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p66Shc regulates podocyte autophagy in high glucose environment through the Notch-PTEN-PI3K/Akt/mTOR pathway.

BACKGROUND AND AIMS
Autophagy has been found to be involved in podocyte injury, which is a key factor in the progression of diabetic kidney disease (DKD). p66Shc is an important protein adaptor that regulates production of reactive oxygen species (ROS) and induction of apoptosis, and is a novel biomarker for oxidative damage of renal tubules. Our preliminary studies showed that p66Shc expression in podocytes of DKD patients is increased, while autophagic flux and podocyte number is decreased in DKD patients. The mechanism by which p66Shc may regulate podocyte autophagy and injury remains unknown. The present study aimed to investigate the molecular function of p66Shc under high glucose condition and its possible therapeutic utility in DKD.


METHODS
We histologically evaluated kidney injury in a streptozocin (STZ)-induced mouse model of diabetes using HE, PAS, PASM, and Masson staining and assessed glomerular structure by transmission electron microscopy. The apoptosis rate of high glucose-treated podocytes was assessed by TUNEL and Annexin V/PI staining. Markers of podocyte autophagy were measured by immunofluorescence and western blotting. DHE/ET fluorescence quantification was used for ROS detection and quantification.


RESULTS
Urine creatinine, serum creatinine, urinary microalbumin, and p66Shc expression were significantly increased in STZ-induced diabetic mice. Cultured MPC5 podocytes subjected to high glucose showed reduced viability, and p66Shc overexpression further accelerated apoptosis. p66Shc knockdown enhanced HG-induced autophagy, while p66Shc overexpression reduced the expression of PTEN and increased the expression of mTOR and phospho-mTOR. LC3 protein expression was higher in cells with p66Shc knockdown, indicating that activation of p66Shc inhibits podocyte autophagy. DAPT, an inhibitor of the Notch pathway, downregulated the expression of p66Shc.


CONCLUSION
These findings indicate that p66Shc inhibits podocyte autophagy and induces apoptosis through the Notch -PTEN-PI3K/Akt/ mTOR signaling pathway in high glucose environment, providing novel evidence for its potential role in DKD treatment.