University of Groningen
Inhibition of tyrosine kinase receptor signaling attenuates fibrogenesis in an ex vivo model of
human renal fibrosis
Bigaeva, Emilia; Stribos, Elisabeth G. D.; Mutsaers, Henricus A. M.; Piersma, Bram; Leliveld,
Anna M.; de Jong, Igle J.; Bank, Ruud A.; Seelen, Marc A.; van Goor, Harry; Wollin, Lutz
Published in:American journal of physiology-Renal physiology
DOI:
10.1152/ajprenal.00108.2019
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below.
Document Version
Final author's version (accepted by publisher, after peer review)
Publication date: 2020
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Bigaeva, E., Stribos, E. G. D., Mutsaers, H. A. M., Piersma, B., Leliveld, A. M., de Jong, I. J., Bank, R. A., Seelen, M. A., van Goor, H., Wollin, L., Olinga, P., & Boersema, M. (2020). Inhibition of tyrosine kinase receptor signaling attenuates fibrogenesis in an ex vivo model of human renal fibrosis. American journal of physiology-Renal physiology, 318(1), F117-F134. https://doi.org/10.1152/ajprenal.00108.2019
Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).
Take-down policy
If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim.
Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
Inhibition of tyrosine kinase receptor signaling attenuates
1
fibrogenesis in an ex vivo model of human renal fibrosis
2 3
Emilia Bigaeva a,1, Elisabeth G.D. Stribos a,b,1, Henricus A.M. Mutsaers a,c, Bram Piersmae, Anna M.
4
Leliveld d, Igle J. de Jong d, Ruud A. Bank e, Marc A. Seelen b, Harry van Goor e, Lutz Wollin f, Peter
5
Olinga a,*, Miriam Boersema a.
6 7
a Department of Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen
8
Research Institute of Pharmacy, the Netherlands 9
b Department of Internal Medicine, Division of Nephrology, University of Groningen, University
10
Medical Center Groningen, the Netherlands 11
c Department of Clinical Medicine, Aarhus University, Denmark
12
d Department of Urology, University of Groningen, University Medical Center Groningen, the
13
Netherlands 14
e Department of Pathology and Medical Biology, University of Groningen, University Medical Center
15
Groningen, the Netherlands 16
f Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany
17 18
1 These authors contributed equally to this work
19
*Corresponding author: 20
Prof. Peter Olinga, Department of Pharmaceutical Technology and Biopharmacy, University of 21
Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, the Netherlands. Tel: +31 50 363 8373. E-22
mail: p.olinga@rug.nl 23
24
Supplemental Material available at 25
URL: https://figshare.com/s/87cd11d7a729a236a8c6 26
DOI: https://doi.org/10.6084/m9.figshare.9999557 27
ABSTRACT 28
Poor translation from animal studies to human clinical trials is one of the main hurdles in the 29
development of new drugs. Here, we used precision-cut kidney slices (PCKS) as a translational model 30
to study renal fibrosis and to investigate whether inhibition of tyrosine kinase receptors, with the 31
selective inhibitor nintedanib, can halt fibrosis in murine and human PCKS. We used renal tissue of 32
murine and human origin to obtain PCKS. Control slices and slices treated with nintedanib were 33
studied to assess viability, activation of tyrosine kinase receptors, cell proliferation, collagen type I 34
accumulation, gene and protein regulation. During culture, PCKS spontaneously develop a fibrotic 35
response that resembles in vivo fibrogenesis. Nintedanib blocked culture-induced phosphorylation of 36
platelet-derived growth factor receptor and vascular endothelial growth factor receptor. Furthermore, 37
nintedanib inhibited cell proliferation, reduced collagen type I accumulation and expression of 38
fibrosis-related genes in healthy murine and human PCKS. Modulation of extracellular matrix 39
homeostasis was achieved already at 0.1 μM, while high concentrations (1 and 5 μM) elicited possible 40
non-selective effects. In PCKS from human diseased renal tissue, nintedanib showed limited capacity 41
to reverse established fibrosis. In conclusion, nintedanib attenuated the onset of fibrosis in both 42
murine and human PCKS by inhibiting the phosphorylation of tyrosine kinase receptors; however, the 43
reversal of established fibrosis was not achieved. 44
45
KEYWORDS 46
Renal fibrosis; precision-cut kidney slices; tyrosine kinase receptor; nintedanib 47
48 49 50
INTRODUCTION 51
Renal fibrosis, defined by the progressive deposition of connective tissue, is a hallmark of chronic 52
kidney disease (CKD), which affects an estimated 10% of the population in developed countries (19). 53
CKD progresses to end-stage renal disease (ESRD), that eventually requires replacement therapy — 54
dialysis or transplantation. Current research investigates strategies to halt CKD progression, or even 55
to reverse renal fibrosis (6, 37). Yet, no effective therapy has been clinically implemented. 56
Pathological activation of various receptor tyrosine kinases (RTKs) — such as platelet-derived growth 57
factor (PDGF), fibroblast growth factor (FGF) and epidermal growth factor (EGF) receptors — plays 58
a key role in renal fibrogenesis (3, 5, 29, 36, 44). The PDGF receptor (PDGFR) is an attractive 59
molecular target for antifibrotic therapies (8), since PDGFR signaling is involved in 60
(trans)differentiation of collagen-producing myofibroblasts (7, 9, 18). The receptors PDGFRα and β 61
are expressed in renal tissue mainly by glomerular mesangial cells, interstitial fibroblasts and vascular 62
smooth-muscle cells (4). Several studies reported an increased expression of both receptors in murine 63
and human renal disease (7, 12). The activation of the PDGFR leads to glomerulosclerosis and 64
(tubulo)interstitial fibrosis (29). Therefore, blocking PDGFR signaling is a promising strategy to halt 65
the progression of renal fibrosis. 66
Nintedanib is a small molecule tyrosine kinase inhibitor, approved in several countries worldwide for 67
the treatment of idiopathic pulmonary fibrosis (IPF) and for the second line treatment of non-small-68
cell lung carcinoma with adenocarcinoma histology. Nintedanib affects signaling pathways of 69
multiple growth factors, including vascular endothelial growth factor (VEGF), FGF and PDGF, as 70
well as Lck and Src non-receptor kinases (32, 41). In a phase II randomized clinical trial, nintedanib 71
showed anti-angiogenic effects and had an acceptable safety profile in patients with advanced renal 72
cell carcinoma (10). To our knowledge, the impact of nintedanib on human renal fibrosis has not been 73
published. 74
The lack of translational models of human renal fibrosis hampers the search for effective antifibrotic 75
therapies (33). In vitro models lack cellular heterogeneity, and animal models have limited 76
implications for human disease. To partly overcome these limitations, we used precision-cut kidney 77
slices (PCKS) as an ex vivo model of renal fibrosis (13, 34, 43). PCKS replicate the organotypic 78
multicellular characteristics, as one slice maintains the complex three-dimensional architecture of the 79
kidney, and have a high translational impact, as both murine and human tissue, healthy and diseased, 80
can be used. In addition, PCKS culture is reproducible and allows for a substantial reduction of 81
animal use, making it a promising preclinical tool for drug development. 82
In this study, we aimed to investigate therapeutic effects of nintedanib in PCKS, and to find whether 83
inhibition of nintedanib’s molecular targets may prevent renal fibrosis in murine and, more 84
importantly, in human kidneys. 85
MATERIALS AND METHODS 87
Ethics statement 88
This study was approved by the Medical Ethical Committee of the University Medical Centre 89
Groningen (UMCG), according to Dutch legislation and the Code of Conduct for dealing responsibly 90
with human tissue in the context of health research (www.federa.org), forgoing the need of written 91
consent for ‘further use’ of coded-anonymous human tissue. 92
The animal experiments were approved by the Animal Ethics Committee of the University of 93
Groningen (DEC 6416AA-001). 94
95
Renal tissue 96
Macroscopically healthy renal cortical tissue (n=9) was obtained from tumor nephrectomies, and 97
fibrotic renal tissue (n=10) was obtained from ESRD nephrectomies or transplantectomies. Table 1 98
