Abstract: FR-PO615

TGFbeta (TGFb)-Stimulated PI 3 Kinase (PI 3 K)/Akt Downregulates DEPTOR to Increase Podocyte Hypertrophy and Matrix Protein Expression

Session Information

Category: Diabetes

  • 501 Diabetes Mellitus and Obesity: Basic - Experimental

Authors

  • Das, Falguni, UTHSCSA, SAN ANTONIO, Texas, United States
  • Ghosh-choudhury, Nandini, UTHSCSA, SAN ANTONIO, Texas, United States
  • Kasinath, Balakuntalam S., University of Texas Health Science Center, San Antonio, Texas, United States
  • Ghosh-Choudhury, Goutam, University of Texas Health Science Center, San Antonio, Texas, United States
Background

TGFb contributes to kidney injury in diabetic nephropathy (DN). mTOR controls renal cell hypertrophy and matrix protein expression. We have recently shown that TGFb regulates the expression of deptor, a negative regulatory component of both mTORC1 and mTORC2. We hypothesized that PI 3 K/Akt signaling may contribute to deptor regulation.

Methods

Recombinant TGFb (2 ng/ml), rat podocytes, pharmacological inhibitors, dominant negative (dn) expression vectors and siRNAs transfection, immunoblotting, immunoprecipitation, protein synthesis and hypertrophy assays, glomeruli from rats with streptozotocin (STZ)-induced DN were employed.

Results

TGFb decreased the expression of deptor in a time-dependent manner, leading to increase in the mTORC1 and mTORC2 activity, as judged by the increase in phosphorylation of their substrates S6 kinase/4EBP-1 (mTORC1) and Akt (Ser-473)/NDRG1 (mTORC2). To explore the mechanism of deptor suppression, we used the PI 3 kinase and Akt inhibitors, Ly 294002 and MK 2206, and PTEN or dominant negative (dn) PI 3 K or dn Akt kinase, which reversed the TGFb-induced deptor downregulation, resulting in inhibition of mTORC1 and mTORC2 activation. TGFb inhibited the deptor-mTOR complex formation in co-immunoprecipitation experiments. Expression of PTEN or dn Akt kinase restored the complex formation in the presence of TGFb. Furthermore, dn PI 3 K or dn Akt kinase significantly inhibited the TGFb-induced hypertrophy. Interestingly, siRNAs against deptor alone induced hypertrophy of podocytes similar to TGFb; additionally they reversed the inhibition of hypertrophy induced by dn PI 3 K or Akt kinase. Moreover, expression of dn PI 3 K or dn Akt kinase significantly inhibited the TGFb-stimulated expression of fibronectin, which was reversed by siDeptor. Finally, we found significantly reduced expression of deptor, concomitant with increased activation of mTORC1 and mTORC2 in the glomeruli of STZ-induced diabetic rats.

Conclusion

Our data for the first time demonstrate the involvement of PI 3 K/Akt signaling in the suppression of deptor by TGFb to maintain high mTOR activity necessary for renal cell hypertrophy and matrix protein expression.

Funding

  • NIDDK Support