miRNA对PAN肾病大鼠nephrin介导蛋白尿调控机制及雷公藤制剂的保护作用
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摘要
研究背景与目的：
蛋白尿是慢性肾脏病进展的独立危险因素,足细胞损伤是发生蛋白尿的关键环节,足细胞损伤与慢性肾脏病进展密切相关。一些miRNA可能通过介导nephrin磷酸化调节足细胞裂隙膜(slit diaphragm, SD)分子的表达,故调控miRNA可以通过改善SD蛋白的表达从而减少尿蛋白。雷公藤多苷可改善SD蛋白表达,保护足细胞,减少蛋白尿,但其分子机制未完全阐明,假设雷公藤制剂可能是通过miRNA介导nephrin磷酸化实现的。本研究以雷公藤制剂治疗尿蛋白的疗效为临床依据,以足细胞SD分子表达为研究靶点,以miRNA为调控因子,阐明miRNA调控SD分子表达的规律,通过雷公藤制剂对PAN诱导的足细胞损伤的形态学改变、肾组织miRNA及足细胞SD分子nephrin、podocin核酸和蛋白表达变化的干预作用,阐明雷公藤制剂治疗尿蛋白的分子靶点,为延缓慢性肾脏病进展提供实验依据。
方法：
芯片检测及机制研究实验分为5组：空白组、模型组、雷至胶囊(二至丸+雷公藤多苷)高剂量组、雷公藤多苷高剂量组,在前面实验基础上,雷公藤制剂干预实验分为8组：空白组、模型组、雷至胶囊高剂量组、雷至胶囊低剂量组、雷公藤多苷高剂量组、雷公藤多苷低剂量组、雷公藤甲素组和缬沙坦组,每组10只。颈静脉注射嘌呤霉素氨基核苷(PAN) 100mg/kg体重建立PAN肾病模型。空白组颈静脉注射等量生理盐水。各组大鼠造模后第2天开始每日固定时间分别进行灌胃给药,持续10天.分别于造模前、造模第3d、9d代谢笼喂养,观察大鼠一般情况、称重、记24h尿量。造模前1d及造模10d尾静脉抽血化验血常规、血生化,在注射PAN后第10天,局麻下摘取肝肾,左肾分别于0.4%福尔马林及戊二醛固定,用于光镜、免疫荧光、TUNEL染色及透射电镜；右肾-70冰箱储存,用于提取肾小球总RNA、用于RT-PCR、miRNA芯片检测及real-time RT PCR验证。RTPCR检测大鼠肾组织dice、nephrin、podocin、synaptopodin mRNA表达,样品总RNA提取后,经miRNA标记、芯片杂交、图像扫描、数据分析,筛选表达差异明显的miRNA,经real-time RT PCT验证的候选miRNA, Western Blotting检测dicer、nephrin、podocin、synaptopodin蛋白表达。
结果：
1.大鼠一般情况：PAN造模后第3d大鼠出现尿量减少、腹水,而浮肿不明显,精神萎靡,进食减少,毛发竖起,体重下降。第5-7天腹水明显增加,表现肾病综合症。死亡率30%(3/10)。而空白组无腹水,尿蛋白正常,提示PAN造模成功。
2.病理改变,PAN模型鼠肾小管上皮细胞变性,严重区域细胞坏死。所有大鼠肾小管管腔内均有透明管型,皮质部管腔轻度扩张。光镜下肾小球及间质无明显病变。电镜下可见足细胞足突融合、消失,而正常对照组足细胞结构正常。
3. miRNA芯片分析：经紫外分光光度法及凝胶电泳检测证实样品总RNA符合要求。各组芯片信号强度相关分析显示：模型组和空白组相关系数0.599,雷至胶囊组和模型组相关系数0.614.两组信号值差异明显,而雷至胶囊组与空白组相关系数0.932.各组内差异不明显。
芯片筛选显示,模型组高表达miRNA106个,低表达miRNA 63个,差异倍数(fold change)在1.8～7.0。雷至胶囊高剂量组高表达的miRNA82个,低表达44个,其中,rno-mir-23a、rno-mir-300-3p等在模型模型组肾皮质高表达的65个miRNA,与雷公藤制剂组相比表达下调,rno-mir-24、mo-mir-30c、rno-mir-22等模型模型组低表达的miRNA,与雷公藤制剂组肾皮质表达上调。
Real-time RT PCR结果显示rno-mir-23a、mo-miR-300-3p在模型组高表达,表达量是空白组的2.472、2.514倍。rno-miR-24、rno-miR-30c在模型组低表达,表达量是空白组的0.312、0.555倍。与芯片结果接近,证实芯片结果可靠。可认为以上miRNA是PAN肾病特异性miRNA.
