A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales

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基于玄府理论的固本通络方对IgA肾病大鼠Podocin mRNA和α-actinin-4 mRNA表达的影响

目的:探析基于玄府理论的固本通络方对IgA肾病大鼠Podocin mRNA和α-actinin-4 mRNA表达的调节及对IgA肾病蛋白尿的治疗作用。方法:将80只SD大鼠随机分为正常组、模型组、中药组和科素亚组,采用牛血清清蛋白+四氯化碳+蓖麻油+脂多糖的方法建立IgA肾病大鼠模型。分别于造模结束后、治疗2周、治疗4周后检测各组大鼠24 h尿蛋白定量、血肌酐、尿素氮水平。治疗2周和4周时,采用光镜、免疫荧光法观察肾小球形态学变化;PCR法检测Prodocin、α-actinin-4 mRNA表达量。结果:治疗前,正常组24 h尿蛋白定量明显少于模型组,差异具有统计学意义(P<0.05)。模型组、中药组及科素亚组肾脏病理示肾小球系膜增生,基底膜增厚,系膜区IgA荧光强度极高。经治疗,与模型组比较,中药组及科素亚组在治疗2周、4周后24 h尿蛋白定量明显下降,差异具有统计学意义(P<0.05);中药组在治疗2周、治疗4周后与科素亚组比较24 h尿蛋白定量均减少,差异具有统计学意义(P<0.05)。与模型组比较,治疗后中药组和科素亚组Podocin mRNA含量明显升高,α-actinin-4 mRNA含量均明显降低,差异具有统计学意义(P<0.05);治疗4周与治疗2周比较,科素亚组Podocin含量明显升高,中药组α-actinin-4 mRNA含量均明显降低,差异具有统计学意义(P<0.01);治疗4周后中药组α-actinin-4 mRNA含量均明显低于科素亚组,差异具有统计学意义(P<0.05)。中药组及科素亚组肾小球病变较治疗前损伤程度轻,荧光强度明显减低。结论:固本通络方可以减轻IgA肾病大鼠蛋白尿与调节足细胞Podocin和α-actinin-4表达有关。     

微小RNA-17-5p在小儿肾病综合征发病中的作用及其机制研究

目的 分析微小RNA-17-5p（miR-17-5p）在小儿肾病综合征（NS）发病中的意义及其通过激活素A（ActA）/Smads通路对肾足细胞凋亡的影响。方法 选取2018年3月至2019年3月收治的55例NS患儿为NS组，另选取50例同期体检的健康儿童为正常对照组，比较两组外周血miR-17-5p表达情况。培养人肾足细胞株，分别转染含miR-17-5p反义寡核苷酸重组质粒（抑制组）、含无意义随机序列的对照载体（阴性对照组），未处理的人肾足细胞为空白组。比较各组细胞凋亡情况，以及转染后细胞miR-17-5p、ActA mRNA、Smads mRNA和相关蛋白表达情况。结果 NS组外周血miR-17-5p水平高于正常对照组（P < 0.001）。与空白组和阴性对照组比较，抑制组细胞凋亡率、miR-17-5p相对表达量更低，ActA、Smad2、Smad3三者的mRNA及蛋白相对表达量更高（P < 0.001）。结论 miR-17-5p在小儿NS外周血的含量升高，低表达miR-17-5p能抑制人肾足细胞的凋亡，其机制可能与上调ActA、Smad2、Smad3三者的mRNA及蛋白表达有关。     

Podocytic degeneration and regeneration in puromycin aminonucleoside nephropathy in the rat

In various types of chronic glomerulonephritis, proliferation of mesangial and endothelial cells is often seen together with extracellular matrix accumulation. In contrast, the proliferation of visceral glomerular epithelial cells (podocytes) has not been detected in any disease in which these cells are the principal target of injury. To examine whether proliferation of podocytes occurs, puromycin aminonucleoside nephropathy was induced in Wistar rats by intraperitoneal injection of puromycin aminonucleoside (15mg/100g of bodyweight). In this nephropathy model of nephrotic syndrome with minimal change, the main lesions consisted of vacuolation and flattened foot processes of podocytes, which are the primary target of puromycin aminonucleoside. Urine protein excretion and proliferating cell nuclear antigen (PCNA) positive cells were examined. The PCNA+ podocytic count reached its maximum on day 3, indicating that the most marked podocytic proliferation occurs just after the injection of PAN. Twenty-four hour urinary excretion in this nephropathy model supported the hypothesis that mitosis of podocytes occurs in association with recovery on day 13.     

大鼠足细胞的原代培养及其Nephrin的特异表达

目的 建立简便的大鼠足细胞原代培养体系,检测其去氧肾上腺素(Nephrin) mRNA及蛋白的表达水平.方法 无菌取肾,分离肾皮质,胶原酶消化法分离肾小球,培养9～10d,胰蛋白酶差异消化法传代,过筛去除肾小球继续培养5～7 d:形态学观察和间接免疫荧光法进行足细胞鉴定;流式细胞仪分析足细胞纯度;实时定量PCR(qRT-PCR)、间接免疫荧光法和Western blot法比较前三代足细胞中Nephrin mRNA及蛋白的表达水平.结果 接种5d后大部分肾小球贴壁,肾小球周围有细胞爬出,其最外缘有树枝状细胞分布,形态学及特异性抗体Nephrin、WT-1鉴定为足细胞;流式细胞仪分析足细胞纯度为94.7％;间接免疫荧光和qRT-PCR均显示:与第二、三代足细胞相比,第一代足细胞中Nephrin mRNA和蛋白表达均较高(均P＜0.05);Western blot示体外培养大鼠足细胞表达两种分子量的Nephrin,且第一代足细胞Nephrin灰度值高于第二、三代足细胞(P＜0.05).结论 酶消化法结合差异过筛法接种可促进肾小球贴壁,有利于培养纯度高的足细胞;大鼠足细胞体外培养条件下能特异性表达Nephrin结构及功能蛋白.     

Urinary podocytes in childhood IgA nephropathy

SUMMARY: We have previously demonstrated the presence of detached podocytes in the urine in various glomerular diseases. In this study, we investigated the pathological significance of detachment of urinary podocytes (u-podocytes) in childhood IgA nephropathy. We investigated 28 children with IgA nephropathy. U-podocytes were detected in the urinary sediment by immunofluorescence. Renal pathological changes and urinalysis, including the u-podocyte test, were analysed. The expression of glomerular basement membrane (GBM) components, integrins and cytoskeleton components on urinary podocytes was investigated using dual immunofluorescence in 12 patients. The u-podocyte score paralleled the acute glomerular changes in renal biopsy samples. The infiltration of glomerular macrophages and polymorphonuclear leucocytes, and the number of urinary leucocytes, correlated highly with the u-podocyte score. Some of the u-podocytes expressed integrins and also components of the GBM. Detachment of podocytes reflects the presence of acute glomerular lesions. Infiltration of glomerular leucocytes is considered to be the cause of the detachment, partly because of the destruction of the GBM.     

Dysregulated Dynein-Mediated Trafficking of Nephrin Causes INF2-related Podocytopathy

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