summarizes patient demographics. Renal tissue was stored in ice-cold University of Wisconsin (UW) 99
organ preservation solution, and cold ischemia time was limited to 2-3 hours. 100
Murine tissue was obtained from male C57BL/6 mice, with an average weight of 28.3 g (± 2.4) and 101
12.1 weeks of age (± 2.2). The animals were housed in filter-top cages with free access to water and 102
food. Kidneys were harvested via a terminal procedure performed under isoflurane/O2 anesthesia
103
(Pharmachemie BV, Haarlem, the Netherlands) and stored in ice-cold UW solution until further use. 104
105
Preparation and treatment of precision-cut kidney slices 106
PCKS were prepared according to the protocol by Poosti et al. (mouse; (30)) and Stribos et al. (human; 107
(34)), using a Krumdieck tissue slicer. Slices were incubated in Williams’ Medium E with GlutaMAX 108
(Life Technologies, Carlsbad, California, USA) containing 10 μg/mL ciprofloxacin and 26 mM 109
glucose, at 37°C in a 80% O2/5% CO2 atmosphere while gently shaken. Culture medium was used
110
without serum supplementation to avoid the associated batch-to-batch variability (i.e., to establish 111
fully controlled environment). Nintedanib was kindly provided by Boehringer Ingelheim (Biberach, 112
Germany). We treated murine or human PCKS with nintedanib (0.1 – 10 μM) for 48h. Analyses were 113
performed using three pooled slices from the same animal/donor (technical replicates) from at least 114
three to five animals or donors (biological replicates). 115
116
Viability of PCKS 117
Viability of the slices was assessed by measuring ATP content using the ATP bioluminescence kit 118
(Roche Diagnostics, Mannheim, Germany), as previously described (39). 119
120
Quantitative real-time PCR and low-density array 121
Total RNA was extracted with the RNeasy mini kit (Qiagen, Venlo, the Netherlands) and reverse 122
transcribed using the Reverse Transcription System (Promega, Leiden, the Netherlands). cDNA was 123
used for quantitative real-time PCR performed with a Viia7 Real-Time PCR system (Applied 124
Biosystems, Bleiswijk, the Netherlands). Gene expression was calculated using the 2-ΔCt method (26)
125
and corrected for GAPDH. The Taqman gene expression assays used in this study are listed in 126
Supplementary Table S1. 127
The expression of 44 genes related to fibrosis (Supplementary Table S2) was examined using a 128
custom-designed low-density array (LDA, Applied Biosystems) (31). cDNA from renal tissue of 129
C57BL/6 control mice that underwent UUO microsurgery was kindly provided by Dr. Bram Piersma. 130
A total of 100 μl reaction mixture containing 6 ng/μl cDNA and 50 μl 2x Taqman Universal PCR 131
Master Mix (Applied Biosystems) was loaded per sample. PCR amplification was performed on a 132
Viia7 Real-Time PCR system (Applied Biosystems). 133
134
Western blotting 135
Total protein was extracted from PCKS with ice-cold RIPA buffer (Thermo Scientific, Waltham, 136
Massachusetts, USA) supplemented with a protease inhibitor cocktail and PhosStop (Sigma-Aldrich, 137
Saint Louis, Missouri, USA). A total of 80-100 μg of protein was separated via SDS-PAGE using 10% 138
polyacrylamide gels and blotted onto polyvinylidene fluoride membranes (Trans-Blot Turbo Transfer 139
System, Bio-Rad, Veenendal, the Netherlands). 2,2,2-trichloroethanol (TCE; Sigma-Aldrich) allowed 140
for visible detection of total protein load (22). Membranes were blocked in 5% non-fat milk/TBST 141
(Bio-Rad) and incubated with the primary antibody (Supplementary Table S3) overnight at 4˚C, 142
followed by incubation with the appropriate HRP-conjugated secondary antibody. Protein bands were 143
visualized using Clarity Western ECL Substrate (Bio-Rad) and ChemiDoc Touch Imaging System 144
(Bio-Rad). Protein expression was corrected for total protein and expressed as a relative value to the 145
control group. 146
147
Phosphoproteomic analysis of RTKs by multiplex 148
A human RTK phosphoprotein magnetic bead panel (Merck Millipore, Billerica, Massachusetts, 149
USA), was used according to manufacturer’s instructions. Total protein was extracted using the 150
supplied lysis buffer supplemented with protease inhibitor cocktail. Samples were diluted to a 151
concentration of 1.5 μg/μl and passed through a 0.45 μm syringe filter (Whatman, Maidstone, UK). 152
Detection was performed with the MAGPIX multiplexing instrument (Luminex, Austin, Texas, USA). 153
Mean fluorescent intensity (MFI) was used for quantification. 154
155
Histology 156
PCKS were fixed in 4% buffered formalin, embedded in paraffin and sectioned 2 μm thick. Tissue 157
damage and renal fibrosis were assessed by Periodic acid–Schiff (PAS) and Picro Sirius Red (PSR) 158
staining. Additionally, we performed immunohistochemistry for Ki-67, α-SMA and collagen type I. 159
After deparaffinisation and antigen retrieval with 0.1 M Tris-EDTA (pH 9.0) in microwave oven for 160
15 min, tissue sections were blocked with 2% rat serum in PBS/2% BSA for 10 min and then 161
incubated with primary antibodies (Supplementary Table S3) for 1h. The antibodies were localized 162
using the appropriate HRP-conjugated secondary and tertiary antibodies and the ImmPact NovaRed 163
kit (Vector, Burlingame, USA), followed by hematoxylin counterstaining. Stained tissue sections were 164
scanned using a Nanozoomer Digital Pathology Scanner (NDP Scan U10074-01, Hamamatsu 165
Photonics K.K., Japan). To quantify the stained areas, the whole-slide images were processed with 166
Aperio ImageScope v12.3 (Aperio Technologies, Vista, CA) by applying the Positive Pixel Count V9 167
algorithm (hue value set to 0). The intensities were measured as percentages − number of positive and 168
strong positive pixels divided by the total number of pixels − and expressed as relative values to the 169 control group. 170 171 Statistics 172
The results are expressed as mean ± standard error of mean (SEM) of minimum 3 independent 173
experiments. Statistics were performed using GraphPad Prism 6.0 (GraphPad Software Inc.) by 174
unpaired Student’s t-test or one-way ANOVA followed by Dunnett’s multiple comparisons test. The 175
protein levels of HSP47 and α-SMA were compared using non-parametric Kruskal-Wallis test, 176
followed by Dunn’s multiple comparisons test. Differences between groups were considered to be 177
statistically different when p < 0.05. For the LDA heatmap, average-linkage clustering was performed 178
using Pearson correlation. The heatmap was generated using the online tool Morpheus 179
(https://software.broadinstitute.org/morpheus/). 180
RESULTS 182
A brief summary of the study workflow is presented in Figure 1A. Murine and human PCKS remained 183
viable during 48h incubation, as reflected by unchanged ATP and total protein content (Figure 1B). In 184
addition, PAS staining revealed typical structural changes in slices due to the culturing in accordance 185
with previously reported results (34, 35). In particular, murine and human PCKS from healthy kidneys 186
showed signs of cellular damage (i.e., pyknosis and anucleosis), tubulointerstitial injury and 187
glomerular injury at 48h. In turn, culturing of human fibrotic PCKS induced further expansion of 188
interstitial ECM, tubular atrophy and glomerular sclerosis. Nintedanib at 5 μM showed no indication 189
towards worsening of the morphology (Figure 1C). 190
191
Nintedanib in murine PCKS 192
Mitigation of fibrosis and inflammation by nintedanib
193
During 48h incubation, spontaneous onset of fibrosis occurred in mPCKS, as reflected by an 194
upregulation of mRNA levels of collagen type I, fibronectin and heat shock protein HSP47 (encoded 195
by Col1a1, Fn1 and Serpinh1, respectively) and by increased protein levels of HSP47 (Figure 2A and 196
B). Nintedanib effectively mitigated fibrogenesis as it clearly reduced Col1a1 gene expression (Figure 197
3A), with an IC50 of 0.7 μM (Supplementary Figure S1A). Furthermore, nintedanib reduced mRNA
198
levels of Fn1 and Acta2 (IC50 of 4.4 μM and 6.2 μM, respectively), while it affected Serpinh1 only at
199
the highest concentration (IC50=5.8 μM). Treatment with 5 μM nintedanib significantly decreased
200
interstitial accumulation of collagen type I and protein expression of HSP47, but did not affect α-SMA 201
(Figure 3B and C). Collagen mRNA and protein levels declined below baseline expression (at 0h) 202
