Ex vivo fibrosis research: 5 mm closer to human studies
Bigaeva, Emiliia
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KINASE RECEPTOR SIGNALLING
ATTENUATES FIBROGENESIS
IN AN EX VIVO MODEL OF
HUMAN RENAL FIBROSIS
Emilia Bigaeva
*Elisabeth G.D. Stribos
*Henricus A.M. Mutsaers
Bram Piersma
Anna M. Leliveld
Igle J. de Jong
Ruud A. Bank
Marc A. Seelen
Harry van Goor
Lutz Wollin
Peter Olinga
Miriam Boersema
*these authors share first authorship
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ABSTRACT
Background: Poor translation from animal studies to human clinical trials is one of the main hurdles
in the development of new drugs. Here, we used precision-cut kidney slices (PCKS) as a translational model to study renal fibrosis and to investigate whether inhibition of tyrosine kinase receptors, with the selective inhibitor nintedanib, can halt fibrosis in murine and human PCKS.
Methods: We used renal tissue of murine and human origin to obtain PCKS. Control slices and slices
treated with nintedanib were studied to assess viability, activation of tyrosine kinase receptors, cell proliferation, collagen type I accumulation, gene and protein regulation.
Results: During culture, PCKS spontaneously develop a fibrotic response that approximates in vivo
fibrogenesis. Nintedanib blocked culture-induced phosphorylation of platelet-derived growth factor receptor and vascular endothelial growth factor receptor. Furthermore, nintedanib inhibited cell proliferation, reduced collagen type I accumulation and expression of fibrosis-related genes in healthy murine and human PCKS. Modulation of extracellular matrix homeostasis was achieved already at 0.1 μM, while high concentrations (1 and 5 μM) elicited possible non-selective effects. In PCKS from human diseased renal tissue nintedanib showed a limited capacity to reverse established fibrosis.
Conclusions: Nintedanib attenuated the onset of fibrosis in both murine and human PCKS by
inhibiting the phosphorylation of tyrosine kinase receptors; however, the reversal of established fibrosis was not achieved.
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INTRODUCTION
Renal fibrosis, defined by the progressive deposition of connective tissue, is a hallmark of chronic kidney disease (CKD), which affects an estimated 10% of the population in developed countries [1]. CKD progresses to end-stage renal disease (ESRD), that eventually requires replacement therapy — dialysis or transplantation. Current research investigates strategies to halt CKD progression, or even to reverse renal fibrosis [2,3]. Yet, no effective therapy has been clinically implemented.
Pathological activation of various receptor tyrosine kinases (RTKs) — such as platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) receptors — plays a key role in renal fibrogenesis [4–8]. The PDGF receptor (PDGFR) is an attractive molecular target for antifibrotic therapies [9], since PDGFR signalling is involved in (trans)differentiation of collagen-producing myofibroblasts [10–12]. The receptors PDGFRα and β are expressed in renal tissue mainly by glomerular mesangial cells, interstitial fibroblasts and vascular smooth-muscle cells [13]. Several studies reported an increased expression of both receptors in murine and human renal disease [10,14]. The activation of the PDGFR leads to glomerulosclerosis and (tubulo)interstitial fibrosis [4]. Therefore, blocking PDGFR signalling is a promising strategy to halt the progression of renal fibrosis.
Nintedanib is a small molecule tyrosine kinase inhibitor, approved in several countries worldwide for the treatment of idiopathic pulmonary fibrosis (IPF) and for the second line treatment of non-small-cell lung carcinoma with adenomacarcinoma histology. Nintedanib affects signalling pathways of multiple growth factors, including vascular endothelial growth factor (VEGF), FGF and PDGF, as well as Lck and Src non-receptor kinases [15,16]. In a phase II randomized clinical trial, nintedanib showed anti-angiogenic effects and had an acceptable safety profile in patients with advanced renal cell carcinoma [17]. To our knowledge, the impact of nintedanib on human renal fibrosis has not been published.
The lack of translational models of human renal fibrosis hampers the search for effective antifibrotic therapies [18]. In vitro models lack cellular heterogeneity, and animal models have limited implications for human disease. To partly overcome these limitations, we used precision-cut kidney slices (PCKS) as an ex vivo model of renal fibrosis [19–21]. PCKS replicate the organotypic multicellular characteristics, as one slice maintains the complex three-dimensional architecture of the kidney, and have a high translational impact, as both murine and human tissue, healthy and diseased, can be used. In addition, PCKS culture is reproducible and allows for a substantial reduction of animal use, making it a promising preclinical tool for drug development.
In this study, we aimed to investigate therapeutic effects of nintedanib in PCKS, and to find whether inhibition of nintedanib’s molecular targets may prevent renal fibrosis in murine and, more importantly, in human kidneys.
MATERIALS & METHODS
Ethics statement
This study was approved by the Medical Ethical Committee of the University Medical Centre Groningen (UMCG), according to Dutch legislation and the Code of Conduct for dealing responsibly with human tissue in the context of health research (www.federa.org), forgoing the need of written consent for ‘further use’ of coded-anonymous human tissue. The animal experiments were approved by the Animal Ethics Committee of the University of Groningen (DEC 6416AA-001).
Renal tissue
Macroscopically healthy renal cortical tissue (n=9) was obtained from tumor nephrectomies, and
fibrotic renal tissue (n=10) was obtained from ESRD nephrectomies or transplantectomies. Table 1
summarizes patient demographics. Renal tissue was stored in ice-cold University of Wisconsin (UW) organ preservation solution, and cold ischemia time was limited to 2-3 hours.
Murine tissue was obtained from male C57BL/6 mice, with an average weight of 28.3 g (± 2.4) and 12.1 weeks of age (± 2.2). The animals were housed in filter-top cages with free access to water and
food. Kidneys were harvested via a terminal procedure performed under isoflurane/O2 anesthesia
(Pharmachemie BV, Haarlem, the Netherlands) and stored in ice-cold UW solution until further use.
