【作者】 邹新蓉；

【导师】 王小琴；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2011， 博士

【摘要】 肾性骨病(ROD)是慢性肾脏病的主要并发症之一,对患者的生存质量和生存时间有着重要影响。由于肾脏结构和功能的破坏导致肾脏羟化酶系的活性降低,1,25 (OH)2 VitD3的生成相对或绝对不足,PTH的合成与释放失控,是ROD发病的核心环节。Klotho基因(KL)是一种与衰老相关的基因,主要在肾小管、脑脉络膜和甲状旁腺表达,尤以肾皮质远曲小管表达最为强烈。KL基因缺陷鼠可出现一系列与人类“肾精亏虚证”相似的衰老表型变化。研究证实,KL在慢性肾脏病(CKD)肾组织中的表达明显下调,而KL下调可导致骨质疏松,说明KL可能参与发生ROD。另外,成纤维细胞生长因子23(FGF-23)、成骨蛋白-1(BMP-7)、骨保护素(OPG)作为调节骨和肠钙磷转运及骨代谢的激素,在ROD发生过程中起重要作用。那么,KL是否通过调控这些蛋白而参与发生ROD呢?如果能够证实这一假说,将进一步阐明ROD的发病机制并为临床治疗提供新靶点。本研究采用大鼠5/6肾切除残余肾模型,探讨KL基因对FGF-23、BMP-7、OPG的调控机制及肾安颗粒(复方淫羊藿苷、黄芪甲苷、大黄葸醌提取物)的干预作用,预期结果不仅能深化对ROD发病机制的认识,还将在一定程度上揭示中药防治ROD的作用机理。实验一大鼠残余肾模型的制备目的：探讨大鼠残余肾并肾性骨病模型的制备方法。方法：Wistar大鼠48只随机分为正常对照组(A组,n=8)、假手术组(B组,n=8)、造模组(C组,n=32)。经适应性喂养1周后,C组大鼠以戊巴比妥钠腹腔注射麻醉后,行2/3左肾切除,1周后再次行右肾切除。各组大鼠均予正常饮食,A组和B组饮蒸馏水,C组左肾切除术后第二天开始加磷酸二氢钠饮水。各组大鼠造模术后1月眼球取血,测定血红蛋白(HGB)、血肌酐(Scr)、尿素氮(BUN)、血磷(P3+)、血钙(Ca2+)、血清碱性磷酸酶(ALP)、血清全段甲状旁腺激素(iPTH)。取血前1天代谢笼收集24h尿,测尿肌酐(UCr)并计算内生肌酐清除率(Ccr)。结果：造模术后1月,A、B两组大鼠活泼好动,毛色整齐光亮,体重增加；C组精神萎靡,安静不喜动,毛色杂乱,体重变化不明显。C组HGB与A组和B组比较明显降低(P<0.05)；C组BUN、Scr与A组和B组比较明显升高(P<0.01),C组Ccr与A组和B组比较明显降低(P<0.01)；C组ALP与A组和B组比较明显升高(P<0.05)；C组iPTH与A组和B组比较显著升高(P<0.01)；C组Ca2+与A组和B组比较明显降低(P<0.05)；C组P3+与A组和B组比较明显升高(P<0.01)。A、B两组间以上指标比较无显著性差异(P>0.05)。结论：5/6肾切除加高磷饮水1月可以导致残余肾模型大鼠钙磷代谢异常,继发PTH升高,提示并发ROD。实验二残余肾模型钙、磷代谢的变化及肾安颗粒的干预作用目的：探讨残余肾模型大鼠钙、磷代谢的变化及肾安颗粒的干预作用。方法：将第一部分实验中的造模组大鼠32只随机分为4组,即模型组(C组,n=8)、阳性药物骨化三醇对照组(D组,n=8)、肾安颗粒中剂量组(E组,n=8)、肾安颗粒高剂量组(F组,n=8)。正常对照组(A组,n=8)、假手术组(B组,n=8)与第一部分相同。D组以骨化三醇溶液灌胃；E、F组分别以中、高浓度肾安颗粒灌胃；A、B、C组以等量生理盐水灌胃。灌胃容量1ml/100g/日,总疗程8周。用药前后检测各组大鼠血HGB、Scr、BUN、P3+、Ca2+、ALP、iPTH,取血前1天代谢笼收集24h尿,测Ucr并计算Ccr。结果：各组大鼠存活情况：用药期间C组和D组各死亡1只,余各组大鼠均存活。用药前,C、D、E、F四组BUN、Scr、P3+与A、B两组比较明显升高,Ccr、Ca2+与A、B两组比较明显降低(P<0.05),ALP与A、B两组比较无明显变化(P>0.05)。用药8周后,A、B两组HGB、BUN、Scr、Ccr、P3+、Ca2+、ALP、iPTH与用药前比较无明显变化(P>0.05)；C组BUN、Scr、P3+、ALP、iPTH与用药前比较明显升高(P<0.05),Ca2+、Ccr、HGB与用药前比较明显降低；D组Ca2+与用药前比较明显升高(P<0.05),P3+较用药前略下降但无统计学意义(P>0..05),D组ALP与用药前比较降低(P<0.05),iPTH与用药前比较明显降低(P<0.01)；E组Ca 2+与用药前比较明显升高(P<0.05),P3+、ALP与用药前比较明显降低(P<0.05),iPTH与用药前比较显著下降(P<0.01)；F组Ca2+与用药前比较升高明显(P<0.05),P3+、ALP与用药前比较明显降低(P<0.05),iPTH与用药前比较显著降低(P<0.01)。用药后,D组Ca2+与C组比较明显升高(P<0.05),ALP、iPTH与C组比较均明显降低(P<0.05),P3+与C组比较无显著性差异(P>0.05)；E组Ca2+与C组比较明显升高(P<0.05),P3+、ALP、iPTH与C组比较均明显降低(P<0.05或P<0.01)；F组Ca2+与C组比较明显升高(P<0.05),P3+、ALP、iPTH与C组比较均显著降低(P<0.05或P<0.01)。用药8周后,E、F两组HGB、CCr与D组比较明显升高(P<0.05),Scr与D组比较明显降低(P<0.05),BUN与D组比较无显著性差异(P>0.05)。E、F两组Ca2+、ALP、iPTH与D组比较均无显著性差异(P>0.05),P3+与D组比较明显下降(P<0.05)；E、F两组间上述各指标比较无显著性差异(P>0.05)。结论：肾安颗粒具有稳定残余肾模型大鼠肾功能、提高HGB、改善钙磷代谢、抑制甲状旁腺机能亢进的作用。实验三KLOTHO基因在残余肾模型大鼠的表达及肾安颗粒的干预作用目的：探讨KL基因在残余肾模型大鼠的表达及肾安颗粒的干预作用。方法：分组方法及用药同第二部分。用药结束后,处死全部大鼠,取肾脏和股骨,肾组织常规包埋切片染色行光学显微镜检查,以双能X射线骨密度仪测定股骨头部的骨密度。采用Real-timePCR检测大鼠肾脏KLothomRNA的表达；采用Western blot检测肾脏KLotho蛋白、股骨FGF-23、BMP-7、OPG蛋白水平；免疫组化法检测股骨BMP-7、OPG表达；免疫荧光检测股骨FGF-23表达。结果：骨密度：C组与A、B两组比较明显降低,E、F、D组与C组比较明显升高(P<0.05),E组与D组比较略有升高,但无统计学意义(P>0.05),F组与D组比较无显著性差异(P>0.05)。KLothomRNA:C组和D组的表达与A组和B组比较明显减低(P<0.01或P<0.05)；E、F组表达与C组比较明显增强(P<0.01)；E、F组两组表达与D组比较明显增强(P<0.05)。Klotho蛋白：A组的表达均高于其它各组,与C组相比明显增高(P<0.01)；E、F组的表达与C组和D组比较明显增高(P<0.05)；E组与F组间比较无差异性(P>0.05)。Western blot及免疫荧光检测FGF-23：C、D、F三组表达均明显高于A组和B组(P<0.05或P<0.01),E组表达与A组和B组比较无明显差异(P>0.05)；D、E、F三组表达明显低于C组(P<0.05)；E组表达低于D组和F组(P<0.05)。