4.与空白组比较,模型组肾组织dicer酶免疫荧光增强,dicer核酸与蛋白表达均升高,而SD分子nephrin、podocin免疫荧光减弱,核酸与蛋白表达下降,相关分析表明二者呈现负相关,提示dicer通过miRNA负向调控SD分子的表达。另外,模型组足细胞骨架蛋白synaptopodin表达亦下降,TUNEL染色显示足细胞凋亡数量增加,凋亡细胞指数明显升高(p<0.01),提示miRNA与足细胞骨架蛋白表达减少及足细胞凋亡增加密切相关。
5.与模型组比较,雷公藤制剂(雷至胶囊、雷公藤多苷不同剂量)干预后,肾组织dicer酶免疫荧光减弱,dicer核酸与蛋白表达下降,而SD分子nephrin、podocin免疫荧光改善,核酸与蛋白表达增加,提示雷公藤制剂对PAN肾病SD分子表达的调控与dicer酶有关,雷公藤制剂可通过dicer-miRNA改善SD分子的表达减少蛋白尿。足细胞骨架蛋白synaptopodin表达改善,TUNEL染色显示足细胞凋亡数量减少,说明雷公藤制剂还通过调控足细胞骨架蛋白及细胞凋亡发挥保护足细胞作用,这一作用与雷公藤制剂调控dicer与miRNA有关。
6.雷公藤制剂各组24h尿蛋白、血总蛋白、白蛋白、血脂、血红蛋白均有不同程度改善,雷至胶囊高剂量组降蛋白尿、改善贫血、降低AST疗效优于雷公藤多苷及其余对照组,各治疗组血肌酐、尿素氮轻度升高(p<0.05)。模型组肾组织病理主要表现为：肾小管上皮细胞变性,严重区域细胞坏死。所有大鼠肾小管管腔内均有透明管型,皮质部管腔轻度扩张。肾小球及间质无明显病变。电镜下可见足突融合、消失,药物有减轻肾小管上皮细胞变性,减少肾小管内透明管型的作用,其中雷至胶囊高剂量和雷公藤多苷高剂量的减轻作用与模型组相比有统计学显著性差异。电镜下雷至胶囊高剂量组足突融合减轻,足突明显恢复。
结论：
1.一次性颈静脉注射PAN1OOmg/kg体重可成功建立典型的急性足细胞损伤模型,效果确切。
2.miRNA芯片筛选显示,PAN肾病肾皮质表达显著上调的miRNA106个,表达下调的miRNA62个,高表达的miRNA基因位点呈簇状分布(clustering),集中分布于19号染色体和6号染色体,提示这2个染色体基因位点可能与PAN肾病的发生密切相关。筛选出PAN肾病特征性miRNA:rno-miR-23a、rno-miR-300-3p在PAN肾病中高表达,提示这些miRNA可能参与PAN肾病足细胞损伤及蛋白尿。rno-miR-24、rno-miR-30c在PAN肾病中低表达,提示这些miRNA可能通过抗凋亡发挥保护作用。
3. dicer通过miRNA负向调控PAN肾病大鼠SD分子(nephron podocin等)及骨架蛋白的表达,还可能通过miRNA参与足细胞凋亡的调控。提示dicer及miRNA可能是调控SD分子及蛋白尿的关键靶点。
4.雷公藤制剂可下调PAN肾病大鼠dicer表达,通过dicer-miRNA途径改善PAN肾病大鼠SD分子nephrin、podocin及骨架蛋白synaptopodin的表达,并能通过miRNA抑制足细胞凋亡发挥保护足细胞作用。提示dicer、rno-miR-24、rno-miR-30c、rno-miR-23a可能是雷公藤制剂治疗PAN肾病蛋白尿的分子治疗靶点。
5.本研究将二至丸联合雷公藤多苷组成的雷至胶囊用于雷公藤减毒研究,国内外未见报道,雷至胶囊(二至丸+雷公藤多苷)降蛋白尿、改善贫血、降低AST疗效优于雷公藤多苷及其余治疗组,其机制可能是雷至胶囊通过降低PAN肾病大鼠蛋白尿、改善贫血、降低AST、减轻足细胞足突融合及肾小管上皮细胞变性等发挥减毒增效作用。
Background and objective:
Proteinuria is a dangerous factor of chronic kidney disease (CKD). Podocyte injury is not only the key link of proteinuria, but also closely related with CKD progress. Some microRNAs may induce slit diaphragm (SD) molecules expression via triggering nephrin phosphorylation to reduce proteinuria. Tripterygium glucosides can be proved to improve SD molecules expression, protect podocyte and reduce proteinuria.but its molecular mechanism is not clear. The hypothesis is that Tripterygium glucosides can improve SD molecules expression via triggering nephrin phosphorylation to reduce proteinuria. With SD molecules expression as target, and miRNAs as regulatory factors, the mechnisme that miRNA regulate SD molecules expression and Tripterygium preparation induce SD molecules expression via miRNA is explored to provide experiment basis for delying CKD progress, by observing morphology change of PAN nephrosis, expression of miRNA, SD molecules (nephrin and podocin) Nucleic acids and proteins with intervention of Tripterygium preparation.