Table 1. Patient demographics
Healthy renal tissue (n=9) Fibrotic renal tissue (n=10)
Gender (% male) 67 40
Age (in years) 66 ± 8 47 ± 15
Nephrectomy side (% left) 37.5 50
Creatinine before nephrectomy
(μmol/L) 81 ± 13 545 ± 403
eGFR before nephrectomy (ml/
min/1.73m²)* 81 ± 9 NA
Time on dialysis (mean in months) NA 116 ± 136
Time since (first) transplantation
(mean in months) NA 132 ± 150
Type renal tissue NA Non-functioning kidney
allograft (n=4), kidney allograft with infected abscess (n=1), non-functioning
native ESRD kidney (n=5). *calculated using the Modification of Diet in Renal Disease (MDRD) formula.
Values are presented as the mean ± standard deviation or otherwise if indicated.
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Preparation and treatment of precision-cut kidney slices
PCKS were prepared according to the protocol by Poosti et al. (mouse; [22]) and Stribos et al. (human; [21]), using a Krumdieck tissue slicer. Slices were incubated in Williams’ Medium E with GlutaMAX (Life Technologies, Carlsbad, California, USA) containing 10 μg/mL ciprofloxacin and 26 mM glucose,
at 37°C in a 80% O2/5% CO2 atmosphere while gently shaken. Nintedanib was kindly provided by
Boehringer Ingelheim (Biberach, Germany). We treated murine or human PCKS with nintedanib (0.1 – 10 μM) for 48h. Analyses were performed using three pooled slices from the same animal/donor (technical replicates) from at least three to five animals or donors (biological replicates).
Viability of PCKS
Viability of the slices was assessed by measuring ATP content using the ATP bioluminescence kit (Roche Diagnostics, Mannheim, Germany), as previously described [23].
Quantitative real-time PCR and low-density array
Total RNA was extracted with the RNeasy mini kit (Qiagen, Venlo, the Netherlands) and reverse transcribed using the Reverse Transcription System (Promega, Leiden, the Netherlands). cDNA was used for quantitative real-time PCR performed with a Viia7 Real-Time PCR system (Applied Biosystems,
Bleiswijk, the Netherlands). Gene expression was calculated using the 2-ΔCt method [24] and corrected
for GAPDH. The Taqman gene expression assays used in this study are listed in Supplementary Table
S1.
The expression of 44 genes related to fibrosis (Supplementary Table S2) was examined using a
custom-designed low-density array (LDA, Applied Biosystems) [25]. cDNA from renal tissue of C57BL/6 control mice that underwent UUO microsurgery was kindly provided by Dr. Bram Piersma. A total of 100 μl reaction mixture containing 6 ng/μl cDNA and 50 μl 2x Taqman Universal PCR Master Mix (Applied Biosystems) was loaded per sample. PCR amplification was performed on a Viia7 Real-Time PCR system (Applied Biosystems).
Western blotting
Total protein was extracted from PCKS with ice-cold RIPA buffer (Thermo Scientific, Waltham, Massachusetts, USA) supplemented with a protease inhibitor cocktail and PhosStop (Sigma-Aldrich, Saint Louis, Missouri, USA). A total of 80-100 μg of protein was separated via SDS-PAGE using 10% polyacrylamide gels and blotted onto polyvinylidene fluoride membranes (Trans-Blot Turbo Transfer System, Bio-Rad, Veenendal, the Netherlands). 2,2,2-trichloroethanol (TCE; Sigma-Aldrich) allowed for visible detection of total protein load [26]. Membranes were blocked in 5% non-fat milk/TBST
(Bio-Rad) and incubated with the primary antibody (Supplementary Table S3) overnight at 4˚C, followed by
incubation with the appropriate HRP-conjugated secondary antibody. Protein bands were visualized using Clarity Western ECL Substrate (Bio-Rad) and ChemiDoc Touch Imaging System (Bio-Rad). Protein expression was corrected for total protein and expressed as a relative value to the control group.
Phosphoproteomic analysis of RTKs by multiplex
A human RTK phosphoprotein magnetic bead panel (Merck Millipore, Billerica, Massachusetts, USA), was used according to manufacturer’s instructions. Total protein was extracted using the supplied lysis buffer supplemented with protease inhibitor cocktail. Samples were diluted to a concentration of 1.5 μg/μl and passed through a 0.45 μm syringe filter (Whatman, Maidstone, UK). Detection was performed with the MAGPIX multiplexing instrument (Luminex, Austin, Texas, USA). Mean fluorescent intensity (MFI) was used for quantification.
Histology
PCKS were fixed in 4% buffered formalin, embedded in paraffin and sectioned 2 μm thick. Tissue damage and renal fibrosis were assessed by Periodic acid–Schiff (PAS) and Picro Sirius Red (PSR) staining. Additionally, we performed immunohistochemistry for Ki-67, α-SMA and collagen type I. After deparaffinisation and antigen retrieval with 0.1 M Tris-EDTA (pH 9.0) in microwave oven for 15 min, tissue sections were blocked with 2% rat serum in PBS/2% BSA for 10 min and then incubated with primary antibodies (Supplementary Table S3) for 1h. The antibodies were localized using the appropriate HRP-conjugated secondary and tertiary antibodies and the ImmPact NovaRed kit (Vector, Burlingame, USA), followed by hematoxylin counterstaining. Stained tissue sections were scanned using a Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01, Hamamatsu Photonics K.K., Japan). To quantify the stained areas, the whole-slide images were processed with Aperio ImageScope v12.3 (Aperio Technologies, Vista, CA) by applying the Positive Pixel Count V9 algorithm (hue value set to 0). The intensities were measured as percentages − number of positive and strong positive pixels divided by the total number of pixels − and expressed as relative values to the control group.
Statistical analysis
The results are expressed as mean ± standard error of mean (SEM) of minimum 3 independent experiments. Statistics were performed using GraphPad Prism 6.0 (GraphPad Software Inc.) by unpaired one-tailed Student’s t-test or one-way ANOVA followed by Dunnett’s multiple comparisons test. The protein levels of HSP47 and α-SMA were compared using non-parametric Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. Differences between groups were considered to be statistically different when p < 0.05. For the LDA heatmap, average-linkage clustering was performed using Pearson correlation. The heatmap was generated using the online tool Morpheus (https:// software.broadinstitute.org/morpheus/).
