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

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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.