Dataset Information

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV ?5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations.

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 ?5 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 ?5 chain was increased in the M1 line only (P = 0.06). Discussion:Sodium 4-phenylbutyrate increases collagen IV ?5 mRNA levels, reduces ER stress and autophagy, and possibly facilitates collagen IV ?5 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.

SUBMITTER: Wang D

PROVIDER: S-EPMC5678609 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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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 Dongmao D Mohammad Mardhiah M Wang Yanyan Y Tan Rachel R Murray Lydia S LS Ricardo Sharon S Dagher Hayat H van Agtmael Tom T Savige Judy J

Kidney international reports 20170327 4

<h4>Introduction</h4>X-linked Alport syndrome (OMIM 301050) is caused by <i>COL4A5</i> 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.<h4>Methods</h4>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 extr ...[more]

PMID: 29142990

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Project description:BackgroundMissense variants in COL4A genes are often found in patients with an Alport syndrome-like presentation, but their pathogenicity is not always clear. We encountered a woman with microscopic hematuria and proteinuria at 33 years of age with a diagnosis of thin basement membrane disease who was approaching end stage kidney disease at 59 years of age. We hypothesized that this patient's kidney disease was within the spectrum of Alport syndrome.MethodsWe used histologic, genetic, and biochemical approaches to investigate the mechanisms of kidney disease. By immunofluorescence, we investigated collagen IV chain composition of the glomerular basement membrane (GBM). We employed targeted sequencing to search for pathogenic variants in COL4A and other relevant genes. We utilized N- and C-terminal split NanoLuciferase assays to determine the effect of a novel COL4A4 variant of uncertain significance (VUS) on collagen IV heterotrimer formation in vitro. We transfected COL4A4 expression constructs with split NanoLuciferase fragment-fused COL4A3 and COL4A5 constructs into human embryonic kidney 293T cells. To assay for α3α4α5(IV) heterotrimer formation and secretion, we measured luminescence in cell lysates and culture supernatants from transfected cells.ResultsImmunostaining suggested that the collagen α3α4α5(IV) network was present throughout the patient's GBMs. DNA sequencing revealed a novel homozygous VUS: COL4A4 c.1180G>A (p. Gly394Ser). In the C-terminal split luciferase-based α3α4α5(IV) heterotrimer formation assays, luminescence levels for G394S were comparable to WT, but in the N-terminal tag assays, the extracellular luminescence levels for G394S were decreased by approximately 50% compared with WT.ConclusionsOur cell-based assay provides a platform to test COL4 VUS and shows that G394S impairs assembly of the α3α4α5(IV) N-terminus and subsequent trimer secretion. These data suggest that the COL4A4-G394S variant is pathogenic and causes an atypical mild form of autosomal recessive Alport syndrome.

Dataset Information

The Chemical Chaperone, PBA, Reduces ER Stress and Autophagy and Increases Collagen IV ?5 Expression in Cultured Fibroblasts From Men With X-Linked Alport Syndrome and Missense Mutations.

Publications

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.

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