CAJ | 학술논문

目的观察肾损伤大鼠低蛋白配伍α酮酸饮食后是否可直接影响肾小球系膜细胞外基质(ECM)的产生和局部肾素-血管紧张素系统(RAS)的表达.方法 30只雄性SD大鼠建立3/4肾切除模型,分组如下(均n=10):正常蛋白饮食组(NPD,18%酪蛋白)、低蛋白饮食组(LPD,6.5%酪蛋白)和低蛋白配伍α酮酸饮食组(LK,5.4%酪蛋白+1%α酮酸).另取10只大鼠行假手术后给予正常蛋白饮食作为对照组(sham).12周后麻醉处死大鼠,收集各组动物血清.取10%浓度的血清干预体外培养的系膜细胞48 h.采用ELISA法检测细胞上清液中血管紧张素II(AngII)、转化生长因子β1(TGF-β1)及纤连蛋白(FN)的水平.采用Western印迹法和实时定量PCR法检测系膜细胞血管紧张素Ⅱ1型受体(ATlR)、FN及TGF-β1的蛋白和基因表达.结果 (1)动物实验:各组大鼠体质量、血清总蛋白及白蛋白水平差异均无统计学意义,肾切除各组大鼠血肌酐及尿蛋白水平均显著高于sham组(均P<0.05).血肌酐水平在肾切除组间差异无统计学意义,但LPD组血尿素氮及尿蛋白水平显著低于NPD组(均P<0.05),而LK组更低(均P<0.05).(2)NIN实验:NPD血清干预组细胞分泌AngⅡ水平[(12.70±0.12)mg/g蛋白比(8.04±0.62)mg/g蛋白]及AT1R基因和蛋白表达水平均显著高于sham组,同时伴有FN[(39.84±0.06)g/g蛋白比(20.58+0.46)g/g蛋白]及TGF-β1[(83.85±3.58)mg/g蛋白比(10.12±0.56)mg/g]基因转录和分泌水平的上升(均P<0.05);LPD及LK血清干预组可显著抑制上述改变(均P<0.05).应用RAS阻断剂氯沙坦可显著降低NPD组中FN及TGF-β1的合成与分泌,对LPD组中二者的表达也有进一步的抑制作用(均P<0.05),但对LK组无明显影响.结论低蛋白配伍α酮酸饮食可维持肾切除大鼠营养平衡,直接抑制系膜细胞RAS的表达,减少细胞外基质的分泌,从而发挥肾脏保护作用.
목적 관찰신손상대서저단백배오α동산음식후시부가직접영향신소구계막세포외기질(ECM)적산생화국부신소-혈관긴장소계통(RAS)적표체.방법 30지웅성SD대서건립3/4신절제모형,분조여하(균n=10):정상단백음식조(NPD,18%락단백)、저단백음식조(LPD,6.5%락단백)화저단백배오α동산음식조(LK,5.4%락단백+1%α동산).령취10지대서행가수술후급여정상단백음식작위대조조(sham).12주후마취처사대서,수집각조동물혈청.취10%농도적혈청간예체외배양적계막세포48 h.채용ELISA법검측세포상청액중혈관긴장소II(AngII)、전화생장인자β1(TGF-β1)급섬련단백(FN)적수평.채용Western인적법화실시정량PCR법검측계막세포혈관긴장소Ⅱ1형수체(ATlR)、FN급TGF-β1적단백화기인표체.결과 (1)동물실험:각조대서체질량、혈청총단백급백단백수평차이균무통계학의의,신절제각조대서혈기항급뇨단백수평균현저고우sham조(균P<0.05).혈기항수평재신절제조간차이무통계학의의,단LPD조혈뇨소담급뇨단백수평현저저우NPD조(균P<0.05),이LK조경저(균P<0.05).(2)NIN실험:NPD혈청간예조세포분비AngⅡ수평[(12.70±0.12)mg/g단백비(8.04±0.62)mg/g단백]급AT1R기인화단백표체수평균현저고우sham조,동시반유FN[(39.84±0.06)g/g단백비(20.58+0.46)g/g단백]급TGF-β1[(83.85±3.58)mg/g단백비(10.12±0.56)mg/g]기인전록화분비수평적상승(균P<0.05);LPD급LK혈청간예조가현저억제상술개변(균P<0.05).응용RAS조단제록사탄가현저강저NPD조중FN급TGF-β1적합성여분비,대LPD조중이자적표체야유진일보적억제작용(균P<0.05),단대LK조무명현영향.결론 저단백배오α동산음식가유지신절제대서영양평형,직접억제계막세포RAS적표체,감소세포외기질적분비,종이발휘신장보호작용.
Objective To observe the changes of renin-angiotensin system (RAS) in cultured mesangial cells by serum from 3/4 nephrectomized rats feeding with low protein diet and α-keto acid. Methods Thirty male SD rats received 3/4 nephrectomy (Nx) were placed on 18%normal protein diet (NPD,n=10),6% low protein diet(LPD,n=10) or 5% low protein plus 1%α-keto acid diet (LK,n=10) flor 12 weeks.Ten male SD sham-operated rats fed with 18% normal protein diet were used as control (sham group).In addition,mesangial cells were cultured in sera (10%) collected from above animals treated with or without losartan (0.02 mmol/L)for 48 hours.ELISA was applied to detect the level of Ang II,TGF-β1 and fibronectin (FN) in cell medium.Westem blotting was used to determine the protein level of ATI receptor (AT1R)and real-time PCR was used to detect the mRNA level of AT1R,TGF-β1 and FN. Results (1) Nutritional indices including body weight,total protein and albumin had no significant difference in each group. (2) Serum creatinine and 24 h pruteinuria were significantly inceased in nephrectomized groups compared to sham group(P<0.05,respectively).24 h proteinuria was greatly lower in LK group than that in NPD and LPD groups(P<0.05,respectively).(3)LK greatly decteased the level of Ang II[NPD(12.70±0.12)mg/g protein;sham(8.04±0.62)mg/g protein]in supernatant as well as the protein and mRNA expression of AT1R in cultured mesangial cells (P<0.05).(4)NPD serum directly induced higher secretion[FN:sham(20.58±0.46)g/g protein,NPD (39.84±0.06)g/g protein;TGF-β1:sham(10.12±O.56)mg/g protein,NPD(83.85±3.58)mg/g protein] and mRNA expression of FN and TGF-β1 compared with sham group (P<0.05).LPD decreased these increment (P<0.05) and LK showed stronger inhibitory effect (P<0.05). (5)Losartan application sharply reduced FN and TGF-β1 production both in supematant and in mRNA expression in NPD serum treated cells (P<0.05,respectively). Conclusion Low protein diet with α-keto acids supplement directly inhibits the RAS in mesangial cells which may contribute to its beneficial effect on the kidney.