TI - Renal tubular epithelial cells from PKD2 ( 1-703 ) transgenic rat display augmented proliferation independent of the JAK2/STAT-1/p21/Cdk2 pathway . AB - The unexpected but significant results above , prompted us to utilize primary renal epithelial cells obtained from a 7.5 week-old mutant PKD2 transgenic rat ( 1-703 ) [abbreviated PKD2 ( 1-703 ) ] , expressing a truncated form of PC-2 lacking the C-terminal region of the protein . The transgenic animals manifest a cystic phenotype characterized by the formation of multiple cysts in the kidneys [19] . Tubular renal epithelial cells were isolated by sequential filtration of renal cells and cultured in low serum-containing medium . The epithelial character of the isolated cells and the absence of contaminating fibroblasts were confirmed by cadherin and vimentin expression respectively ( Figure 5A ) . In contrast to the cell lines examined , primary tubular epithelial cells ( TECs ) isolated from PKD2 ( 1-703 ) transgenic rat , demonstrated an increase in cellular proliferation compared with their normal counterparts . Specifically , Western blot analysis on whole cell lysates demonstrated that TECs isolated from the PKD2 ( 1-703 ) rat have significantly higher levels of PCNA than TECs isolated from normal Sprague-Dawley rats ( Figure 5A ) . In addition , the percentage of cells in the G0/G1 phase of the cell cycle was lower in the mutant cells than in normal cells as judged by cell cycle analysis ( 906 + - 093 to 841 + - 128 ) . In concert , the percentage of G2/M-phase mutant cells was higher than G2/M-phase normal cells ( 506 + - 031 to 129 + - 137 ) ( Figure 5B ) . Despite the higher proliferative activity of mutant cells , p21 levels and STAT-1 PHOSphorylation remain unaltered ( Figure 5A ) , suggesting that PKD2 -induced proliferation is STAT-1/p21-independent . We then hypothesized that alternative pathways might be responsible for PKD2 -induced proliferation in this system . To this end , we performed a genome -wide gene expression analysis on TECs isolated from two normal Sprague Dawley rats and three PKD2 ( 1-703 ) rats . Differentially expressed genes were identified with ANOVA . We concentrated only on genes involved in the cell cycle regulation (Figure 6A) . From all the cell cycle genes listed in figure 6A , only two differ significantly in expression between normal and mutant cells , those being Cdk2 and cyclin -dependent kinase inhibitor 1C or p57KIP2 . On the contrary , p21 did not show any significant difference , confirming the Western blot results ( Figure 6A and 5A ) . The chip data were verified by quantitative real-time PCR analysis after normalization using two housekeeping genes , HPRT and GAPDH . In agreement with the chip data , p57 mRNA levels were downregulated in the mutant animals as compared with their normal counterparts ( normalized fold change 47 + - 019 ) . Similarly , Cdk2 mRNA levels were augmented in the mutant cells ( normalized fold change 12 + - 0015 ) ( Figure 7A ) . Cdk2 protein upregulation and p57 protein downregulation were also verified by immunoblotting . Consistent with the microarray data , Cdk2 protein levels were significantly elevated in mutant primary cultures ( normalized fold change 22 + - 006 ) . Similarly , p57 levels were downregulated in mutant TECs ( normalized fold change 19 + - 02 ) ( Figure 7B ) . On the contrary , Western blot analysis demonstrated , as expected , that p57 protein levels remain unchanged in HEK293 stable clones and NRK-52E transfectants ( Figure 7C ) . It should be noted that p57 levels in the cell lines examined is expressed at very low levels and it was barely detectable by Western blot . Given that in the PKD2 ( 1-703 ) transgenic rat the cysts originate predominantly from the proximal tubule segment of the nephron , we wanted to exclude the possibility that proximal tubule cells are overrepresented in the primary mutant TECs culture , thus confounding the interpretation of the results . In order to do that , lysates from normal and mutant TECs were immunobloted with anti-Megalin antibody , a proximal tubule marker [35] . As shown on figure 5A , Megalin protein levels were equivalent among normal and mutant TECs suggesting that the proportion of cells of proximal origin was comparable among the different cultures and did not create a sampling bias .