Marianne Terrasse1,2, Florence Jouan2, Thibault Dolley-Hitze1,2, Yannick Arlot-Bonnemains2, Marc-Antoine Belaud-Rotureau2,3, Nathalie Rioux-Leclercq2,4, Nolwenn Lorcy1,2and Cécile Vigneau1,2
1CHU Pontchaillou, Service de Néphrologie, 2 rue Henri Le Guilloux 35033 Rennes Cedex, France
2Université Rennes 1, CNRS UMR6290, KyCa, 2 avenue du Professeur Léon Bernard, CS, 34317, 35043 Rennes Cedex, France
3CHU Pontchaillou, Service de cytogénétique, 2 rue Henri Le Guilloux 35033 Rennes Cedex, France
4CHU Pontchaillou, Service d'anatomie et cytologie pathologiques, 2 rue Henri Le Guilloux 35033 Rennes Cedex, France
Received: 14 July, 2015;Accepted: 26 October, 2015; Published: 28 October, 2015
Dr. Marianne Terrasse, Université Rennes 1, CNRS UMR6290, KyCa, 2 avenue du Professeur Léon Bernard, CS, Tel: 0033 2 99 28 43 96; Tel: 0033 2 99 28 43 96; Fax: 0033 2 99 28 41 50; Fax: 0033 2 99 28 41 50; E-mail:
Terrasse M, Jouan F, Dolley-Hitze T, Arlot-Bonnemains Y, Belaud-Rotureau MA, et al. (2015) Anti-VEGF Therapy Induces Proteinuria through Endothelial Disorganization Leading to Nephrin Decrease in Podocytes. Int J Immunother Cancer Res 1(1): 021-028.10.17352/2455-8591.000006
© 2015 Terrasse M et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Glomerular slit diaphragm; Targeted anti-VEGF therapy; Sunitinib; Podocytes; Actin cytoskeleton
Background: VEGF is involved in cancer development by stimulating neo-angiogenesis and tumor proliferation. Anti-angiogenic therapies, especially tyrosine kinase inhibitors such as sunitinib, have significantly improved cancer prognosis. Nevertheless, renal side effects, such as proteinuria and thrombotic microangiopathy, have been reported. The underlying physiopathological mechanisms remain unclear, but animal models and clinical similarities with preeclampsia suggest that such therapies affect the function of the endothelial and epithelial layers of the glomerular basement membrane, with activation of the endothelin signaling system and loss of glomerular slit diaphragm integrity. The aim of this in vitro study was to determine sunitinib effects on normal podocytes and glomerular endothelial cells.
Methods: The glomerular microvascular endothelial (GMVEC) and human glomerular visceral epithelial (hGVE) cell lines were incubated with various doses of sunitinib. The MTT Cell Proliferation Assay was used to assess cell proliferation. Expression of nephrin (a major slit diaphragm protein) and endothelin was evaluated by immunofluorescence or western blotting assays.
Results: Sunitinib inhibited GMVEC and hGVE cell proliferation in a dose-dependent manner. In GMVEC cells, endothelin transcription and secretion were increased after incubation with sunitinib. Conversely, in hGVE cells, sunitinib did not affect nephrin expression. However, conditioned medium from GMVEC cells incubated with sunitinib modified nephrin expression when added to the culture medium of hVGE cells. This effect was inhibited by pre-incubating hGVE cells with an endothelin inhibitor.
Conclusion: This study suggests an indirect toxicity of sunitinib on podocytes through endothelin. Therefore, sunitinib-induced renal side effects could be controlled with endothelin inhibitors.
Vascular endothelial growth factor (VEGF) plays a key role in tumor angiogenesis and in metastasis spreading. In the last decade, anti-angiogenic therapies targeting the VEGF pathway have revolutionized the treatment of metastatic cancer . They include the anti VEGF monoclonal humanized antibody bevacizumab and tyrosine kinase inhibitors, such as sunitinib, that target especially VEGF receptors (VEGF-R) . Such therapies have significantly improved progression-free survival and overall survival [3-5]. Therefore, sunitinib is now recommended as first-line therapy for patients with metastatic renal cell carcinoma, breast cancer and gastrointestinal stromal tumors .
