Set7 Methyltransferase and Phenotypic Switch in Diabetic Glomerular Endothelial Cells

Abstract

Key Points:

1. Set7 knockout improves diabetic glomerular structure and function and prevents diabetes-induced endothelial–mesenchymal transition (EDMT) by regulating Igfbp5.

2. Set7 knockdown prevents, and (R)-PFI-2 hydrochloride reverses, diabetes-induced EDMT by regulating insulin growth factor binding protein 5.

3. Set7 regulates the phenotypic EDMT switch, and inhibiting the methyltransferase attenuates glomerular injury in diabetic kidney disease.

Background: Hyperglycemia influences the development of glomerular endothelial cell damage, and nowhere is this more evident than in the progression of diabetic kidney disease (DKD). While the Set7 lysine methyltransferase is a known hyperglycemic sensor, its role in endothelial cell function in the context of DKD remains poorly understood.

Methods: Single-cell transcriptomics was used to investigate Set7 regulation in a mouse model of DKD, followed by validation of findings using pharmacological and short hairpin RNA inhibition inhibition of Set7.

Results: Set7 knockout (Set7KO) improved glomerular structure and albuminuria in a mouse model of diabetes. Analysis of single-cell RNA-sequencing data showed dynamic transcriptional changes in diabetic renal cells. Set7KO controls phenotype switching of glomerular endothelial cell populations by 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 kidney and circulating hyperglycemic cells exposed to TGFβ1. We showed that the highly selective Set7 inhibitor (R)-PFI-2 hydrochloride 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 corresponded in human glomerular endothelial cells with (R)-PFI-2 hydrochloride inhibition or Set7 short hairpin RNA silencing.

Conclusions: Set7 regulates the phenotypic endothelial–mesenchymal transition switch and suggests that targeting the lysine methyltransferase could protect glomerular cell injury in DKD.

Podcast: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2024_04_25_ASN0000000000000345.mp3

Graphical abstract

Figure 1

Set7KO improves glomerular structure and function in a model of diabetes. (A) Experimental design. Diabetes was induced in Set7WT (Set7^+/+ApoE^−/−) and Set7KO (Set7^−/−ApoE^−/−) mice using STZ. The kidney cortex was isolated after 10 weeks of diabetes and assessed by histological staining. Comparative scRNA-seq of the cortex identified Set7-dependent genes, and target validation was performed in human cell lines. Increased (B) UAE, (C) UCAR, (D) Kim1 expression, (E) glomerular collagen I and (F) collagen IV, and (G) mesangial area expansion in DKD were reduced by Set7KO. Mesangial area was determined by PAS staining. Data represented as mean±SEM. *P < 0.05, **P < 0.01, ***P < 0.001. n≥6 per group. Ctrl, control; Diab, diabetes; DKD, diabetic kidney disease; Kim1, kidney injury molecule 1; KO, knockout; PAS, periodic acid–Schiff; PFI-2, (R)-PFI-2 hydrochloride; scRNA-seq, single-cell RNA-sequencing; STZ, streptozocin; UAE, urinary albumin excretion; UCAR, urine albumin–creatinine ratio; Veh, vehicle; WT, wild type.

Figure 2

Influence of Set7 deletion in diabetic kidney transcriptome revealed by scRNA-seq. (A) scRNA-seq cell clusters in the diabetic kidney were visualized by t-distributed stochastic neighbor embedding (tSNE) after analysis using Seurat and R package. (B) Composition of the cell clusters in each experimental group. Significant changes in cell composition between experimental groups (ANOVA) included the proximal tubule (P < 0.05) and GEN (P = 0.066) clusters, as calculated using the propeller method. (C) GSEA identifies four reactome pathways that belong to OXPHOS, rRNA processing, EMO, and PPARα activation. These pathways were significantly changed by Set7 in diabetes. (D) Distribution of diabetic Set7-dependent gene expression was visualized by tSNE. Color gradient indicates the number of transcripts detected per cell. BL, B-lymphocyte; CDI, collecting duct intercalated; DCT, distal convoluted tubule; EMO, extracellular matrix organization; ER, erythroid; GEN, glomerular endothelial; GSEA, gene set enrichment analysis; LH, loop of Henle; MAC, macrophage; MG, mesangial; MSC, mesenchymal; NK, natural killer; OXPHOS, respiratory electron transport; PDC, podocyte; PPARα, peroxisome proliferator-activated receptor alpha; PTC, proximal tubule cell; rRNA, ribosomal RNA; TL, T-lymphocyte.

