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Role of TGF-β in Ischemia and Reperfusion Injury Induced Renal Fibrosis in Human Proximal Tubular Epithelial Cells.

R. Kapoor, N. Sheerin, J. Kirby.

Institute of Cellular Medicine, Newcastle University, Newcastle Upon Tyne, United Kingdom.

Meeting: 2016 American Transplant Congress

Abstract number: C118

Keywords: Fibrosis, Reactive oxygen species, Renal injury, Renal ischemia

Session Information

Date: Monday, June 13, 2016

Session Name: Poster Session C: Ischemia Reperfusion Injury and Organ Preservation

Session Time: 6:00pm-7:00pm

 Presentation Time: 6:00pm-7:00pm

Location: Halls C&D

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Ischemia-reperfusion injury (IRI) leads to an irreversible damage to the tubular epithelium and promotes acute kidney injury (AKI) that is associated with progressive loss of kidney function that further seeds the development of chronic kidney disease. The role of TGF-β in stimulating extracellular matrix deposition and promoting renal fibrosis has been well established. Our study was designed to evaluate the role of TGF-β in IRI in HKC8 (human proximal tubular epithelial cells) and further determine if TGF-β is produced or released by these cells.

HKC8 cells were treated with ALK-5 inhibitor followed by treatment with H2O2 and CoCl2. Western blot and immunofluorescence was performed to study the expression of α-SMA and E-Cadherin .HKC8 cells were treated with H2O2 and CoCl2 and supernatants collected were transferred to control cultured HKC8 cells. Following this, changes in protein expression was determined. Similar study was performed using Smad-luciferase reporter assay using Smad-luciferase transfected HKC8 cells.

Mimicking ischemia using CoCl2 treatment induced a significant increase (p<0.01) in α-SMA and a decrease (p<0.001) in E-cadherin expression as compared to control and ALK-5 inhibitor treated cells. H2O2 treatmentsignificantly increased (p<0.01) α-SMA and decreased E-Cadherin expression as compared to control and ALK-5 inhibitor treated cells. Media transfer studies showed a similar trend of epithelial and fibrotic marker expression in HKC8 cells. CoCl2 and H2O2 increased luciferase activity in HKC8 cells transfected with smad3/luciferase reporter plasmid compared with control and ALK-5 inhibitor treated cells suggesting involvement of TGF-β in IRI induced kidney fibrosis. Similar study was conducted using supernatants obtained from CoCl2 and H2O2 stimulated cells.

Inducing Ischemia and reperfusion conditions lead to an increase in the fibrotic cell marker α-SMA and a decrease in epithelial cell marker E-cadherin suggesting a loss of epithelial cell phenotype in HKC8 cells. The findings provide new insights into TGF-β involvement following IRI induced AKI and may provide a basis for understanding the underlying molecular pathways leading to chronic kidney failure.

CITATION INFORMATION: Kapoor R, Sheerin N, Kirby J. Role of TGF-β in Ischemia and Reperfusion Injury Induced Renal Fibrosis in Human Proximal Tubular Epithelial Cells. Am J Transplant. 2016;16 (suppl 3).

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To cite this abstract in AMA style:

Kapoor R, Sheerin N, Kirby J. Role of TGF-β in Ischemia and Reperfusion Injury Induced Renal Fibrosis in Human Proximal Tubular Epithelial Cells. [abstract]. Am J Transplant. 2016; 16 (suppl 3). https://atcmeetingabstracts.com/abstract/role-of-tgf-in-ischemia-and-reperfusion-injury-induced-renal-fibrosis-in-human-proximal-tubular-epithelial-cells/. Accessed March 3, 2021.

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