when mPCKS were treated with 5 and 10 μM nintedanib. Spontaneous onset of fibrosis in mPCKS 203
was accompanied by an inflammatory response after 48h (Figure 2C). We observed a significant 204
decrease in mRNA levels of tumor necrosis factor, interleukin-1 beta and interleukin-6 (encoded by 205
Tnf, Il-1β and Il-6, respectively) in the presence of nintedanib, while Cxcl1 (chemokine (C-X-C motif)
206
ligand 1) expression was not affected (Figure 3D). 207
208
Antiproliferative activity of nintedanib in mPCKS
Previous studies reported the antiproliferative effects of nintedanib (15, 40, 41). We evaluated these 210
effects in mPCKS by Ki-67 immunohistochemistry, a marker of cell proliferation (Figure 3E). We 211
observed a 3.5-fold culture-induced increase in proliferation that was attenuated by 5 μM nintedanib 212
by approximately 30%. 213
214
Low density array for genes related to ECM homeostasis
215
To investigate whether the observed onset of fibrogenesis in mPCKS approximates in vivo 216
fibrogenesis, we measured and compared the expression of 43 genes related to ECM homeostasis in 217
kidneys from mice with renal injury induced by unilateral ureteral obstruction (UUO) and mPCKS. 218
Figure 4A shows that both obstruction of murine kidneys (for 3 or 7 days) and culture of mPCKS 219
introduced considerable changes in the expression of ECM-related genes: the majority of the tested 220
genes were upregulated in the obstructed kidneys and cultured mPCKS. We performed a detailed 221
statistical analysis on a subset of these samples and compared transcriptional changes that occur 222
during the obstruction of murine kidneys for 7 days and during 48h culture of mPCKS (Figure 5). The 223
analysis revealed that 31 out of 43 tested genes (72%) were regulated in the same direction (i.e., 224
upregulated) in the UUO kidneys and mPCKS, while only three genes (Eln, Fmod, Pcolce) were 225
regulated in the opposite direction. Additionally, seven genes were regulated during culture of 226
mPCKS, but not during 7d UUO, and included P4ha1, P4ha3, P4hb, Leprel1, Leprel2, Loxl1 and 227
Mmp13). Among tested transcripts, only expression of Adamts14 was unaltered by the obstruction or
228
culturing. Taken together, culturing of PCKS for 48h induced changes in ECM homeostasis that for 229
the large part mirrored changes observed in UUO kidneys, indicating that PCKS resemble in vivo 230
fibrogenesis. 231
Next, we investigated the impact of nintedanib on mPCKS. Figure 4B shows that nintedanib 232
manifested its inhibitory activity already at the lowest concentration (0.1 μM). A detailed statistical 233
analysis of the subset of these samples showed that 0.1 μM nintedanib significantly reduced 234
expression of 80% of the tested ECM-related genes, including collagen subtypes Col1a1, Col1a2, 235
Col3a1, Col4a1, Col5a1 and Col6a1 and ECM degradation matrix metalloproteinases encoded by
236
Mmp2, Mmp9 and Mmp13 (Figure 6). In turn, treatment of mPCKS with nintedanib at 5 μM
significantly inhibited only 59% of ECM-related transcripts. This was accompanied by the observation 238
that at high concentrations — 1 and 5 μM — nintedanib regulated different mRNA clusters than at 239
lower concentrations — 0.1 and 0.5 µM. In particular, seven genes (16 %) were regulated significantly 240
differently (i.e., not in the same direction) by nintedanib at 0.1 μM vs. 5 μM (Figure 6C). Among these 241
genes, Adamts3, Pcolce, Slc39a13, Ddr1 and Ddr2 were downregulated by nintedanib at low 242
concentration but not regulated at high concentration. Two transcripts – Dcn and Leprel1 – “switched” 243
their expression from unaltered or downregulated after the treatement with 0.1 μM nintedanib to being 244
upregulated by 5 μM nintedanib. 245
246
Nintedanib in healthy human PCKS 247
Targeted inhibition of gene expression and tyrosine kinase receptor activation
248
Culturing of the slices led to a significant downregulation of VEGFR1, VEGFR2 and FGFR2 mRNA 249
(Figure 7A). Nintedanib reduced expression of PDGFRB, VEGFR1 and VEGFR3 in hPCKS already at 250
0.1 μM (Figure 7B). The treatment did not affect expression of VEGFR2; however, it increased 251
FGFR2 expression at 5 μM.
252
Four phospho-RTKs (RTKs) were upregulated during incubation of hPCKS: PDGFRα, p-253
PDGFRβ, p-VEGFR1, and p-VEGFR2 (Figure 7C). This suggests that the onset of fibrosis in healthy 254
human PCKS was associated with the activation of PDGF and VEGF signaling pathways. Figure 7D 255
shows that nintedanib reduced phosphorylation of p-PDGFRα and p-VEGFR1 at 0.3 μM (by 39.8% 256
and 55%, respectively) and of PDGFRβ and VEGFR2 at 0.1 μM (by 45.3% and 23%). In contrast, the 257
activation of VEGFR3 and FGFR1 in hPCKS was neither affected by 48h incubation, nor by the 258
treatment with nintedanib (Figure 7C and D). 259
260
Mitigation of fibrosis and inflammation markers by nintedanib
261
In line with previously published data (34), fibrogenesis was initiated during incubation of hPCKS, as 262
revealed by the increase in COL1A1 and SERPINH1 transcription and protein levels of HSP47 (Figure 263
8A and B). Expression of ACTA2 significantly dropped after 48h, while FN1 expression remained 264
unchanged. Treatment with nintedanib resulted in a concentration-dependent inhibition of all tested 265
fibrosis markers, except for ACTA2 (Figure 9A). The IC50 was 0.6 μM for COL1A1, 1.5 μM for
266
SERPINH1 and 1.7 μM for FN1 (Supplementary Figure S1B). Nintedanib at 5 μM reduced the
267
accumulation of collagen type I to the baseline levels (at 0h) and affected protein level of HSP47 268
(Figure 9B and C). In concordance with gene expression, nintedanib had no influence on α-SMA 269
expression. 270
Similar to mPCKS, a substantial increase in mRNA levels of cytokines, such as TNF, IL-1B, IL-6 and 271
CXCL8/IL-8, occurred in hPCKS during culture period (Figure 8C). Nintedanib significantly reduced
272
the expression of these inflammation markers at the highest tested concentration (5 μM) (Figure 9D). 273
Interestingly, IL-1B mRNA level was inhibited at 0.5 μM. 274
275
Antiproliferative activity of nintedanib in hPCKS
276
We observed comparable results of Ki-67 expression in hPCKS, as in mPCKS: expression increased 277
during culture (fold induction of 10.4), and nintedanib reduced proliferation by approximately 73% 278
(Figure 9E). 279
280
Nintedanib in established fibrosis PCKS 281
Characterization of fibrotic human PCKS
282
Fibrotic hPCKS (fhPCKS) showed high basal gene expression of COL1A1, SERPINH1 and FN1, as 283
well as clear inflammatory profile compared to healthy kidneys (Figure 10A and B). Histologic 284
analysis confirmed the fibrotic phenotype by showing an extensive tubular atrophy, ECM 285
accumulation and interstitial fibrosis (Figure 10C and D). 286
To analyze the processes occurring during culture of fhPCKS, we studied viability and gene 287
expression up to 72h. Similar to healthy human PCKS (34), ATP content of fhPCKS increased during 288
first 24h, after which levels plateaued (Figure 10E). As described earlier, healthy PCKS develop a 289
fibrotic response during incubation. We observed a different pattern in fhPCKS: mRNA expression of 290
COL1A1, SERPINH1 and FN1 remained unchanged (Figure 10F). On the other hand, elevated
291
collagen type I deposition and highly increased protein expression of HSP47 might indicate that 292
fibrogenesis was still ongoing during incubation (Figure 10G and H). Similar to hPCKS, ACTA2 293
expression dropped in fhPCKS during culture, while α-SMA protein expression remained unchanged. 294
Regarding the inflammation markers, fhPCKS showed unaffected gene expression of TNF and IL-1B 295
during culture. Levels of IL-6 and CXCL8/IL-8 increased at 24h and then gradually declined (Figure 296
10I). 297
298
Effect of nintedanib on fibrosis and inflammation markers
299
Treatment of fhPCKS with nintedanib did not affect fibrosis markers on gene expression. Only the 300
highest tested concentration (5 μM) numerically, but not statistically significantly, reduced expression 301
of COL1A1 and SERPINH1 (Figure 11A). We detected non-significant effects on interstitial 302
accumulation of collagen type I and protein expression of α-SMA, however, nintedanib at 5 μM 303
significantly affected HSP47 (Figure 11B and C). Interestingly, 1 and 5 μM nintedanib downregulated 304
IL-1B in fhPCKS by 82.5% and 86.3%, respectively (Figure 11D).