Western blot及免疫组化检测BMP-7：在大鼠股骨中,C、D、F三组表达明显低于A组和B组(P<0.05或P<0.01),E组表达略低于A组和B组但无统计学意义(P>0.05)；D、E、F三组表达与C组比较明显升高(P<0.05)；E、F两组表达高于D组(P<0.05)。Western blot及免疫组化检测OPG:C组表达明显低于A组和B组(P<0.05)；D、E、F三组表达明显高于C组(P<0.05)；D、E、F三组间比较无显著性差异(P>0.05)。KL表达与FGF-23、BMP-7、OPG表达的关系：在模型组,随着KLotho表达的下调,FGF-23表达升高,BMP-7、OPG表达降低；肾安干预后,随着KLotho表达的上调,FGF-23表达下降,BMP-7、OPG表达升高。结论：KL基因在残余肾模型肾组织中的表达降低,与FGF-23呈明显负相关,与BMP-7、OPG呈明显正相关,肾安颗粒可能通过上调KL基因的表达,下调FGF-23,刺激成骨细胞BMP-7及OPG的合成,调节肾、骨、肠对钙、磷的转运,从而改善ROD。更多还原

【Abstract】 Renal osteodystrophy(ROD) is the necessary complication of CKD (chronic kidney disease), which lowers the life quality and reduces survival time of patients. With the lower activity of kidney hydroxylase due to the destruction of structure and function of kidney, the production of 1,25 (0H)2 VitD3 is relatively or absolutely in sufficient, and synthesis and release of PTH is out of control, which is the core link of ROD. Klotho gene (KL) is related to senility, and expressed in brain choroids, Parathyroid gland and kidney, especially in renal cortical expression of distal tubule strongest. KL gene defect mice could exhibit senility phenotypic variation, which is similar with mankind syndrome of deficiency of kidney essence. It is approved that the expression of KL in kidney tissues decline dramatically in CKD, and the lower of KL could result in osteoporosis, which means KL may participate in producing ROD. In addition, bone morphogenic protein-1 (BMP-1), fibroblast growth factor-23 (FGF-23), and osteoprotegerin (OPG) are treated as bone hormones which are used to regulate calcium and phosphate metabolism in bone and gut and influence the generate process of ROD. Well, whether by virtue of protein regulation KL plays an important role in production of ROD? Once the hypothesis is approved, the pathogenesis of ROD could be further clarified and a new target would be provided for clinical treatment. In this paper, Rat 5/6 nephrectomy remnant kidney model is employed, regulation mechanisms of KL gene to BMP-1, FGF-23, and OPG are discussed, intervention of Shen’an Granular, such as compound-icariin、astragalin-1、rhubarb anthraquinone, is investigated. Anticipated results not only can deepen the understanding of the pathogenesis of ROD, but also can show the action mechanism of ROD prophylaxis and treatment with Chinese medicine to a certain extent.Part I Making the rat model of remnant kidneyObjective To investigate a new kind of method of setting up animal model of remnant kidney and renal osteodstrophy.Methods 48 Wistar rats were randomly divided into normal rats group(A group,n=8), pseudo- operation group(B group,n=8),model group(C group,n=32). After one week adaptive feeding, maked intraperitoneal injection of Sodium pentobarbital,the rats of C group were 2/3 left nephrectomized,which were feeding the high-phosphorus water next day, then right nephrectomized one week later. All of the rats were given normal diet, A group and B group drinking distilled water; Acquired the blood of rats eyeball a month after the 2/3 left nephrectomized,then