Work

FO068The LMX1βR246Q Mutation Induces Podocyte Injury Through Dysregulation of Cholesterol Transport Gene Expression

Year: 2019

Type: article

Abstract: INTRODUCTION: We previously reported a heterozygous missense mutation of the LIM Homeobox Transcription Factor 1 Beta (LMX1β) as a cause of nail-platella-like renal disease (NPLRD) in two families with hereditary FSGS. Currently, there are no targeted therapies for LMX1βR246Q-induced glomerulopathy. We hypothesized that LMX1βR246Q disrupts the expression of disease-relevant molecular targets within key signaling pathways that may be amenable to pharmacotherapy. To test this hypothesis, we performed an unbiased, whole-transcriptomic analysis in our established LMX1βWT and LMX1βR246Q-overexpressing podocyte lines to uncover potential therapeutic targets. METHODS: We conducted RNA-seq, qPCR, immunoblot, Oil Red O staining, electron microscopy, cholesterol efflux assays and apoptosis assays in our established LMX1βWT, LMX1βR246Q- and VIVIT overexpressing podocytes to evaluate the effect of the LMX1βR246Q mutation on cholesterol transport gene expression, cholesterol efflux and viability. RESULTS: Using RNA-seq, we determined that LMX1βR246Q-overexpressing podocytes express significantly reduced levels of ATP-binding Cassette Transporter Family A1 (ABCA1) and ABCG1 relative to LMX1βWT-overexpressing podocytes. These results were confirmed by qRCR. Protein expression of ABCA1 and ABCG1 was similarly reduced. Consistent with these findings, LMX1βR246Q-overexpressing podocytes displayed significantly increased lipid droplet accumulation, decreased basal and Apo-AI-stimulated cholesterol efflux and increased apoptosis relative to LMX1βWT-overexpressing podocytes. Evaluation of the putative promoter regions for ABCA1 and ABCG1 revealed no LMX1β binding sequences (i.e. FLAT-E or FLAT-F) but did reveal multiple candidate NFAT binding sequences. LMX1βR246Q-overexpressing podocytes displayed decreased NFAT4 expression and ABCA transporter expression was decreased in VIVIT-overexpressing podocytes. Oil Red O staining of proband renal biopsy tissue showed intraglomerular lipid staining and electron microscopy imaging showed electron-dense globules consistent with the accumulation of lipid droplets in podocyte phagolysosomes. CONCLUSIONS: The LMX1βR246Q mutation may cause FSGS, in part, through the dysregulation of NFAT-dependent lipid trafficking gene expression. This study provides the first demonstration of a potential role for impaired lipid trafficking in the pathobiology of familial FSGS and suggests that lipid depleting therapies (e.g. cyclodextrins) may have a role in the treatment of some forms of familial FSGS.

Source: Nephrology Dialysis Transplantation

DOI: 10.1093/ndt/gfz096.fo068