#1995 Runt-related Transcription Factor 1 (RUNX1) is a mediator of acute kidney injury

Abstract

Abstract Background and Aims A better understanding of the pathogenesis of acute kidney injury (AKI) may lead to new therapeutic approaches. Kidney transcriptomics analyses of murine folic acid-induced AKI (FA-AKI) identified Runx1 as the most upregulated RUNX family gene. RUNX1 is a key regulator of hematopoiesis, associated to leukemias and other non-immunological cancers and modulates myoblast proliferation during muscle regeneration and cardiac remodelling after myocardial infarction. In kidney diseases, RUNX1 is overexpressed in polycystic kidney disease and kidney cancer and favours fibrosis. Method We examined the expression of RUNX1 in folic acid-AKI (FA-AKI), in bacterial lipopolysaccharide (LPS)-induced cytokine storm-AKI (CS-AKI) and in human AKI. For this, female 12- to 14-week-old C57BL/6J wild type (WT) mice, received a single intraperitoneal (i.p) injection of acid folic (250 mg/kg), LPS (5 mg/kg) or vehicle. In cultured murine tubular MCT cells, we explored the expression and role of RUNX1 in response to the cytokine TWEAK or LPS. The chemical inhibitor of RUNX1 Ro5-3335 and a specific small interfering RNA (siRNA) against RUNX1 were used in cultured cells to explore the role of RUNX1 in TWEAK and LPS-induced inflammation. Ro5-3335 was also used in murine AKI (FA-AKI and CS-AKI) to test its potential as a therapeutic target. In all experimental models, plasma samples were collected to assess renal function. Kidneys were collected for RNA (RT-PCR), protein (Western blot, ELISA, immunohistochemistry) and histopathologic studies. Results Kidney transcriptomics identified Runx1 as the most upregulated Runx gene in FA-AKI at 24 h and the most overactive transcription factor in upstream regulator analysis. RUNX1 overexpression in FA-AKI was validated at mRNA and protein levels and localized mainly to tubular cell nuclei. CS-AKI also upregulated kidney RUNX1 in nuclei. In a transcriptomics array of murine tubular cells stimulated with TWEAK for 6 h, Runx1 was upregulated compared with non-stimulated cells, while Runx2 and Runx3 were not. RT-PCR confirmed an early upregulation of Runx1 (3 h) followed by a progressive return to baseline by 24 h. TWEAK also increased total and nuclear RUNX1 protein levels from 6 h onwards. Since kidney RUNX1 is also increased in CS-AKI, we tested whether LPS regulated RUNX1 in cultured tubular cells. LPS transiently increased Runx1 mRNA expression and it also increased whole cell and nuclear RUNX1 protein levels. Mechanistically, RUNX1 bound to the Il-6 gene promoter in response to TWEAK and LPS, and RUNX1 targeting with the chemical inhibitor Ro5-3335 or a specific siRNA prevented the TWEAK- and LPS-induced upregulation of IL-6. NFκB1 p50 pathway is involved in the RUNX1/IL-6 axis since the specific p50 inhibitor SN50 decreased the nuclear accumulation of RUNX1 and IL-6 expression in TWEAK- or LPS-stimulated MCT cells. In vivo, preventive Ro5-3335 improved kidney function and reduced inflammation in FA-AKI and CS-AKI. Moreover, in FA-AKI, Ro5-3335 also prevented the expression of profibrotic genes. However, Ro5-3335 administration after the insult only improved kidney function in CS-AKI. Kidney transcriptomics identified inflammatory genes and transcription factors such as Yap1 and p53 as key targets of Ro5-3335 in CS-AKI. Conclusion RUNX1 contributes to AKI by driving the expression of genes involved in inflammation and represents a novel therapeutic target in AKI.