性而产生肾脏保护作用。方法：30只雄性SD大鼠行5／6肾大部切除术建立慢性肾功能衰竭模型，1周后给予不同蛋白含量饲料喂养，根据喂养分组如下：（1）正常蛋白组（NPD组）：予18％酪蛋白；（2）低蛋白组（LPD组）：予6％酪蛋白；（3）低蛋白+α-酮酸组（LK组）：予5％酪蛋白+1％α-酮酸，每组10只大鼠。另取10只雄性SD大鼠行假手术后予以正常蛋白（18％酪蛋白）含量饲料作对照组（Sham组）。12周后麻醉处死。留取血、尿和肾脏组织标本。应用放免法测定皮质匀浆中肾素和血管紧张素Ⅱ（AngⅡ）的水平，应用ELISA方法测定血浆中AngⅡ的水平。应用real-time PCR、免疫组织化学和免疫印迹法分别检测肾脏局部肾素和AngⅡ的Ⅰ型受体（AT₁）的基因和蛋白表达。比色法测定肾组织匀浆液中的丙二醛（MDA）、超氧化物歧化酶（SOD）、谷胱甘肽髓过氧化物酶（GSH-Px）和过氧化氢酶（CAT）的水平。结果：1．肾素-血管紧张素系统的改变：NPD、LPD和LK组大鼠的血浆和皮质匀浆中AngⅡ水平均较Sham组[血浆AngⅡ：（0.073±0.001）pg／ml：皮质匀浆AngⅡ：（20.48±2.34）pg／ml]显著升高（p值均＜0.05），但仅皮质匀浆中AngⅡ的水平在LPD组（38.5±5.87）和LK组（30.23±4.23）中较NPD组中（68.92±10.23）显著降低（p值均＜0.05）。皮质匀浆中肾素水平仅在LK组（0.16±0.01）中较NPD组中（0.20±0.01）显著降低（p＜0.05）。肾脏局部肾素蛋白的表达在LPD组（0.4824±0.0640）和LK组（0.3363±0.0267）均较NPD组（0.7805±0.0905）显著减少（p值均＜0.05），分别为NPD组的61.81％和43.09％。肾素基因的水平在LK组中（0.64±0.10）较NPD组（1.11±0.18）和LPD组（1.06±0.15）显著降低（p值均＜0.05），分别是NPD组的57.65％和LPD组的60.37％。肾脏局部AT₁蛋白的表达在LPD组（0.14±0.02）和LK组（0.13±0.01）均较NPD组（0.19±0.02）显著减少（p值均＜0.05），分别为NPD组的74％和68％。AT_1a基因的水平在LK组中（0.66±0.17）分别较NPD组（1.16±0.30）和LPD组（0.91±0.29）显著降低43.11％和27.47％（p值均＜0.05）。2．肾脏局部氧化应激：NPD、LPD和LK组大鼠的组织匀浆液中MDA含量均显著高于Sham组（3.9458±0.3953）（p值均＜0.05），但其中LK组（5.7220±0.2922）和LPD组（8.9340±0.2363）组织匀浆液中的MDA含量均较NPD组（11.3703±0.7423）显著减少，LK组的组织匀浆液中MDA含量又较LPD组进一步减少（p值均＜0.05）；而NPD、LPD和LK组大鼠的组织匀浆液中SOD、GSH-Px和CAT水平均显著低于Sham组[SOD：（152.3860±3.7769）；GSH-Px：（88.5448±2.7205）；CAT：（1189.2600±95.4250）]（p值均＜0.05），但在LK组[SOD：（88.5448±2.7205）；GSH-Px：（860.9982±18.0659）；CAT：（917.6600±30.1156）]和LPD组[SOD：（62.7137±2.3850）；GSH-Px：（755.9942±17.3367）；CAT：（715.0025±22.9055）]的组织匀浆液中其水平均较NPD组[SOD：（40.8243±3.0096）；GSH-Px：（589.5017±21.2464）；CAT：（464.0778±20.6500）]显著升高，在LK组的组织匀浆液中含量又较LPD组进一步减少（p值均＜0.05）。3．相关性分析：组织匀浆液中MDA含量（r=0.892）、肾组织TGF-β₁表达（r=0.716）、GSI（r=0.807）和ECM增生评分（r=0.673）均与组织匀浆液中AngⅡ的水平呈正相关（p值均＜0.001），而组织匀浆液中SOD活力（r=0.978）、GSH-Px活力（r=-0.965）和CAT活力（r=-0.891）均与组织匀浆液中AngⅡ的水平呈负正相关（p值均＜0.001）。结论：低蛋白合并α-酮酸饮食可能通过抑制肾脏局部RAS兴奋性，改善5／6肾大部切除大鼠肾脏局部氧化应激状态，减轻肾组织纤维化病变，发挥肾脏保护作用。更多还原

【Abstract】 PARTⅠThe effects of short-time application of low protein diet supplement withα-keto acidson refractory hyperphosphatemia in maintenance hemodialysis patientsObjectiveHyperphosphatemia is one of the most common complications in maintenance hemodialysis（MHD） patients, which closely relates to cardiovascular complications and renalosteodystrophy disease. Protein restriction will certainly bring to reduced phosphrousintake, but there is little experience of treating hyperphosphatemia in hemodialysis patientswith low protein diet （LPD） because of worring about malnutrition. Ketosteril^?, a mixtureofα-ketoanalogs, is often combined with LPD to add essential amino acids whichdeficiency in chronic kidney disease （CKD） patients, as well as be used as a potentphosphrous binder. The purpose of the study is to evaluate the effects of short-timeapplication of low protein diet supplement withα-keto acids on refractoryhyperphosphatemia in MHD patients.MethodsAll planned menu was designed and individualized by the dietitian according to body massindex （BMI） with total calorie intake of 30～35kcal/kg/d, protein intake of 0.8g/kg/d.