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Kidney Week

Abstract: PO0605

An In Vitro Model to Elucidate the Synthesis of Extracellular Matrix Proteins Involved in Renal Interstitial Fibrosis

Session Information

  • CKD Mechanisms - 1
    October 22, 2020 | Location: On-Demand
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2103 CKD (Non-Dialysis): Mechanisms

Authors

  • Møller, Alexandra L., Nordic Bioscience, Herlev, Denmark
  • Reese-Petersen, Alexander L., Nordic Bioscience, Herlev, Herlev , Denmark
  • Rasmussen, Daniel Guldager Kring, Nordic Bioscience, Herlev, Herlev , Denmark
  • Genovese, Federica, Nordic Bioscience, Herlev, Herlev , Denmark
  • Karsdal, Morten Asser, Nordic Bioscience, Herlev, Herlev , Denmark

Group or Team Name

  • Fibrosis, Renal and Cardiovascular Research
Background

Accumulation of extracellular matrix (ECM) proteins is a hallmark of renal fibrosis, which can lead to altered tissue homeostasis, kidney failure, and ultimately death. Many different cell types are involved in this process, but fibroblasts are the main source of ECM proteins such as fibronectin, collagen type I (COL I), III (COL III), and VI (COL VI). Recently it was suggested that a fragment of COL VI released during collagen maturation is, in fact, a bioactive molecule (endotrophin; ETP) with signaling potential, indicating that collagens are not just passive structural proteins. In this study, we investigated the effect of transforming growth factor (TGF)-β and ETP on the synthesis of different ECM proteins by human renal fibroblasts in the scar-in-a-jar (SiaJ) cell model.

Methods

Cells were seeded in 48-well plates at 30.000 cells/well and incubated for 24h in DMEM + 10% FBS for adherence. Cells were starved by incubating them for further 24h in DMEM + 0.4% FBS. Fresh medium was added at day 0 with 225/150 mg/mL Ficoll 70/400 and 1% ascorbic acid, containing 0.02 nM TGF-β or either 12 or 30 nM ETP. Medium was changed and collected on days 3, 6, 10, and 13. Biomarkers of COL I (PRO-C1), III (PRO-C3), VI (PRO-C6), and fibronectin (FBN-C) formation were assessed in the medium by enzyme-linked immunosorbent assays developed at Nordic Bioscience.

Results

Stimulating renal fibroblasts with 0.02 nM TGF-β significantly increased the formation of COL I (P<0.0001), III (P<0.0001), and fibronectin (P<0.0001) compared to unstimulated cells. Interestingly, TGF-β treatment suppressed the formation of COL VI compared to untreated cells. Stimulating with 30 nM ETP significantly increased the formation of COL I (P<0.0001) and III (P<0.0001) compared to unstimulated cells. 12 nM ETP significantly increased the synthesis of fibronectin compared to unstimulated cells (P<0.0001).

Conclusion

Different growth factors induce different protein expression profiles in fibroblasts. Interestingly, ETP, which originates from the ECM, drives renal fibrosis through increasing COL I and III as well as fibronectin. This SiaJ model, combined with the investigated biomarkers of ECM formation, could be used to elucidate the mechanisms behind acute and sustained matrix production profiles.