Targeting Set7 in an experimental model of diabetic nephropathy

Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of mouse single-cell RNA-sequencing data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of GEN cell populations through transcriptional regulation of the insulin growth factor binding protein 5 (IGFBP5). Chromatin immunoprecipitation assays confirmed that the expression of the IGFBP5 gene was associated with mono- and dimethylation of histone H3 lysine 4 (H3K4me1/2). This generalizability was investigated in human renal and circulating hyperglycemic cells exposed to TGFb1. We showed that the highly selective Set7 inhibitor PFI-2 attenuated indices associated with renal cell damage and mesenchymal transition, specifically (1) reactive oxygen species production, (2) IGFBP5 gene regulation, and (3) expression of mesenchymal markers. Furthermore, renal benefit observed in Set7KO diabetic mice closely corresponds in human glomerular endothelial cells with PFI-2 inhibition or Set7 shRNA silencing. Overall design: Single cell RNA analysis (Drop-seq) of diabetic kidney tissue from streptozotocin-induced Setd7 deficient (Setd7-/-) mice. Mouse models included: Diabetic (Setd7-/- ApoE-/-); Control (Setd7-/- ApoE-/-); Diabetic (Setd7+/+ ApoE-/-); Control (Setd7+/+ ApoE-/-). For target validation experiments, human immortalized glomerular endothelial (GEN) cells were cultured with or without 15 mM (R)-PFI-2 (PFI-2; Cayman Chemicals) dissolved in DMSO for 24 hours before exposing them to 5.5 or 25 mM D-glucose in the presence or absence of 5 ng/ml TGF-b1 (R&D Systems) for 48 hours at 37°ockdown of Set7 was performed in GEN cells using MISSION shRNA expressing lentivirus vector (Sigma). Cells transduced with MISSION Non-target shRNA control vector (Sigma) were used as a control. GEN treatment groups included: GEC_NG (5.5 mM D-glucose); GEC_PFI (25 mM D-glucose + 15 mM (R)-PFI-2 + 5 ng/ml TGF-b1); GEC_TGF (25 mM D-glucose + 5 ng/ml TGF-b1); KD_TGF (25 mM D-glucose + Set7 shRNA + 5 ng/ml TGF-b1); NT_TGF (25 mM D-glucose + MISSION Non-target shRNA + 5 ng/ml TGF-b1). Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and shRNA inhibition of Set7.