The objective of this K08 Award proposal is to enable the candidate to become an independent investigator in renal pathology and molecular biology in the field of chronic kidney disease (CKD). The candidate proposes a research plan under the multidisciplinary mentorship of Drs. Becker, Hullett (biochemistry), Oberley (pathology), Aiken (molecular biology) and Bakken (faculty development). The laboratory efforts will be supplemented with coursework in molecular biology, pathology and biochemistry designed to enhance the candidate's training as a physician-scientist. CKD is a worldwide public health problem with poor outcomes and high costs. Emerging evidence suggests that tubular epithelial-myofibroblast transdifferentiation (EMT) is an important event in renal tubulointerstitial fibrosis, a common final pathway to various injuries to the kidney. EMT involves the transformation of tubular epithelial cells (TEC) into cells with mesenchymal morphology, formation of actin stress fibers, loss of cell adhesions and increased cell migration. Heat shock protein 27 (HSP27) is a stress protein that exerts its cytoprotective effects by apoptosis inhibition, control of the redox status and modulation of actin filament dynamics. Despite its cytoprotective roles and interactions with actin filaments, HSP27 has not been studied in EMT.
The aims of this proposal are (1) to study HSP27 mRNA and protein levels and localization during EMT (2) to dissect the the signaling events that lead to HSP27 activation (3) to investigate whether overexpression of HSP27 delays/arrests EMT. We have successfully reproduced EMT in NRK52E cells (rat proximal TEC cell line) after six days of treatment with TGF-(1. Following the extraction of mRNA and protein from cultured cells, HSP27 mRNA and protein levels will be studied at various time-points by real-time PCR, immunoblot (IB) and immunohistochemistry (IHC) techniques, together with markers of EMT (m-smooth muscle actin and ecadherin). HSP27 cellular localization will be analyzed by fluorescence and immunogold electron microscopy (EM). HSP27-actin interactions and the mitogen-activated protein kinase (MAPK) pathway will be assessed by IB, IHC and and EM using phalloidin and total/phosphospecific antibodies, with inhibitors of the MAPK pathway. Overexpression of HSP27 will be obtained by recombinant virus-mediated HSP27 gene transfer. This proposal would allow us to determine whether HSP27 overexpression can prevent/delay EMT.
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