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RESULTS
A brief summary of the study workflow is presented in Figure 1A. Murine and human PCKS remained
viable during 48h incubation, as reflected by unchanged ATP and total protein content (Figure 1B). In
addition, PAS staining revealed typical structural changes in slices due to the culturing in accordance with previously reported results [21,27]. Nintedanib at 5 μM showed no indication towards worsening
of the morphology (Figure 1C).
Nintedanib in murine PCKS
Mitigation of fibrosis and inflammation by nintedanib
During 48h incubation, spontaneous onset of fibrosis occurred in mPCKS, as reflected by an upregulation of mRNA levels of collagen type I, fibronectin and heat shock protein HSP47 (encoded
by Col1a1, Fn1 and Serpinh1, respectively) and by increased protein levels of HSP47 (Supplementary
Figure S1A and B). Nintedanib effectively mitigated fibrogenesis as shown by a clear reduction of
Col1a1 gene expression (Figure 2A), with an IC50 of 0.7 μM (Supplementary Figure S2A). Furthermore,
nintedanib reduced mRNA levels of Fn1 and Acta2 (IC50 of 4.4 μM and 6.2 μM, respectively), while it
affected Serpinh1 only at the highest concentration (IC50=5.8 μM). Treatment with 5 μM nintedanib
significantly decreased interstitial accumulation of collagen type I and protein expression of HSP47,
but did not affect α-SMA (Figure 2B and C). Collagen mRNA and protein levels declined below baseline
expression (at 0h) when mPCKS were treated with 5 and 10 μM nintedanib. Spontaneous onset of
fibrosis in mPCKS was accompanied by an inflammatory response after 48h (Supplementary Figure
S1C). We observed a significant decrease in mRNA levels of tumor necrosis factor, interleukin-1 beta
and interleukin-6 (encoded by Tnf, Il-1β and Il-6, respectively) in the presence of nintedanib, while
Cxcl1 (chemokine (C-X-C motif) ligand 1) expression was not affected (Figure 2D).
Antiproliferative activity of nintedanib in mPCKS
Previous studies reported the antiproliferative effects of nintedanib [16,28,29]. We evaluated these
effects in mPCKS by Ki-67 immunohistochemistry, a marker of cell proliferation (Figure 2E). We
observed a 3.5-fold culture-induced increase in proliferation that was attenuated by 5 μM nintedanib by approximately 30%.
Low density array for genes related to ECM homeostasis
We measured and compared the expression of 44 genes related to ECM homeostasis in kidneys from
mice with renal injury induced by unilateral ureteral obstruction (UUO) and mPCKS (Figure 3A). At
0h, PCKS exhibited gene expression profile similar to the sham-operated kidneys. Obstruction for 3 or 7 days resulted in an upregulation of the majority of tested genes. Hierarchical clustering analysis clearly separated 0h PCKS and 48h PCKS: while 0h PCKS clustered together with sham-operated
kidney samples, 48h PCKS clustered closer to 3dUUO and 7dUUO kidneys (Figure 3A). This suggests
that culturing of PCKS for 48h induced changes in ECM homeostasis that relatively resembled changes observed in UUO kidneys, indicating that PCKS approximate in vivo fibrogenesis.
Figure 1. (A) Schematic illustration of the workflow. Renal tissue of murine or human origin was used to obtain cylindrical
cores. By placing the tissue cores in Krumdieck tissue slicer, filled with ice-cold Krebs-Henseleit buffer, we prepared precision-cut kidney slices with a wet weight of 4-5 mg and estimated thickness of 250-300 µm. Slices were subsequently incubated in 12-well plates (1 slice per well) in culture medium with or without nintedanib for 48h at 37ºC. Medium was refreshed at 24h. At the end of culture period, samples were collected by pooling three slices (from each animal/donor) for each type of analysis. Visualization of kidney slices prepared from healthy tissue is represented by the blue color, from diseased tissue – by the orange color. Same color code is applied to all figures. (B) Viability of murine, human and fibrotic human PCKS treated with nintedanib for 48h was measured by ATP and total protein content. Data are shown as values relative to non-treated control slices at 48h and are expressed as mean (± SEM), n=4-5, *p < 0.05. (C) Representative images of Periodic acid–Schiff (PAS) staining of untreated slices at 0h and 48h as well as of slices treated with 5 µM nintedanib (scale bar = 100 µm).
A B 0h cont rol 48h cont rol 48h ni nt edani b 5 μM mPCKS hPCKS fhPCKS 0 1 5 10 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive A TP val ue Viability mPCKS 0 0.1 0.3 0.5 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive A TP val ue Viability hPCKS 0 0.1 0.3 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive A TP val ue Viability fhPCKS 0 1 5 10 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive p ro tei n co nt en t Protein mPCKS 0 0.1 0.3 0.5 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive p ro tei n co nt en t Protein hPCKS 0 0.1 0.3 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive p ro tei n co nt en t Protein fhPCKS Krumdieсk Tissue Slicer Treatment: +/- nintedanib Incubation: 1 slice/well for 48 hours;
80% O2/ 5% CO2; 370C; shaking WT C57BL/6 Healthy kidneys Tissue cores (60-200 slices) Analysis Precision-cut kidney slices (PCKS) Healthy kidneys Transplantectomy Fibrotic kidneys 200-300 μm 5 mm C
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Figure 2. Antifibrotic, anti-inflammatory and antiproliferative effect of nintedanib in healthy mouse kidney.
Murine PCKS were cultured in the presence of nintedanib (1, 5 or 10 µM) for 48h. (A) mRNA expression of fibrosis markers after 48h incubation. (B) Representative sections with interstitial collagen type I accumulation visualized by immunohistochemistry (scale bar = 50 µm) and quantitative analysis of staining intensity. (C) Protein levels of HSP47 and α-SMA at 48h with representative Western blot images. (D) mRNA expression of inflammation markers after 48h incubation. (E) Expression of cell proliferation marker Ki-67 in murine PCKS during culture and after 48h treatment with 5 µM nintedanib was visualized by immunohistochemistry and quantified as relative intensity values.
Data are expressed as mean (± SEM), n=3-5, *p < 0.05.
