Colon tumors in the AOM-treated S and S.LEW congenic rats.

<p>(A) Blue arrows point to colon tumors of various sizes observed in experimental rats receiving AOM injection. (B) Scatter plot showing the distribution of the total number of colon tumors in all experimental rats. (C) Scatter plot comparing the percentage of colon tumors in different sizes between the S rats and the S.LEW congenic (C) rats. Each data point in the scatter plot represents one rat. The data points from rats that did not develop visible colon tumors are not included in the plot. All values were expressed as mean ± SEM. **: <i>P</i> < 0.01.</p

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in the AOM-treated S and S.LEW congenic rats.

<p>The top significantly up-regulated (A) and down-regulated (B) KEGG pathways of differentially expressed genes in the S.LEW congenic rats compared with the S rats (n = 3/group). “Enrichment Score” = “-log₁₀(Fisher p-value)” of the corresponding pathway in the microarray analysis.</p

TUNEL assay of colon tissues from the AOM-treated S and S.LEW congenic rats (n = 3/group).

<p>All nuclei were stained with DAPI (blue). Cells with red stained nuclei were apoptotic cells. (A) Representative tissue section of the negative control, in which the TUNEL reaction mixture was replaced with the Label Solution without the terminal transferase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153519#sec002" target="_blank">Methods</a> for details). (B) Representative tissue section from the S rat. (C) Representative tissue section from the S.LEW congenic strain. TUNEL staining was also scored by three independent operators blinded to the study groups.</p

Gene expression profiles in the AOM-treated S and S.LEW congenic rats.

<p>(A) Heatmap of differentially expressed genes with a p-value cut-off 0.05 and a fold-change cut-off 2.0 between the S.LEW congenic (C) and S rats (n = 3/group). S1, S2, S3 are 3 individual S rats. C1, C2, C3 are 3 individual S.LEW congenic rats. Gene expression level is shown as a function of color, with lower expression in green and higher expression in red. (B) Heatmap of differentially expressed genes with a p-value cut-off 0.005 and a fold-change cut-off 2.5 between the S.LEW congenic (C) and S rats (n = 3/group). S1, S2, S3 are 3 individual S rats. C1, C2, C3 are 3 individual S.LEW congenic rats. Gene expression level is shown as a function of color, with lower expression in green and higher expression in red. (C) The volcano plot showing the significant up- and down-regulated genes in the S.LEW congenic strain compared with the S (n = 3/group). The horizontal green line represents a p-value with the cut-off 0.05. All the gray and red squares above this horizontal green line represent the differentially expressed genes with statistical significance (p < 0.05). The vertical green lines mark the limits for fold-change with the cut-off value of 2.0, whereby the red squares outside of the area between the two vertical green lines represent the genes with more than 2 fold-change and the gray squares between the two vertical green lines represent the genes with less than 2 fold-change. The black square denotes <i>Mbd2</i> with a 1.38 fold increased expression in the congenic strain. (D) Real-time PCR data confirming that colon <i>Mbd2</i> was expressed significantly higher in the S.LEW congenic (C) rats compared with the S rats (n = 6/group).</p

Pleiotropic Effect of a High Resolution Mapped Blood Pressure QTL on Tumorigenesis

<div><p>This study is focused on a translationally significant, genome-wide-association-study (GWAS) locus for cardiovascular disease (QT-interval) on human chromosome 17. We have previously validated and high resolution mapped the homologous genomic segment of this human locus to <42.5 kb on rat chromosome 10. This <42.5 kb segment in rats regulates both QT-interval and blood pressure and contains a single protein-coding gene, rififylin (<i>Rffl</i>). The expression of <i>Rffl</i> in the hearts and kidneys is differential between Dahl S and S.LEW congenic rats, which are the strains used for mapping this locus. Our previous study points to altered rate of endocytic recycling as the underlying mechanism, through which <i>Rffl</i> operates to control both QT-interval and blood pressure. Interestingly, <i>Rffl</i> also contributes to tumorigenesis by repressing caspases and tumor suppressor genes. Moreover, the expression of Methyl-CpG Binding Domain Protein 2 (<i>Mbd2</i>) in the hearts and kidneys is also higher in the S.LEW congenic strain than the background (control) Dahl S strain. <i>Mbd2</i> can repress methylated tumor suppressor genes. These data suggest that the S.LEW congenic strain could be more susceptible to tumorigenesis. To test this hypothesis, the S and S.LEW strains were compared for susceptibility to azoxymethane-induced colon tumors. The number of colon tumors was significantly higher in the S.LEW congenic strain compared with the S rat. Transcriptomic analysis confirmed that the chemical carcinogenesis pathway was significantly up-regulated in the congenic strain. These studies provide evidence for a GWAS-validated genomic segment on rat chromosome 10 as being important for the regulation of cardiovascular function and tumorigenesis.</p></div

Intrarenal Dopaminergic System Is Dysregulated in SS-<i>Resp18^mutant</i> Rats

The genetic and molecular basis of developing high blood pressure and renal disease are not well known. Resp18mutant Dahl salt-sensitive (SS-Resp18mutant) rats fed a 2% NaCl diet for six weeks have high blood pressure, increased renal fibrosis, and decreased mean survival time. Impairment of the dopaminergic system also leads to hypertension that involves renal and non-renal mechanisms. Deletion of any of the five dopamine receptors may lead to salt-sensitive hypertension. Therefore, we investigated the interaction between Resp18 and renal dopamine in SS-Resp18mutant and Dahl salt-sensitive (SS) rats. We found that SS-Resp18mutant rats had vascular dysfunction, as evidenced by a decrease in vasorelaxation in response to sodium nitroprusside. The pressure–natriuresis curve in SS-Resp18mutant rats was shifted down and to the right of SS rats. SS-Resp18mutant rats had decreased glomerular filtration rate and dopamine receptor subtypes, D1R and D5R. Renal dopamine levels were decreased, but urinary dopamine levels were increased, which may be the consequence of increased renal dopamine production, followed by secretion into the tubular lumen. The increased renal dopamine production in SS-Resp18mutant rats in vivo was substantiated by the increased dopamine production in renal proximal tubule cells treated with L-DOPA. Overall, our study provides evidence that targeted disruption of the Resp18 locus in the SS rat dysregulates the renal dopaminergic system

Gene Ontology for Molecular Function (GO:MF) in the AOM-treated S and S.LEW congenic rats.

<p>The top significantly up-regulated (A) and down-regulated (B) GO:MF terms of differentially expressed genes in the S.LEW congenic rats compared with the S rats (n = 3/group). “Enrichment Score” = “-log₁₀(Fisher p-value)” of the corresponding molecular function in the microarray analysis.</p

Shorter QT-intervals in <i>Rffl-lnc1</i> disruption model 1 compared with S rats.

<p>(A) Representative ECG recordings from individual S and <i>Rffl-lnc1</i> disruption model 1. (B) Shorter QT-intervals in <i>Rffl-lnc1</i> disruption model 1 compared with S rats (n = 5–6 rats/group). Experimental rats were maintained on low-salt diet after weaning and surgically implanted with CTA-F40 transmitters at about 18 weeks of age. Surgical rats were individually housed and allowed to recover for at least 3 days before ECG recording. Data plotted was obtained by telemetry recording once every 5 minutes continuously and averaged for 4-hour intervals. All values are expressed as mean ± SEM. *: <i>P</i> < 0.05.</p