RNA Sequencing of Left-ventricular Tissue Highlights Novel Pathways Impacted by the Loss of miR-146b-5p in a Rat Model of Chronic Kidney Disease

Chronic kidney disease (CKD) mediates pathological changes in heart and vascular tissues, yet many molecular mechanisms by which renal injury directly affects cardiovascular pathology (CVD) are unknown. miRNAs are proposed to be powerful regulators of pathophysiological signaling networks; having the potential to radically change the landscape of mRNA transcript expression and protein abundance in the context of health and disease. We have profiled the expression of miRNAs in a rat model of CKD – the 5/6 nephrectomy (5/6Nx) – with the objective to identify miRNAs with strong effects on CKD-mediated CVD. One such miRNA, miR-146b-5p (miR-146b), is highly upregulated in 5/6Nx rats compared to sham-operated controls at 7-weeks post-5/6Nx in both the kidney (5.4 fold increase; p<0.05) and the heart (1.6 fold increase, p<0.05), a time-point at which we see maximal renal and cardiac pathology. To examine the effects of miR-146b upregulation in our model of CKD, we performed RNA sequencing on cardiac tissue at 7 weeks post-5/6Nx in both wild-type (WT) Sprague-Dawley (SD) rats and a unique CRISPR-Cas9-mediated miR-146b knockout strain (miR-146b -/- ) created on the SD background. Of 3,018 genes differentially regulated by more than 1.5-fold (absolute value) in miR-146b -/- vs. WT rats after 5/6Nx, 135 had significantly altered expression (adjusted p-value <0.05). We filtered all differentially expressed genes through Ingenuity Pathway Analysis to identify affected signaling networks in miR-146b -/- vs. WT rats that correlate with observed pathological phenotypes. The expression pattern of differentially regulated genes in these networks indicates predicted activation of pathways that contribute to tissue fibrosis and mineralization, reactive oxygen species signaling, and the inflammatory and immune responses. These pathways are enriched with predicted miR-146b target genes and correlate closely with observed phenotypes in miR-146b -/- 5/6Nx rats; including increased renal fibrosis and mineralization of cardiovascular tissue, and the well-known role of miR-146b in negative feedback regulation of NFκB signaling. These preliminary analyses uncover unique networks of genes regulated by miR-146b that may contribute to CKD-mediated CVD.