P300-mediated H3K18 acetylation triggers necroptosis via modulation of KRT18 transcription in diabetic nephropathy

Abstract

Diabetic nephropathy (DN) is a major cause of end-stage renal disease. While glomerular damage is a known aspect of its pathology, tubular epithelial cell necroptosis also plays a crucial role in disease progression. Epigenetic modifications, particularly histone acetylation, have garnered attention for their role in the regulation of kidney damage-related gene expression. This study explores whether the histone acetyltransferase P300 regulates KRT18 expression via histone H3 lysine 18 acetylation (H3K18ac), driving tubular epithelial cell necroptosis and accelerating DN progression. We establish an STZ-induced diabetic nephropathy mouse model and a high glucose-treated HK-2 cell model. Western blot analysis, qPCR, immunohistochemistry, and AO/PI staining are employed to assess the expression levels of P300, H3K18ac, KRT18, and necroptosis-related proteins (RIPK1 and MLKL). Functional validation of the P300-KRT18 axis is performed using shRNA interference, overexpression, and the small molecule inhibitor C646. Both in vivo and in vitro models show significant upregulation of P300, H3K18ac, and KRT18, coupled with RIPK1/MLKL pathway activation and increased cell death. P300 knockdown or C646 treatment effectively inhibits H3K18ac and KRT18 expression, reducing necroptosis; KRT18 knockdown also alleviates P300 overexpression-induced cell death. Co-transfection with P300 overexpression and KRT18 interference demonstrates that KRT18 is a key downstream effector of P300-mediated necroptosis. In conclusion, P300 upregulates KRT18 expression through H3K18 acetylation, subsequently activating the RIPK1/MLKL pathway and promoting tubular epithelial cell necroptosis. The P300-KRT18 axis may serve as a novel epigenetic therapeutic target for DN, suggesting that epigenetic regulation could be a viable intervention strategy to delay DN progression.

1. Introduction

Diabetic nephropathy (DN) is one of the most common and severe chronic complications of diabetes and has become a leading cause of end-stage renal disease (ESRD) [1,2]. Beyond glomerular damage induced by hyperglycemia, increasing evidence indicates that tubulointerstitial injury plays a crucial role in the progression of DN, with programmed cell death of tubular epithelial cells being particularly significant in the pathological process [3,4].

Necroptosis, a form of programmed cell death distinct from apoptosis, is characterized by the involvement of the RIPK1/MLKL signaling pathway, leading to cell membrane rupture and the release of inflammatory mediators [5–7]. While necroptosis has been noted in several kidney diseases, such as ischemia-reperfusion injury, IgA nephropathy, and DN [8–10], its specific upstream regulatory mechanisms, especially in relation to epigenetic modifications, remain largely unexplored.

P300 is a well-known histone acetyltransferase (HAT) that regulates chromatin conformation and gene transcription through acetylation of histone H3 at lysine 18 (H3K18ac) and other sites [11]. Although the role of P300 in kidney diseases has recently garnered attention, direct evidence linking P300-mediated histone acetylation to tubular cell necroptosis is still lacking [12,13]. Keratin 18 (KRT18), a major epithelial-specific intermediate filament protein, is well established for its critical roles in maintaining cellular mechanical integrity and serving as a biomarker in various carcinomas [14,15]. However, in contrast to its extensively studied structural and oncogenic functions, the potential role of KRT18 in renal diseases, particularly in DN, remains largely unexplored. Although KRT18 is abundantly expressed in renal tubular epithelial cells [16], it is completely unknown whether and how it participates in tubular necroptosis. Moreover, the possibility that KRT18 could be a direct functional target under epigenetic regulation, thereby contributing to necroptosis, represents a significant knowledge gap.