A representative H&E stained kidney sec tion from PKD2 rats of different ages is depicted on Figure 2A. Renal fibrosis was also graded in Azan stained kidney sections of PKD2 rats at different ages. As expected, fibrosis in the cortex clearly increases with increasing age demonstrating a gradual destruction of the renal parenchyma. Proliferation related genes become deregulated at later stages of cystogenesis Cysts were demonstrated to appear at birth, and therefore the question arose as to which genetic factors were involved at these initial stages of cyst formation. Consequently, our investigation included gene expression profiling of whole kidney homogenates, per formed at early stages of the disease, in PKD2 rats at 0, 6 and 24 days. Hence, three WT SD and three PKD2 male rats at the age of 0, 6 and 24 days were sacrificed, their kidneys excised and RNA isolated from whole kidney homogenates.
Differentially expressed genes were identified by microarray analysis using the Affymetrix GeneChip Rat Expression Array Rae230 2. The microarray data revealed a total of 1011 statistically selleck significant differentially expressed genes at all three time selelck kinase inhibitor points between mutant and wild type rats. From those 39 genes were differentially expressed at 0 days. At 6 days there were 249 genes and at 24 days, 763 genes differentially expressed. Interestingly, none of the genes deregulated at day 0 were proliferation or cell cycle related. At day 6 only two genes involved in cell cycle regulation, namely ANAPC4 and CCND1, were found to be significantly downregulated in mutant animals. On the other hand, five cell cycle related genes appear to be up regulated in mutant animals 24 days after birth. Most importantly, known proliferation genes such as c Myc were augmented in the kidneys of mutant rats 24 days after birth at the time point where cystic burden seems to plateau.
In order to verify the microarray results, the expression of classical proliferation cell cycle related markers such as PCNA, c Myc and Ki 67 was validated by quantitative real time PCR analysis at selected time points. Consis tently, PCNA and Ki 67 mRNA levels were similar among wild type and mutant rats at both time points. In agreement with the

microarray results, c Myc mRNA appears to be significantly upre gulated in mutant rat kidneys only at 24 days after birth. However, PCNA and c Myc protein levels were comparable among the two different groups at 0, 6, 12 and 24 days as judged by western blot analysis. In order to verify these results we performed Ki 67 staining on kidney sections from 0 days old SD and Pkd2 mutant rats. As shown on Figure 6 the num ber of Ki 67 positive cells in renal tubules was similar between wild type and mutant animals.