α-ketoacids were administered at the dose of 12 pills per day throughout the 8-week study. Thenutrition status was evaluated through the three-day-diaries （recorded at week 1, 2, 4 and 8）,MNA score （minimal nutritional assessment）, somatometric measurement indices （armmuscle circumference, triceps skin-fold thickness, dry weight, body mass index） andbiochemical indices （serum creatinine, protein catabolic rate, total protein, albumin, plasmaamino acids profile）. High sensitivity C reactive protein （hs CRP）, urea clearance rate （Kt/V）and CO₂ combinding power （CO₂-CP） before and after the trial were measured to observethe inflammation, dialysis adequacy and metabolic acidosis, respectively. Serum calcium,phosphrous and intact parathyroid hormone （iPTH） were detected before and after the trialto estimate the effects on calcium and phosphrous metabolism. ResultsThe average calories intake, protein intake and phosphate intake during the study were30.10±3.70kcal/kg/d, 0.86±0.15g / kg/d and 669.55±134.51 mg/d, respectively. There is nosignificant difference in somatometric measurement indices and biochemical indicesbetween pre-trial and post-trial （p＞0.05）. No difference was observed in hs CRP and Kt/Vbefore and after the trial （p＞0. 05）, while CO₂-CP was significantly higher after the trial thanbefore [ （25.34±2.81 vs. 18.49±1.77）mmol/L, p＜0.001]. Serum phosphate level and calciumphosphateproduct were significantly decreased in the end of the study compared to thosebefore the trial [P: （5.59±1.20vs7.26±1.42）mg/dl, p＜0.001; Ca×P: （52.94±12.80 vs 70.60±12.39） mg²/dl², p＜0.001]。There was no marked change of serum calcium and iPTH afterthe trial [Ca: （9.44±1.04vs9.80±1.00） mg/dl;iPTH: （454.23±36.51vs531.28±48.00） pg/ml]（p＞0.05）.ConclusionLow protein diet supplement withα-keto acids could be a safe and efficient therapy tomanage the refractory hyperphosphatemia of the MHD patients. A large scaled, multicenters,randomized controlled clinical trial is needed to confirm whether a long-termapplication of such treatment has the similar benefits. PARTⅡIn vivo study of the renal protective mechanisms oflow protein diet withα-keto acids supplement1. Study of the effects of low protein diet withα-keto acids supplementon renal function of 5/6 nephrectomized rats and its mechanismsObjectiveLow protein diet withα-keto acids supplement therapy is often been used in advancedchronic kidney disease patients to slow the progression of the disease, but it has been usedwith controversary on its safty and little known mechanisms. The purpose of the study is toobserve the effects of low protein diet withα-keto acids supplement on renal function of 5/6nephrectomized rats and its possible mechanisms.MethodsChronic renal failure model was established by 5/6 nephrectomy （Nx） in 30 male Sprague-Dawleyrats, then the animals were randomly assigned to the following diet groups: normalprotein group （NPD:18% casein protein）, low protein group （LPD:6% casein protein） andsupplemented low protein group （LK: 