MO063: A New Tool for Preclinical Research and Drug Discovery: Extracellular Matrix Remodeling Quantification in Human Precision-Cut Kidney Slices

Abstract

Abstract BACKGROUND AND AIMS Accumulation of extracellular matrix (ECM) proteins is a hallmark of renal fibrosis, leading to altered tissue homeostasis, kidney failure and ultimately death. Current treatment strategies are ineffective in halting renal fibrogenesis, and drug discovery is hampered by the lack of clinically relevant biomarkers. Thus, there is an urgent need for early and pharmacodynamic biomarkers of renal fibrosis to support the development of new therapeutic treatments. In this study, we explored the use of non-invasive biomarkers of ECM turnover in human precision-cut kidney slices (PCKS): we observed the release of these markers over time in the slice supernatants and measured the changes following treatment with a prostaglandin E2 (PGE2) EP1 receptor antagonist. METHOD PCKS were prepared from healthy and fibrotic human kidney tissue obtained from patients treated at Aarhus University Hospital, Skejby. The slices were cultured in 48-well plates in medium (300 µl/well) and incubated for up to 72 hours to stimulate the development of renal fibrosis. The medium was collected at hours 0, 24, 48 and 72. Biomarkers of collagen type I (PRO-C1), III (PRO-C3), fibronectin (FN; FBN-C) and α-SMA formation and MMP-mediated degradation of collagen type I (C1M), III (C3M) and IV (Tumstatin; TUM) were measured in the medium by ELISA assays developed at Nordic Bioscience. mRNA expression levels of COL1A1, FN and α-SMA were measured by qPCR at all time points. Viability was assessed by measuring the ATP content of the slices at all time points. The anti-fibrotic effect of SC-19 220—a PGE2 EP1 receptor antagonist—was studied in fibrotic PCKS at hours 24 and 48 after treatment. RESULTS Fibrosis developed spontaneously in human kidney slices, as shown by increasing levels of PRO-C1 (P < 0.0001), PRO-C3 (P < 0.0001), FBN-C (P < 0.0001) and α-SMA (P < 0.0001) over time. Levels of C1M (not significant) and C3M (P < 0.01) did not increase to the same extent, and levels of TUM decreased over time (P < 0.001), indicating that this model reflects the process of fibrogenesis over fibrolysis. In addition, protein levels of PRO-C1 and FBN-C correlated significantly with mRNA levels of COL1A1 (r = 0.92, P = 0.0088) and FN (r = 0.86, P = 0.0277), respectively. Treatment with SC-19 220 reduced the gene expression of COL1A1, FN and α-SMA, and at a protein level it decreased levels of PRO-C1, PRO-C3 and FBN-C. PCKS retained constant ATP levels up to 72 hours. CONCLUSION In conclusion, we demonstrated that quantification of ECM remodeling in human PCKS via noninvasive biomarkers can be a valuable preclinical tool to study renal fibrogenesis and the effect of anti-fibrotic treatments. This study highlights that the EP1 receptor is a promising target for halting the production of ECM proteins. Since this is a human model, results may be translated to clinical care with higher confidence.