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Abstract: TH-PO855

Early B Cell Factor 1 Plays a Role in the Maintenance of Glomerular Filtration Barrier Integrity

Session Information

Category: Diabetic Kidney Disease

  • 601 Diabetic Kidney Disease: Basic

Authors

  • Andrews, Darrell C., University College Dublin, Dublin, Ireland
  • Brennan, Eoin P., University College Dublin, Dublin, Ireland
  • Beverly-Staggs, Laura L., Mount Desert Island Biological Lab, Salisbury Harbor, Maine, United States
  • Schroder, Patricia Ann, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine, United States
  • Conlon, Peter J., Beaumont Hospital, Dublin 9, Co Dublin, Ireland
  • Schiffer, Mario, Hannover Medical School, Hannover, Germany
  • Godson, Catherine, The Conway Institute of Biomolecular and Biomedical, Belfield, Dublin, Ireland

Group or Team Name

  • The GENIE Consortium
Background

Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD) and organ failure worldwide. Current therapies are inadequate and at best slow progression but do not arrest or reverse it. Proteinuria is associated with DKD progression and reflects loss of integrity of the glomerular filtration barrier (GFB). Defining the mechanisms underlying a compromised GFB during the progression of DKD is key to developing effective therapeutic strategies. We identified EBF1 as associated with macroalbuminuria and ESRD by our GWAS analysis in a case (n=2,861 individuals with T1DM) vs control (n=12,418 individuals with T1DM and no evidence of renal disease 15 years post-diagnosis) approach. Here we investigate the contribution of EBF1 to proteinuria associated with DKD using a zebrafish model of GFB integrity and elaborate on its role in maintaining a functional GFB.

Methods

To investigate a possible causative role for EBF1 in loss of GFB integrity with resulting proteinuria Tg(l-fabp:DBP:EGFP) zebrafish embryos were injected with control or EBF1 morpholinos. Morpholinos targeting the podocyte gene CD2AP were used as a positive control. Tg(l-fabp:DBP:EGFP) transgenic zebrafish express a fluorescent plasma protein with a molecular weight equivalent to human albumin. Compromised GFB integrity leading to loss of the fusion protein from the circulation can be quantified by fluorescence imaging of the retinal plexus. The ultrastructure of GFB was visualised using electron microscopy. While RNA sequencing analysis was used to map the transcriptome of EBF1 knockdown fish vs control.

Results

EBF1 knockdown led to a significant loss of retinal fluorescence compared to control and equivalent to that of CD2AP at 96hpf. Knockdown also caused generalised edema compared to control, further supporting a role for this gene in kidney function. EM analysis revealed loss of EBF1 caused pronounced podocyte foot process effacement and screening of a cohort of cell lines confirmed high levels of expression of EBF1 in podocytes. RNA sequencing analysis combined with Ingenuity Pathway Analysis revealed desregulated transcripts and key pathways affected by EBF1 knockdown which were validated by real time PCR.

Conclusion

These findings suggest that EBF1 plays a key role in maintaining GFB integrity.

Funding

  • NIDDK Support