Targeting N6-Methyladenine of Tubular Mitochondrial DNA Against Hypertensive CKD

Background: Hypertension is a significant global health issue that contributes to chronic kidney disease (CKD). Mitochondrial dysfunction in renal tubular epithelial cells (TECs) plays a crucial role in the progression of CKD. However, the involvement of mitochondrial DNA (mtDNA) methylation in hypertensive CKD and its potential as a therapeutic target remains largely unexamined.

Methods: A hypertensive CKD mouse model was induced by continuous infusion of AngII (angiotensin II). Renal mtDNA methylation types and levels were evaluated using nanopore Sequencing and dot blot assays. The impact of N⁶-methyladenine (6mA) modification, catalyzed by METTL4 (methyltransferase-like protein 4), on hypertensive CKD was assessed using TEC-specific Mettl4 knockout mice.

Results: Utilizing high-confidence Nanopore Sequencing, we identified 6mA as the predominant methylation type in renal mtDNA, rather than 5-methylcytosine. In mice with hypertensive CKD, renal mtDNA 6mA levels significantly increased, while 5-methylcytosine levels remained stable. METTL4, the only known mammalian methyltransferase for 6mA, was upregulated in the renal tubules of hypertensive CKD mice and patients with hypertension. AngII stimulation increased METTL4 expression and mtDNA 6mA levels in primary TECs. Activated c-Jun/AP-1 (activator protein-1) directly promoted Mettl4 transcription in AngII-treated primary TECs. METTL4-catalyzed mtDNA 6mA impeded mitochondrial transcription initiation complex assembly, thereby halting mtDNA transcription, disrupting mitochondrial function, and resulting in the transition of TECs into a proinflammatory and profibrotic phenotype. TEC-specific Mettl4 gene deletion in mice exhibited reduced mtDNA 6mA, preserved mtDNA transcription, improved tubular mitochondrial function, and alleviation of hypertensive CKD.

Conclusions: This study reveals that METTL4-catalyzed mtDNA 6mA contributes to renal mitochondrial dysfunction and hypertensive CKD, offering novel therapeutic strategies from the perspective of mitochondrial Epigenetics.