Murine Precision-Cut Kidney Slices as an ex vivo Model to Evaluate the Role of Transforming Growth Factor-β1 Signaling in the Onset of Renal Fibrosis

Abstract

Renal fibrosis is characterized by progressive accumulation of extracellular matrix (ECM) proteins, resulting in loss of organ function and eventually requiring renal replacement therapy. Unfortunately, no efficacious treatment options are available to halt renal fibrosis and translational models to test pharmacological agents are not always representative. Here, we evaluated murine precision-cut kidney slices (mPCKS) as a promising ex vivo model of renal fibrosis in which pathophysiology as well as therapeutics can be studied. Unique to this model is the use of rodent as well as human renal tissue, further closing the gap between animal models and clinical trials. Kidneys from C57BL/6 mice were used to prepare mPCKS and slices were incubated up to 96h. Viability, morphology, gene expression of fibrosis markers (Col1a1, Acta2, Serpinh1, Fn1, and Pai-1), inflammatory markers (Il1b, Il6, Cxcl1), and protein expression (collagen type 1, α-smooth muscle actin, HSP47) were determined. Furthermore, to understand the role of the transforming-growth factor β (TGF-β) pathway in mPCKS, slices were incubated with a TGF-β receptor inhibitor (LY2109761) for 48 h. Firstly, viability and morphology revealed an optimal incubation period of 48 h. Secondly, we demonstrated an early inflammatory response in mPCKS, which was accompanied by subsequent spontaneous fibrogenesis. Finally, LY2109761 showed great antifibrotic capacity in mPCKS by decreasing fibrosis markers on mRNA level as well as by reducing HSP47 protein expression. To conclude, we here present an ex vivo model of renal fibrosis, which can be used to further unravel the mechanisms of renal fibrogenesis and to screen antifibrotic therapy efficacy.

Predictive Value of Precision-Cut Kidney Slices as an Ex Vivo Screening Platform for Therapeutics in Human Renal Fibrosis

“…Figure 2a provides a quick comparison of the magnitude of the culture-induced transcriptional changes across PCKS without detailing marker genes, while Figure 2b-d and Figure S2 provide detailed expression data. In line with previous reports [22,42], preparation and culturing of the slices triggered the early onset of fibrosis and inflammation and activated both the TGFβ and PDGF pathways in PCKS prepared from healthy murine and human kidneys. In particular, 13 out of 14 tested genes were upregulated at 48 h in mPCKS, while hPCKS showed culture-induced upregulation of eight genes, namely COL1A1, SERPINH1, PLOD2, TGFB, SERPINE1, PDGFRB, TNF, and IL-6 ( Figure 2 and Figure S2).…”

“…We showed that injury inflicted by slice preparation and sustained by culturing for 48 h affects the transcriptional program of PCKS, dictating faster changes in gene regulation in murine PCKS compared to human PCKS. Independent of the species, PCKS responded to culturing by an increased expression of ECM-and inflammation-related gene markers accompanied by the activation of TGFβ and/or PDGF signaling pathways, in line with previous reports [22,42]. Species differences in injurious responses included unchanged mRNA levels of fibronectin, TGFβ receptor 1, PDGFB ligand and interleukin (IL)-1B in human PCKS as opposed to their increased transcription in mouse PCKS, which might suggest a delayed response of human PCKS, and, therefore, a longer incubation time (beyond 48 h) might be needed to induce changes in these transcripts.…”

Implementation of pre-clinical methodologies to study fibrosis and test anti-fibrotic therapy

“…Normal mouse precision cut kidney slices (PCKS) cultured in 80% oxygen have increased inflammatory gene expression within 3 hours, whilst fibrosis gene expression was elevated by 96 hours. Treatment with LY2109761, a TGFβ receptor inhibitor, blunted the fibrogenic response confirming that spontaneous fibrosis was in part TGFβ-dependant [31]. Using this system, successful myofibroblast-specific targeting of an anti-fibrotic IFNγ-peptide conjugate (PPB-PEG-IFNγ) limited TGFβ-induced fibrogenic gene expression and collagen deposition [32], providing evidence that peptide therapies can penetrate tissue slices and exert biological effects.…”

Comparative study of nanoparticle uptake and impact in murine lung, liver and kidney tissue slices

“…With this in mind, in this study we aimed at comparing nanoparticle impact in lungs, liver, and kidneys, selected as three of the major organs for nanosafety assessment (Olinga and Schuppan 2013;Sauer et al 2014;Stribos et al 2017).…”