examined HGB, Scr,BUN, P3+, Ca2+, ALP, iPTH, Collected the urinary of all of the 24hours before acquiring blood. Examined urine creatinine and calculated creatinine clearance rate.Results Making model one month later, Both of A and B groups were lively, bright and tidy coat, weight gain; C group were apathetic, quiet and do not like to move, messy fur, body weight did not change significantly.Compared with A and B group, HGB of C group was decreased obviously (p<0.05);While, BUN and Scr, otherwise (p<0.01); Contrasted to A group, Ccr of C group was decreased dramatically (p<0.01); ALP of C group was increased dramatically (p<0.05); Compared with A group, iPTH of C group was increased obviously (p<0.01);While, Ca2+ otherwise(p<0.05); P3+ of C group was increased contrasted to A group (p<0.01). These Laboratory parameters was no significant difference between A and Bgroup(P>0.05).Conclusions 5/6 nephrectomy combined with high phosphorus water one month can lead to disfunction of calcium and phosphorus metabolism of remnant kidney rats, then PTH increase, which demonstrates renal osteodstrophy arising.PartⅡThe influences of calcium and phosphorus metabolism of rat model of remnant kidney and the interference effect of Shen’an GranularObjective To probe the interference effect of Shen’an Granular in the calcium and phosphorus metabolism of rat model of remnant kidney.Methods Wistar rats of model group above, were divided into 4 groups:model group (C group,n=8), calcitriol group (D group,n=8), moderate-dose Shen’an Granular group (E group,n=8);high-dose Shen’an Granular group(F group,n=8). A,B group same as part-1. D group feeding with calcitriol. E group feeding with moderate-dose Shen’an Granular.F group feeding with high-dose Shen’an Granular. A,B,C feeding with same dose normal saline. The total course of treatment was 8 weeks. Respectively examined HGB, Scr,BUN, P3+, Ca2+, ALP, iPTH of each group before and after medication, Collected the urinary of all of the 24hours before acquiring blood. Examined urine creatinine and calculated creatinine clearance rate.Results Survival of the rats:each of C and D group had one died during the medication, the remaining were survival. Before medication,such as Scr, P3+ and BUN of C, D, E, F group were higher than A,B group, While Ca2+ and Ccr otherwise (p<0.05);After 8 weeks of medication, there was no significant difference between before and after treatment about the Laboratory parameters, which were HGB、BUN、Scr、Ccr、P3+、Ca2+、ALP and iPTH in A,B group (p>0.05); In D group, Ca2+ was increased after medication (p<0.05); While, P3+ and ALP otherwise (p<0.05);as well as iPTH(p<0.05); In E group, Ca2+ was increased after medication (p<0.05); While, P3+ and ALP otherwise (p<0.05);as well as iPTH(p<0.01);In F group, Ca2+ was increased after medication (p<0.05); While, P3+ and ALP otherwise (p<0.05);as well as iPTH(p<0.01); Compared with C group, Ca2+ of D group was increased obviously after medication (p<0.05);While, P3+ and ALP, iPTH, otherwise (p<0.05); Compared with C group, Ca2+ of E group was increased obviously (p<0.05);While, P3+ and