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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08DK067981-04
Application #
7231699
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Rankin, Tracy L
Project Start
2004-09-10
Project End
2009-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
4
Fiscal Year
2007
Total Cost
$116,827
Indirect Cost
Name
University of Wisconsin Madison
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Djamali, Arjang; Muth, Brenda L; Ellis, Thomas M et al. (2013) Increased C4d in post-reperfusion biopsies and increased donor specific antibodies at one-week post transplant are risk factors for acute rejection in mild to moderately sensitized kidney transplant recipients. Kidney Int 83:1185-92
Vidyasagar, Aparna; Reese, Shannon R; Hafez, Omeed et al. (2013) Tubular expression of heat-shock protein 27 inhibits fibrogenesis in obstructive nephropathy. Kidney Int 83:84-92
Djamali, A; Reese, S; Hafez, O et al. (2012) Nox2 is a mediator of chronic CsA nephrotoxicity. Am J Transplant 12:1997-2007
Dahlberg, Rebecca; Muth, Brenda; Samaniego, Milagros et al. (2010) One-year serum albumin is an independent predictor of outcomes in kidney transplant recipients. J Ren Nutr 20:392-7
Conley, James; Olafsson, Andrew; Djamali, Arjang (2010) Do statins delay the incidence of ESRD in diabetic patients with moderate CKD? J Nephrol 23:321-7
Djamali, Arjang; Vidyasagar, Aparna; Yagci, Gokhan et al. (2010) Mycophenolic acid may delay allograft fibrosis by inhibiting transforming growth factor-beta1-induced activation of Nox-2 through the nuclear factor-kappaB pathway. Transplantation 90:387-93
Djamali, A; Vidyasagar, A; Adulla, M et al. (2009) Nox-2 is a modulator of fibrogenesis in kidney allografts. Am J Transplant 9:74-82
Djamali, Arjang; Samaniego, Millie (2009) Fibrogenesis in kidney transplantation: potential targets for prevention and therapy. Transplantation 88:1149-56
Kukla, Aleksandra; Adulla, Madhurima; Pascual, Julio et al. (2008) CKD stage-to-stage progression in native and transplant kidney disease. Nephrol Dial Transplant 23:693-700
Vidyasagar, Aparna; Reese, Shannon; Acun, Zeki et al. (2008) HSP27 is involved in the pathogenesis of kidney tubulointerstitial fibrosis. Am J Physiol Renal Physiol 295:F707-16

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