Figure 3

Set7 regulates the GEN transcriptome. (A) Increased Set7 expression in the kidney cortex derived from diabetic mice. Expression of Set7 mRNA in the kidney cortex was assessed by qRT-PCR. n=5 per group. Data are represented as mean±SEM. ***P < 0.001 versus control Set7WT mice. (B) Cell distribution of Set7 expression in the kidney cortex by scRNA-seq. (C) Set7KO regulates the expression of GEN and mesenchymal markers in the diabetic kidney. Color gradient indicates significant (P < 0.05) log fold changes for each gene: red (elevated), blue (reduced), and white (no significant change; P > 0.05). (D) The top 15 reactome pathways that are significantly changed by diabetes (control versus diabetic Set7WT) and Set7 deletion in diabetes (Diab Set7KO; diabetic Set7WT versus diabetic Set7KO) are illustrated in GEN and mesenchymal cell clusters using GSEA. qRT-PCR, quantitative real-time reverse-transcription PCR.

Figure 4

Set7-dependent regulation of the GEN transcriptome in diabetic mouse kidney. (A) Identification of the novel GEN cell population in diabetic Set7KO mouse kidney. Reclustering of the GEN and mesenchymal populations in our scRNA-seq data. (B) Top 40 genes significantly changed in the GEN (Igfbp5 high) and GEN (Igfbp5 low) clusters. (C) Distribution of Igfbp5 and Plat gene markers in each experimental group is illustrated. (D) Composition of the cell clusters in each experimental group. Significant changes in cell composition between experimental groups (ANOVA) included GEN (Igfbp5 high, P < 0.05) and GEN (Igfbp5 low, P < 0.01) and calculated using the propeller method. (E) Reactome pathway exchanges observed in the GEN (Igfbp5 high) and GEN (Igfbp5 low) cell clusters. EMT, epithelial-to-mesenchymal transition; IGFBP5, insulin growth factor binding protein 5; UMAP, uniform manifold projection.

Figure 5

PFI-2 attenuates diabetes-related pathways and genes in human renal cells. (A) Chemical structure of the SET7 inhibitor PFI-2. (B) Molecular docking was used to evaluate the binding characteristics of PFI-2 within the substrate peptide-binding groove of the SET7 protein. The binding affinity (kcal/mol) and key residues are shown. The italicized residues were predicted to form hydrogen bonds with PFI-2. Polar residues are in dark blue, while negatively charged residues are in red. (C) The per-residue RMSD (Å) was calculated and the conformational changes that occur in the post-SET loop (shown in shade). (D) Inhibition of Set7 histone methyltransferase activity by PFI-2. FLAG tagged Set7 protein (FLAGSet7) was incubated with [H³]-S-adenosyl-methionine and histone H3 peptide in the presence of PFI-2. Tritiated histone H3 peptide was measured by liquid scintillation. Activity is presented as percentage, 100% corresponding to FLAGSet7 with DMSO vehicle control. Data are presented as mean±SEM, n=3. (E) Protein-peptide docking was performed using the HPEPDOCK server. The histone H3 peptide was modified to contain an unmethylated lysine residue (K4) and was docked to structures of SET7 in the absence (left and middle) and presence of PFI-2 (right) within the peptide-binding groove. (F) Schematic of the experimental design to assess the Set7 inhibitor PFI-2 in human cell lines under diabetic conditions. Cells were treated with PFI-2 for 24 hours before hyperglycemic stimulation with TGFβ1 for 48 hours. (G) Set7 inhibition by PFI-2 in human cell lines. Demethylation of Rpl29 and Set7 levels were quantified by Li-COR Odyssey. Gapdh was used as loading control. (H) PFI-2 attenuates the expression of TGFβ1-induced genes in human cell types. The expression of genes shown in brackets belong to the four core pathways: OXPHOS (MDH2, NDUFB2), PPARα (ACOX1, ANGPTL4), rRNA (NHP2), and EMO (PDGFA, COL4A3). Gene expression was assessed by qRT-PCR. The assessment of ANGPTL4 expression was used as a TGFβ1 response gene. n=6 per group. Data are represented as mean±SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal glucose control; #P < 0.05, ##P < 0.01, ###P < 0.001 versus diabetic condition (HG+TGFβ1). HG, high glucose; RMSD, root-mean-square deviation; SAH, S-adenosyl homocysteine; SAM, S-adenosyl methionine.

