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Abstract: TH-PO884

PI3 Kinase/Akt Node Acts as a Driver for High Glucose-Induced miR-214 Expression to Induce a Positive Feedback Loop via PTEN for Matrix Protein Expression in Proximal Tubular Epithelial Cells (PTECs)

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Maity, Soumya, UTHSCSA, San Antonio, Texas, United States
  • Das, Falguni, UTHSCSA, San Antonio, Texas, United States
  • Ghosh-choudhury, Nandini, UTHSCSA, San Antonio, Texas, United States
  • Kasinath, Balakuntalam S., UTHSCSA, San Antonio, Texas, United States
  • Ghosh-Choudhury, Goutam, UTHSCSA, San Antonio, Texas, United States
Background

We have recently shown that high glucose (HG) increased the expression of miR-214 to regulate renal fibrosis by directly targeting the 3’ UTR of PTEN tumor suppressor protein (Am J Physio Cell Physiol 313: C430, 2017). The mechanism by which HG regulates miR-214 expression is not known. We hypothesized involvement of PI 3 kinase (PI 3 K)/Akt cascade in this process.

Methods

Human PTECs, activation-specific phospho-antibodies, shRNA, dominant negative kinases, real time qRT-PCR and immunoblotting were employed.

Results

In human PTECs, HG increased the expression of miR-214. To address the role of PI 3 K, we used its inhibitor Ly294002 (Ly). Ly significantly inhibited the HG-stimulated expression of miR-214. Similarly, expression of dominant negative PI 3 K markedly blocked the miR-214 expression in response to HG. Inhibition of Akt, the downstream kinase of PI 3 K, by MK2206 significantly suppressed the HG-induced miR-214 expression. In diabetic renal pathology a significant role of mechanistic target of rapamycin complex 1 (mTORC1) has been established. Rapamycin significantly attenuated the miR-214 expression by HG. Furthermore, shRNA against raptor, required for mTORC1 activation, blocked the HG stimulated miR-214 expression, similar to rapamycin. Since miR-214 downregulates PTEN to drive tubular matrix protein expression, we examined the role of PI 3 K/Akt in PTEN protein expression. Downregulation of PTEN by HG was reversed by dominant negative PI 3 K or Akt, which led to inhibition of mTORC1 activity as judged by phosphorylation of its substrate S6 kinase at Thr-389. Consequently, the HG-induced fibronectin expression was blocked by the dominant negative PI 3 K and Akt. Interestingly, plasmid-derived expression of miR-214 attenuated the increment of PTEN by the dominant negative PI 3 K or Akt in the presence of HG, resulting in increase in mTORC1 activity and expression of fibronectin.

Conclusion

These results are the first demonstration for a key role of PI 3 K/Akt signaling in HG-induced miR-214 expression. Furthermore, our data give evidence for a positive feedback loop involving the PI 3 K/Akt/mTORC1/miR-214 and PTEN for driving tubular fibrosis.

Funding

  • Veterans Affairs Support