Abstract: FR-PO1026
Antisense Oligonucleotide-Mediated Terminal Intron Retention of Endoglin to Reduce Extracellular Matrix Production in Chronic Allograft Dysfunction
Session Information
- CKD Mechanisms: From Mendel to Mars
November 03, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: CKD (Non-Dialysis)
- 2303 CKD (Non-Dialysis): Mechanisms
Authors
- Gerrits, Tessa, Leiden University Medical Center, Department of Pathology, Leiden, Zuid-Hollend, Netherlands
- Bijkerk, Roel, Leiden University Medical Center, Department of Nephrology, Leiden, Netherlands
- Bruijn, Jan A., Leiden University Medical Center, Department of Pathology, Leiden, Zuid-Hollend, Netherlands
- Scharpfenecker, Marion, Leiden University Medical Center, Department of Pathology, Leiden, Zuid-Hollend, Netherlands
- Baelde, Hans J., Leiden University Medical Center, Department of Pathology, Leiden, Zuid-Hollend, Netherlands
Background
Chronic allograft dysfunction (CAD) is a chronic, progressive, and irreversible process associated with progressive interstitial fibrosis and tubular atrophy. Transforming growth factor-beta (TGF-β) is considered an important cytokine in the development of interstitial fibrosis. The TGF-β co-receptor endoglin (ENG) tends to be upregulated in several human organs when chronic damage and fibrosis is present, including the kidney. ENG has two membrane bound isoforms occurring by alternative splicing. The long endoglin isoform (l-ENG) was previously shown to enhance the extent of renal fibrosis in an UUO mouse model, while the short endoglin isoform (s-ENG) inhibited renal fibrosis in this model.
Methods
We isolated mRNA from kidney biopsy material of patients with CAD (n=12) to investigate expression of ENG and short endoglin (s-ENG). As a control, kidneys that were excluded for transplantation were used (n=6). Furthermore, we designed antisense oligonucleotides (ASOs) to achieve higher levels of s-ENG by terminal intron retention of the ENG pre-mRNA and transfected these in the human kidney fibroblast cell line TK173.
Results
ENG mRNA is 2.3 fold (p<0.05) upregulated in the interstitium of patients with CAD compared to mRNA from controls. Interestingly, mRNA levels of s-ENG were not upregulated. Therefore, the fraction of s-ENG from the total ENG mRNA was significantly lower in CAD patients compared to controls (2.4% vs 4.3%; p<0.05).
We showed that ASOs enhanced the splicing to the short endoglin isoform (2 fold; p<0.05). Combining multiple ASOs did not amplify this enhancement. After transfection with various ASOs, the ASO covering the exonic splicing enhancer decreased CCN2, COL1A1 and FN1 mRNA expression after TGF-β1 stimulation (respectively 20%, 30%, 20%; p<0.05).
Conclusion
ENG mRNA levels are upregulated in CAD, but s-ENG which is suggested to inhibit renal fibrosis, does not increase accordingly. With ASOs targeting ENG splicing we were able to alter the ENG isoform ratio towards s-ENG and reduce TGF-β1 downstream signaling. These results open the way to explore the potential of ENG ASOs as a therapy to reduce interstitial fibrosis and thereby slowing down the progression to end stage kidney disease.