The chemical chaperone, PBA, reduces ER stress and autophagy and increases collagen IV α5 expression in cultured fibroblasts from men with X-linked Alport syndrome and missense mutations

Wang, D., Mohammad, M., Wang, Y., Tan, R., Murray, L. S., Ricardo, S., Dagher, H., van Agtmael, T. and Savige, J. (2017) The chemical chaperone, PBA, reduces ER stress and autophagy and increases collagen IV α5 expression in cultured fibroblasts from men with X-linked Alport syndrome and missense mutations. Kidney International Reports, 2(4), pp. 739-748. (doi: 10.1016/j.ekir.2017.03.004)

Abstract

Introduction: X-linked Alport syndrome (OMIM 301050) is caused by COL4A5 missense variants in 40% of families. This study examined the effects of chemical chaperone treatment (sodium 4-phenylbutyrate) on fibroblast cell lines derived from men with missense mutations. Methods: Dermal fibroblast cultures were established from 2 affected men and 3 normals. Proliferation rates were examined, the collagen IV α5 chain localized with immunostaining, and levels of the intra- and extracellular chains quantitated with an in-house enzyme-linked immunosorbent assay. COL4A5 mRNA was measured using quantitative reverse transcriptase polymerase chain reaction. Endoplasmic reticulum (ER) size was measured on electron micrographs and after HSP47 immunostaining. Markers of ER stress (ATF6, HSPA5, DDIT3), autophagy (ATG5, BECN1, ATG7), and apoptosis (CASP3, BAD, BCL2) were also quantitated by quantitative reverse transcriptase polymerase chain reaction. Measurements were repeated after 48 hours of incubation with 10 mM sodium 4-phenylbutyrate acid. Results: Both COL4A5 missense variants were associated with reduced proliferation rates on day 6 (P = 0.01 and P = 0.03), ER enlargement, and increased mRNA for ER stress and autophagy (all P values < 0.05) when compared with normal. Sodium 4-phenylbutyrate treatment increased COL4A5 transcript levels (P < 0.01), and reduced ER size (P < 0.01 by EM and P < 0.001 by immunostaining), ER stress (p HSPA5 and DDIT3, all P values < 0.01) and autophagy (ATG7, P < 0.01). Extracellular collagen IV α5 chain was increased in the M1 line only (P = 0.06). Discussion: Sodium 4-phenylbutyrate increases collagen IV α5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV α5 extracellular transport. Whether these actions delay end-stage renal failure in men with X-linked Alport syndrome and missense mutations will only be determined with clinical trials.

• Item Type: Articles
• Additional Information: This work was supported by grants from the Macquarie/ Pederson Family Trust/Kidney Foundation of Canada/ Alport Syndrome Foundation, from the Alport Syndrome Foundation of Australia to JAS, and from the Research Councils UK Fellowship (EP/E50036611) to TVA.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Van Agtmael, Professor Tom
• Authors: Wang, D., Mohammad, M., Wang, Y., Tan, R., Murray, L. S., Ricardo, S., Dagher, H., van Agtmael, T., and Savige, J.
• College/School: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
• Journal Name: Kidney International Reports
• Publisher: Elsevier
• ISSN: 2468-0249
• Published Online: 27 March 2017
• Copyright Holders: Copyright © 2017 International Society of Nephrology
• First Published: First published in Kidney International Reports 2(4): 739-748
• Publisher Policy: Reproduced under a Creative Commons license