Loss of miR‐146b‐5p Exacerbates Pathological Left‐Ventricular Remodeling in a Rat Model of CKD
Authors: Paterson Mark , Chuppa Sandra , Kriegel Alison J. 
Published: November 2023

Type 4 cardiorenal syndrome (CRS4) is a complex disorder in which primary chronic kidney disease (CKD) contributes to secondary cardiovascular disease (CVD). CKD patients experience a significant increase in relative risk of cardiac events and premature cardiovascular mortality compared to the general population. While the link between the kidney and the heart is well‐established, the mechanisms by which CKD mediates CVD are not well understood. microRNA (miR) research is a rapidly growing area as miRs have been shown to be important mediators of pathophysiology. We have utilized a 5/6 nephrectomy (5/6Nx) model of CKD in Sprague‐Dawley (SD) rats to investigate the role miRs play in progressive CKD‐mediated cardiac dysfunction. We have previously reported several miRs to be differentially expressed in left ventricular (LV) tissue at 7 weeks after 5/6Nx (p<0.05, fold change >2, vs. sham‐operated controls). In this study, we focus on the role of rno‐miR‐146b‐5p (miR‐146b, fold change 3.59, 5/6Nx/sham) in our rat model of CRS4. Studies in humans have identified miR‐146b as highly expressed in kidney fibrosis, CKD, vascular inflammatory disease, and coronary artery disease. We have developed the first reported line of miR‐146b null mutant rats (miR‐146b−/−) on the SD background using CRISPR‐Cas9‐mediated gene editing to examine the functional response to miR‐146b knockout. Briefly, we perform 5/6Nx on WT and miR‐146b−/− at 10 weeks of age and follow the animals for 7 weeks post‐5/6Nx. Urine was collected for 24 hours and transthoracic echocardiography was performed on all animals prior to 5/6Nx and upon follow‐up every two weeks throughout the recovery period to evaluate kidney function, LV remodeling and cardiac function. At 7 weeks post‐5/6Nx, a time point where significant pathological cardiac remodeling and pathology has been observed in SD rats by our laboratory, tissues are collected for histological analysis and downstream molecular assays. Confirming the functional knockout of miR‐146b, abundance of mature miR‐146b decreased by >95% in miR‐146b−/− animals (% change from WT 3.52±0.23; p<0.05; n=4). Abundance of its related miR‐family member, miR‐146a‐5p, was unchanged in LV tissue (% change from WT = 85.85±5.37; p=0.18; n=4), further confirming the specificity of the knockout and revealing no compensatory upregulation of the isomiR. Analysis of LV tissue 7 weeks after 5/6Nx has shown miR‐146b−/− rats develop more severe LV fibrosis compared to their wild‐type (WT) littermates (% fibrotic area 2.97±0.72 vs. 1.16±0.25; p=0.077; n=3–4). At this same time‐point, echocardiographic analysis of LV structure and function reveals miR‐146b−/− animals develop increased systolic ventral wall thickness (3.84±0.29 vs. 2.80±0.26 mm, p<0.05; n=3) along with increased fractional shortening (49.92±1.07 vs. 37.66±3.59, p<0.05; n=3) compared to WT 5/6Nx littermates. These data, taken together, suggest loss of miR‐146b‐5p in the rat exacerbates pathological cardiac remodeling. Future studies will investigate the specific mechanism by which miR‐146b regulates heart function in CRS4.

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13SDG17100095