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

循环因子是一种假定的足细胞毒素，被认为是导致原发性局灶节段性肾小球硬化(FSGS)患者肾病综合征(NS)快速复发的原因，尽管已有令人鼓舞的方法可用，但尚未完全确定(Savin等人，JASN，2000)。在这项研究中，我们试图确定FSGS因素的“特征”。作为第一步，我们研究了条件永生化小鼠足细胞系经移植后复发FSGS患者(n=3)的血浆体外刺激后基因表达的改变，并与移植后未复发FSGS患者(n=2)、非FSGS患者(n=2；MCNS和HSPN)和健康对照组的血浆进行了比较。细胞与5%v/v的血浆孵育48h后，提取总RNA。DNA酶处理后，用每个阵列基因的特异引物反转录制备随机标记的cDNAs探针。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，如果表达水平增加或减少一倍或一半，则认为发生了上调/下调。选定的结果通过RT-PCR和实时定量PCR检测得到进一步确认和量化。当比较与不同血浆样本孵育的细胞的基因表达谱时，值得注意的是，整合素连接激酶(ILK)仅在复发的FSGS患者中上调。综上所述，我们的初步结果表明ILK可能在移植后复发的FSGS的发病机制中发挥作用。

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.