Single-cell transcriptomics reveals a mechanosensitive injury signaling pathway in early diabetic nephropathy

Abstract

Background: Diabetic nephropathy (DN) is the leading cause of end-stage renal disease, and histopathologic glomerular lesions are among the earliest structural alterations of DN. However, the signaling pathways that initiate these glomerular alterations are incompletely understood.

Methods: To delineate the cellular and molecular basis for DN initiation, we performed single-cell and bulk RNA sequencing of renal cells from type 2 diabetes mice (BTBR ob/ob) at the early stage of DN.

Results: Analysis of differentially expressed genes revealed glucose-independent responses in glomerular cell types. The gene regulatory network upstream of glomerular cell programs suggested the activation of mechanosensitive transcriptional pathway MRTF-SRF predominantly taking place in mesangial cells. Importantly, activation of MRTF-SRF transcriptional pathway was also identified in DN glomeruli in independent patient cohort datasets. Furthermore, ex vivo kidney perfusion suggested that the regulation of MRTF-SRF is a common mechanism in response to glomerular hyperfiltration.

Conclusions: Overall, our study presents a comprehensive single-cell transcriptomic landscape of early DN, highlighting mechanosensitive signaling pathways as novel targets of diabetic glomerulopathy.

Fig. 1

scRNA-seq of BTBR ob/ob mouse kidneys with early DN. a Experimental scheme. b UMAP plot of annotated cell types. c Dot plot of defining marker genes for each cell type. d Total number of significant DEGs (nDEGs) in each cell type. Top enriched pathways in glomerular e and PT cell types f at 6 and 12 weeks. UMAP, uniform manifold approximation and projection; Ctrl, control; DN, diabetic nephropathy; Podo, podocyte; EC, endothelial cell; Mesan, mesangial cell; Int, interstitial cell; PT, proximal tubule; dLOH, descending limb of loop of Henle; aLOH, ascending limb of loop of Henle; DCT, distal convoluted tubule; CNT, connecting tubule; PC, collecting duct principal cell; IC-A, A-type collecting duct intercalated cell; IC-B, B-type collecting duct intercalated cell; Trans, transition cell; Imm, immune cell; Mitotic, mitotic cell

Fig. 2

Shared features of human and experimental DN. a Heatmap displaying the Pearson correlation coefficient between scRNA-seq and bulk RNA-seq data. b Overview of significant glomerular DEGs detected in both single-cell and bulk RNA-seq datasets, as well as in the ERCB patient dataset. cHeatmap showing the significant regulation of DEGs identified in microdissected glomeruli and in glomerular cells from DN patients and mice, respectively. Nonsignificant genes are shown in grey. ERCB, European Renal cDNA Bank

Fig. 3

Mechanosensitive transcriptional regulators are activated in DN glomeruli. a Dot plot displaying the commonly changed transcriptional regulations in both single-cell and bulk RNA-seq data estimated by IPA. b Heatmap showing the activity z scores of transcription factors estimated by SCENIC. c Heatmap showing the log2FC (DN vs. Ctrl) values of MRTF transcriptional target genes in mice glomerular cells. Nonsignificant genes are shown in gray (a, c). d Immunofluorescence staining of the mouse kidney paraffin sections showing the expression and localization of MRTFA and MRTFB in control and DN glomeruli. Glomerular endothelial cells were labeled by EMCN (endomucin), podocytes were labeled by NPHS1 (Nephrin), nuclei were counterstained with HOECHST. Arrows indicate mesangial cells, which were negative for EMCN and NPHS1

Fig. 4

MRTF transcriptional target genes are upregulated in DN patients with type 2 diabetes. a Heatmap showing the log2FC (DN vs. Ctrl) values of MRTF transcriptional target genes in microdissected glomeruli from Pima early DN patients. b Pearson correlations between MRTF target gene expression levels and mesangial volumes in Pima early DN patients. c Heatmap showing the log2FC (DN vs. Ctrl) values of MRTF transcriptional target genes in microdissected glomeruli from ERCB patient datasets. Nonsignificant genes are shown in gray. DN, diabetic nephropathy; HT, hypertensive nephropathy; IgA, IgA nephropathy; FSGS, focal segmental glomerulosclerosis; MGN, membranous nephropathy; SLE, lupus nephritis; RPGN, ANCA-associated glomerulonephritis; MCD, minimal change disease

Fig. 5

Immunofluorescence staining confirms the activation of MRTFB in mesangial cells during early DN. Immunofluorescence staining of the human kidney samples (ctrl: n = 2, DN: n = 5). Representative images showing the expression and localization of MRTFA and MRTFB in control and DN glomeruli. Glomerular endothelial cells were labeled by CD34, podocytes were labeled by NPHS1 (Nephrin), and nuclei were counterstained with HOECHST. Arrows indicate mesangial cells, which were negative for CD34 and NPHS1

Fig. 6

Mesangial cells exhibit dominant signaling networks in DN glomeruli. a Dot plot showing mesangial marker genes encoding transmembrane proteins responsible for the perception and transduction of mechanical signals. MSCs, mechanosensitive ion channels; GPCRs, G protein-coupled receptors. b Change of cell–cell interactions in all pairs of kidney cell types in DN vs. Ctrl mice. c Change of cell–cell interactions in all pairs of glomerular cell types in DN vs. Ctrl mice. d Increased cell–cell interactions for mesangial cell signaling to podocytes and glomerular endothelial cell in DN mice

Fig. 7

Kidney ex vivo perfusion activates mechanosensitive signaling pathways. a Experimental scheme of mouse kidney ex vivo perfusion. b Heatmap showing the log2FC values of MRTF transcriptional target genes in perfused versus control glomeruli. c Top enriched pathways of MRTF transcriptional target genes. d Experimental scheme of pig kidney ex vivo perfusion. e UMAP plot of annotated cell types from snRNA-seq of perfused and control pig kidney tissue. g Immunofluorescence staining against MEIS1 in the pig kidney cortex. Nuclei were counterstained with DAPI. G, glomerulus. Dashed lines indicate the area of glomerulus. Scale bar: 100 μm. f Dot plot displaying defining marker genes for each cell type. STROMA, stromal cells. h Heatmap showing z scores of mechanosensitive transcriptional regulators estimated by IPA. i Dot plot displaying MRTF transcriptional target genes significantly changed in perfused kidney tissue. Nonsignificant regulation or genes are shown in gray g, h