0 1 5 10 0.0000 0.0002 0.0004 0.0006 0.0008 0.0010 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Tnf * * * 0 1 5 10 0.00 0.05 0.10 0.15 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Col1a1 * * * 0 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) α-SMA Nintedanib [μM] HSP47 (47 kD) α-SMA (42 kD) 0 1 0 5
0h control 48h control 48h nintedanib 5 μM
A B D 0 1 5 10 0.000 0.005 0.010 0.015 0.020 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Cxcl1 0 1 5 10 0.0000 0.0005 0.0010 0.0015 0.0020 0.0025 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Acta2 * * 0 1 5 10 0.00 0.10 0.20 0.30 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Fn1 * * 0 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) HSP47 * 0 5 0.0 0.5 1.0 1.5 (0h) Nintedanib [µM] R el at ive i nt en si ty ( fo ld ) Collagen I * 0 1 5 10 0.000 0.001 0.002 0.003 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Il-1b * * 0 1 5 10 0.000 0.005 0.010 0.015 0.020 0.025 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Il-6 * * 0 1 5 10 0.00 0.02 0.04 0.06 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) Serpinh1 *
0h control 48h control 48h nintedanib 5 μM
0 48
0 2 4 6
Time of incubation [hours]
R el at ive i nt en si ty ( fo ld ) Ki-67 mPCKS * 0 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive i nt en si ty ( fo ld ) Ki-67 mPCKS * E C
Figure 3. TaqMan Low density array of extracellular matrix homeostasis genes. (A) Heatmap of the expression
patterns of extracellular matrix (ECM) related genes demonstrating the similarities in fibrogenic processes between the murine UUO model and the PCKS model. Induction of fibrogenesis in PCKS cultured for 48h resembles the changes in UUO kidneys at day 3 and 7, while non-cultured PCKS (0h) had a similar expression profile compared to sham-operated kidneys. (B) Heatmap of ECM modulation profiles in murine PCKS at 0h, 48h and treated with nintedanib (0.1-5 μM) for 48h. Average-linkage hierarchical clustering was performed using Pearson correlation; n=3-6. Full gene names are listed in Supplementary Table S2.
We further investigated the impact of nintedanib on fibrosis. Figure 3B shows that nintedanib
suppressed culture-induced upregulation of ECM related genes already at the lowest concentration. Nintedanib (0.1 μM) inhibited the expression of almost all collagen subtypes (Col1a1, Col1a2, Col4a1,
Col5a1, Col6a1) except for Col3a1. Furthermore, at high concentrations — 1 and 5 μM — nintedanib
regulated different mRNA clusters than at lower concentrations — 0.1 and 0.5 µM. For instance, genes related to collagen formation Adamts3, Lox, Loxl2, Loxl4, Pcolce2 and P4hb were downregulated by nintedanib at low concentrations but increased in expression at high concentrations, which resembled the expression profile of untreated slices at 48h. In addition, ECM degradation matrix metalloproteinases encoded by Mmp2, Mmp9 and Mmp13 genes were highly upregulated during culture and inhibited by 5 μM nintedanib.
row min row max
ECM component Collagen processing ECM remodeling ECM receptor
SHAM 3d SHAM 3d SHAM 3d SHAM 7d SHAM 7d SHAM 7d PCKS 0h PCKS 0h PCKS 0h UUO 3d UUO 3d UUO 3d UUO 3d UUO 3d UUO 3d UUO 7d UUO 7d UUO 7d UUO 7d UUO 7d UUO 7d PCKS 48h PCKS 48h PCKS 48h
Leprel1 P4hb Plod1 Plod3 Ddr1 Adamts14 Pcolce Eln Fmod P4ha3 Plod2 Dcn P4ha1 Slc39a13 Leprel2 Mmp13 Loxl4 P4ha2 Col6a1 Lepre1 Loxl2 Pcolce2 Col5a1 Lox Bgn Col3a1 Fkbp10 Bmp1 Fn1 Timp1 Mmp9 Loxl1 Adamts3 Adamts2 Col1a1 Loxl3 Col1a2 Col4a1 Ctsk Mmp2 Serpinh1 Mmp14 Ddr2
row min row max
0h 0h 0h 0h 48h Control 48h Control 48h Control 48h 0.1 uM 48h 0.1 uM 48h 0.1 uM 48h 0.5 uM 48h 0.5 uM 48h 0.5 uM 48h 1 uM 48h 1 uM 48h 5 uM 48h 5 uM 48h 5 uM
id P4ha3 Adamts3 P4hb Slc39a13 Ddr1 P4ha1 Plod1 P4ha2 Lepre1 Leprel2 Loxl3 Adamts2 Bgn Mrc2 Loxl1 Col1a2 Col1a1 Col6a1 Col5a1 Fkbp10 Plod2 Serpinh1 Lox Pcolce2 Loxl2 Col4a1 Col3a1 Plod3 Bmp1 Mmp14 Fn1 Timp1 Mmp9 Mmp13 Loxl4 Ddr2 Mmp2 Ctsk Dcn Adamts14 Leprel1 Pcolce Eln Fmod id A B
5
Nintedanib in healthy human PCKS
Targeted inhibition of gene expression and tyrosine kinase receptor activation
Culturing of the slices led to a significant downregulation of VEGFR1, VEGFR2 and FGFR2 (Supplementary
Figure S3A). Nintedanib reduced expression of PGDFRB and VEGFR1 in hPCKS already at 0.1 μM (Figure
4A), and inhibited VEGFR3 (Supplementary Figure S3C). The treatment did not affect expression of
VEGFR2; however, it increased FGFR2 expression at 5 μM.
Five phospho-RTKs (p-RTKs) were upregulated during incubation of hPCKS: p-PDGFRα, p-PDGFRβ,
p-VEGFR1, p-VEGFR2 and p-FLT3 (Supplementary Figure S3B). Figure 4B shows that nintedanib
reduced phosphorylation of p-PDGFRα and p-VEGFR1 at 0.3 μM (by 39.8% and 55%, respectively) and of PDGFRβ and VEGFR2 at 0.1 μM (by 45.3% and 23%). In contrast, the activation of VEGFR3 and FGFR1 in hPCKS was neither affected by 48h incubation, nor by the treatment with nintedanib (Supplementary Figure S3B and D).
