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

Akita mice showed increased AGT expression, oxidative stress, tubular cell size, and luminal dilation in proximal tubules, all of which were reduced in Akita AgtRT KO mice.

• Type 1 diabetic Akita mice and Akita mice with angiotensinogen (Agt) deletion in renal tubules (Akita AgtRT KO) were studied at 42 weeks
• Renal tubule-specific knockout of angiotensinogen was used to inactivate the intrarenal renin-angiotensin system
• Proximal tubule morphological changes including increased tubular cell size and luminal dilation were observed in Akita mice and normalized in Akita AgtRT KO mice

Elevated senescence markers in distal tubules of Akita mice were normalized by tubular AGT deletion.

• Senescence-associated β-galactosidase (SA-β-gal) activity was elevated in distal tubules of Akita mice
• Senescence-associated secretory phenotype (SASP) and increased senescence marker p16 expression were observed in distal tubules of Akita mice
• All senescence markers were normalized in Akita AgtRT KO mice

Human CKD/DKD datasets confirmed a positive correlation between AGT and CDKN2A/p16 expression.

• Analysis of human chronic kidney disease and diabetic kidney disease datasets was performed
• AGT expression positively correlated with CDKN2A/p16 expression in these datasets
• This finding supports the translational relevance of the mouse model findings

Akita mice exhibited elevated NOX4 expression and mitochondrial cristae disruption in proximal tubules.

• NADPH oxidase 4 (NOX4) expression was elevated in proximal tubules of Akita mice compared to Akita AgtRT KO mice
• Mitochondrial cristae disruption was observed in proximal tubules of Akita mice
• Akita mice also showed significant oxidative DNA damage, renal inflammation, fibrosis and apoptosis compared to Akita AgtRT KO mice

High glucose and angiotensin II triggered NOX4-mediated mitochondrial oxidative stress and dysfunction in proximal tubular HK-2 cells in vitro.

• In vitro experiments used human proximal tubular cells (HK-2 cells)
• Both high glucose and angiotensin II (Ang II) were required to trigger NOX4-mediated mitochondrial oxidative stress
• Mitochondrial dysfunction was observed downstream of NOX4 activation in this in vitro model

Angiotensin II induced p16-dependent senescence in distal tubular MDCK cells in vitro.

• Madin-Darby canine kidney (MDCK) cells were used as a distal tubular cell model
• Ang II induced senescence in MDCK cells in a p16-dependent manner
• This finding links intrarenal RAS activation directly to distal tubule senescence

Conditioned medium from senescent MDCK cells triggered epithelial-to-mesenchymal transition in HK-2 cells, which was reversed by losartan or N-acetylcysteine.

• Conditioned medium from senescent MDCK cells was applied to HK-2 proximal tubular cells
• Epithelial-to-mesenchymal transition (EMT) was induced in HK-2 cells by the conditioned medium from senescent cells
• Both losartan (an angiotensin II receptor blocker) and N-acetylcysteine (an antioxidant) reversed the EMT induction
• This suggests SASP from senescent distal tubular cells promotes profibrotic changes in proximal tubular cells