However, anti-VEGF drugs can induce several adverse effects (gastrointestinal disorders, skin toxicity and hypertension) that may require dose reduction or discontinuation in half of the patients. Proteinuria after treatment with bevacizumab is observed in 21% to 63% of patients , but renal adverse effects may have been underestimated. Indeed, diagnostic kidney biopsies, which are rarely performed in these patients, have highlighted some cases of immuno-allergic interstitial nephritis , focal segmental glomerulosclerosis , acute tubular necrosis , and more frequently thrombotic microangiopathy (TMA). These lesions are potentially reversible after drug discontinuation [11-14]. Recently, we included 22 patients in the RARe database (Registry of patients with major kidney side effects during treatment with anti-VEGF drugs) . Analysis of the renal biopsies from these patients indicated the presence of acute or chronic TMA in 21. Specifically, the observed lesions (glomerular endothelial cell swelling, loss of endothelial fenestrae and effacement of foot processes) were reminiscent of the alterations observed in preeclampsia . Indeed, there are clinical and pathophysiological similarities between preeclampsia and the renal side effects of anti-VEGF treatments .
During preeclampsia, expression of the soluble form of VEGF-R1 (sFLT-1) increases, whereas VEGF serum level decreases . By binding and neutralizing VEGF, sFLT1 inhibits the VEGF signaling pathway. This leads to disorganization of vascular endothelial (VE) cadherin a key component of endothelial cell-cell junctions , and consequently to increased vascular permeability and proteinuria. It has been reported that VEGF-A phosphorylates VEGF-R2 and VE-cadherin, ultimately resulting in disassembly of intercellular junctions and increased permeability of the glomerular slit diaphragm .
Moreover, histological analysis of kidney biopsies from patients with preeclampsia revealed the presence of TMA, with reduced formation of endothelial fenestrae. In mice, injection of sFLT-1 induces a “preeclampsia-like syndrome” with acute hypertension, edema, TMA histological lesions and reduction in nephrin expression . In kidney biopsies from patients treated with anti-VEGF agents, VEGF, synaptopodin and nephrin expression levels are decreased , whereas the endothelin signaling system, which stimulates VEGF production , is activated [23,24]. Similarly, in preeclampsia, expression of nephrin and synaptopodin is reduced . Loss of nephrin could be due to shedding from the cell surface through endothelin-1 release by endothelial glomerular cells .
These studies suggest that the proteinuria induced by anti-VEGF drugs is a consequence of the dysregulation of the slit diaphragm, leading to endothelial and epithelial cell dysfunction. Therefore, the aim of this study was to determine the in vitro effects of sunitinib on normal podocytes and glomerular endothelial cells. Cell-cell interactions in cells incubated with sunitinib were studied to test the hypothesis of endothelin-1 (ET-1) implication, as described in preeclampsia.
Materials and Methods
Cell lines and cell culture methods
The human glomerular visceral epithelial cell line hGVE (i.e., podocytes) was generously given by Pr. Rondeau (INSERM U702, Paris, France) . Cells were cultured in Dulbecco's Modified Eagle Medium/Nutriment Mixture F-12 (DMEM/F-12; Gibco®, Carlsbad, USA) with 5% fetal bovine serum (FBS) (PAA Laboratories®, Pasching, Austria), 1% Penicillin- Streptomycin solution (10000 U/mL penicillin + 10000µg/mL streptomycin, Gibco®) and in extempo 1% of 100X Insulin-Transferrin-Selenium-X Supplement (Gibco®). Cells were cultured in 25cm³ or 75cm³ tissue culture flasks.
The commercial glomerular microvascular endothelial cell line (GMVEC) (ACBRI 128 – CellSystems®) was isolated from normal human renal cortex. Cells were cultured in EGM™2 SingleQuots® (Lonza® Walkersville, USA), containing 5% FBS, 0.04% hydrocortisone, 0.4% human fibroblast growth factor, 0.1% VEGF, 0.1% ascorbic acid, 0.1% human epidermal growth factor, 0.1% arginine 3 – insulin-like growth factor, and 0.1% GA-1000 solution (30 mg/ml gentamycin + 15 µg/ml amphotericin).
Sunitinib: kindly supplied by Pfizer® (New-York, USA), was dissolved in dimethylsulfoxide (DMSO) to a 10mM stock solution stored at -20°C. Supernatants of GMVEC cells incubated with sunitinib or DMSO (called conditioned medium) were used to treat hGVE cells. The endothelin receptor-A (ET-RA) antagonist BQ-123 (Sigma-Aldrich®, Saint-Louis, USA) was dissolved in sterile water to a 0.4mM or 400µM stock solution and stored at -20°C.
MTT Cell Proliferation Assay: To analyze cell proliferation, 2000 cells/well were plated in 96-well plates. Three days later, cells were incubated or not with sunitinib and proliferation was assessed after 24, 48 and 72 hours using the colorimetric MTT Cell Proliferation Assay (Sigma-Aldrich®, Saint-Louis, USA), according to the manufacturers protocol. Absorbance at 550nm was measured with a FLUO-star Omega spectrophotometer (BMG Labtech®, Offenbourg, Germany). Experiments were done in triplicate.