Mitigation of fibrosis and inflammation markers by nintedanib
In line with previously published data [21], fibrogenesis was initiated during incubation of hPCKS, as revealed by the increase in COL1A1 and SERPINH1 transcription and protein levels of HSP47 (Supplementary Figure S4A and B). Expression of ACTA2 significantly dropped after 48h, while FN1 expression remained unchanged. Treatment with nintedanib resulted in a concentration-dependent
inhibition of all tested fibrosis markers, except for ACTA2 (Figure 4C). The IC50 was 0.6 μM for COL1A1,
1.5 μM for SERPINH1 and 1.7 μM for FN1 (Supplementary Figure S2B). Nintedanib at 5 μM reduced the
accumulation of collagen type I to the baseline levels (at 0h) and affected protein level of HSP47 (Figure
4D and E). In concordance with gene expression, nintedanib had no influence on α-SMA expression.
Figure 4. Effects of nintedanib in healthy human kidney. Human PCKS were cultured in the presence of nintedanib
(0.1 – 5 µM) for 48h. (A) Tyrosine kinase receptors (RTKs) mRNA expression was measured by qPCR. (B) Phosphorylation of RTKs was measured by multiplex magnetic bead assay and expressed as relative mean fluorescence intensity (MFI).
(C) mRNA expression of fibrosis markers after 48h incubation. (D) Representative sections with interstitial collagen type I
accumulation visualized by immunohistochemistry (scale bar = 100 µm) and quantitative analysis of staining intensity. (E) Protein levels HSP47 and α-SMA at 48h with representative Western blot images. (F) mRNA expression of inflammation markers after 48h incubation. (G) Expression of cell proliferation marker Ki-67 in human PCKS during culture and after 48h treatment with 5 µM nintedanib was visualized by immunohistochemistry and quantified as relative intensity values. Data are expressed as mean (± SEM), n=4-5, *p < 0.05.
FLT1, Fms related tyrosine kinase 1; KDR, kinase insert domain receptor; PDGFRB, platelet derived growth factor receptor beta; PDGFRA, platelet derived growth factor receptor alpha; VEGFR1, vascular endothelial growth factor receptor 1; VEGFR2, vascular endothelial growth factor receptor 2; COL1A1, collagen type I alpha 1; SERPINH1, serine proteinase inhibitor clade H (Heat Shock Protein 47) member 1; FN1, fibronectin 1; ACTA2, alpha 2 smooth muscle actin; HSP47, heat shock protein 47; α-SMA, alpha smooth muscle actin; TNF, tumor necrosis factor; IL-1B, interleukin 1 beta; IL-6, interleukin 6; CXCL8, C-X-C motif chemokine ligand 8 (IL-8, interleukin 8); hPCKS, human precision-cut kidney slices.
0 0.1 0.3 0.5 1 5 0.00 0.05 0.10 0.15 0.20 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) KDR (VEGFR2)
0h control 48h control 48h nintedanib 5 μM
D E 0 0.1 0.3 0.5 1 5 0.00 0.05 0.10 0.15 0.20 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) ACTA2 Nintedanib [μM] HSP47 (47 kD) α-SMA (42 kD) 0 0.1 0.3 0.5 1 0 5 0 0.1 0.3 0.5 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) α-SMA 0 0.1 0.3 0.5 1 5 0.00 0.05 0.10 0.15 0.20 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) SERPINH1 * 0 0.1 0.3 0.5 1 5 0.00 0.02 0.04 0.06 0.08 0.10 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) FN1 * * 0 5 0.0 0.5 1.0 1.5 (0h) Nintedanib [µM] R el at ive i nt en si ty ( fo ld ) COL I * 0 0.1 0.3 0.5 1 5 0.000 0.002 0.004 0.006 0.008 0.010 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) TNF * 0 0.1 0.3 0.5 1 5 0.000 0.005 0.010 0.015 0.020 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-1B * * * 0 0.1 0.3 0.5 1 5 0.00 0.05 0.10 0.15 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-6 * 0 0.1 0.3 0.5 1 5 0.00 0.20 0.40 0.60 0.80 1.00 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) CXCL8/ IL-8 * 0 0.1 0.3 0.5 1 5 0.00 0.02 0.04 0.06 0.08 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) COL1A1 * * * F 0 0.1 0.3 0.5 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) HSP47 *
0h control 48h control 48h nintedanib 5 μM
G 0 48 0 5 10 15 20 25
Time of incubation [hours]
R el at ive i nt en si ty ( fo ld ) Ki-67 hPCKS * 0 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive i nt en si ty ( fo ld ) Ki-67 hPCKS * 0 0.1 0.3 0.5 1 5 0.00 0.02 0.04 0.06 0.08 0.10 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) PDGFRB * * * * 0 0.1 0.3 0.5 1 5 0.00 0.01 0.02 0.03 0.04 0.05 (0h) Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) FLT1 (VEGFR1) * * * * * 0 0.1 0.3 0.5 1 5 0 50 100 150 (0h) Nintedanib [µM] % rel at ive M FI Phospho-PDGFRA * * * * 0 0.1 0.3 0.5 1 5 0 50 100 150 (0h) Nintedanib [µM] % rel at ive M FI Phospho-PDGFRB * * * * * 0 0.1 0.3 0.5 1 5 0 50 100 150 (0h) Nintedanib [µM] % rel at ive M FI Phospho-VEGFR1 * * * * 0 0.1 0.3 0.5 1 5 0 50 100 150 (0h) Nintedanib [µM] % rel at ive M FI Phospho-VEGFR2 * * * * * B C A
5
Similar to mPCKS, a substantial increase in mRNA levels of cytokines, such as TNF, IL-1B, IL-6 and CXCL8/
IL-8, occurred in hPCKS during culture period (Supplementary Figure S4C). Nintedanib significantly reduced the expression of these inflammation markers at the highest tested concentration (5 μM) (Figure 4F). Interestingly, IL-1B mRNA level was inhibited at 0.5 μM.
Antiproliferative activity of nintedanib in hPCKS
We observed comparable results of Ki-67 expression in hPCKS, as in mPCKS: expression increased during culture (fold induction of 10.4), and nintedanib reduced proliferation by approximately 73% (Figure 4G).