Figure 6

Set7-dependent transcriptional regulation of IGFBP5 in human GEN cells. (A) PFI-2 attenuates TGFβ1-induced ROS levels in GEN cells. Cellular and mitochondrial ROS production was measured by fluorescence intensity using the fluorogenic dyes DCFDA and MitoSOX Red (Mitochondrial Superoxide Indicator). n=8 per group. (B) PFI-2 diminishes TGFβ1-induced EDMT in hyperglycemic GEN cells. The expression of the endothelial (CDH5, PECAM1, PLAT, and IGFBP5) and mesenchymal markers (VIM, EDN1, TAGLN, and THBS2) were assessed by qRT-PCR. n=6 per group. (C) Set7 inhibition by shRNA in human GEN cells. Whole-cell extracts prepared from controls (non-target vector) or Set7 knockdown (shSet7) in GEN cells were analyzed by immunoblot. Rpl29 methylation and Set7 protein content were quantified by Li-COR Odyssey. Gapdh was used as loading control. n=3 per group. (D) Specific reduction of IGFBP5 in shSet7 GEN cells. PLAT and IGFBP5 gene expression were assessed by qRT-PCR. n=6 per group. (E) Set7 regulates H3K4 methylation content of the human IGFBP5 gene. The regulatory promoter and enhancer elements derived from the GeneHancer database are illustrated. ChIP regions (R1, R2, and R3) were assessed for H3K4me1 and H3K4me2. (F and G) Soluble chromatin was fractionated from GEN cells and immunopurified with H3K4me1 or H3K4me2 antibodies. qPCR was used to assess DNA enrichment. (F) PFI-2 attenuates TGFβ1-induced H3K4 methylation at the IGFBP5 gene in human GEN cells. (G) shSet7 regulates H3K4 methylation in human GEN cells. n=3 per group. Normoglycemia (NG); hyperglycemia/TGFβ1 (HG/TGF); hyperglycemia/TGFβ1/PFI-2 (HG/TGF/PFI). Data are represented as mean±SEM. *P < 0.05, **P < 0.01, ***P < 0.001; #P < 0.05, ##P < 0.01, ###P < 0.001 versus diabetic condition (HG+TGFβ1). ChIP, chromatin immunoprecipitation; DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate; EDMT, endothelial–mesenchymal transition; qPCR, quantitative real-time PCR; ROS, reactive oxygen species; shRNA, short hairpin RNA.

Figure 7

Targeting Set7 in diabetic GEN transcriptome. Set7-mediated endothelial transcriptomes in the GEN population derived from (A) diabetic mice and (B) human GEN cells stimulated with TGFβ1 and HG (TGF/HG) conditions. (C) scRNA-seq analysis demonstrates the transcriptional expression index (TEI) of Set7-dependent diabetic pathways in diabetic mice include the following: IGFBPs regulation, TGFβ receptor signaling, Rho GTPases-activated ROCKs, EMO, smooth muscle contraction, JAK-STAT signaling after IL-12 stimulation, cooperation of prefoldin and TriC/CCT in actin and tubulin folding, and NR1H2- and NR1H3-mediated signaling pathways are identified. *P < 0.05, **P < 0.01 versus nondiabetic mice control; †P ≤ 0.05, ††P < 0.01, †††P < 0.001 versus diabetic Set7WT mice. (D) Influence of Set7 inhibition by PFI-2 or shRNA in human GEN cells identifies Set7-dependent diabetic genes and pathways. Genes were filtered by P value < 0.05 in either TGF/HG or Set7 inhibition in TGF/HG; §§P < 0.01, §§§P < 0.001 versus nondiabetic condition (NG); #P < 0.05, ##P < 0.01, ###P < 0.001 versus diabetic (TGF/HG) condition.

Figure 8

Schematic model of Set7-mediated regulation of phenotypic switch. In diabetic GEN cells, Set7 activates IGFBP5 expression by regulating H3K4me1 and H3K4me2. The mesenchymal-like cells are hypothesized to transition and activate ROS, EMO, JAK-STAT, and EMT pathways. Genetic knockout and pharmacological inhibition of Set7 attenuates ROS production and improves fibrosis and proinflammatory pathways and the expression of EDMT genes implicated in DKD.