Abstract: TH-PO079

Glomerular Enhancer of Zeste Homolog-2 (EZH2) Histone Methyltransferase Reduces Glomerular Endothelial Glycocalyx during Diabetic Nephropathy by Regulating Hyaluronan Synthesis

Session Information

Category: Glomerular

  • 1002 Glomerular: Basic/Experimental Pathology

Authors

  • Sol, Marloes, University Medical Center Groningen, University of Groningen, Groningen, Groningen, Netherlands
  • Qiu, Jiedong, Medical Faculty Mannheim, University of Heidelberg, , Mannheim, Germany
  • Van der vlag, Johan, Radboud University Nijmegen Medical Centre, Nijmegen, Gelderland, Netherlands
  • van den Born, Jacob, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
  • Yard, Benito, Medical Faculty Mannheim, University of Heidelberg, , Mannheim, Germany
  • Hillebrands, Jan-luuk, University Medical Center Groningen, University of Groningen, Groningen, Groningen, Netherlands
  • Kamps, Jan A.A.M., University Medical Center Groningen, University of Groningen, Groningen, Groningen, Netherlands
  • Krenning, Guido, University Medical Center Groningen, University of Groningen, Groningen, Groningen, Netherlands
Background

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. The glomerular endothelial glycocalyx is the first barrier that prevents leakage of circulating proteins. Injury to the glycocalyx evokes proteinuria and kidney failure. The polycomb group methyltransferase Enhancer of Zeste Homolog 2 (EZH2) inhibits expression of its target genes through methylation of lysine 27 on histone 3 (H3K27Me3). We recently performed a target screen for genes involved in glycocalyx turnover, which indicated that EZH2 inhibits glycocalyx synthesis in glomerular endothelial cells. We hypothesized that EZH2 activity is increased in the glomerular endothelium during DN thereby reducing glycocalyx synthesis.

Methods

H3K27me3 was analyzed in glomerular endothelial cells by immunofluorescence in BTBRob/ob mice, a mouse model for DN. Glycocalyx in these mice was measured by the binding of fluorescently-labeled wheat germ agglutinin. In glomerular endothelial cells, EZH2 was silenced by RNAi. Gene expression was assessed by Quantitative Real-time PCR.

Results

H3K27me3 in glomerular endothelial cells was increased 1.5-fold compared to non-diabetic mice (p=0.026). Albumin-creatinine ratios of BTBRob/ob mice correlated with the increase in H3K27me3 (p=0.044; r2=0.674). A 2-fold loss of glomerular glycocalyx was observed in BTBRob/ob mice (p=0.002). Silencing of EZH2 in glomerular endothelial cells led to a decrease in H3K27me3 and an 8-fold increase in the hyaluronan synthesizing enzyme HAS1 (p<0.001). ENCODE database analysis revealed a binding site for EZH2 in the HAS1 gene, suggesting that HAS1 is a direct target of EZH2. Interestingly, the hyaluronan degrading enzymes, HYAL1 (p=0.002), HYAL2 (p=0.015), and HYAL3 (p=0.014) were all decreased upon knockdown of EZH2.

Conclusion

In conclusion, our data suggests that EZH2-mediated epigenetic changes reduce endothelial glycocalyx via reduction of hyaluronan in DN.