Nintedanib in established fibrosis PCKS
Characterization of fibrotic human PCKSFibrotic hPCKS (fhPCKS) showed high basal gene expression of COL1A1, SERPINH1 and FN1, as well
as clear inflammatory profile compared to healthy kidneys (Figure 5A and B). Histologic analysis
confirmed the fibrotic phenotype by showing an extensive tubular atrophy, ECM accumulation and
interstitial fibrosis (Figure 5C and D).
To analyze the processes occurring during culture of fhPCKS, we studied viability and gene expression up to 72h. Similar to healthy human PCKS [21], ATP content of fhPCKS increased during first 24h, after
which levels plateaued (Figure 5E). As described earlier, healthy PCKS develop a fibrotic response
during incubation. We observed a different pattern in fhPCKS: mRNA expression of COL1A1, SERPINH1
and FN1 remained unchanged (Figure 5F). On the other hand, elevated collagen type I deposition
and highly increased protein expression of HSP47 might indicate that fibrogenesis was still ongoing
during incubation (Figure 5G and H). Similar to hPCKS, ACTA2 expression dropped in fhPCKS during
culture, while α-SMA protein expression remained unchanged. Regarding the inflammation markers, fhPCKS showed unaffected gene expression of TNF and IL-1B during culture. Levels of IL-6 and CXCL8/
IL-8 increased at 24h and then gradually declined (Figure 5I).
Figure 5. Characterization of fibrotic human precision-cut kidney slices. PCKS were prepared from fibrotic human
renal tissue obtained during ESRD nephrectomies or transplantectomies. (A) Baseline (prior incubation) mRNA expression of fibrosis markers in fibrotic compared to healthy tissue slices. (B) Baseline (prior incubation) mRNA expression of inflammation markers in fibrotic compared to healthy tissue slices. (C) Representative photomicrographs of human healthy and fibrotic PCKS prior incubation (scale bar = 250 μm). Histologic analyses by PAS and PSR staining showing extensive tubular atrophy and interstitial fibrosis, while α-SMA and collagen type I immunohistochemistry further confirmed the fibrotic phenotype. (D) Quantitative analysis of collagen type I immunohistochemistry in healthy and fibrotic PCKS (prior incubation). (E) Viability of fibrotic PCKS during incubation presented as the average of pmol ATP per μg total protein. (F) Effect of incubation on mRNA expression of fibrosis markers. (G) Representative images of immunohistochemistry of collagen type I (scale bar = 100 μm) in fibrotic PCKS during 72h culture with quantitative analysis. (H) Protein levels of HSP47 and α-SMA (n=5) during incubation with representative Western blot images. (I) Effect of incubation on mRNA expression of inflammation markers in fhPCKS. Data are expressed as mean (± SEM), n=9 for healthy PCKS and n=8-9 for fibrotic PCKS, *p < 0.05.
Healthy Fibrotic 0.00 0.05 0.10 0.15 0.20 0.25 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) COL1A1 * Healthy Fibrotic 0.00 0.20 0.40 0.60 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) ACTA2 Healthy Fibrotic 0.00 0.05 0.10 0.15 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) SERPINH1 Healthy Fibrotic 0.00 0.02 0.04 0.06 0.08 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) CXCL8/ IL-8 * Healthy Fibrotic 0.000 0.005 0.010 0.015 0.020 0.025 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) TNF * Healthy Fibrotic 0.00 0.02 0.04 0.06 0.08 0.10 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-1B * Healthy Fibrotic 0.000 0.005 0.010 0.015 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-6 * A B Healthy Fibrotic 0.00 0.50 1.00 1.50 2.00 m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) FN1 * H eal thy k idney Fi br ot ic k id ne y
PAS PSR α-SMA COL I
C Healthy Fibrotic 0 10 20 30 St ain in g in te ns ity , % COL I * D 0 24 48 72 0 5 10 15
Incubation time (hours)
A TP /P rot ei n ( pm ol /ug) Viability fhPCKS E 0 24 48 72 0.00 0.10 0.20 0.30 0.40 0.50
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) COL1A1 0 24 48 72 0.00 0.05 0.10 0.15 0.20
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) SERPINH1 0 24 48 72 0.000 0.005 0.010 0.015 0.020
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) TNF 0 24 48 72 0.00 0.05 0.10 0.15 0.20
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-1B F 0 24 48 72 0.00 0.20 0.40 0.60
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) ACTA2 * * * 0 24 48 72 0.00 0.50 1.00 1.50
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) FN1 0 24 48 72 0.00 0.10 0.20 0.30
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-6 * * 0 24 48 72 0.00 0.50 1.00 1.50 2.00
Incubation time (hours)
m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) CXCL8/ IL-8 * * I G 0h 24h 48h 72h 0 24 48 72 0 1 2 3 4
Time of incubation [hours]
R el at ive i nt en si ty ( fo ld ) COL I Hours HSP47 (47 kD) α-SMA (42 kD) 0 24 48 72 0 24 48 72 0.0 0.5 1.0 1.5 2.0
Incubation time (hours)
R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) α-SMA H 0 24 48 72 0 200 400 600
Incubation time (hours)
R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) HSP47 * *
5
Effect of nintedanib on fibrosis and inflammation markersTreatment of fhPCKS with nintedanib did not affect fibrosis markers on gene expression. Only the highest tested concentration (5 μM) numerically (but not statistically significantly) reduced expression
of COL1A1 and SERPINH1 (Figure 6A). We detected non-significant effects on interstitial accumulation
of collagen type I and protein expression of α-SMA, however, nintedanib at 5 μM significantly affected
HSP47 (Figure 6B and C). Interestingly, 1 and 5 μM nintedanib downregulated IL-1B in fhPCKS by 82.5%
and 86.3%, respectively (Figure 6D).
Antiproliferative activity of nintedanib in fhPCKS
Similar to hPCKS, culture for 48h induced Ki-67 expression in fhPCKS (Figure 6G), while nintedanib
inhibited cell proliferation by approximately 48%.
Figure 6. Antifibrotic and anti-inflammatory effect of nintedanib in fibrotic human precision-cut kidney slices.
