MO010COLLAGEN TYPE VI PRODUCTION BY KIDNEY FIBROBLASTS IS DEPENDENT ON THE RIGHT STIMULI AND MAY SELF-PERPETUATE FIBROSIS
Authors: Møller Alexandra Louise , G. K. Rasmussen Daniel , A. Karsdal Morten , Genovese Federica 
Published: May 2021

Abstract

Background and Aims
Accumulation of extracellular matrix (ECM) proteins is a hallmark of kidney 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 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 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 different pro-fibrotic stimulants on COL VI production and the effect of ETP itself on human kidney fibroblasts in the scar-in-a-jar (SiaJ) cell model.

Method
Cells were seeded in 48-well plates at 30.000 cells/well and incubated for 24h in DMEM + 10% FBS for adherence. Cells were then starved by incubating them for further 24h in DMEM + 0.4% FBS. To induce fibrogenesis, fresh medium was added at day 0 with 225/150 mg/mL Ficoll 70/400 and 1% ascorbic acid, containing either 7-, 0.7-, or 0.07 nM PDGF-AA, 8-, 0.8-, or 0.08 nM PDGF-BB, 4-, 0.4-, or 0.04 nM PDGF-CC, 7-, 0.7-, or 0.07 nM PDGF-DD, 0.9-, or 0.09 nM CTGF, 0.02 nM TGF-β 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), and VI (PRO-C6) formation were assessed in the medium by enzyme-linked immunosorbent assays developed at Nordic Bioscience.

Results
The stimulation of kidney fibroblasts with PDGF-AA, -BB, -CC, and -DD caused an increase in PRO-C6 compared to the unstimulated cells at every time point (P&lt;0.0001). The increase in formation peaked at day 10, and a dose-dependent increase in COL VI levels was observed with PDGF-DD treatment. Interestingly, CTGF treatment did not enhance the synthesis of COL VI at any time point, and TGF-β treatment suppressed PRO-C6 levels compared to the untreated cells (not significant). The stimulation with 30 nM ETP caused an increase in PRO-C1 (P&lt;0.0001) and PRO-C3 (P&lt;0.0001) compared to the unstimulated cells on days 6, 10, and 13. The increase in collagen formation peaked at day 10 for both markers, with a 7.28-fold increase for COL I and a 4.13-fold increase for COL III.

Conclusion
The production of COL VI, an important mediator of fibrosis and inflammation through its bioactive fragment endotrophin, shows a differential expression after stimulation of kidney fibroblasts with different pro-fibrotic growth factors. Interestingly, members of the PDGF family induced COL VI production, whereas CTGF and TGF-β did not. Moreover, we confirmed that ETP itself could stimulate kidney fibroblasts to produce more ECM proteins; hence COL VI may self-perpetuate fibrosis. This SiaJ model, combined with ECM formation biomarkers, could be used to elucidate the mechanisms behind acute and sustained matrix production profiles in vivo.