Methods:
Eighty male wistar rats were randomly divided into eight groups, including control group, model group, leizhi capsule high-dosage group, leizhi capsule low-dosage group, Tripterygium glucosides high-dosage group, Tripterygium glucosides low-dosage group, triptolid group and valsartan group(with each grou ten rats). PAN nephrosis medel was made by jugular vein injection of PAN (100mg/kg body weight, dissolve, in physiological saline),while control group rats were made by jμgular vein injection of physiological saline with equal volume. By irrigation stomach once a day for ten days, All rats had been given medicines as follows:
physiological saline 2 ml for control group and model group, Tripterygium glucosides 2mg dissolved in Erzhi(containing and Droμght Ephraim grass) solusion lml or lmg dissolved in Erzhi(containing and Droμght Ephraim grass) solusion 0.5ml for Leizhi capsule high-dosage or low-dose group, Tripterygium glucosides lmg/kg/d orlmg/200g/d for Tripterygium glucosides high-dosage or low-dose group, triptolid 2mg/200g/d for triptolid group and valsartan valsartan 1.5 mg/200g/d for valsartan group. The blood and urine samples were collected, and renal tissues were processed after killed. The 24h urinary protein excretion and blood biochemistry parameters were measured by routine methods. The glomerular morphology and podocyte ultrastructure were observed by light microscopy and transmission electron microscopy respectively. The foot processwidth was examind by morphometric method. The nephrin, podocin, dicer and synaptopodin expression and distribution change were determined by indirect immunofluorescence staining. TUNEL dyeing was for detecting apoptosis podocytes.
Nephrin, podocin,dicer and synaptopodin mRNA and proteins were detected by RT PCR and Western Blotting respectively.miRNA expression profile was detected by Exiqon miRNA Array, including:prepare the RNA Sample and RNA Sample QC, miRNA labeling, miRNA array hybridization, miRNA array scanning and analysis. Real time RT-PCR analysis for mature miRNAs was used to validate 4 differentially expressed miRNAs between control and model group in microRNA microarray assays
Results:
1. PAN nephrosis rats were made successfully by jμgular vein injection of PAN (100mg/kg body weight). In day 5, model rats were in low spirits, with decreased urine volume, ascites, malnutrition and wight loss. From day 7 to day 10, the nephrotic syndromes were worst in model rats, but without skin edema. Some rats died of serious ascites, the mortality is 30%(3/10).
