Results

Confirmation of PKD2 expression in Pkd2 KO and PKD2 TG MEFs
Mouse Pkd2 mRNA was revealed in KO wild-type (KOWT) MEF cells but not in KO MEF cells. Also, Pkd2 mRNA was expressed in TG wild-type (TGWT) MEF cells and PKD2 TG MEF cells. Human PKD2 mRNA was only expressed in PKD2 TG MEF cells obtained from PKD2 TG embryos following injection of the human PKD2 transgene (Fig. 1A). Consistently, the expression level of polycystin-2 was lower in KO MEFs than KOWT MEFs as well as higher in TG MEFs than TGWT MEFs (Fig. 1B).

Microarray gene expression analysis
We used a mouse 30 K whole gene oligonucleotide microarray to identify mRNAs whose expression was altered by the Pkd2 KO and PKD2 TG MEF cells. The normalized log ratios corresponding to each time point were exported to the software for clustering algorithm. We used Axon's Acuity 3.1 (Axon Instruments, Union City, CA, USA) for using the 'gene shaving' algorithm. To estimate the number of clusters in a dataset, we used 'Gap Statistics' in Acuity 3.1. The cluster number estimated by gap statistics was used for an input parameter in gene shaving. After gene shaving, we used a single linkage hierarchical clustering. The single linkage hierarchical clustering method divides 8 'gene shaving' clusters (Fig. 2). The majority of genes whose expression was appreciably altered encoded proteins involved in critical biological processes, such as metabolism, transcription, cell adhesion, cell cycle, and signal transcription. Forty-five genes whose expression was changed by 2-fold or greater were identified (Table 1).

Verification of candidate genes by quantitative RT-PCR and immunoblot analysis
To verify the results obtained by cDNA microarray analysis, expression patterns for selected genes were confirmed at the RNA and protein levels. We selected seven genes among the 45 genes (Fig. 3A). As result of realtime RT-PCR, the expression patterns of PKCα, ITGα4, and AQP1 were identical to microarray analysis in Pkd2 KO MEF cells (Fig. 3B). In contrast, 6 genes, except for TGF-β2, were identically expressed in PKD2 TG MEF cells (Fig. 3C). Especially, the AQP1 gene may be significantly regulated by differential Pkd2 gene expression level. Furthermore, to confirm the AQP1 protein expression level, western blot analysis was performed using MEF cells as well as kidney tissues obtained from Pkd2 KO (or heterozygote) and PKD2 TG mice. As result, AQP1 protein level was consistent with mRNA levels in both MEFs and tissues (Fig. 4).