EP300 drives renal fibrosis in diabetic nephropathy via histone acetyltransferase-mediated nephrocystin-4 expression

Abstract

BACKGROUND

Diabetic nephropathy (DN) is a major microvascular complication of diabetes, and progressive renal fibrosis is the central feature that drives long-term kidney function decline. Epigenetic regulation is increasingly recognized in metabolic diseases, but the contribution of the histone acetyltransferase EP300 to diabetic renal fibrosis remains unclear. Nephrocystin-4 (NPHP4), a cilia-related gene, has been implicated in tubular injury and fibrotic changes, yet its regulation and role in DN have not been defined.

AIM

To determine whether EP300 promotes renal fibrosis in DN by activating NPHP4 through H3K27 acetylation and to evaluate the functional consequences of targeting this pathway in cell and animal models.

METHODS

Transcriptomic analysis of human diabetic kidney samples was performed using RNA sequencing, with EP300 expression patterns validated by real-time quantitative polymerase chain reaction (RT-qPCR). Chromatin immunoprecipitation-RT-qPCR was used to assess EP300 binding and histone H3 lysine 27 acetylation (H3K27ac) levels at the NPHP4 promoter. In vitro studies were conducted in HK-2 cells cultured under high-glucose conditions (30 mmol/L) with EP300 or NPHP4 knockdown, followed by functional assays including cell viability measured by the Cell Counting Kit-8 assay, apoptosis assessed by TUNEL staining, and fibrotic marker expression analyzed by western blotting for alpha-smooth muscle actin and fibronectin. In vivo experiments involved diabetic mice treated with the EP300 inhibitor C646, AAV-mediated NPHP4 silencing, or combination therapy. Renal function was subsequently evaluated by measuring serum creatinine, blood urea nitrogen, and 24-hour proteinuria, along with histological assessment of fibrosis using Masson staining and immunostaining.

RESULTS

Our results revealed that EP300 was significantly upregulated in the kidneys of DN patients, and it transcriptionally activated NPHP4 by enriching H3K27ac at its promoter, as demonstrated by chromatin immunoprecipitation-RT-qPCR. In high glucose-stimulated HK-2 cells, knockdown of either EP300 or NPHP4 effectively attenuated fibrosis marker expression (alpha-smooth muscle actin and fibronectin) and restored the balance between apoptosis and proliferation. In a DN mouse model, pharmacological inhibition of EP300 (C646) or AAV-mediated knockdown of NPHP4 substantially improved renal function (reduced serum creatinine, blood urea nitrogen, and proteinuria) and attenuated renal fibrosis, with combination therapy showing the most pronounced benefits. Immunohistochemical and immunofluorescence analyses further validated that the EP300–NPHP4 axis critically regulates renal fibrotic progression.

CONCLUSION

In conclusion, our study identifies the EP300/H3K27ac/NPHP4 axis as a novel epigenetic pathway driving renal fibrosis in DN. These findings provide mechanistic insight into the diabetic renal fibrotic process and highlight the therapeutic potential of targeting EP300 or NPHP4 in the treatment of DN.

Keywords: EP300/nephrocystin-4; Diabetic nephropathy; Fibrosis; Histone H3 lysine 27 modification; Targeted intervention

Core Tip: Renal fibrosis is a key pathological process driving diabetic nephropathy progression, yet its epigenetic regulation remains unclear. This study identifies EP300 as a histone acetyltransferase that activates nephrocystin-4 transcription through Histone H3 lysine 27 acetylation, promoting renal fibrosis. Inhibition of the EP300-nephrocystin-4 axis alleviates fibrotic injury and improves renal function in diabetic mice, providing new mechanistic insight and a potential therapeutic target for diabetic nephropathy.