Runx2 Deletion in Myeloid Cells Exaggerates Kidney Fibrosis Through Increase in Siglec-F+ Neutrophils

Background: Runt-related transcription factor 2 (Runx2) is widely known as a transcription factor for bone formation and vascular calcification. Our previous study demonstrated that Runx2 in myeloid cells, including neutrophils and macrophages, prevents adverse cardiac fibrosis after myocardial infarction; however, the pathophysiological role of myeloid Runx2 in kidney fibrosis is unknown. Methods: Runx2 expression in human and murine fibrotic kidneys was examined using immunohistochemistry and flow cytometry with anti-Runx2 antibody. Myeloid cell-specific Runx2 knockout (CKO) mice were subjected to unilateral ureteral obstruction (UUO). Kidney fibrosis was evaluated using Masson’s trichrome staining, measuring hydroxyproline level, and immunofluorescence analysis with anti-α-SMA antibody. For single-cell RNA sequencing (scRNA-seq), myeloid cells were isolated from murine kidneys subjected to UUO by magnetic cell sorting. To examine the effects of Siglec-F+ neutrophils on kidney fibrosis, CKO mice were subjected to UUO and treated with the Siglec-F depletion antibody or isotype control antibody (40 ug/head, i.p. at day 2 and 4 after UUO). Results: Public scRNA-seq data, immunohistochemistry, and flow cytometry demonstrated that Runx2 expression was upregulated in myeloid cells of murine fibrotic kidneys. Runx2 expression was also upregulated in human kidneys with end-stage renal disease. CKO mice showed increased fibrosis area and hydroxyproline content compared with control mice day 7 after UUO (fibrosis area: fl/fl; 8.30±0.87 um2/field, CKO; 10.87±1.23 um2/field, n = 5 for fl/fl, n = 7 for CKO). Additionally, the α-SMA-positive area was higher in CKO mice than in control mice. scRNA-seq revealed that Runx2 knockout in myeloid cells affected neutrophil properties, such as increased Siglec-F-positive neutrophils. Treatment with the Siglec-F depletion antibody reduced kidney fibrosis in CKO mice after UUO. Conclusion: Runx2 deletion in myeloid cells exaggerates kidney fibrosis through an increase in the number of Siglec-F+ neutrophils, providing a novel insight into the pathogenesis of kidney fibrosis.