2. Morphologic changes in light microscope include epithelial cells degenerationrenal tubular and transparent cast, but there are no obvious changes in glomerulus and renal interstitial.Meanwhile, the degree of podocte processes effacement was obvious in model groups cample in electronic microscope.
3. miRNA array detection shows 106 miRNA upregulated and 62 miRNA down regulated in PAN nephrosis rat. Fold change (model vs. control group) vary from 1.8 to 7.0. For leizhi capsule high-dose group and model sample, therr are 90 miRNA differentially expressed, with 65 up and 25 down. The most important finding in our study is the discovery of the specific miRNA related to PAN nephrosis (rno-miR23a, rno-miR-24, rno-miR-30c and rno-miR-300-3p, which haved been validated by Real time RT-PCR analysis.
4. Compared with control sample, miRNA mature key enzyme dicer up- regulated in PAN nephrosis rats. Expression profile of nephrin, podocin and synaptopodin mRNA and protein reduced in model sample. In addition, apoptosis of podocyte increase in PAN sample. So dicer is not only closely related with SD molecules expression (negative regulation), but also correlates well with scaffolding proteins synaptopodin and podocyte apoptosis via miRNAs.
5.Compared with model sample, Immune Fluorescence intensity of dicer, expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in samples treated with Tripterygium wilfordii Hook. preparation. This sμggests that dicer correlates with SD molecules and scaffolding proteins synaptopodin expression with intervention of Tripterygium wilfordii Hook. preparation. Tripterygium glucosides improve SD molecules expression and reduce proteinuria by triggering dicer-miRNA. Protection effect on podocyte of Tripterygium preparation is related with miRNA induceing podocyte apotosis.
6. The nephrotic syndrome of PAN rats treated with Tripterygium preparation was significantly improved compared with that in PAN nephrosis control. Their urinary protein excretion was decreased, plasma albumin was increased, and high cholesterol/ triglyceride levels were remission, and anemia improced. Serum creatinine increased a little (p<0.05). In light microscop, epithelial cells degenerationrenal of renal tubular and transparent cast improved inn both leizhi capsule and Tripterygium glucosides samples(p<0.05).Meanwhile, the degree and area of podocte processes effacement was significantly reduced in both leizhi capsule and Tripterygium glucosides groups in electronic microscope(p<0.05).
Conclusion:
1. PAN nephrosis rats can be made successfully by jμgular vein injection of PAN (100mg/kg body weight).
2. miRNA array detection shows 106 miRNA upregulated and 62 miRNA down regulated in PAN nephrosis rat, among these up-regulated miRNAs,two miRNA clusters on two different chromosome:one cluster(rno-miR-24 and rno-miR-23a) in chromosome 19,the other cluster (rno-miR-300,rno-miR-381, rno-miR-487b, rno-miR-376c and rno-miR-495) in chromosome 6. This sμggests PAN nephrosis correlates with chromosome 6 and chromosome 19. miRNAs of PAN nephrosis were screening. Up-regulated miRNAs(rno-miR-23a. rno-miR-300-3p) may trigger podocyte injury and proteinuria, while down-regulated miRNAs(rno-miR-24、rno-miR-30c) may be protective factors by anti-apoptosis.
3. Dicer may regulate negatively expression profile of nephrin, podocin and synaptopodin mRNA and protein, participating in regulate podocyte apoptosis via miRNA. It is sμggested that dicer and these miRNAs are probably key regulated targets.
4. Dicer can be down-regulated in PAN induced rats treated with Tripterygium preparation. Expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in samples treated with Tripterygium wilfordii Hook by triggering dicer-miRNA, which play the podocyte protection role by inhibit podocyte apotosis.so dicer and these miRNAs(rno-miR-24、rno-miR-30c、rno-miR-23a) may be are probably key molecules therapeutic targets.
5. Leizhi capsule(Erzhi pil plus Tripterygium glucosides) can reduce toxity and increase effects of Tripterygium glucosides by reducing proteinuria, improving anemia, lowering AST, improving podocte processes effacement, and reducing epithelial cells degenerationrenal of renal tubular.