305 306
Antiproliferative activity of nintedanib in fhPCKS
307
Similar to hPCKS, culture for 48h induced Ki-67 expression in fhPCKS (Figure 11E), while 308
nintedanib inhibited cell proliferation by approximately 48%. 309
DISCUSSION 310
PCKS provide a unique opportunity to translate the obtained results from rodent models to human, 311
which is important for clinical drug development (38). In this study we expanded the experimental 312
application of PCKS: we used healthy renal tissue of murine and human origin, and explored PCKS 313
from human fibrotic renal tissue as a model of established fibrosis. With this translational ex vivo 314
model, we investigated the effects of nintedanib in healthy and diseased tissue. PCKS model replicates 315
some of the main characteristics of CKD, such as cellular damage, tubulointerstitial fibrosis, (local) 316
inflammation, accumulation of ECM proteins and dysregulated matrix turnover, as shown in this study 317
and previously reported by others (30, 34, 35). We demonstrated that spontaneous induction of 318
fibrogenesis in PCKS resembles in vivo fibrogenesis, making this model suitable to study the 319
mechanism of action and efficacy of antifibrotic compounds. We showed that nintedanib blocks the 320
expression and phosphorylation of tyrosine kinase receptors and inhibits cell proliferation. 321
Additionally, nintedanib attenuated the onset of fibrosis not only in murine, but also in human PCKS, 322
although reversal of established fibrosis could not be achieved. 323
Nintedanib engaged its intended targets in human kidneys: it inhibited gene expression of PDGFRβ, 324
VEGFR1 and 3, as well as culture-induced phosphorylation of PDGFRα and β, VEGFR1 and 2 325
starting at 0.1 μM — a concentration that is in the range of its maximum human exposure of 0.07 µM 326
in patients with IPF after standard dosing (11, 28, 42). The attenuation of PDGFR-α and β signaling 327
by nintedanib is of therapeutic interest for renal fibrosis, as PDGF signaling leads to the differentiation 328
of pericytes and resident fibroblasts to profibrotic ECM-producing myofibroblasts (9). The literature 329
presents conflicting results on the role of VEGFR signaling in fibrosis and peritubular capillary 330
restoration (2, 20, 24, 27). Therefore, beneficial inhibition of VEGFR signaling by nintedanib in renal 331
fibrosis is subject to further studies. 332
The inhibitory effects of nintedanib on RTKs were investigated by Liu et al. in a unilateral ureteral 333
obstruction (UUO) mouse model (25). They reported that nintedanib effectively blocked UUO-334
induced phosphorylation of PDGFRβ, VEGFR2, FGFR1 and FGFR2. In human PCKS, both FGFR1 335
and FGFR2 were not affected by nintedanib, possibly due to the differences in fibrogenic processes in 336
murine and human tissues. 337
PCKS develop an early inflammatory response during culture, followed by the onset of fibrosis (34). 338
Nintedanib exerted anti-inflammatory activity in mPCKS and hPCKS. The observed effects are in line 339
with earlier studies of nintedanib in mouse models of lung fibrosis (23, 40). This anti-inflammatory 340
activity of nintedanib might translate into attenuation of renal injury. 341
Nintedanib also exerted antifibrotic effects in mPCKS and hPCKS, demonstrated by a marked 342
reduction of collagen 1a1 mRNA and protein expression. Furthermore, in mPCKS nintedanib 343
modulates ECM homeostasis even at the lowest concentration. Downregulation of these genes might 344
lead to the altered secretion and fibril formation of collagen, as reported in primary human lung 345
fibroblasts treated with TGF-β (21). High concentrations of nintedanib regulate different mRNA 346
clusters than low concentrations, reflected by a partial switch from inhibitory profile at 0.1 and 0.5 µM 347
to the induction of some ECM related genes at 1 and 5 µM. This could be explained by possible non-348
selective activity of nintedanib at high concentrations, while low concentrations have a more specific 349
kinase inhibitory profile (14). Despite that nintedanib at 1 and 5 µM has an altered impact on ECM 350
homeostasis, its overall effect remains antifibrotic. 351
The demonstrated attenuation of fibrosis concurs with previous results: nintedanib reduced lung 352
fibrosis in bleomycin- or silica-treated mice and rats (1, 40, 41) and showed antifibrotic effects in 353
various mouse models of systemic sclerosis (16, 17). Liu et al. (25) found that administration of 354
nintedanib for 7 days after UUO injury attenuated renal fibrosis. Nintedanib inhibited TGF-β1 induced 355
renal fibroblasts-to-myofibroblasts transition and expression of ECM proteins in vitro in renal 356
interstitial fibroblasts, indicating that nintedanib affects early events of TGF- β signaling. Wollin et al. 357
also reported that nintedanib at higher concentrations possesses anti-TGF-β activity (40, 41). We 358
hypothesize that the observed antifibrotic effects of nintedanib in PCKS might be attributed to a 359
combination of RTK inhibition and, perhaps non-selective, anti-TGF-β activity. 360
The culture of mPCKS, hPCKS and fhPCKS induced a strong spontaneous proliferative response and 361
in line with published data (15, 40, 41), nintedanib effectively inhibited culture-induced cell 362
proliferation in PCKS. 363
Our newly established translational PCKS model with tissue from fibrotic human kidneys showed a 364
clear fibrotic phenotype compared to renal tissue from control donors. Culture of fhPCKS did not 365
further increase the assessed markers of fibrosis, although an inflammatory peak was observed after 366
the first day of culture. The pre-existing fibrotic phenotype of fhPCKS might explain the difference 367
with healthy PCKS. 368
Nintedanib showed diminished reduction in fibrosis markers in fhPCKS compared to hPCKS, most 369
likely due to an increased interindividual variability (underlying primary renal disease, dialysis time, 370
time since transplantation and medication) in the fibrotic kidney slices. Nintedanib seems to be more 371
effective in preventing or halting the onset of fibrosis in healthy PCKS rather than in reversing the 372
established fibrosis as modeled by fhPCKS. Our data in human diseased tissue is in conflict with the 373
data in murine diseased tissue: delayed administration of nintedanib to UUO mice by Liu et al. (25) 374
resulted in partial reversal of established renal fibrosis. However, even prolonged kidney obstruction 375
in mice is not as severe as the late-stage fibrosis seen in CKD patients, emphasizing the need for 376
models that more closely resemble human pathology. PCKS, especially the culture of fhPCKS, can 377
serve as a model for human CKD. Nevertheless, limitations of the ex vivo PCKS model are: (1) the 378
relatively short culture period (48-72h) is not always sufficient to detect post-translational events; (2) 379