ALP, iPTH, otherwise (p<0.05); Compared with C group, Ca2+ of Fgroup was increased obviously (p<0.05);While, P3+ and ALP, iPTH, otherwise (p<0.05); Compared with D group, the P3+, Ca2+, ALP, iPTH of E and F group (p<0.01) had no significant difference (P>0.05).After 8 weeks mediction, Compared with D group, HGB,Ccr of E,F group were to be better dramatically(p<0.05); While, Scr otherwise (p<0.05); as well as BUN, Ca2+, ALP, iPTH(P> 0.05);and P3+ decreased significantly(p<0.05); These Laboratory parameters were no significant difference between E and F group(P>0.05).Conclusions Shen’an Granular can stabilize renal function, increase HGB,and also improve calcium and phosphorus metabolism of rat model of remnant kidney and inhibit hyper-parathyroidism. PartⅢThe expression of Klotho gene in the rat model of remnant kidney and the interference effect of Shen’an GranularObjective To probe the influences of the expression of Klotho gene in the rat model of remnant kidney and the interference effect of Shen’an GranularMethods The drug and grouping method was same as part-2, After treatment, all rats were executed, then acquired renal and femoral bone, routine embedded sections of kidney stained line of optical microscopy, measured the density of the femoral head by dual energy X-ray. Examine the expression of Klotho mRNA of Wistar rats by RT-PCR,and Western blot was administered to measure the expression of kindey’s Klotho and the thighbone’FGF-23,BMP-7,OPG, in the level of protein. Immunohistochemistry was used to detect the expression of thighbone’ BMP-7 and OPG.Detected the expression of thighbone’FGF-23 by immunofluorescence.Results Compared with C group, the expression of bone density in E,F,D group was to be better dramatically(p<0.05), E group was slightly higher than D group,but had no significant difference (P>0.05),as well as the comparison between F group and D group(P>0.05); Contrasted to A group and B group, KlothomRNA of C group and D group were obviously decreased (P<0.01 or P<0.05), KlothomRNA of E group and F group were obviously inecreased, compared with C group(P<0.01),as well as D group(p<0.05), the expression of Klotho protein of renal cortex,which was highest in A group (P<0.05),and significantly higher compared with C group (P<0.01), between E group and F groups showed no difference (P> 0.05). By Western blot and immunofluorescence detection of FGF23, the expression of FGF-23 in the E and F groups and D group were lower than C group (P< 0.05) otherwise of BMP-7 (P<0.05), the E and F group’BMP-7 was above D group in the level of protein (P<0.05). the expression of OPG in C group, was lower than the A group (P<0.05),while,each durg group was above it (P<0.05). between D and E,F group had no significant difference (P>0.05).Conclusions The expression of Klotho gene in the rat model of renal osteodstrophy is suppressed, the low Klotho gene is closely related to FGF-23、BMP-7、OPG,Shen’an granular can produce favorable effects on the high-expression of Klotho gene and osteoblast factor, regulating calcium and phosphorus metabolism, improving renal osteodystrophy.更多还原