蛋白尿是慢性肾脏病进展的独立危险因素,足细胞损伤是发生蛋白尿的关键环节,足细胞损伤与慢性肾脏病进展密切相关。一些miRNA可能通过介导nephrin磷酸化调节足细胞裂隙膜(slit diaphragm, SD)分子的表达,故调控miRNA可以通过改善SD蛋白的表达从而减少尿蛋白。雷公藤多苷可改善SD蛋白表达,保护足细胞,减少蛋白尿,但其分子机制未完全阐明,假设雷公藤制剂可能是通过miRNA介导nephrin磷酸化实现的。本研究以雷公藤制剂治疗尿蛋白的疗效为临床依据,以足细胞SD分子表达为研究靶点,以miRNA为调控因子,阐明miRNA调控SD分子表达的规律,通过雷公藤制剂对PAN诱导的足细胞损伤的形态学改变、肾组织miRNA及足细胞SD分子nephrin、podocin核酸和蛋白表达变化的干预作用,阐明雷公藤制剂治疗尿蛋白的分子靶点,为延缓慢性肾脏病进展提供实验依据。
方法：
芯片检测及机制研究实验分为5组：空白组、模型组、雷至胶囊(二至丸+雷公藤多苷)高剂量组、雷公藤多苷高剂量组,在前面实验基础上,雷公藤制剂干预实验分为8组：空白组、模型组、雷至胶囊高剂量组、雷至胶囊低剂量组、雷公藤多苷高剂量组、雷公藤多苷低剂量组、雷公藤甲素组和缬沙坦组,每组10只。颈静脉注射嘌呤霉素氨基核苷(PAN) 100mg/kg体重建立PAN肾病模型。空白组颈静脉注射等量生理盐水。各组大鼠造模后第2天开始每日固定时间分别进行灌胃给药,持续10天.分别于造模前、造模第3d、9d代谢笼喂养,观察大鼠一般情况、称重、记24h尿量。造模前1d及造模10d尾静脉抽血化验血常规、血生化,在注射PAN后第10天,局麻下摘取肝肾,左肾分别于0.4%福尔马林及戊二醛固定,用于光镜、免疫荧光、TUNEL染色及透射电镜；右肾-70冰箱储存,用于提取肾小球总RNA、用于RT-PCR、miRNA芯片检测及real-time RT PCR验证。RTPCR检测大鼠肾组织dice、nephrin、podocin、synaptopodin mRNA表达,样品总RNA提取后,经miRNA标记、芯片杂交、图像扫描、数据分析,筛选表达差异明显的miRNA,经real-time RT PCT验证的候选miRNA, Western Blotting检测dicer、nephrin、podocin、synaptopodin蛋白表达。
结果：
1.大鼠一般情况：PAN造模后第3d大鼠出现尿量减少、腹水,而浮肿不明显,精神萎靡,进食减少,毛发竖起,体重下降。第5-7天腹水明显增加,表现肾病综合症。死亡率30%(3/10)。而空白组无腹水,尿蛋白正常,提示PAN造模成功。
2.病理改变,PAN模型鼠肾小管上皮细胞变性,严重区域细胞坏死。所有大鼠肾小管管腔内均有透明管型,皮质部管腔轻度扩张。光镜下肾小球及间质无明显病变。电镜下可见足细胞足突融合、消失,而正常对照组足细胞结构正常。
3. miRNA芯片分析：经紫外分光光度法及凝胶电泳检测证实样品总RNA符合要求。各组芯片信号强度相关分析显示：模型组和空白组相关系数0.599,雷至胶囊组和模型组相关系数0.614.两组信号值差异明显,而雷至胶囊组与空白组相关系数0.932.各组内差异不明显。
芯片筛选显示,模型组高表达miRNA106个,低表达miRNA 63个,差异倍数(fold change)在1.8～7.0。雷至胶囊高剂量组高表达的miRNA82个,低表达44个,其中,rno-mir-23a、rno-mir-300-3p等在模型模型组肾皮质高表达的65个miRNA,与雷公藤制剂组相比表达下调,rno-mir-24、mo-mir-30c、rno-mir-22等模型模型组低表达的miRNA,与雷公藤制剂组肾皮质表达上调。
Real-time RT PCR结果显示rno-mir-23a、mo-miR-300-3p在模型组高表达,表达量是空白组的2.472、2.514倍。rno-miR-24、rno-miR-30c在模型组低表达,表达量是空白组的0.312、0.555倍。与芯片结果接近,证实芯片结果可靠。可认为以上miRNA是PAN肾病特异性miRNA.