the availability of freshly resected human renal tissue is limited; (3) interorgan interactions cannot be 380
directly assessed; and (4) circulating immune cells that contribute to the fibrogenesis are absent, 381
because slices lack blood circulation, even though PCKS retain native vascular network. Considering 382
the significant role of infiltrating inflammatory cells and associated cytokines in renal fibrosis, a co-383
culture system of PCKS with immune cells (or subsets of immune cells) can be established for future 384
studies, which will better reflect the immunological interactions during the fibrogenic process in renal 385
tissue. 386
Taken together, our results demonstrate the pharmacological effects of nintedanib in an ex vivo model 387
of renal fibrosis, facilitating translation from animal studies to the clinic. Treatment of PCKS with 388
nintedanib inhibited cell proliferation and attenuated the onset of inflammation and fibrosis, although 389
reversal of established fibrosis could not be achieved. Nintedanib successfully inhibited PDGFR and 390
VEGFR phosphorylation, demonstrating the potential use of these receptors as therapeutic targets to 391
attenuate renal fibrosis. Therefore, along with the benefit of reducing animal use, human PCKS might 392
provide direct and clinically relevant insights into human renal disease and therapeutic strategies. 393
ACKNOWLEDGEMENTS 394
The authors thank the abdominal transplantation surgeons of the University Medical Center Groningen 395
for providing the human renal tissue. 396
397
CONFLICT OF INTEREST 398
The authors declare the following competing financial interest: Dr. Lutz Wollin is an employee of 399
Boehringer Ingelheim Pharma GmbH & Co. 400
401
AUTHOR CONTRIBUTIONS 402
E.B., E.G.D.S., P.O. and M.B. designed the study; E.B., E.G.D.S. and M.B. carried out experiments 403
and analyzed the data; B.P., R.A.B., H.v.G. and L.W. provided additional analytical tools and 404
chemicals for this study; A.M.L. and I.J.d.J. helped with human tissue procurement; E.B. and E.G.D.S. 405
wrote the manuscript with critical review from H.A.M.M., R.A.B., M.A.S., H.v.G, L.W., P.O. and 406
M.B. All of the authors approved the final version of the manuscript for publication. 407
408
FUNDING 409
This work was kindly supported by ZonMw (the Netherlands Organization for Health Research and 410
Development), grant number 114025003 and by Lundbeckfonden, grant number R231-2016-2344 411
(received by H.A.M.M). 412
REFERENCES 414
1. Ackermann M, Kim YO, Wagner WL, Schuppan D, Valenzuela CD, Mentzer SJ, Kreuz S, Stiller
415
D, Wollin L, Konerding MA. Effects of nintedanib on the microvascular architecture in a lung fibrosis
416
model. Angiogenesis 20: 359–372, 2017.
417
2. Bábíčková J, Klinkhammer BM, Buhl EM, Djudjaj S, Hoss M, Heymann F, Tacke F, Floege J,
418
Becker JU, Boor P. Regardless of etiology, progressive renal disease causes ultrastructural and
419
functional alterations of peritubular capillaries. Kidney Int 91: 70–85, 2017.
420
3. Beyer C, Distler JHW. Tyrosine kinase signaling in fibrotic disorders. Biochim Biophys Acta - Mol
421
Basis Dis 1832: 897–904, 2013.
422
4. Boor P, Ostendorf T, Floege J. PDGF and the progression of renal disease. Nephrol Dial Transplant
423
29: i45–i54, 2014.
424
5. Buhl EM, Djudjaj S, Babickova J, Klinkhammer BM, Folestad E, Borkham-Kamphorst E,
425
Weiskirchen R, Hudkins K, Alpers CE, Eriksson U, Floege J, Boor P. The role of PDGF-D in
426
healthy and fibrotic kidneys. Kidney Int 89: 848–861, 2016.
427
6. Cernaro V, Trifirò G, Lorenzano G, Lucisano S, Buemi M, Santoro D. New therapeutic strategies
428
under development to halt the progression of renal failure. Expert Opin Investig Drugs 23: 693–709,
429
2014.
430
7. Chen Y-T, Chang F-C, Wu C-F, Chou Y-H, Hsu H-L, Chiang W-C, Shen J, Chen Y-M, Wu K-D,
431
Tsai T-J, Duffield JS, Lin S-L. Platelet-derived growth factor receptor signaling activates pericyte–
432
myofibroblast transition in obstructive and post-ischemic kidney fibrosis. Kidney Int 80: 1170–1181,
433
2011.
434
8. van Dijk F, Olinga P, Poelstra K, Beljaars L. Targeted Therapies in Liver Fibrosis: Combining the
435
Best Parts of Platelet-Derived Growth Factor BB and Interferon Gamma. Front Med 2: 72, 2015.
436
9. Duffield JS. Cellular and molecular mechanisms in kidney fibrosis. J Clin Invest 124: 2299–2306, 2014.
437
10. Eisen T, Loembé A-B, Shparyk Y, MacLeod N, Jones RJ, Mazurkiewicz M, Temple G, Dressler H,
438
Bondarenko I. A randomised, phase II study of nintedanib or sunitinib in previously untreated patients
439
with advanced renal cell cancer: 3-year results. Br J Cancer 113: 1140–1147, 2015.
440
11. Eisen T, Shparyk Y, Macleod N, Jones R, Wallenstein G, Temple G, Khder Y, Dallinger C,
441
Studeny M, Loembe A-B, Bondarenko I. Effect of small angiokinase inhibitor nintedanib (BIBF 1120)
442
on QT interval in patients with previously untreated, advanced renal cell cancer in an open-label, phase
II study. Invest New Drugs 31: 1283–1293, 2013.
444
12. Floege J, Eitner F, Alpers CE. A New Look at Platelet-Derived Growth Factor in Renal Disease. J Am
445
Soc Nephrol 19: 12–23, 2008.
446
13. Genovese F, Kàrpàti ZS, Nielsen SH, Karsdal MA. Precision-Cut Kidney Slices as a Tool to
447
Understand the Dynamics of Extracellular Matrix Remodeling in Renal Fibrosis. Biomark Insights 11:
448
77–84, 2016.
449
14. Hilberg F, Roth GJ, Krssak M, Kautschitsch S, Sommergruber W, Tontsch-Grunt U, Garin-Chesa
450
P, Bader G, Zoephel A, Quant J, Heckel A, Rettig WJ. BIBF 1120: Triple Angiokinase Inhibitor with
451
Sustained Receptor Blockade and Good Antitumor Efficacy. Cancer Res 68: 4774–4782, 2008.
452
15. Hostettler KE, Zhong J, Papakonstantinou E, Karakiulakis G, Tamm M, Seidel P, Sun Q, Mandal
453
J, Lardinois D, Lambers C, Roth M. Anti-fibrotic effects of nintedanib in lung fibroblasts derived
454
from patients with idiopathic pulmonary fibrosis. Respir Res 15: 157, 2014.
455
16. Huang J, Beyer C, Palumbo-Zerr K, Zhang Y, Ramming A, Distler A, Gelse K, Distler O, Schett
456
G, Wollin L, Distler JHW. Nintedanib inhibits fibroblast activation and ameliorates fibrosis in
457
preclinical models of systemic sclerosis. Ann Rheum Dis 75: 883–890, 2016.
458
17. Huang J, Maier C, Zhang Y, Soare A, Dees C, Beyer C, Harre U, Chen C-W, Distler O, Schett G,
459
Wollin L, Distler JHW. Nintedanib inhibits macrophage activation and ameliorates vascular and
460
fibrotic manifestations in the Fra2 mouse model of systemic sclerosis. Ann Rheum Dis 76: 1941–1948,
461
2017.
462
18. Humphreys BD, Lin S-L, Kobayashi A, Hudson TE, Nowlin BT, Bonventre J V, Valerius MT,
463
McMahon AP, Duffield JS. Fate Tracing Reveals the Pericyte and Not Epithelial Origin of
464
Myofibroblasts in Kidney Fibrosis. Am J Pathol 176: 85–97, 2010.
