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Title: Extracellular signal-regulated kinase 5 in human podocytes : the effects of a diabetic milieu
Author: Badshah , Irbaz Isaac
ISNI:       0000 0004 5367 8827
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2015
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INTRODUCTION: Podocytes are specialised cells integral to the normal functioning kidney. As a consequence of diabetes podocyte dysfunction, phenotype impairment, and podocytopaenia occur. The ERK5 MAPK is involved in cell survival, proliferation, differentiation, and motility; processes central to the pathologies of diabetic nephropathy. AIMS: To establish an in vitro cellular model for studying the MEK5-ERK5 pathway in human podocytes and its regulation by diabetes-associated stimuli. METHODS: Conditionally immortalised human podocytes were treated with EGF (1.25-10 ng/ml), TGF B1 (1.25-10 ng/ml), high o-glucose (15, 30 mM), BSA and AGE-BSA (50,200 Ilg/ml). MEK5 was inhibited with BIX02188 (10 IlM), EGFR with AG1478 (10 IlM), ALK5 with SB431542 (10 IlM), Ras signalling with farnesylthiosalicylic acid (10 IlM), p38 with SB202190 (2 .5 IlM), proteasome action with MG132 (10 IlM). Proteins investigated by western blotting and immunopre,cipitation; gene expression by qPCR; phenotype and protein subcellular localisation by immun ~fl uo rescence; cell number by MTS proliferation assay; motility by scratch assay; barrier function by electric cell -substrate impedance sensing; apoptosis by flow cytometry. RESULTS: Podocytes expressed ERK5 which wa~ predominantly nuclear. EGF-induced ERK5 phosphorylation was AG1478- and BIX02188-sensitive. TGF - ~l- induced ERK5 phosphorylation was SB431542-, AG1478- and BIX02188-sensitive. EGF reduced synaptopodin immunofluorescence; TGF- ~l decreased nuclear P-cadherin and increased a-SMA; BIX02188 prevented these changes. EGF- and TGF-~l- induced a BIX02188-sensitive increase in cell number. Podocyte motility decreased with BIX02188 and TGF- ~1. BIX02188 reduced transcellular impedance which was alleviated with EGF, but prevented the TGF - ~l-induced reduction. Although high o-glucose increased p-ERK5, ERK5 protein was reduced. AGE-BSA decreased p-ERK5 and induced ERK5 SUMOylation. TGF- ~l in a SB202190-sensitive manner, BIX02188, high o-glucose, and BSA increased apoptosis. CONCLUSION: This work describes the novel expression and altered phosphorylat ion of ERK5 within human podocytes in a diabetic environment. A MEK5 inhibitor prevented certain growth factor-induced phenotypic alterations. Diabetic insults dysregulated ERK5 and along with a MEK5 inhibitor induced apoptosis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available