5% casein protein+1%α-keto acids）. Ten maleSprague-Dawley sham-operated rats giving 18% casein protein served as control group（Sham）. All rats were killed at the end of the 12^th week with blood and urine samplescollected. Serum albumin （Alb）, total protein （TP）, blood urea nitrogen （BUN）, serumcreatinine （Scr）, triglyceride （TG）, cholesterol （CHO）, high density lipoprotein（HDL）, lowdensity lipolprotein （LDL） and fasting blood glucose （FBG） were measured by routinebiochemistry. Fasting serum insulin was detected by radioimmunoassay. 24h urine proteinexcretion was detected with coomassie brilliant blue combined techniques. Electronicmicroscope was used to observe the structure of glomerular filtration membrane.Proteomics was used to identify the differentially-expressed protein in serum among thefour groups. The assays of malonaldehyde （MDA）, superoxide dismutase （SOD）,glutathione peroxidase （GSH-Px） in serum were measured by colorimetric method.Immunohistochemistry and western blot were used to detect the protein expression ofTGF-β₁ in residual kidney. Pathological changes of the residual kidney were investigatedwith periodic acid schiff （PAS） staining. Results1. General status: nutritional indices including weight, Alb and TP were not significantdifferent among the four groups （p＞0.05）. Scr was significantly higher in the Nx ratsthan in Sham rats [（NPD: 58.67±4.80vs.LPD:59.40±3.65 vs.LK:58.60±4.56 vs.Sham:34.67±5.47） umol/L, p＜0.05], but was of no difference among the three Nx groups（p＞0.05）. BUN was obviously lower in LPD （7.26±1.29） and LK group （6.63±2.20）than in NPD group （11.03±2.45）, （p＞0.05）. Proteinuria level of Nx groups wasmarkedly higher than that of the Sham group [（17.70±8.10）mg/24h], and that of the LKgroup [（38.30±5.50）mg/24h] was lower than those of NPD [（94.23±20.15）mg/24h] andLPD [（58.70±8.18）mg/24h] groups（p＜0.05）. The glomerular filtration membrane ofNPD group manifested foot process fusion and endothelial cell fenestrationdisappearance, but it was kept almost normal in LPD and LK group;2. 328 kinds of serological protein in four groups were analyzed by proteomics. When theNx groups were compared to Sham group, there were five kinds of protein expressionelevated, while eight kinds of protein expression declined （eg: anti-oxidative stressrelated protein GSH-Px, et al）. Five types of protein expressed more in LPD group thanin NPD group （eg: GSH-Px, et al）, while ten types of protein expressed less in LPDgroup （eg: acute reactive protein ITIH4, et al）. There were fourteen kinds of elevatedprotein （eg: nutrition related protein serotrasferrin and anti-oxidative stress relatedprotein GSH-Px, et al） and seven kinds of reduced protein （eg: ITIH4 and ApoE, et al）in LK group compared to NPD group. Compared with LPD group, nine types of proteinlevel （eg: GSH-Px, et al） raised up while six kinds of protein level （eg: ApoE andfibrosis related protein laminin） stepped down in LK group;3. Fat and carbohydrate metabolism: the FBG was of no difference among the fourgroups（p＞0.05）, while