Intrarenal renin–angiotensin system inactivation ameliorates tubulopathy via attenuation of mitochondrial oxidative stress, dysfunction and senescence in diabetes

Intrarenal renin-angiotensin system (iRAS) activation has been implicated in tubulopathy in diabetic kidney disease (DKD), its underlying mechanisms remain unclear. Type 1 diabetic Akita mice and Akita mice with angiotensinogen (Agt) deletion in renal tubules (Akita AgtRT KO) and their respective controls were studied at 42 weeks. Akita mice exhibit increased AGT expression, oxidative stress, tubular cell size and luminal dilation in proximal tubules (PTs), while reduced in Akita AgtRT KO mice. Elevated senescence-associated β-galactosidase (SA-β-gal) activity and senescence-associated secretory phenotype (SASP) along with increased senescence marker p16 expression in distal tubules (DTs) were also observed in Akita mice, all normalized in Akita AgtRT KO mice. Human CKD/DKD datasets confirmed AGT positively correlated with CDKN2A/p16 expression. Akita mice also showed elevated NADPH oxidase 4 (NOX4) expression and mitochondrial cristae disruption in PTs, accompanied by significant oxidative DNA damage, renal inflammation, fibrosis and apoptosis cf. Akita AgtRT KO mice. In vitro, high glucose and angiotensin II (Ang II) triggered NOX4-mediated mitochondrial oxidative stress and dysfunction in proximal tubular (HK-2) cells. In addition, Ang II induced p16-dependent senescence in distal tubular (Madin-Darby canine kidney, MDCK) cells. Conditioned medium from senescent MDCK cells triggered epithelial-to-mesenchymal transition in HK-2 cells, which was reversed by losartan or N-acetylcysteine. These findings suggest that iRAS promotes tubulopathy in DKD through NOX4-induced mitochondrial oxidative stress and dysfunction in PTs and oxidative DNA damage-induced senescence in DTs, providing new therapeutic targets.