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Signaling pathways in cell models of Fabry disease nephropathy

Labilloy, Anatália (2015) Signaling pathways in cell models of Fabry disease nephropathy. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Chronic Kidney Disease is a leading cause of morbidity, impaired quality of life and premature death in patients with Fabry disease, being of major public health significance. At the cellular level, besides within lysosomes, glycosphingolipids that accumulate in Fabry disease due to alpha-galactosidase A (α-gal A) deficiency localize to membrane microdomains, which play crucial roles in protein clustering, membrane trafficking, and especially cell signaling. The mechanisms by which increased levels of these glycosphingolipids and consequent changes in microdomain dynamics and lysosomal dysfunction all result in cellular and organ injury are not well understood. To effectively study Fabry disease disease mechanisms at the cellular level, I first established and characterized an epithelial kidney cell model of Fabry disease in Madin-Darby canine kidney (MDCK) cells using small interfering RNA (siRNA). I then examined protein dynamics at the plasma membrane of a model raft-associated protein, GFP-GPI, in this model system. Number and Brightness Analysis in live cells showed a significant increase in the oligomeric size of antibody-induced clusters in α-gal A silenced cells compared to control cells (5.08 ± 0.45 vs 2.74 ± 0.24, respectively). To explore possible consequences of these findings in signaling pathways that are relevant to human disease, I first generated human kidney cell models of Fabry disease in immortalized podocytes and tubule epithelial cells (HK-2) applying the genome editing technique of clustered, regularly interspaced, short palindromic repeats and associated endonuclease 9 from S. pyogenes (CRISPR/Cas9). I compared abundance and phosphorylation of relevant signaling proteins through a high-throughput phosphorylation profiling for Fabry disease and control immortalized human podocytes. Fabry disease podocytes showed significant changes in total protein abundance and/or phosphorylation in 59 proteins. Pathway analysis predicted differential signaling of several canonical pathways in Fabry disease podocytes. These studies provided for the first time an understanding of raft protein dynamics and signaling in kidney cells deficient for α-gal A, potentially opening new avenues for biomarker discovery and drug development for Fabry disease nephropathy.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Labilloy, Anatáliaand101@pitt.eduAND101
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairWeisz, Ora Aweisz@pitt.eduWEISZ
Committee MemberFinegold, David Ndnf@pitt.eduDNF
Committee MemberKiselyov, Kirillkiselyov@pitt.eduKISELYOV
Committee MemberUrban, Zsolturbanz@pitt.eduURBANZ
Committee MemberFerrell, Robert Erferrell@pitt.eduRFERRELL
Date: 30 September 2015
Date Type: Publication
Defense Date: 15 May 2015
Approval Date: 30 September 2015
Submission Date: 20 May 2015
Access Restriction: 5 year -- Restrict access to University of Pittsburgh for a period of 5 years.
Number of Pages: 158
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Human Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Fabry disease, glycosphingolipids, lipid rafts, alpha-galactosidase A, signaling, nephropathy
Date Deposited: 30 Sep 2015 13:30
Last Modified: 15 Nov 2016 14:28
URI: http://d-scholarship.pitt.edu/id/eprint/25253

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