fasting serum insulin and HOMA-IR in Nx groups weresignificantly higher than in Sham group [Insulin:（12.48±2.24）;HOMA-IR:（4.51±0.61）],but those in LK group [Insulin: （18.71±3.92）; HOMA-IR:（10.23±2.02）] weresignificantly lower than in LPD [Insulin: （27.49±4.15）; HOMA-IR:（14.68±0.10）] andNPD [Insulin:（41.86±6.13）; HOMA-IR: （19.93±1.52）] groups（p＜0,05）; There was nosignificantly differences of HDL in the four goups （p＞0.05）, while TG （NPD:1.25±0.46;LPD:1.20±0.36; LK:0.92±0.33）, CHO（NPD:2.89±0.65; LPD: 2.56±0.51; LK:2.47±0.52） and LDL （NPD:0.33±0.12; LPD:0.28±0.10; LK: 0.25±0.08） in Nx groups weresignificantly higher than in Sham group[TG （0.71±0.16）; CHO（1.53±0.11 ）; LDL（0.16±0.03）], but only TG and CHO in LK group were significantly lower than in LPD and NPD groups （p＜0.05）;4. Circualr oxidative stress: Serological MDA of the Nx groups was significantly higherthan that of Sham group （3.1992±0.1781） （p＜0.05）, but was reduced lower in LK（4.6056±0.1217） and LPD group （5.2752±0.1572） than in NPD group （6.4898±0.2621）,which was reduced further lower in LK group than in LPD group （p＜0. 05）.SerologicalSOD and GSH-Px of the Nx groups were notably lower than those of Sham group [SOD:（46.5054±2.4142）; GSH-Px:（332.2500±33.8189）], （p＜0.05）, but they were elevatedhigher in LK [SOD: （28.0876±1.0904）; GSH-Px:（173.7770±4.0926）] and LPD group[SOD: （22.4170±1.2948）; GSH-Px:（117.5703±5.4685）] than in NPD group [SOD:（15.2142±1.9010）; GSH-Px:（63.0785±4.8479）], which were elevated further higher inLK group than in LPD group （p＜0. 05）;5. Renal fibrosis: TGF-β₁ protein expression in NPD group （0.6459±0.0442） wasremarkably higher than that in Sham group （0.0671±0.0172）, which was significantlydown-regulated in LPD （0.3763±0.0314） and LK group （0.3128±0.0296）, （p＜0.05）.Glomerular sclerosis index （GSI） and excelluar matrix score of the Nx groups weresignificantly higher than those of Sham group, but improved lessen in LPD and LKgroup than in NPD group [GSI （NPD: 37.50±6.31 vs. LPD: 18.32±3.22 vs. LK: 14.91±2.33 vs. Sham:2.71±1.02）; ECM score （NPD: 0.176±0.05 vs LPD: 0.078±0.02 vs LK:0.056±0.02 vs Sham: 0.032±0.01）; p＜0. 05].ConclusionsLow protein diet withα-ketoacids supplement could keep stable nutritional status as well asameliorate the lipids metabolism disturbance and insulin resistance in 5/6 nephrectomizedrats. The therapy could also exhibit renal protective effects of lessening azotemia, reducingfoot process fusion and urinary protein excretion at the same time. Improvement of circularoxidative stress and renal fibrosis change may both be involved. 2. Effects of low protein diet withα-keto acids supplement onrenin-angiotensin system（RAS） in 5/6 nephrectomized ratsObjectiveThe activation of renal local RAS, one of key factors contributing to the progression ofchronic kidney disease, may lead to enhanced oxidative stress and aggravated renal fibrosis.It has been proved that high protein diet will result in increased renin secretion. The purposeof the study is to observe the effects of low protein diet