#2584 Mapping Progression of DKD in ReninAAV UNx db/db mice utilizing time-series RNA sequencing

Abstract

Abstract Background and Aims The Renin adeno-associated virus (AAV) uninephrectomized (UNx) db/db mouse model of diabetic kidney disease (DKD) recapitulates hallmarks of DKD with hypertension, including albuminuria and glomerulosclerosis. Despite previous characterization of the model, gaps persist in our understanding regarding temporal patterns of gene expression and pathophysiology in the glomeruli. This longitudinal study characterizes isolated glomeruli from ReninAAV UNx db/db mice during progression of DKD with hypertension. Method Female db/db (BKS.Cg-Dock7m +/+ Leprdb/J) received a single i.v. dose of LacZAAV (control) or ReninAAV. Untreated db/m mice (BKS.Cg-Dock7m +/+ Leprdb/J) served as healthy controls. One week after AAV injection, ReninAAV mice underwent UNx, recovered for three weeks, and were subsequently randomized and stratified into 4 groups according to fed blood glucose and body weight. ReninAAV UNx mice were terminated at 4, 8, 12 and 16 weeks (w4-w16) after UNx. Control groups were terminated at 16 weeks. Endpoints included urine and plasma biochemistry, kidney histology (i.e. Collagen 1a1 and Glomerulosclerosis) and RNA sequencing of glomeruli isolated by laser capture microdissection. Results ReninAAV UNx db/db mice showed progressive kidney fibrosis (Col1a1%-area) and albuminuria (uACR) compared to LacZAAV db/db and db/m mice across all timepoints investigated. Furthermore, a significant increase in glomerulosclerosis was observed from w12 and onwards compared to LacZAAV and db/m mice. When compared to LacZAAV mice, more than 1,700 glomerular differentially regulated genes and more than 250 uniquely regulated genes were found at each timepoint, including markers of fibrosis, inflammation and glomerular injury. Several glomerular drug targets exhibited time-specific regulated in ReninAAV UNx db/db mice (e.g. Slc5a2, Egfr, Agtr1a). Regulated genes were segmented into nine clusters with distinct temporal patterns. Among these, a continuous increase in expression of genes associated with extracellular matrix (ECM), small molecule transport and temporal stimulation of metabolic pathways. Finally, a correlation analysis revealed strong associations between ECM and ECM-related pathways vs. glomerulosclerosis and albuminuria. Conclusion The ReninAAV UNx db/db mouse model of hypertensive DKD demonstrates a progressive increase in fibrosis and albuminuria paralleled by dynamic changes in glomerular gene expression signatures. Notably, the identification of temporal patterns of gene expression and pathway regulation provides novel insights into DKD pathophysiology and progression. This underscores the ReninAAV UNx db/db mouse as an applicable model of human DKD in preclinical target and drug discovery.