465
19. James MT, Hemmelgarn BR, Tonelli M. Early recognition and prevention of chronic kidney disease.
466
Lancet 375: 1296–1309, 2010.
467
20. Kang DH, Joly AH, Oh SW, Hugo C, Kerjaschki D, Gordon KL, Mazzali M, Jefferson JA, Hughes
468
J, Madsen KM, Schreiner GF, Johnson RJ. Impaired angiogenesis in the remnant kidney model: I.
469
Potential role of vascular endothelial growth factor and thrombospondin-1. [Online]. J Am Soc Nephrol
470
12: 1434–47, 2001. http://www.ncbi.nlm.nih.gov/pubmed/11423572 [8 Mar. 2019].
471
21. Knüppel L, Ishikawa Y, Aichler M, Heinzelmann K, Hatz R, Behr J, Walch A, Bächinger HP,
472
Eickelberg O, Staab-Weijnitz CA. A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone.
Inhibition of Collagen Fibril Assembly. Am J Respir Cell Mol Biol 57: 77–90, 2017.
474
22. Ladner CL, Yang J, Turner RJ, Edwards RA. Visible fluorescent detection of proteins in
475
polyacrylamide gels without staining. Anal Biochem 326: 13–20, 2004.
476
23. Lee HY, Hur J, Kim IK, Kang JY, Yoon HK, Lee SY, Kwon SS, Kim YK, Rhee CK. Effect of
477
nintedanib on airway inflammation and remodeling in a murine chronic asthma model. Exp Lung Res 43:
478
187–196, 2017.
479
24. Lin S-L, Chang F-C, Schrimpf C, Chen Y-T, Wu C-F, Wu V-C, Chiang W-C, Kuhnert F, Kuo CJ,
480
Chen Y-M, Wu K-D, Tsai T-J, Duffield JS. Targeting Endothelium-Pericyte Cross Talk by Inhibiting
481
VEGF Receptor Signaling Attenuates Kidney Microvascular Rarefaction and Fibrosis. Am J Pathol 178:
482
911–923, 2011.
483
25. Liu F, Wang L, Qi H, Wang J, Wang Y, Jiang W, Xu L, Liu N, Zhuang S. Nintedanib, a triple
484
tyrosine kinase inhibitor, attenuates renal fibrosis in chronic kidney disease. Clin Sci 131: 2125–2143,
485
2017.
486
26. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative
487
PCR and the 2−ΔΔCT Method. Methods 25: 402–408, 2001.
488
27. Long DA, Norman JT, Fine LG. Restoring the renal microvasculature to treat chronic kidney disease.
489
Nat Rev Nephrol 8: 244–250, 2012.
490
28. Mross K, Stefanic M, Gmehling D, Frost A, Baas F, Unger C, Strecker R, Henning J,
Gaschler-491
Markefski B, Stopfer P, de Rossi L, Kaiser R. Phase I Study of the Angiogenesis Inhibitor BIBF 1120
492
in Patients with Advanced Solid Tumors. Clin Cancer Res 16: 311–319, 2010.
493
29. Ostendorf T, Eitner F, Floege J. The PDGF family in renal fibrosis. Pediatr Nephrol 27: 1041–1050,
494
2012.
495
30. Poosti F, Pham BT, Oosterhuis D, Poelstra K, van Goor H, Olinga P, Hillebrands J-L. Precision-cut
496
kidney slices (PCKS) to study development of renal fibrosis and efficacy of drug targeting ex vivo. Dis
497
Model Mech 8: 1227–1236, 2015.
498
31. Remst DFG, Blom AB, Vitters EL, Bank RA, van den Berg WB, Blaney Davidson EN, van der
499
Kraan PM. Gene Expression Analysis of Murine and Human Osteoarthritis Synovium Reveals
500
Elevation of Transforming Growth Factor β-Responsive Genes in Osteoarthritis-Related Fibrosis.
501
Arthritis Rheumatol 66: 647–656, 2014.
502
32. Roth GJ, Binder R, Colbatzky F, Dallinger C, Schlenker-Herceg R, Hilberg F, Wollin S-L, Kaiser
R. Nintedanib: From Discovery to the Clinic. J Med Chem 58: 1053–1063, 2015.
504
33. Stribos EGD, Hillebrands J-L, Olinga P, Mutsaers HAM. Renal fibrosis in precision-cut kidney
505
slices. Eur J Pharmacol 790: 57–61, 2016.
506
34. Stribos EGD, Luangmonkong T, Leliveld AM, de Jong IJ, van Son WJ, Hillebrands J-L, Seelen
507
MA, van Goor H, Olinga P, Mutsaers HAM. Precision-cut human kidney slices as a model to
508
elucidate the process of renal fibrosis. Transl Res 170: 8-16.e1, 2016.
509
35. Stribos EGD, Seelen MA, van Goor H, Olinga P, Mutsaers HAM. Murine Precision-Cut Kidney
510
Slices as an ex vivo Model to Evaluate the Role of Transforming Growth Factor-β1 Signaling in the
511
Onset of Renal Fibrosis. Front Physiol 8: 1026, 2017.
512
36. Strutz F, Zeisberg M, Hemmerlein B, Sattler B, Hummel K, Becker V, Müller GA. Basic fibroblast
513
growth factor expression is increased in human renal fibrogenesis and may mediate autocrine fibroblast
514
proliferation. Kidney Int 57: 1521–1538, 2000.
515
37. Tampe D, Zeisberg M. Potential approaches to reverse or repair renal fibrosis. Nat Rev Nephrol 10:
516
226–237, 2014.
517
38. Westra IM, Mutsaers HAM, Luangmonkong T, Hadi M, Oosterhuis D, de Jong KP, Groothuis
518
GMM, Olinga P. Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset
519
of liver fibrosis. Toxicol Vitr 35: 77–85, 2016.
520
39. Westra IM, Oosterhuis D, Groothuis GMM, Olinga P. Precision-cut liver slices as a model for the
521
early onset of liver fibrosis to test antifibrotic drugs. Toxicol Appl Pharmacol 274: 328–338, 2014.
522
40. Wollin L, Maillet I, Quesniaux V, Holweg A, Ryffel B. Antifibrotic and Anti-inflammatory Activity of
523
the Tyrosine Kinase Inhibitor Nintedanib in Experimental Models of Lung Fibrosis. J Pharmacol Exp
524
Ther 349: 209–220, 2014.
525
41. Wollin L, Wex E, Pautsch A, Schnapp G, Hostettler KE, Stowasser S, Kolb M. Mode of action of
526
nintedanib in the treatment of idiopathic pulmonary fibrosis. Eur Respir J 45: 1434–1445, 2015.
527
42. Wollin SL, Bonella F, Stowasser S. Idiopathic pulmonary fibrosis: current treatment options and
528
critical appraisal of nintedanib. Drug Des Devel Ther 9: 6407, 2015.
529
43. Zhang S, Liu Q, Xiao J, Lei J, Liu Y, Xu H, Hong Z. Molecular validation of the precision-cut kidney
530
slice (PCKS) model of renal fibrosis through assessment of TGF-β1-induced Smad and p38/ERK
531
signaling. Int Immunopharmacol 34: 32–36, 2016.
