#2607 Generation of a tissue resident macrophage depleted reporter rat to examine cardiovascular homeostasis and kidney disease
Authors: Hur Heeyoun , Hadoke Patrick , Pridans Clare , Denby Laura 
Published: May 2024

Cardiovascular disease (CVD) and chronic kidney disease (CKD) are currently the 1st and 10th leading cause of global mortality, respectively. By 2050, CKD is predicted to be the 5th leading cause, with most of those individuals dying of CVD. Recent single cell RNA sequencing data has highlighted the plasticity of macrophage populations in both renal and cardiovascular injury and repair. Many of these subpopulations of macrophages remain uncharacterized.

We have generated the Red FIRE rat, which is a cross between the Csf1r-mApple reporter rat and the super-enhancer FIRE knockout rat. The Csf1r-mApple reporter rat introduces a fluorescent signal to cells of the monocyte-macrophage lineage and the KO of FIRE (fms-intronic regulatory element), while the FIRE rat has fms-intronic regulatory element (FIRE), which is the superenhancer in the Csf1r locus knocked out, resulting in depletion of tissue resident macrophages.

These red FIRE rats will allow us to visualize all Csf1r expressing cells (macrophages) and in the FIRE rats deplete populations of tissue resident macrophages (TRM) (including those in the kidney and heart). This will enable us to visualize and study TRM populations in homeostasis and injury. We hypothesise that the red FIRE rats will show a more severe phenotype in injury and have altered vascular function (local and systemic).

Male and female (8–10-week-old) rats underwent a 6- or 12-week 5/6 subtotal nephrectomy (5/6 STNx) in groups of 4-8. Baseline, midpoint and endpoint urine and serum were collected for analysis, as well as blood pressure measurements and echocardiograms to monitor kidney and heart health/decline. At the end of the experiment, kidneys and the heart were collected for flow cytometry, immunofluorescence, RT-qPCR, and histological staining. The thoracic aorta, mesentery, and common renal arteries were collected for functional analysis using isometric (wire)myography.

The knockout of the FIRE super enhancer resulted in depletion of kidney TRMs (Fig. 1) as visualised by immunofluorescent staining of both wild type and macrophage-depleted (red FIRE) rat tissue. Figure 1A shows the composite image and individual stains; DAPI, IBa1 (pan macrophage marker), and Csf1r-mApple (tissue resident macrophages) on a naïve heart. Representative composite images also compare STNx hearts (Fig. 1B) and kidneys (Fig. 1C) in both wild type and macrophage-depleted rats. This allowed for comparison of these rats in both injury and homeostasis.

Figure 1: Immunofluorescent staining of the uninjured heart of a FIRE wild type (WT), mApple positive rat (A), the injured kidney (B) and heart (C) of mApple positive, FIRE wild type (Control) or mApple positive, FIRE knock out (FIRE) rats post 12-week 5/6 subtotal nephrectomy (5/6 STNx). DAPI: nuclei, IBa1: all macrophages, csf1r (mApple): tissue resident macrophages. Representative imagining, figure made using FIJI.

Our preliminary data suggests previously undescribed roles for tissue-resident macrophages in rats i.e. ion handling, vascular homeostasis and roles in CKD and repair. These results could help reveal new specific druggable targets that could be exploited for treatments which could halt progression of disease and thus reduce end stage renal failure patient numbers, healthcare costs and premature CVD-related deaths.