Macrophages communicate with mesangial cells through the CXCL12/DPP4 axis in lupus nephritis pathogenesis

Abstract

Lupus nephritis (LN) occurs in 50% of cases of systemic lupus erythematosus (SLE) and is one of the most serious complications that can occur during lupus progression. Mesangial cells (MCs) are intrinsic cells in the kidney that can regulate capillary blood flow, phagocytose apoptotic cells, and secrete vasoactive substances and growth factors. Previous studies have shown that various types of inflammatory cells can activate MCs for hyperproliferation, leading to disruption of the filtration barrier and impairment of renal function in LN. Here, we characterized the heterogeneity of kidney cells of LN mice by single-nucleus RNA sequencing (snRNA-seq) and revealed the interaction between macrophages and MCs through the CXC motif chemokine ligand 12 (CXCL12)/dipeptidyl peptidase 4 (DPP4) axis. In culture, macrophages modulated the proliferation and migration of MCs through this ligand-receptor interaction. In LN mice, treatment with linagliptin, a DPP4 inhibitor, effectively inhibited MC proliferation and reduced urinary protein levels. Together, our findings indicated that targeting the CXCL12/DPP4 axis with linagliptin treatment may serve as a novel strategy for the treatment of LN via the CXCL12/DPP4 axis.

Fig. 1. Biochemical and pathological alterations of LN mice.

A–C The 24-h urine protein concentrations, and relative anti-dsDNA and anti-nuclear antibody levels in LN mice at 4, 6, 10, and 12 weeks. D HE staining of kidneys at different weeks. Scale bar = 20 μm, n = 3 for each group.

Fig. 2. snRNA-seq landscape of kidneys in LN mice.

A Schematic workflow for 10X Genomics snRNA-seq. B t-SNE analysis (left) showed that there were 10 different cell clusters in the renal cortex. The distributions of renal cells at 6 and 10 weeks are shown on the right side. Different cell clusters are color-coded. C Changes in the proportions of each cell cluster from 6 to 10 weeks. D t-SNE map showing the expression levels of single marker genes in proximal tubules, endothelial cells, macrophages, MCs, distal tubules, and podocytes. E Expression heatmap of the top three DEGs in the indicated cell clusters. F The expression of Lama2 on MCs was verified by co-immunolocalization staining with Fhl2. Scale bar = 5 μm, n = 3 for each group.

Fig. 3. Molecular and temporal profiles of MC subtype heterogeneity.

A t-SNE analysis showing that there were four subclusters of MCs. Different cell subclusters are color-coded. B Changes in the numbers of cells in each mesangial subcluster in both the 6- and 10-week samples. C Bubble plots showing the expression of marker genes in subclusters of MCs. D Top 15 enriched GO BP terms of DEGs in Mes 0–3. E Distribution of MCs in the cell trajectory: the right panel is the pseudo-time trajectory of each MC sub-cluster.

Fig. 4. Cell–cell communication networks in the kidneys of LN mice by CellPhoneDB analysis.

A Network visualization of ligand–receptor connectivity in LN mice. Nodes represent clusters; the larger the node, the more the interactions between the cell and other cell types. The lines represent the interactions between nodes, and the thickness of each line is proportional to the strength of the ligand–receptor pair between cell types. B Ligand–receptor relationship between macrophages and MC subclusters when MCs are the receptor cells. C Immunohistochemical co-localization of Fn1 and DPP4 in the kidneys of LN mice. Scale bar = 5 μm, n = 5 for each group. D DPP4 immunofluorescence staining of DPP4 in glomerular MCs. Scale bar = 20 μm, n = 5 for each group.

Fig. 5. Macrophages regulate MCs via the CXCL12/DPP4 axis.

A CCK8 assays showing the effects of CXCL12 and linagliptin on MC viability. B EdU assays were performed to detect the proliferation of MCs treated with CXCL12 and linagliptin. Scale bar = 75 μm, n = 3 for each group. C Wound healing and D Transwell assays were performed to detect the migration of MCs treated with CXCL12 and linagliptin. 10×: Scale bar = 75 μm. 20×: Scale bar = 50 μm, n = 3 for each group.

Fig. 6. CXCL12 promotes the proliferation and migration of MCs via DPP4 in vitro.

A Co-culture pattern map of macrophages and MCs. B qRT-PCR assays were performed to detect the expression of CXCL12 in macrophages after siRNA interference. C EdU assays were performed to characterize the proliferation of MCs when knocking down CXCL12 in macrophages and adding linagliptin. Scale bar = 75 μm, n = 3 for each group. D Transwell assays were performed to characterize the migration of MCs when knocking down CXCL12 in macrophages and adding linagliptin. 10×: Scale bar = 75 μm. 20×: Scale bar = 50 μm, n = 3 for each group.

Fig. 7. Therapeutic effect of linagliptin on LN mice.

A Flowchart of LN mice treated with saline and linagliptin. Linagliptin treatment group (n = 10 for each group) (3 mg/kg/d) and control group (n = 10 for each group) (0.1 mL/10 g). B Quantitative analysis of 24-h proteinuria in LN mice after 0, 2, 3, and 5 weeks of gavage treatment. C–E Serum levels of ANA, complement C3 and anti-dsDNA in the treatment and control groups after 3 and 5 weeks of gavage treatment. F Blood glucose levels in the treatment and control groups.

Fig. 8. HE staining and pAKT expression in the kidney of LN mice after gavage treatment.

A HE staining of kidneys in the treatment and control groups after 3 and 5 weeks of gavage treatment. 20×: Scale bar = 50 μm. 40×: Scale bar = 20 μm, n = 10 for each group. B The expression of pAKT on MCs was verified by co-immunolocalization staining with Fn1. Scale bar = 10 μm, n = 3 for each group.

Fig. 9. Schematic illustrating the interaction between macrophages and glomerular MCs in LN.

In the early stage of LN, macrophages regulate MCs through the CXCL12/DPP4 axis to initiate the pathogenesis and affect MC proliferation and migration. As the disease progresses, the proliferation and migration of the MCs and endothelial cells will damage the structure and function of the glomerulus, block Baumann’s capsule, affect the glomerular filtration function, destabilize the microvascular endothelial stability, aggravate hematuria, and ultimately lead to renal injury.