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Abstract: PO1231

Suppressed Autophagy Drives Increased Cellular Metabolic Activity in Human ADPKD Cells

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Atwood, Daniel, University of Colorado, Aurora, Colorado, United States
  • Pokhrel, Deepak, University of Colorado, Aurora, Colorado, United States
  • Edelstein, Charles L., University of Colorado, Aurora, Colorado, United States
Background

We published that the autophagy phenotype in Pkd1RC/RC mouse kidneys is characterized by decreases in crucial autophagy proteins (Cell Signal 2020). We attempt to determine the mechanistic role of suppressed autophagy as it relates to cell metabolic activity (viability and proliferation) in ADPKD cells.

Methods

Human primary immortalized cultured cells were used: normal renal cortical tubular epithelium (RCTE, PKD1+/+) and ADPKD cyst-lining epithelium (9-12, PKD1-/-). To measure autophagic flux, cells were treated with lysosomal inhibitor chloroquin (C) and LC3-II (immunoblot), a marker of autophagosomes or mCherry LC3 (fluorescence) was analyzed. MTT assay was used to measure cellular metabolic activity (cell viability and proliferation). Relative densitometry units (RDU) were measured on immunoblots.

Results

There was an increase in MTT and a decrease in AnnV in 9-12 vs RCTE cells. MTT OD was 0.36 in RCTE vs 0.44 in 9-12 (p<0.01). AnnexinV (AnnV) (% gated), marker of apoptosis, was 67 in RCTE vs 18 in 9-12 (p<0.0001). The increase in LC3-II with C in RCTE was not seen in 9-12 indicating suppressed autophagic flux. LC3-II (RDU) -/+ C was 0.6 vs 1.4 in RCTE (P<0.01) and 0.9 vs 1.1 (NS) in 9-12. mCherry (% gated) -/+ C was 6 vs 16 (p<0.05) in RCTE and 6 vs 13 (NS) in 9-12 cells. p62, marker of autophagic cargo, was increased in 9-12 vs RCTE. p62 (RDU) was 0.7 in RCTE and 1.2 in 9-12 (p<0.05). Cells were treated with an ATG7 shRNA (SH) to inhibit a crucial autophagy protein. There was a 50% decrease in LC3-II and ATG7 protein in SH-treated 9-12 cells. SH resulted in an increase in MTT and a decrease in AnnV indicating that suppressed autophagy drives MTT and inhibits apoptosis. MTT OD was 0.7 with scrambled shRNA (SCR) and 0.9 with SH (p<0.05). AnnV was 26 with SCR vs 16 with SH (p<0.05). Tat-Beclin peptide (TAT), a specific autophagy inducer, resulted in a decrease in MTT in 9-12 suggesting that autophagy decreases MTT. MTT OD was 0.5 with Veh vs. 0.2 with TAT (p<0.001). TAT did not affect AnnV.

Conclusion

In ADPKD cyst lining epithelial cells there is increased MTT, suppressed autophagic flux, and decreased apoptosis. Autophagy inhibition increased MTT and autophagy induction decreased MTT. Suppressed autophagy drives increased cellular metabolic activity in ADPKD cells. The effect of autophagy inhibition/induction on cyst growth in vivo merits further study.

Funding

  • Veterans Affairs Support