Abstract: FR-PO170

Downregulation of Akt Induces Mitochondrial Injury in Proteinuric States

Session Information

  • Mitochondriacs and More
    November 03, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Cell Biology

  • 202 Apoptosis, Proliferation, Autophagy, Cell Senescence, Cell Transformation

Authors

  • Erkan, Elif, Children's Hospital of Cincinnati, CIncinnati, Ohio, United States
  • Lu, Lu, Cincinnati Children's Hospital, Cincinnati, Ohio, United States
Background

Nephrotic range proteinuria contributes to progression of glomerular diseases by causing tubulointerstitial injury. High concentrations of albumin in the glomerular filtrate triggers proximal tubular cell apoptosis, a precursor for tubular atrophy. We propose that downregulation of prosurvival serine/thereonine kinase, protein kinase B (Akt) induces apoptosis in proximal tubule epithelial cells by causing mitochondrial injury.

Methods

In-vitro albumin overload is induced by incubating human kidney proximal tubule epithelial (HKC-8) cells with 10mg/ml of human albumin for 24 hours.
C57BL/6 B6 mouse with targeted deletion of Akt1/2 in proximal tubule epithelial cells(Akt1/2 lox/lox Sglt2 cre) is generated and subjected to intraperitoneal albumin injections (10 mg/g body weight) for 6 weeks. Intraperitoneal albumin and saline injected Sglt2 cre negative mice are utilized as negative controls.
Apoptosis is evaluated by fluorogenic caspase-3 assay, caspase 9 staining and TUNEL assays. Mitochondrial and cytosolic translocation of cytochrome-c and Bax is assessed by Western Blotting.

Results

Albumin overload caused caspase-3 activation in association with downregulation in expression of pSer473 Akt and pThr 308 Akt and Akt substrate phospho Fork head box O1(FOXO 1) and fork head box O3 (FOXO 3) proteins in HKC-8 cells. Expression of FOXO target BIM is upregulated in association with albumin induced apoptosis. Furthermore overexpression of Akt by constitutively active Akt construct mitigates albumin induced-apoptosis where as treatment with pan Akt inhibitor MK-2206 potentiates the apoptotic response in HKSC-8 cells.
In-vivo albumin overload results in cytochrome-c release in the cytoplasm of Akt1/2 lox/lox Sglt2 cre mice in association with tubular apoptosis. This response was associated with translocation of proapoptotic Bax to the mitochondria and increase in BIM expression.

Conclusion

We concluded that Akt plays an important role in protection of proximal tubule epithelial cells from apoptosis. We propose that in proteinuric states, inhibition of Akt phosphorylation causes nuclear translocation of FOXO 1 and FOXO 3 and an increase in transcription of BIM leading to cytochrome-c release to cytoplasm and apoptosis in proximal tubule epithelial cells.
We postulate that therapeutic interventions that are targeted to increase tubular Akt expression can attenuate tubulointerstitial injury in proteinuric states.