532
44. Zhuang S, Liu N. EGFR signaling in renal fibrosis. Kidney Int Suppl 4: 70–74, 2014.
FIGURE LEGENDS 534
Figure 1. (A) Schematic illustration of the workflow. Renal tissue of murine or human origin was 535
used to obtain cylindrical cores. By placing the tissue cores in Krumdieck tissue slicer, filled with ice-536
cold Krebs-Henseleit buffer, we prepared precision-cut kidney slices with a wet weight of 4-5 mg and 537
estimated thickness of 250-300 µm. Slices were subsequently incubated in 12-well plates (1 slice per 538
well) in culture medium with or without nintedanib for 48h at 37ºC. Medium was refreshed at 24h. At 539
the end of culture period, samples were collected by pooling three slices (from each animal/donor) for 540
each type of analysis. Visualization of kidney slices prepared from healthy tissue is represented by the 541
blue color, from diseased tissue – by the orange color. Same color code is applied to all figures. (B) 542
Viability of murine, human and fibrotic human PCKS treated with nintedanib for 48h was measured 543
by ATP and total protein content. Data are shown as values relative to non-treated control slices at 48h 544
and are expressed as mean (± SEM), n=4-5, *p<0.05. (C) Representative images of Periodic acid– 545
Schiff (PAS) staining of untreated slices at 0h and 48h as well as of slices treated with 5 µM 546
nintedanib (scale bar = 100 µm). 547
548
Figure 2. Spontaneous fibrogenic and inflammatory response in murine PCKS during culture. 549
(A) mRNA expression of fibrosis markers. (B) Protein levels of HSP47 and α-SMA with 550
representative Western blot images. (C) mRNA expression of inflammation markers. Data are 551
expressed as mean (± SEM), n=4-5. Gene expression levels were compared using unpaired Student’s 552
t-test; protein levels were compared using non-parametric Mann-Whitney test, *p<0.05 553
Col1a1, collagen type I alpha 1; Serpinh1, serine proteinase inhibitor clade H (Heat Shock Protein 47) 554
member 1; Fn1, fibronectin 1; Acta2, alpha 2 smooth muscle actin; HSP47, heat shock protein 47; α-555
SMA, alpha smooth muscle actin; Tnf, tumor necrosis factor; Il-1b, interleukin 1 beta; Il-6, interleukin 556
6; Cxcl1, C-X-C motif chemokine ligand 1. 557
558
Figure 3. Antifibrotic, anti-inflammatory and antiproliferative effect of nintedanib in healthy 559
mouse kidney. Murine PCKS were cultured in the presence of nintedanib (1, 5 or 10 µM) for 48h. (A) 560
mRNA expression of fibrosis markers after 48h incubation. (B) Representative sections with 561
interstitial collagen type I accumulation visualized by immunohistochemistry (scale bar = 50 µm) and 562
quantitative analysis of staining intensity. (C) Protein levels of HSP47 and α-SMA at 48h with 563
representative Western blot images. (D) mRNA expression of inflammation markers after 48h 564
incubation. (E) Expression of cell proliferation marker Ki-67 in murine PCKS during culture and after 565
48h treatment with 5 µM nintedanib was visualized by immunohistochemistry and quantified as 566
relative intensity values. 567
Data are expressed as mean (± SEM), n=3-5, *p<0.05. 568
Col1a1, collagen type I alpha 1; Serpinh1, serine proteinase inhibitor clade H (Heat Shock Protein 47) 569
member 1; Fn1, fibronectin 1; Acta2, alpha 2 smooth muscle actin; HSP47, heat shock protein 47; α-570
SMA, alpha smooth muscle actin; Tnf, tumor necrosis factor; Il-1b, interleukin 1 beta; Il-6, interleukin 571
6; Cxcl1, C-X-C motif chemokine ligand 1; mPCKS, murine precision-cut kidney slices. 572
573
Figure 4. Transcriptional changes in the extracellular matrix homeostasis genes in murine 574
kidney slices as measured by TaqMan low density array. (A) Heatmap illustrating log2 fold
575
changes in the expression of extracellular matrix (ECM) related genes in murine kidneys subjected to 576
3 days or 7 days of ureteral obstruction (UUO) and in murine PCKS during 48h culture. Fold changes 577
are relative to the average expression in the corresponding control group (i.e., 3d UUO relative to 3d 578
SHAM; 7d UUO relative to 7d SHAM; 48h mPCKS relative to 0h mPCKS). (B) Heatmap of ECM 579
modulation profiles in murine PCKS at 0h, 48h and treated with nintedanib (0.1-5 μM) for 48h. Fold 580
changes are relative to the average expression in 0h PCKS group. 581
Red and blue indicate relatively high and low expression, respectively (grey color indicates 582
undetermined values). Average-linkage hierarchical clustering (supervised in A, unsupervised in B) 583
was performed using Pearson correlation. Complementary statistical analyses performed on ΔCt 584
values are shown in Supplementary Figure S3 and S4. Full gene names are listed in Supplementary 585
Table S2. 586
587
Figure 5. Detailed analysis of the transcriptional changes in the extracellular matrix homeostasis 588
determined by TLDA in the UUO mouse kidneys and murine PCKS. (A) Heatmap that illustrates 589
changes in the gene expression patterns (log2 fold changes) in kidneys from mice subjected to 7 days
590
UUO and in murine PCKS (mPCKS) cultured for 48h, as these groups were included for the statistical 591
analysis. Red and blue indicate relatively high and low expression, respectively (grey color indicates 592
undetermined values). Supervised average-linkage hierarchical clustering was performed using 593
Pearson correlation. (B) Statistical analysis (on the ΔCt values) of the transcriptional changes in ECM 594
homeostasis in the obstructed for 7d murine kidneys and in cultured for 48h PCKS (as determined by 595
TLDA). Fold changes were calculated as relative to the corresponding control (i.e., 7d UUO vs. 7d 596
SHAM; 48h mPCKS vs. 0h mPCKS). Fold changes in red indicate significant upregulation (with p < 597
0.05), fold changes in blue indicate significant downregulation (with p < 0.05), while fold changes in 598
black indicate no significant regulation of a gene. Data are expressed as mean ± SEM; experimental 599
groups (7d UUO vs. 7d SHAM; 48h mPCKS vs. 0h mPCKS) were compared by unpaired two-tailed 600
Student’s t-test. (C and D) Number of genes that were not regulated or statistically significantly 601
altered in expression during 7d UUO or during 48h culture of mPCKS. 602
603
Figure 6. Detailed analysis of the transcriptional changes in the expression of extracellular 604
matrix related genes in murine PCKS treated with nintedanib for 48h. (A) Heatmap that 605
illustrates changes in the gene expression patterns (log2 fold changes) in mPCKS cultured in the
606
absence of nintedanib, or treated with nintedanib at 0.1 μM or 5 μM for 48h, as these groups were 607
included for the statistical analysis. Fold changes were calculated as relative to the average expression 608
in the untreated PCKS (48h). Red and blue indicate relatively high and low expression, respectively 609
(grey color indicates undetermined values). Unsupervised average-linkage hierarchical clustering was 610
performed using Pearson correlation. (B) Statistical analysis (on the ΔCt values) of the transcriptional 611
changes in the expression of ECM-related genes in mPCKS after treatment with nintedanib at the 612
lowest (0.1 μM) and the highest (5 μM) tested concentration (as determined by TLDA). Fold changes 613
were calculated as relative to the untreated slices (48h). Fold changes in blue indicate significant 614
downregulation (with p < 0.05), fold changes in red indicate significant upregulation (with p < 0.05), 615
while fold changes in black indicate no significant change in the expression of a gene. Data are 616
expressed as mean ± SEM; experimental groups (48h untreated mPCKS, mPCKS treated with 0.1 μM 617
nintedanib and mPCKS treated with 5 μM nintedanib) were compared by one-way ANOVA followed 618
by Dunnett’s multiple comparisons test. While color indicates significance for comparisons: 48h 619
untreated mPCKS vs. nintedanib 0.1 μM and 48h untreated mPCKS vs. nintedanib 5 μM, (*) denotes 620
statistical differences between mPCKS treated with nintedanib 0.1 μM vs. nintedanib 5 μM. (*) 621