4.与空白组比较,模型组肾组织dicer酶免疫荧光增强,dicer核酸与蛋白表达均升高,而SD分子nephrin、podocin免疫荧光减弱,核酸与蛋白表达下降,相关分析表明二者呈现负相关,提示dicer通过miRNA负向调控SD分子的表达。另外,模型组足细胞骨架蛋白synaptopodin表达亦下降,TUNEL染色显示足细胞凋亡数量增加,凋亡细胞指数明显升高(p<0.01),提示miRNA与足细胞骨架蛋白表达减少及足细胞凋亡增加密切相关。
5.与模型组比较,雷公藤制剂(雷至胶囊、雷公藤多苷不同剂量)干预后,肾组织dicer酶免疫荧光减弱,dicer核酸与蛋白表达下降,而SD分子nephrin、podocin免疫荧光改善,核酸与蛋白表达增加,提示雷公藤制剂对PAN肾病SD分子表达的调控与dicer酶有关,雷公藤制剂可通过dicer-miRNA改善SD分子的表达减少蛋白尿。足细胞骨架蛋白synaptopodin表达改善,TUNEL染色显示足细胞凋亡数量减少,说明雷公藤制剂还通过调控足细胞骨架蛋白及细胞凋亡发挥保护足细胞作用,这一作用与雷公藤制剂调控dicer与miRNA有关。
6.雷公藤制剂各组24h尿蛋白、血总蛋白、白蛋白、血脂、血红蛋白均有不同程度改善,雷至胶囊高剂量组降蛋白尿、改善贫血、降低AST疗效优于雷公藤多苷及其余对照组,各治疗组血肌酐、尿素氮轻度升高(p<0.05)。模型组肾组织病理主要表现为：肾小管上皮细胞变性,严重区域细胞坏死。所有大鼠肾小管管腔内均有透明管型,皮质部管腔轻度扩张。肾小球及间质无明显病变。电镜下可见足突融合、消失,药物有减轻肾小管上皮细胞变性,减少肾小管内透明管型的作用,其中雷至胶囊高剂量和雷公藤多苷高剂量的减轻作用与模型组相比有统计学显著性差异。电镜下雷至胶囊高剂量组足突融合减轻,足突明显恢复。
结论：
1.一次性颈静脉注射PAN1OOmg/kg体重可成功建立典型的急性足细胞损伤模型,效果确切。
2.miRNA芯片筛选显示,PAN肾病肾皮质表达显著上调的miRNA106个,表达下调的miRNA62个,高表达的miRNA基因位点呈簇状分布(clustering),集中分布于19号染色体和6号染色体,提示这2个染色体基因位点可能与PAN肾病的发生密切相关。筛选出PAN肾病特征性miRNA:rno-miR-23a、rno-miR-300-3p在PAN肾病中高表达,提示这些miRNA可能参与PAN肾病足细胞损伤及蛋白尿。rno-miR-24、rno-miR-30c在PAN肾病中低表达,提示这些miRNA可能通过抗凋亡发挥保护作用。
3. dicer通过miRNA负向调控PAN肾病大鼠SD分子(nephron podocin等)及骨架蛋白的表达,还可能通过miRNA参与足细胞凋亡的调控。提示dicer及miRNA可能是调控SD分子及蛋白尿的关键靶点。
4.雷公藤制剂可下调PAN肾病大鼠dicer表达,通过dicer-miRNA途径改善PAN肾病大鼠SD分子nephrin、podocin及骨架蛋白synaptopodin的表达,并能通过miRNA抑制足细胞凋亡发挥保护足细胞作用。提示dicer、rno-miR-24、rno-miR-30c、rno-miR-23a可能是雷公藤制剂治疗PAN肾病蛋白尿的分子治疗靶点。
5.本研究将二至丸联合雷公藤多苷组成的雷至胶囊用于雷公藤减毒研究,国内外未见报道,雷至胶囊(二至丸+雷公藤多苷)降蛋白尿、改善贫血、降低AST疗效优于雷公藤多苷及其余治疗组,其机制可能是雷至胶囊通过降低PAN肾病大鼠蛋白尿、改善贫血、降低AST、减轻足细胞足突融合及肾小管上皮细胞变性等发挥减毒增效作用。
Background and objective:
Proteinuria is a dangerous factor of chronic kidney disease (CKD). Podocyte injury is not only the key link of proteinuria, but also closely related with CKD progress. Some microRNAs may induce slit diaphragm (SD) molecules expression via triggering nephrin phosphorylation to reduce proteinuria. Tripterygium glucosides can be proved to improve SD molecules expression, protect podocyte and reduce proteinuria.but its molecular mechanism is not clear. The hypothesis is that Tripterygium glucosides can improve SD molecules expression via triggering nephrin phosphorylation to reduce proteinuria. With SD molecules expression as target, and miRNAs as regulatory factors, the mechnisme that miRNA regulate SD molecules expression and Tripterygium preparation induce SD molecules expression via miRNA is explored to provide experiment basis for delying CKD progress, by observing morphology change of PAN nephrosis, expression of miRNA, SD molecules (nephrin and podocin) Nucleic acids and proteins with intervention of Tripterygium preparation.
