Abstract: TH-PO897
Proteasomal Activation and the p38 and ERK Pathways Promote Loss of NF-E2 Expression and Induction of Pro-Fibrotic Signaling in TGF-b Treated Tubule Cells
Session Information
- Diabetic Kidney Disease: Basic - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Diabetic Kidney Disease
- 601 Diabetic Kidney Disease: Basic
Author
- Rane, Madhavi J., University of Louisville, Louisville, Kentucky, United States
Background
TGF-β is a critical mediator of diabetes-induced renal fibrosis. Preliminary studies found TGF-β (10 ng/ml; 24 h) treatment of human renal proximal tubule (HK-11) cells decreased Nuclear Factor-Erythroid derived 2 (NF-E2) protein expression and increased pro-fibrotic signaling in the kidney. Current studies identified signaling mechanisms by which TGF-β/diabetes regulates NF-E2 expression and renal fibrosis which may lead to generation of new therapies.
Methods
HK-11 cells were treated with 10 ng/ml of TGF-b for 24 h or pre-treated with p38 and MEK/ERK MAPK inhibitors namely, (0.3 µM) SB203580 and (25 µM) PD98059, or proteasome inhibitor MG132 (0.125 µM, 0.25 µM, 0.5 µM and 1 µM), for an hour prior to treatment with TGF-b. Cell lysates were immunoblotted with appropriate antibodies. HK-11 cells were transfected with pUSe vector/pUse-NF-E2 or with control siRNA or NF-E2 siRNA for 24 h followed by treatment with vehicle/TGF-b for additional 24 h. Cell lysates were immunoblotted with appropriate antibodies. Kidney homogenates from STZ-type 1 diabetic mice, OVE26 type 1 diabetic mice treated with vehicle/MG132, and db/db type 2 diabetic mice, were immunoblotted with appropriate antibodies.
Results
NF-E2 expression was decreased in kidney homogenates from STZ, OVE26, and db/db mice. Moreover, TGF-β (10 ng/ml; 24 h) treatment of human renal proximal tubule (HK-11) cells decreased NF-E2 expression and increased p38 and ERK activation and expression of pro-fibrotic proteins including, CTGF, FN and PAI-1. Over-expression of NF-E2 inhibited CTGF, FN, and PAI-1 expression in TGF-b treated HK-11 cells. Silencing NF-E2 expression induced CTGF expression. TGF-β treatment did not have any effect on NF-E2 transcription or on miRNA-361-5p in HK-11 cells. However, blockade of proteasome, in HK-11 cells and OVE26 mice preserved NF-E2 expression and inhibited pro-fibrotic signaling. Moreover, dual blockade of p38 and MEK/ERK prevented NF-E2 degradation and pro-fibrotic signaling.
Conclusion
Blocakade of p38 and MEK/ERK pathways and proteasomal activation during diabetes and TGF-b treatment of HK-11 cells prevents NF-E2 degradation and attenuates pro-fibrotic signaling in kidney cells.