Fibrotic human PCKS were cultured in the presence of nintedanib (0.1 – 5 μM) for 48h. (A) mRNA expression of fibrosis markers after 48h incubation. (B) Representative sections with interstitial collagen type I accumulation visualized by immunohistochemistry (scale bar = 100 µm) and quantitative analysis of staining intensity. (C) Protein levels HSP47 and α-SMA at 48h with representative Western blot images. (D) mRNA expression levels of inflammation markers after 48h incubation. (E) Expression of cell proliferation marker Ki-67 in fibrotic human PCKS during culture and after 48h treatment with 5 µM nintedanib was visualized by immunohistochemistry and quantified as relative intensity values.
Data are expressed as mean (± SEM), n=4-5, *p < 0.05.
48h control 48h nintedanib 5 μM B C Nintedanib [μM] HSP47 (47 kD) α-SMA (42 kD) 0 0.1 0.3 1 5 0 0.1 0.3 1 5 0.00 0.05 0.10 0.15 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) SERPINH1 0 0.1 0.3 1 5 0.00 0.10 0.20 0.30 0.40 0.50 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) FN1 0 0.1 0.3 1 5 0.00 0.02 0.04 0.06 0.08 0.10 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) ACTA2 0 0.1 0.3 1 5 0.000 0.005 0.010 0.015 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) TNF 0 0.1 0.3 1 5 0.00 0.10 0.20 0.30 0.40 0.50 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-6 0 0.1 0.3 1 5 0.00 0.50 1.00 1.50 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) CXCL8/ IL-8 0 0.1 0.3 1 5 0.0 0.5 1.0 1.5 2.0 2.5 Nintedanib [µM] R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) α-SMA 0 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive i nt en si ty ( fo ld ) COL I 0 0.1 0.3 1 5 0.000 0.005 0.010 0.015 0.020 0.025 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) IL-1B * * 0 0.1 0.3 1 5 0.00 0.10 0.20 0.30 Nintedanib [µM] m R N A exp ressi on , 2 -Δ Ct (nor m al iz ed t o G apdh) COL1A1 D 0 0.1 0.3 1 5 0.0 0.5 1.0 1.5 2.0 Nintedanib [µM] R el at ive p ro tei n exp ressi on (nor m al iz ed t o t ot al pr ot ei n) HSP47 * A
0h control 48h control 48h nintedanib 5 μM
E 0 48 0 1 2 3
Time of incubation [hours]
R el at ive i nt en si ty ( fo ld ) Ki-67 fhPCKS * 0 5 0.0 0.5 1.0 1.5 Nintedanib [µM] R el at ive i nt en si ty ( fo ld ) Ki-67 fhPCKS *
5
DISCUSSION
PCKS provide a unique opportunity to translate the obtained results from rodent models to human, which is important for clinical drug development [30]. In this study we expanded the experimental application of PCKS: we used healthy renal tissue of murine and human origin, and explored PCKS from human fibrotic renal tissue as a model of established fibrosis. With this translational ex vivo model, we investigated the effects of nintedanib in healthy and diseased tissue. We demonstrated that the onset of fibrogenesis in PCKS approximates in vivo fibrogenesis, making this model suitable to study the mechanism of action and efficacy of antifibrotic compounds. We showed that nintedanib blocks the expression and phosphorylation of tyrosine kinase receptors and inhibits cell proliferation. Additionally, nintedanib attenuated the onset of fibrosis not only in murine, but also in human PCKS, although reversal of established fibrosis could not be achieved.
Nintedanib engaged its intended targets in human kidneys: it inhibited gene expression of PDGFRβ, VEGFR1 and 3, as well as phosphorylation of PDGFRa and β, VEGFR1 and 2 starting at 0.1 μM — a concentration that is in the range of its maximum human exposure of 0.07 µM in patients with IPF after standard dosing [31–33]. The attenuation of PDGFRa and β signalling by nintedanib is of therapeutic interest for renal fibrosis, as PDGF signalling leads to the differentiation of pericytes and resident fibroblasts to profibrotic ECM-producing myofibroblasts [11]. The literature presents conflicting results on the role of VEGFR signalling in fibrosis and peritubular capillary restoration [34– 37]. Therefore, beneficial inhibition of VEGFR signalling by nintedanib in renal fibrosis is subject to further studies.
The inhibitory effects of nintedanib on RTKs were investigated by Liu et al. in a unilateral ureteral obstruction (UUO) mouse model [38]. They reported that nintedanib effectively blocked UUO-induced phosphorylation of PDGFRβ, VEGFR2, FGFR1 and FGFR2. In human PCKS, both FGFR1 and FGFR2 were not affected by nintedanib, possibly due to the differences in fibrogenic processes in murine and human tissues.
PCKS develop an early inflammatory response during culture, followed by the onset of fibrosis [21]. Nintedanib exerted anti-inflammatory activity in mPCKS and hPCKS. The observed effects are in line with earlier studies of nintedanib in mouse models of lung fibrosis [28,39]. This anti-inflammatory activity of nintedanib might translate into attenuation of renal injury.
Nintedanib also exerted antifibrotic effects in mPCKS and hPCKS, demonstrated by a marked reduction of collagen 1a1 mRNA and protein expression. Furthermore, in mPCKS nintedanib modulates ECM homeostasis even at the lowest concentration. Downregulation of these genes might lead to the altered secretion and fibril formation of collagen, as reported in primary human lung fibroblasts treated with TGFβ [40]. High concentrations of nintedanib regulate different mRNA clusters than low concentrations, reflected by a partial switch from inhibitory profile at 0.1 and 0.5 µM to the induction of some ECM related genes at 1 and 5 µM. This could be explained by possible non-selective activity of nintedanib at high concentrations, while low concentrations have a more specific kinase inhibitory
profile [41]. Despite that nintedanib at 1 and 5 µM has an altered impact on ECM homeostasis, its overall effect remains antifibrotic.