Methods:
Eighty male wistar rats were randomly divided into eight groups, including control group, model group, leizhi capsule high-dosage group, leizhi capsule low-dosage group, Tripterygium glucosides high-dosage group, Tripterygium glucosides low-dosage group, triptolid group and valsartan group(with each grou ten rats). PAN nephrosis medel was made by jugular vein injection of PAN (100mg/kg body weight, dissolve, in physiological saline),while control group rats were made by jμgular vein injection of physiological saline with equal volume. By irrigation stomach once a day for ten days, All rats had been given medicines as follows:
physiological saline 2 ml for control group and model group, Tripterygium glucosides 2mg dissolved in Erzhi(containing and Droμght Ephraim grass) solusion lml or lmg dissolved in Erzhi(containing and Droμght Ephraim grass) solusion 0.5ml for Leizhi capsule high-dosage or low-dose group, Tripterygium glucosides lmg/kg/d orlmg/200g/d for Tripterygium glucosides high-dosage or low-dose group, triptolid 2mg/200g/d for triptolid group and valsartan valsartan 1.5 mg/200g/d for valsartan group. The blood and urine samples were collected, and renal tissues were processed after killed. The 24h urinary protein excretion and blood biochemistry parameters were measured by routine methods. The glomerular morphology and podocyte ultrastructure were observed by light microscopy and transmission electron microscopy respectively. The foot processwidth was examind by morphometric method. The nephrin, podocin, dicer and synaptopodin expression and distribution change were determined by indirect immunofluorescence staining. TUNEL dyeing was for detecting apoptosis podocytes.
Nephrin, podocin,dicer and synaptopodin mRNA and proteins were detected by RT PCR and Western Blotting respectively.miRNA expression profile was detected by Exiqon miRNA Array, including:prepare the RNA Sample and RNA Sample QC, miRNA labeling, miRNA array hybridization, miRNA array scanning and analysis. Real time RT-PCR analysis for mature miRNAs was used to validate 4 differentially expressed miRNAs between control and model group in microRNA microarray assays
Results:
1. PAN nephrosis rats were made successfully by jμgular vein injection of PAN (100mg/kg body weight). In day 5, model rats were in low spirits, with decreased urine volume, ascites, malnutrition and wight loss. From day 7 to day 10, the nephrotic syndromes were worst in model rats, but without skin edema. Some rats died of serious ascites, the mortality is 30%(3/10).
