Role of the post-translational ISGylation of interferon regulatory factor 9 in diabetes-induced podocyte injury

Abstract

Introduction: Diabetic kidney disease (DKD) is the most common cause of kidney failure, with podocyte injury being a key event in its progression. Here, by performing single-cell regulatory network inference and clustering analysis between DKD and normal kidney samples, we found a significant increase in interferon regulatory factor 9 (IRF9) transcriptional activity in the podocytes of DKD kidneys. However, the role of IRF9 in DKD remains poorly understood.

Methods: Kidney tissue from patients with DKD and high glucose-cultured conditionally immortalized human podocyte cell line (HPC) were employed to investigate IRF9 expression and localization. RNA sequencing was performed to elucidate the role of IRF9 in podocyte injury. Co-immunoprecipitation, in situ proximity ligation assay, and surface plasmon resonance were performed to confirm the binding between IRF9 and interferon-stimulated gene 15 (ISG15). ISGylation and ubiquitination assays were used to examine the potential effects of ISG15 on IRF9 expression. Podocyte-specific IRF9 deletion and overexpression mice and podocyte-specific ISG15-knockdown mice were established to assess kidney injury in vivo.

Results: IRF9 protein expression was increased in the podocytes of patients with DKD, without changes in IRF9 mRNA expression, and IRF9 expression was negatively correlated with eGFR and positively correlated with 24-hour urinary protein. In cultured human podocytes, high glucose conditions stimulated IRF9 protein expression without affecting IRF9 mRNA expression. Silencing IRF9 alleviated high glucose-induced podocyte injury, whereas IRF9 overexpression exacerbated podocyte injury. IRF9 induced podocyte injury via inflammasome activation and pyroptosis. Interestingly, high glucose exposure increased IRF9 protein expression by stimulating ISG15 expression, which promoted IRF9 ISGylation and subsequently inhibited IRF9 ubiquitination to increase IRF9 protein stability. In vivo, the podocyte-specific deletion of IRF9 or podocyte-specific knockdown of ISG15 attenuated podocyte and glomerular injury in streptozotocin-induced diabetic mice fed a high-fat diet; in contrast, the podocyte-specific overexpression of IRF9 exacerbated podocyte and glomerular injury in these diabetic mice.

Conclusions: Targeting ISG15-IRF9 could be a potential novel therapy for podocyte injury in DKD.

Keywords: IRF9; ISG15; ISGylation; diabetic kidney disease; podocytes; ubiquitination.