The demonstrated attenuation of fibrosis concurs with previous results: nintedanib reduced lung fibrosis in bleomycin- or silica-treated mice and rats [16,28,42] and showed antifibrotic effects in various mouse models of systemic sclerosis [43,44]. Liu et al. [38] found that administration of nintedanib for 7 days after UUO injury attenuated renal fibrosis. Nintedanib inhibited TGFβ1 induced renal fibroblasts-to-myofibroblasts transition and expression of ECM proteins in vitro in renal interstitial fibroblasts, indicating that nintedanib affects early events of TGFβ signalling. Wollin et al. also reported that nintedanib at higher concentrations possesses anti-TGFβ activity [16,28]. We hypothesize that the observed antifibrotic effects of nintedanib in PCKS might be attributed to a combination of RTK inhibition and, perhaps non-selective, anti-TGFβ activity.
The culture of mPCKS, hPCKS and fhPCKS induced a strong spontaneous proliferative response and in line with published data [16,28,29], nintedanib effectively inhibited culture-induced cell proliferation in PCKS.
Our newly established translational PCKS model with tissue from fibrotic human kidneys showed a clear fibrotic phenotype compared to renal tissue from control donors. Culture of fhPCKS did not further increase the assessed markers of fibrosis, although an inflammatory peak was observed after the first day of culture. The pre-existing fibrotic phenotype of fhPCKS might explain the difference with healthy PCKS.
Nintedanib showed diminished reduction in fibrosis markers in fhPCKS compared to hPCKS, most likely due to an increased interindividual variability (underlying primary renal disease, dialysis time, time since transplantation and medication) in the fibrotic kidney slices. Nintedanib seems to be more effective in preventing or halting the onset of fibrosis in healthy PCKS rather than in reversing the established fibrosis as modelled by fhPCKS. Our data in human diseased tissue is in conflict with the data in murine diseased tissue: delayed administration of nintedanib to UUO mice by Liu et al. [38] resulted in partial reversal of established renal fibrosis. However, even prolonged kidney obstruction in mice is not as severe as the late-stage fibrosis seen in CKD patients, emphasizing the need for models that more closely resemble human pathology. PCKS, especially the culture of fhPCKS, can serve as a model for human CKD. Nevertheless, limitations of the ex vivo PCKS model are: (1) relatively short culture period (48-72h) is not always sufficient to detect post-translational events; (2) circulating immune cells that contribute to the fibrogenesis are absent; and (3) interorgan interactions cannot be directly assessed.
5
Taken together, our results demonstrate the pharmacological effects of nintedanib in an ex vivo model of renal fibrosis, facilitating translation from animal studies to the clinic. Treatment of PCKS with nintedanib inhibited cell proliferation and attenuated the onset of inflammation and fibrosis, although reversal of established fibrosis could not be achieved. Nintedanib successfully inhibited PDGFR and VEGFR phosphorylation, demonstrating the potential use of these receptors as therapeutic targets to attenuate renal fibrosis. Therefore, along with the benefit of reducing animal use, human PCKS might provide direct and clinically relevant insights into human renal disease and therapeutic strategies.
ACKNOWLEDGEMENTS
The authors thank the abdominal transplantation surgeons of the University Medical Center Groningen for providing the human renal tissue. This work was kindly supported by ZonMw (the Netherlands Organization for Health Research and Development), grant number 114025003 and by Lundbeckfonden, grant number R231-2016-2344 (received by H.A.M.M).
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SUPPLEMENTARY MATERIALS
Supplementary Figure S1. Spontaneous fibrogenic and inflammatory response in murine PCKS during culture. (A) mRNA expression of fibrosis markers. (B) Protein levels of HSP47 and α-SMA with representative Western blot images.
(C) mRNA expression of inflammation markers. Data are expressed as mean (± SEM), n=4-5. Gene expression levels were compared using unpaired Student’s t-test; protein levels were compared using non-parametric Mann-Whitney test, *p < 0.05.
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Supplementary Figure S2. IC20 and IC50 of nintedanib in murine and human PCKS. Nintedanib-induced inhibition
of mRNA expression of fibrosis markers after 48h incubation in murine PCKS (A) and in human PCKS (B). IC20 and IC50 values were calculated by fitting the data to a four-parameter log(inhibitor) vs response curve. Data points are the mean (± SEM), n=3-5.
Supplementary Figure S3. Tyrosine kinase receptors expression and activation in human PCKS. In hPCKS cultured
for 48h, (A) the mRNA expression of tyrosine kinase receptors was measured by rt-qPCR, and (B) phosphorylation was measured by multiplex magnetic bead assay and expressed as relative mean fluorescence intensity (MFI) to 0h control. As an addition to Figure 3, in hPCKS treated with nintedanib (0.1 – 5 µM) for 48h, (C) the mRNA expression of FLT4 and FGFR2 was measured by rt-qPCR and (D) phosphorylation of VEGFR3, FGFR1 and FLT3 was measured by multiplex magnetic bead assay. Data are expressed as mean (± SEM), n=4-5, *p < 0.05.
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Supplementary Figure S4. Spontaneous fibrogenic and inflammatory response in human PCKS during culture. (A) mRNA expression of fibrosis markers. (B) Protein levels of HSP47 and α-SMA with representative Western blot images. (C) mRNA expression of inflammation markers. Data are expressed as mean (± SEM), n=4-5. Gene expression levels were
compared using unpaired Student’s t-test; protein levels were compared using non-parametric Mann-Whitney test, *p < 0.05.
Supplementary Table S1. List of Taqman gene expression assays used for qRT-PCR
Hum an COL1A1 Hs00164004_m1 ACTA2 Hs00426835_g1 SERPINH1 Hs01060397_g1 FN1 Hs01549976_m1 IL6 Hs00985639_m1 TNF Hs01113624_g1 IL1B Hs01555410_m1 IL8 Hs00174103_m1 FLT1 Hs01052961_m1 KDR Hs00911700_m1 FLT4 Hs00176607_m1 FGFR2 Hs01552918_m1 PDGFRB Hs01019589_m1 TGFBR1 Hs00610320_m1 Mouse Col1a1 Mm00801666_g1 Acta2 Mm00725412_S1 Serpinh1 Mm00438058_g1 Fn1 Mm01256744_m1 Il1b Mm00434228_m1 Il6 Mm04207460_m1 Cxcl1 Mm04207460_m1 Tnf Mm00443258_m1