2. Morphologic changes in light microscope include epithelial cells degenerationrenal tubular and transparent cast, but there are no obvious changes in glomerulus and renal interstitial.Meanwhile, the degree of podocte processes effacement was obvious in model groups cample in electronic microscope.
3. miRNA array detection shows 106 miRNA upregulated and 62 miRNA down regulated in PAN nephrosis rat. Fold change (model vs. control group) vary from 1.8 to 7.0. For leizhi capsule high-dose group and model sample, therr are 90 miRNA differentially expressed, with 65 up and 25 down. The most important finding in our study is the discovery of the specific miRNA related to PAN nephrosis (rno-miR23a, rno-miR-24, rno-miR-30c and rno-miR-300-3p, which haved been validated by Real time RT-PCR analysis.
4. Compared with control sample, miRNA mature key enzyme dicer up- regulated in PAN nephrosis rats. Expression profile of nephrin, podocin and synaptopodin mRNA and protein reduced in model sample. In addition, apoptosis of podocyte increase in PAN sample. So dicer is not only closely related with SD molecules expression (negative regulation), but also correlates well with scaffolding proteins synaptopodin and podocyte apoptosis via miRNAs.
5.Compared with model sample, Immune Fluorescence intensity of dicer, expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in samples treated with Tripterygium wilfordii Hook. preparation. This sμggests that dicer correlates with SD molecules and scaffolding proteins synaptopodin expression with intervention of Tripterygium wilfordii Hook. preparation. Tripterygium glucosides improve SD molecules expression and reduce proteinuria by triggering dicer-miRNA. Protection effect on podocyte of Tripterygium preparation is related with miRNA induceing podocyte apotosis.
6. The nephrotic syndrome of PAN rats treated with Tripterygium preparation was significantly improved compared with that in PAN nephrosis control. Their urinary protein excretion was decreased, plasma albumin was increased, and high cholesterol/ triglyceride levels were remission, and anemia improced. Serum creatinine increased a little (p<0.05). In light microscop, epithelial cells degenerationrenal of renal tubular and transparent cast improved inn both leizhi capsule and Tripterygium glucosides samples(p<0.05).Meanwhile, the degree and area of podocte processes effacement was significantly reduced in both leizhi capsule and Tripterygium glucosides groups in electronic microscope(p<0.05).
Conclusion:
1. PAN nephrosis rats can be made successfully by jμgular vein injection of PAN (100mg/kg body weight).
2. miRNA array detection shows 106 miRNA upregulated and 62 miRNA down regulated in PAN nephrosis rat, among these up-regulated miRNAs,two miRNA clusters on two different chromosome:one cluster(rno-miR-24 and rno-miR-23a) in chromosome 19,the other cluster (rno-miR-300,rno-miR-381, rno-miR-487b, rno-miR-376c and rno-miR-495) in chromosome 6. This sμggests PAN nephrosis correlates with chromosome 6 and chromosome 19. miRNAs of PAN nephrosis were screening. Up-regulated miRNAs(rno-miR-23a. rno-miR-300-3p) may trigger podocyte injury and proteinuria, while down-regulated miRNAs(rno-miR-24、rno-miR-30c) may be protective factors by anti-apoptosis.
3. Dicer may regulate negatively expression profile of nephrin, podocin and synaptopodin mRNA and protein, participating in regulate podocyte apoptosis via miRNA. It is sμggested that dicer and these miRNAs are probably key regulated targets.
4. Dicer can be down-regulated in PAN induced rats treated with Tripterygium preparation. Expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in samples treated with Tripterygium wilfordii Hook by triggering dicer-miRNA, which play the podocyte protection role by inhibit podocyte apotosis.so dicer and these miRNAs(rno-miR-24、rno-miR-30c、rno-miR-23a) may be are probably key molecules therapeutic targets.
5. Leizhi capsule(Erzhi pil plus Tripterygium glucosides) can reduce toxity and increase effects of Tripterygium glucosides by reducing proteinuria, improving anemia, lowering AST, improving podocte processes effacement, and reducing epithelial cells degenerationrenal of renal tubular.
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