withα-keto acids supplement onrenin-angiotensin system in 5/6 nephrectomized rats.MethodsExperimental animal modeling composition and grouping were the same as the part 1. Therenin and angiotensinⅡin tissue were measured by radioimmunoassay, whileangiotensinⅡin plasma were detected with the ELISA kit. Immunohistochemistry andwestern blot were used to locate and quantitate the protein expression of renin and AT₁.Real-time PCR was used to detect the gene expression of renin and AT_1a, the main subtypesof AT₁ receptor. The assays of malonaldehyde （MDA）, superoxide dismutase （SOD）,glutathione peroxidase （GSH-Px） and catalase （CAT） in tissue were measured bycolorimetric method.Results1. Change of RAS: the AngⅡlevel both in plasma and in tissue were higher in the Nxgroups than in Sham group [plasma:（0.073±0.001）pg/ml;tissue:（20.48±2.34） pg/ml,p＜0.05], but only in tissue the AngⅡlevel was lower in LPD group （38.57±5.87） and LKgroup （30.23±4.23） than in NPD group （68.92+10.23） （p＜0.05）.The renin level in tissuewas lower in LK group （0.16±0.01） than in NPD group （0.20±0.01） （p＜0.05）. The reninprotein expressed much less in LPD group （0.4824±0.0640） and LK group （0.3363±0.0267） than that in NPD group （0.7805±0.0905） （p＜0.05）. Renin mRNA level in LKgroup （0.64±0.10） was significantly lower than that in NPD group （1.11±0.18） and LPDgroup （1.06±0.15） （p＜0. 05）. The AT₁ protein expression in LPD group （0.14±0.02） andLK group （0.13±0.01） was much lesser than that in NPD group （0.19±0.02） （p＜0.05）.AT_1a mRNA level in LK group （0.66±0.17） was significantly lower than that in NPDgroup （1.16±0.30） and LPD group （0.91±0.29） （p＜0.05）. 2. Renal local oxidative stress: MDA in tissue of the Nx groups was significantly higherthan in Sham group （3.9458±0.3953）, which was reduced lower in LK （5.7220±0.2922）and LPD group （8.9340±0.2363） than in NPD group（11.3703±0.7423）, and was reducedeven lower in LK group than in LPD group （p＜0.05）. SOD, GSH-Px and CAT in tissueof the Nx groups were significantly lower than in Sham group[SOD:（152.3860±3.7769）;GSH-Px: （88.5448±2.7205）; CAT: （1189.2600±95.4250）], （p＜0.05）, while those wereelevated higher in LK [SOD:（88.5448±2.7205）; GSH -Px: （860.9982±18.0659）; CAT:（917.6600±30.1156）] and LPD [SOD:（62.7137±2.3850）; GSH-Px:（755.9942±17.3367）;CAT:（715.0025±22.9055）] group than in NPD group [SOD: （40.8243±3.0096）; GSH-Px:（589.5017±21.2464）; CAT: （464.0778±20.6500）] （p＜0.05）, which were elevated evenhigher in LK group than in LPD group （p＜0.05）.3.Correlation analysis: MDA in tissue （r=0.892）, TGF-β₁ expression （r=0.716）, GSI（r=0.807） and ECM score （r=0.673） were positively related to AngⅡlevel in tissue （p＜0.001）, but SOD （r=-0.978）, GSH-Px （r=-0.965） and CAT （r=-0.891） in tissue werenegatively related to AngⅡlevel in tissue （p＜0.001）.ConclusionsLow protein diet withα-keto acids supplement therapy may exhibit renal protective effectsof improving oxidative stress and renal fibrosis pathology through inhibition the acitivity oflocal renin-angiotensin system in 5/6 nephrectomized rats.更多还原