RNA was extracted using the RNeasy® Mini Kit according to the manufacturer's protocol (Qiagen®, Venlo, Netherlands). RNA concentration was determined in duplicate using a Nano Drop ND-1000® spectrophotometer (Thermo Scientific®, Wilmington, USA).
Quantitative polymerase chain reaction (qPCR)
Reverse transcription (RT): For each sample, 2µg of RNA were mixed with 7µL reaction mix and RNAse-free water to a final volume of 25µL. The reaction mix contained 5µL 5X reaction buffer (250mM Tris-HCl, 375mM KCl, 15mM MgCl2, 50mM DTT) (Promega® M531A), 0.5µL of 10mM dNTP mix (Promega® U151B), 1µL of 500μg/ml Random Primers (Promega® C118A), 0.2µL of 40U/μL RNasin Plus RNase Inhibitor (Promega® N261A) and 0.3µL of 200U/μL M-MLV Reverse Transcriptase® (M170A, Promega®, Madison, USA). RT was performed in triplicate in a PTC 200-Peltier Thermal Cycler (MJ Research), according to the manufacturer's instructions. Complementary DNA samples were stored at -20°C.
Sequence-specific oligonucleotide primers: Primers were purchased from Eurogentec® (Seraing, Belgium) or Sigma®. GAPDH: forward, 5'-CTGACTTCAACAGCGACACC-3' and reverse, 5'-TAGCCAAATTCGTTGTCATACC-3'; ET-1: forward, 5'TCTCTGCTGTTTGTGGCTTG-3' and reverse, 5'-GAGCTCAGCGCCTAAGACTG-3'; VEcadherin: forward, 5'-ACCCCCACAGGAAAAGAATC-3' and reverse, 5'ACACACTTTGGGCTGGTAGG-3'; ET-RA: forward, 5'-GCGCTCTTAGTGTTGACAGGT3' and reverse, 5'-GAATCCCAATTCCCTGAACA-3'; synaptopodin (SYNPO): forward, 5'AGGGAGGACCTAGCAGACG-3' and reverse, 5'-GTCAGCTGGGCTGCAATC-3'.
Relative quantification by real-time PCR was performed using the Applied 7900HT Fast Real-Time PCR System (Applied Biosystems™) and the Power SYBR® Green PCR Master Mix (Applied Biosystems™, Foster City, USA). Experiments were done in triplicate. Double-distilled water blanks and samples reverse transcribed without reverse transcriptase served as negative controls for each run.
Western blot analysis
Protein extraction: Cells were rinsed twice with phosphate-buffered saline (PBS: 10mM sodium phosphate, pH 7.5, 0.9% saline), and then proteins were extracted with cold lysis buffer (20mM Tris-HCl pH 7.5 (Sigma Aldrich®, Saint-Louis, USA), 100mM NaCl, 5mM MgCl2, 0.5mM dithiothreitol, 20mM β-glycerophosphate, 0.2% Nonidet-P40, 10% glycerol, 10µL/mL Protease Inhibitor Cocktail Set IV® (Calbiochem®, Gibbstown, USA) and 200µL/mL Phosphatase Inhibitor Cocktail Set III® (Calbiochem®, Gibbstown, USA)). Extractions were performed at 4°C for 30min and by nitrogen freezing and heating to 37°C for 30min, and then samples were centrifuged at 13.000rpm for 30min.
Protein concentration: The protein concentration of each sample was determined with the Bradford Reagent B6916 (Sigma-Aldrich®) and bovine serum albumin (BSA) (Sigma®) as protein standard. Absorbance was determined by spectrophotometry on a Jenway 6051 Colorimeter (Jenway®, Felsted, UK).
Electrophoresis: Protein extracts (20µg of each sample) were diluted with 3X Laemmli Buffer (3% SDS, 15% β-mercaptoethanol, 30% glycerol, 0.03% BBP, 1.5M Tris HCl pH 7.5, 3mM EDTA) and denatured at 95°C for 5 min. They were then were separated on 10% SDSpolyacrylamide gels in 10% of 10X Tris-Glycine Buffer (BioRad®, Munich, Germany) and 0.1% SDS using a Power PAC 1000 BioRad® generator following the manufacturer's instructions. Protein Molecular Weight Marker Odyssey® was used as reference. Proteins were then transferred to polyvinylidene fluoride membranes (Thermo Scientific®) with transfer buffer (10X Tris-Glycine, BioRad®).
