Fig. 1: Overview of experimental pipeline and characterisation of cell models.

a Schematic representation of the experimental pipeline. Conditionally immortalised human glomerular endothelial cells (GECs), podocytes (Pods), mesangial cells (MCs) and proximal tubular cells (PTCs) were studied in vitro in a basal and insulin-resistant environment (consisting of 1 ng/ml TNFα, 1 ng/ml IL-6, 25 mM glucose and 100 nmol/l insulin). Insulin-sensitive cell lines were established via stable overexpression of the human insulin receptor (IR). The cellular transcriptome and proteome were studied simultaneously using RNA sequencing and tandem-mass-tagged mass spectrometry (n = 5 biological repeats per cell line and condition) and integrated transcriptome and proteome data were analysed using univariate and multivariate statistical models and gene set enrichment analysis (GSEA). Further targeted analysis and validation were performed using single-cell and bulk transcriptomics data from human DKD biopsies. Figure partly created in BioRender. Lay, A. (2022) BioRender.com/x18l854 and BioRender. Lay, A. (2024) BioRender.com/m22n059. b Western blotting of total protein lysates demonstrated suppression of insulin-stimulated (15-min, 10 or 100 nmol/L) IR and Akt phosphorylation in all cell lines exposed to diabetic, insulin resistant, milieu (‘DM’). GECs displayed no evidence of IR downregulation (representative of n = 4 biological replicates). c–f Percentage increase in cellular uptake of [3H]2-deoxy-d-glucose in insulin-stimulated (15-min, 100 nmol/L) GECs (n = 6), Pods (n = 3), MCs (n = 4) and PTCs (n = 5) [all biological repeats] vs. unstimulated cells, with and without exposure to a diabetic, insulin resistant, milieu (‘DM’), unpaired two-tailed t-test, data are presented as mean values ± SEM.