Analysis by cDNA array of altered gene expression in mouse cultured podocytes in response to plasma from recurrent-focal segmental glomerulosclerosis patients

通过 cDNA 阵列分析小鼠培养足细胞响应复发性局灶节段性肾小球硬化患者血浆而改变的基因表达

• 批准号: 14570775
• 负责人: HATTORI Motoshi
• 金额: $ 2.18万
• 依托单位: Tokyo Women's Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14570775/
• 关键词: primary FSGS; proteinuria; podocyte; FSGS factors; cDNA array; 液性因子; CDNAアレイ

Circulating factors, a putative podocyte toxin postulated to be responsible for rapid recurrence of nephrotic syndrome (NS) in patients with primary focal segmental glomerulosclerosis (FSGS), are yet to be fully identified despite the availability of encouraging approaches (Savin et al., JASN, 2000). In this study, we attempted to identify a "signature" of FSGS factors. Using the cDNA array technique as the first step, we investigated the modification of gene expression in a conditionally immortalized mouse podocyte cell line after in vitro stimulation with plasma from patients with recurrent FSGS after transplantation (n=3), compared to plasma from patients with non-recurrent FSGS after transplantation (n=2), non-FSGS NS (n=2 ; MCNS and HSPN) and healthy controls. The cells were incubated with 5% v/v of plasma for 48 hours, after which total RNA was extracted. After DNAase treatment, randomly labeled cDNA probes were prepared by reverse transcription using specific primers of each arrayed gene. Denatured probes were hybridized to the cDNA array and analyzed using a bio-imaging analyzer, and up/down-regulation was considered to have occurred if there was more or less than doubling or half of the expression level. Selected findings were further confirmed and quantified by RT-PCR and a real-time PCR assay. When the gene expression profiles from cells incubated with different plasma samples were compared, it was noteworthy that integrin-linked kinase (ILK) was up-regulated only in the relapsing FSGS patients. In conclusion, our preliminary results indicate that ILK may play a role in the pathogenesis in the development of recurrent FSGS after transplantation.

循环因子是一种假定的足细胞毒素，被认为是原发性局灶节段性肾小球硬化症（FSGS）患者中肾病综合征（NS）快速复发的原因，尽管有令人鼓舞的方法，但尚未完全确定（Savin et al.，January，2000）。在这项研究中，我们试图确定一个“签名”的FSGS因素。使用cDNA阵列技术作为第一步，我们研究了在移植后复发性FSGS患者的血浆（n=3）体外刺激后，与移植后非复发性FSGS患者的血浆（n=2）、非FSGS NS（n=2 ; MCNS和HSPN）和健康对照相比，条件永生化小鼠足细胞系中基因表达的修饰。将细胞与5%v/v血浆孵育48小时，之后提取总RNA。DNA酶处理后，随机标记的cDNA探针通过逆转录使用每个阵列基因的特异性引物制备。将变性的探针与cDNA阵列杂交，并使用生物成像分析仪进行分析，如果表达水平的两倍或一半以上或以下，则认为发生了上调/下调。通过RT-PCR和实时PCR测定进一步证实和定量选定的结果。当与不同血浆样品孵育的细胞的基因表达谱进行比较时，值得注意的是，整合素连接激酶（ILK）仅在复发性FSGS患者中上调。总之，我们的初步研究结果表明，ILK可能发挥作用的发病机制，在移植后复发FSGS的发展。

项目成果

Hattori M et al.: "A combined low-density lipoprotein apheresis and prednisone therapy for steroid-resistant primary focal segmental glomerulosclerosis in children"American Journal of Kidney Diseases. 42(6). 1121-1130 (2003)

Hattori M 等人：“低密度脂蛋白血浆分离术和泼尼松联合疗法治疗儿童类固醇抵抗性原发性局灶节段性肾小球硬化症”美国肾脏病杂志。

Hattori M et al.: "Induction of integrin-Linked kinase(ILK) in mouse cultured podocytes after stimulation with plasma from reccurrent focal segmental glomeruloshlerais"J Am Soc Nephrol. 14. 375A (2003)

Hattori M 等人：“用复发性局灶节段性肾小球肾小球血浆刺激后，小鼠培养的足细胞中整合素连接激酶 (ILK) 的诱导”J Am Soc Nephrol。

Hattori M et al.: "Analysis by cDNA array of altered gene expression in mouse cultured podocytes in response to plasma from focal segmental glomerulosclerosis patients"Journal of the American Society of Nephrology. 13. 123A (2002)

Hattori M 等人：“通过 cDNA 阵列分析小鼠培养的足细胞响应局灶节段性肾小球硬化症患者血浆而改变的基因表达”美国肾病学会杂志。

Hattori M et al.: "Analysis by cDNA array of altered gene expression in mouse cultured podocytes in response to plasma from focal segmental glomerulosclerosis patients."J Am Soc Nephrol. 13. 123A (2002)

Hattori M 等人：“通过 cDNA 阵列分析小鼠培养的足细胞响应局灶节段性肾小球硬化患者血浆而改变的基因表达。”J Am Soc Nephrol。

Hattori M et al.: "Analysis by cDNA array of altered gene expression in mouse cultured podocytes in response to plasma from focal segmental glomerulosclerosis patients"J Am Soc Nephrol. 13. 123A (2002)

Hattori M 等人：“通过 cDNA 阵列分析小鼠培养的足细胞响应局灶节段性肾小球硬化患者血浆而改变的基因表达”J Am Soc Nephrol。