Autophagy Knockout Results in ERK Activation, Increased Proliferation, and Apoptosis of Cyst-Lining Epithelial Cells and Worse Polycystic Kidney Disease

Background: Studies remain contradictory and the mechanistic role of autophagy in cyst growth. Autophagy has not been detailed in rodent and cell models translational to the human disease. Methods: Mice were treated with chloroquine at a dose of 60mg/kg from 50-120 days. An array of mTOR and autophagy proteins was measured in the kidney by immunoblot analysis. Apoptosis and proliferation were measured by TUNEL and PCNA IHC respectively. Cells were treated with 25 or 50uM tat-beclin1 peptide for 4 or 24hrs Results: Suppressed autophagic flux (lack of increase in LC3-II in kidney 2 hrs after IP bafilomycin injection) was observed in 150 d old, but not 70 or 120 d old Pkd1RC/RC kidneys. Pharmacological inhibition of autophagy with chloroquine from an early age suppressed autophagic flux in Pkd1RC/RC kidneys, but had no effect on PKD severity. Knockout of autophagy (ATG7) in kidney tubule specific Pkd1 knockout mice, a rapid model of cyst growth, had no effect on cyst growth or kidney function. Knockout of ATG7 in Pkd1RC/RC mice, a slower and more clinically-relevant model of PKD, resulted in activation of p-ERK, a surge of apoptosis and proliferation of cyst lining epithelial cells and severely worse PKD (See Table). In human PKD 9-12 cells, autophagy induction with Tat-beclin 1 peptide resulted in increased conversion of LC3-I to LC3-II, decreased p-ERK and decreased proliferation and apoptosis (See table). Conclusion: In vivo, autophagy knockout activates p-ERK resulting in a surge of proliferation and apoptosis in cyst lining epithelial cells and worse PKD. In vitro, autophagy induction resulted in decreased p-ERK decreased proliferation and apoptosis. In conclusion, there is an abnormal autophagy, ERK, proliferation/apoptosis axis in cells lining the cyst in ADPKD. Cre-lox technology was used to create ATG7, RC/RC double knockout mice Funding: Veterans Affairs Support - In Vivo Pkd1RC/RC Pkd1RC/RC ATG7-/- In Vitro VEH Tat-Beclin1 25uM 2K/BW (%) 2.1 3.6# PCNA/β-actin 1.2 1.0* Cyst Index (%) 9 26*** MTT OD(590) 0.4 0.2# Cyst # 160 491*** AnnexinV (% gated) 23 3.5# PCNA+ per cysts 5 22*** LC3-I/LC3-II (RDU) 0.4 1.0*** TUNEL+ per cyst 1.1 30.1** p/t-ERK (RDU) 2.3 1.1** LC3-I/LC3-II (RDU) 0.9 4** p/t-ERK (RDU) 0.9 1.4* *P<0.01,**P<0.01, ***P<0.001, #P>0.0001. RDU=relative densitometry units on immunoblot