colored in red indicate genes that were considered as “switched” in their expression between 0.1 μM 622
and 5 μM nintedanib treatment groups. Ns, not significant; n.a., not available (e.g. due to 623
undetermined values). (C) Number of genes that were statistically significantly inhibited by nintedanib 624
0.1 μM and 5 μM, as well as number of genes that “switched” their expression when mPCKS were 625
exposed to the highest tested concentration of nintedanib compared to the lowest tested concentration. 626
627
Figure 7. Tyrosine kinase receptors (RTKs) expression and activation in human PCKS after 48h 628
culture and after nintedanib treatment. (A) RTKs mRNA expression in hPCKS after 48h culture, 629
as measured by rt-qPCR. (B) RTKs mRNA expression in hPCKS after 48h treatment with nintedanib 630
(0.1 – 5 µM). (C) Phosphorylation of RTKs in hPCKS was measured by multiplex magnetic bead 631
assay and expressed as relative mean fluorescence intensity (MFI) to 0h control. (D) Phosphorylation 632
of RTKs in hPCKS treated with nintedanib (0.1 – 5 µM) for 48h, shown as mean MFI relative to the 633
untreated hPCKS. Data are expressed as mean (± SEM), n=4-5, *p<0.05 634
FLT1, Fms related tyrosine kinase 1; FLT4, Fms related tyrosine kinase 4; KDR, kinase insert domain 635
receptor; PDGFRB, platelet derived growth factor receptor beta; PDGFRA, platelet derived growth 636
factor receptor alpha; VEGFR1, vascular endothelial growth factor receptor 1; VEGFR2, vascular 637
endothelial growth factor receptor 2; VEGFR3, vascular endothelial growth factor receptor 3; FGFR1, 638
fibroblast growth factor receptor 1; FGFR2, fibroblast growth factor receptor 2. 639
640
Figure 8. Spontaneous fibrogenic and inflammatory response in human PCKS during culture. 641
(A) mRNA expression of fibrosis markers. (B) Protein levels of HSP47 and α-SMA with 642
representative Western blot images. (C) mRNA expression of inflammation markers. Data are 643
expressed as mean (± SEM), n=4-5. Gene expression levels were compared using unpaired Student’s 644
t-test; protein levels were compared using non-parametric Mann-Whitney test, *p<0.05 645
COL1A1, collagen type I alpha 1; SERPINH1, serine proteinase inhibitor clade H (Heat Shock Protein 646
47) member 1; FN1, fibronectin 1; ACTA2, alpha 2 smooth muscle actin; HSP47, heat shock protein 647
47; α-SMA, alpha smooth muscle actin; TNF, tumor necrosis factor; IL-1B, interleukin 1 beta; IL-6, 648
interleukin 6; CXCL8, C-X-C motif chemokine ligand 8 (IL-8, interleukin 8). 649
650
Figure 9. Effects of nintedanib in healthy human kidney. Human PCKS were cultured in the 651
presence of nintedanib (0.1 – 5 µM) for 48h. (A) mRNA expression of fibrosis markers after 48h 652
incubation. (B) Representative sections with interstitial collagen type I accumulation visualized by 653
immunohistochemistry (scale bar = 100 µm) and quantitative analysis of staining intensity. (C) Protein 654
levels HSP47 and α-SMA at 48h with representative Western blot images. (D) mRNA expression of 655
inflammation markers after 48h incubation. (E) Expression of cell proliferation marker Ki-67 in 656
human PCKS during culture and after 48h treatment with 5 µM nintedanib was visualized by 657
immunohistochemistry and quantified as relative intensity values. 658
Data are expressed as mean (± SEM), n=4-5, *p<0.05. 659
COL1A1, collagen type I alpha 1; SERPINH1, serine proteinase inhibitor clade H (Heat Shock Protein 660
47) member 1; FN1, fibronectin 1; ACTA2, alpha 2 smooth muscle actin; HSP47, heat shock protein 661
47; α-SMA, alpha smooth muscle actin; TNF, tumor necrosis factor; IL-1B, interleukin 1 beta; IL-6, 662
interleukin 6; CXCL8, C-X-C motif chemokine ligand 8 (IL-8, interleukin 8); hPCKS, human 663
precision-cut kidney slices. 664
665
Figure 10. Characterization of fibrotic human precision-cut kidney slices. PCKS were prepared 666
from fibrotic human renal tissue obtained during ESRD nephrectomies or transplantectomies. (A) 667
Baseline (prior incubation) mRNA expression of fibrosis markers in fibrotic compared to healthy 668
tissue slices. (B) Baseline (prior incubation) mRNA expression of inflammation markers in fibrotic 669
compared to healthy tissue slices. (C) Representative photomicrographs of human healthy and fibrotic 670
PCKS prior incubation (scale bar = 250 μm). Histologic analyses by PAS and PSR staining showing 671
extensive tubular atrophy and interstitial fibrosis, while α-SMA and collagen type I 672
immunohistochemistry further confirmed the fibrotic phenotype. (D) Quantitative analysis of collagen 673
type I immunohistochemistry in healthy and fibrotic PCKS (prior incubation). (E) Viability of fibrotic 674
PCKS during incubation presented as the average of pmol ATP per μg total protein. (F) Effect of 675
incubation on mRNA expression of fibrosis markers. (G) Representative images of 676
immunohistochemistry of collagen type I (scale bar = 100 μm) in fibrotic PCKS during 72h culture 677
with quantitative analysis. (H) Protein levels of HSP47 and α-SMA (n=5) during incubation with 678
representative Western blot images. (I) Effect of incubation on mRNA expression of inflammation 679
markers in fhPCKS. Data are expressed as mean (± SEM), n=9 for healthy PCKS and n=8-9 for 680
fibrotic PCKS, *p<0.05. 681
COL1A1, collagen type I alpha 1; SERPINH1, serine proteinase inhibitor clade H (Heat Shock Protein 682
47) member 1; FN1, fibronectin 1; ACTA2, alpha 2 smooth muscle actin; HSP47, heat shock protein 683
47; α-SMA, alpha smooth muscle actin; TNF, tumor necrosis factor; IL-1B, interleukin 1 beta; IL-6, 684
interleukin 6; CXCL8, C-X-C motif chemokine ligand 8 (IL-8, interleukin 8); PAS, periodic acid-685
Schiff; PSR, Picro Sirius red. 686
687
Figure 11. Antifibrotic and anti-inflammatory effect of nintedanib in fibrotic human precision-688
cut kidney slices. Fibrotic human PCKS were cultured in the presence of nintedanib (0.1 – 5 μM) for 689
48h. (A) mRNA expression of fibrosis markers after 48h incubation. (B) Representative sections with 690
interstitial collagen type I accumulation visualized by immunohistochemistry (scale bar = 100 µm) 691
and quantitative analysis of staining intensity. (C) Protein levels HSP47 and α-SMA at 48h with 692
representative Western blot images. (D) mRNA expression levels of inflammation markers after 48h 693
incubation. (e) Expression of cell proliferation marker Ki-67 in fibrotic human PCKS during culture 694
and after 48h treatment with 5 µM nintedanib was visualized by immunohistochemistry and quantified 695
as relative intensity values. 696
Data are expressed as mean (± SEM), n=4-5, *p<0.05. 697
COL1A1, collagen type I alpha 1; SERPINH1, serine proteinase inhibitor clade H (Heat Shock Protein 698
47) member 1; FN1, fibronectin 1; ACTA2, alpha 2 smooth muscle actin; HSP47, heat shock protein 699
47; α-SMA, alpha smooth muscle actin; TNF, tumor necrosis factor; IL-1B, interleukin 1 beta; IL-6, 700
interleukin 6; CXCL8, C-X-C. 701
702
Supplementary Figure S1. IC20 and IC50 of nintedanib in murine and human PCKS. 703
Nintedanib-induced inhibition of mRNA expression of fibrosis markers after 48h incubation in murine 704
PCKS (A) and in human PCKS (B). IC20 and IC50 values were calculated by fitting the data to a 705
four-parameter log(inhibitor) vs response curve. Data points are the mean (± SEM), n=3-5. 706
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.
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] Re la ti v e AT P v a lu e Viability mPCKS 0 0.1 0.3 0.5 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] Re la ti v e AT P v a lu e Viability hPCKS 0 0.1 0.3 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] Re la ti v e AT P v a lu e Viability fhPCKS 0 1 5 10 0.0 0.5 1.0 1.5 Nintedanib [µM] Re la ti v e p ro te in c o n te n t Protein mPCKS 0 0.1 0.3 0.5 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] Re la ti v e p ro te in c o n te n t Protein hPCKS 0 0.1 0.3 1 5 0.0 0.5 1.0 1.5 Nintedanib [µM] Re la ti v e p ro te in c o n te n 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