Immunodetection: Membranes were blocked in 5% dry milk/0.1% Tris-Buffered Saline (50mM Tris-HCl pH 7.4; 150mM NaCl)/0.05% Tween-20 (TBST) for 1hr and then with primary antibodies diluted in 5% dry milk/0.1% TBST at 4°C overnight. Table 1 lists the used primary antibodies. After several rinses with 5% dry milk/0.1% TBST, the appropriate peroxidase-conjugated secondary antibodies were added at room temperature for 1hr. Membranes were washed three times with 0.1% TBST and developed with SuperSignal®
West Dura Extended Duration Substrate (ThermoScientific™, Rockford, USA), using the Curix 60® apparatus (AGFA®, Mortsel, Belgium). Actin expression was tested as loading control. Signal intensity was quantified using the Imagequant™ TL software (GE Healthcare Bio-Sciences AB®, Uppsala, Sweden).
Immunofluorescence staining (IF)
Cells were grown on glass slides. GMVEC cells were fixed at 4°C with 95% ethanol for 30min, followed by acetone at room temperature. hGVE cells were fixed at -20°C with methanol for 3min. For VE-cadherin expression analysis, cells were permeabilized in 0.2% Triton X-100 in PBS for 3 minutes and blocked with 3% BSA IF buffer (PBS, 3% BSA, 0.1% Tween 20) for 1 hour followed by 15 minutes incubation with 1% BSA IF buffer (PBS, 1% BSA, 0.1% Tween 20). Cells were then incubated with primary antibodies against VEcadherin or nephrin at room temperature in a humidified chamber for 1h30. After washing with 1% BSA IF buffer, cells were incubated with Alexa-fluor® 555 donkey anti-mouse IgG (H+L) (1:1000, Molecular Probes, USA). For actin staining, cells were incubated with rhodamine phalloidin conjugated to the red-orange fluorescent dye tetramethylrhodamine (1:100) at room temperature shielded from the light for 1h30. Nuclei were counterstained with DAPI/Anti-fade solution (Q-Biogene, MP Biomedicals, USA) and cells analyzed with a fluorescence microscope (LEICA® DMRXA, Germany).
Enzyme-linked immunosorbent assay (ELISA)
The human ET-1 QuantiGlo ELISA kit (R&D Systems®, Abingdon, United Kingdom) was used according to the manufacture's specifications. ET-1 concentration in aliquots of supernatants from GMVEC cells incubated or not with sunitinib was measured in duplicate. Results were analyzed with the Ascent Software, Multiskan RC (Thermo LabSystems®, Cergy-Pontoise, France), at the wavelength of 450nm.
Cells were plated on eight-well Labtech® chamber slides (30,000 cells/well). The following day, treatment was initiated. At the end of the treatment, cells were rinsed with PBS and fixed in 3.65% formaldehyde (36.7% Formol, Sigma-Aldrich®). Cells were then incubated with 10% hydrogen peroxide for 5min, followed, if needed, by permeabilization by incubation in 0.1% Triton X-100 in PBS for 3min. Blocking was performed with 3% BSA/PBS for 1h, followed by rinses with 1% BSA/PBS for 10min. Cells were stained with primary antibodies diluted in PBS for 1h30. Commercially available kits were used for secondary antibodies (Dual Link System-HRP, Dako®). Antibody reactions were revealed with 3.3diaminobenzidine (DAKO®, Glostrup, Denmark), followed by counterstaining with 0.5X hematoxylin. Slides were mounted with Faramount Aqueous Mounting Medium (DAKO®) and stored at room temperature. Images were acquired with a Leica® DFC 295 microscope.
Results are representative of at least three independent experiments performed in triplicate, unless otherwise mentioned. Results are expressed as the mean ± standard deviation (SD). Statistical analysis of the data was performed using the Student's t-test for independent variables and the R software (Bell Laboratories, USA). Differences were considered significant when p<0.05.
Sunitinib reduces proliferation of hGVE and GMVEC cells
To test the in vitro effects of sunitinib on glomerular epithelial and endothelial cells, hGVE and GMVEC cells were plated in 96-well plates (2000 cells/well) and incubated with different concentrations (0.15µM, 1.5µM, and 5µM) of sunitinib or vehicle alone (DMSO; control cells). Cell proliferation/viability was then assessed with the MTT Cell Proliferation Assay at 24h (D1), 48h (D2), 72h (D3) and 96h (D4) after addition of sunitinib. Compared with control cells, sunitinib reduced proliferation/viability of both hGVE and GMVEC cells in a dose-dependent manner (Figure 1). The 5µM concentration was too toxic, leading to a high rate of cell mortality (data not shown) and therefore was not used in the subsequent experiments.