ABSTRACT: To identify the transcriptional regulatory mechanisms associated with the biological anti-edematous and anti-inflammatory effect of spironolactone, we performed an analysis of the regulated genes in the neuroretina of GK rats aged 14 months and treated with a formulation of Spironolacton-loaded Microspheres (Sp-MSs) that released spironolactone for at least 2 months (mean size 30µm, 1µg/mg MSs/day). RNA-sequencing was performed on the neural retina and non-loade microspheres (NL-MSs) served as control treatment. The heatmaps of genes differentially regulated by Sp-MSs individuals as compared to NL-MSs showed a clear difference between the two conditions with 114 genes significantly regulated between the two groups, 27 significantly down regulated and 87 significantly up regulated (Log2FC ≥ 0,5, adjusted p<0.05). Pathway enrichment analysis using hallmark gene-set function highlighted 9 significantly regulated pathways: unfolded protein response, UV response, G2M checkpoint, allograft rejection, interferon gamma response, MTORC1 signaling, oxidative phosphorylation, MYC targets and adipogenesis (Figure EV 3B, C). Genes encoding proteins known to intervene in vascular permeability and retinal edema were counter regulated by Sp-MSs including Vldlr, Sesn2, Adcyap1, Dusp8, Pten, Slc7a1, Tjp1, Dlg1, Sema7a, although Vegf was rather up-regulated, suggested an anti-edematous mechanism independent from VEGF. The effect of Sp-MSs was confirmed by quantitative PCR for number of these genes demonstrating that spironolactone acts as a transcriptional regulator to normalize the diabetic retina.
