The renal toxicity induced by uremic toxins is thought to have a specific pathologic role in renal disease progression. Indoxyl sulfate (IS) and p-cresol sulfate (PCS) are protein-bound uremic toxins that are not dialyzable.1 Several pathologic mechanisms such as oxidative stress, inflammatory reaction, and renin-angiotensin-aldosterone system activation have been proposed to contribute to chronic kidney injury induced by IS and PCS.2,3 Animal studies have indicated that IS and PCS cause renal fibrosis.3 Pathologic tissue remodeling has been suggested to have a detrimental role in the tissue fibrosis. The matrix metalloproteinases (MMPs) play an important role in tissue remodeling associated with various physiologic and pathologic processes. MMPs can act on a wide range of extracellular matrix proteins.4 EGF receptor (EGFR) and its signaling cascade activation have crucial roles in enhancing MMP expression and function in organogenesis and cancer development.5,6 Evidence indicates that IS and PCS induce tissue remolding in vivo.3,7 However, the molecular mechanism underlying the tissue remodeling induced by IS and PCS is still unknown. This study aimed to find the putative pathologic linkage between the protein-bound uremic toxins, EGFR, and MMPs to clarify the molecular mechanism underlying renal tissue remolding induced by IS and PCS.
The functional domains of EGFR are illustrated in Figure 1A. Two EGFR structure templates, active (Protein Data Bank entry code 1IVO)8 and inactive (Protein Data Bank entry code 1NQL),9 were used for molecular simulation and docking. Utilizing molecular simulation, a putative interdomain pocket of active EGFR (1IVO), which is located just beneath the EGF binding site, was identified (Figure 1B). The docking analysis indicated that IS and PCS could dock in the interdomain pocket of active EGFR (1IVO) (Figure 1, C and D). However, the conformation of the interdomain pocket of inactive EGFR (1NQL) was significantly different from that of active EGFR (1IVO). Protein superimposition revealed that the structures of the interdomain pocket were different between 1NQL and 1IVO (Figure 1, E and F). The docking analysis demonstrated that IS and PCS failed to target the interdomain pocket of inactive EGR (1NQL) (Figure 1, G and H). To evaluate whether IS and PCS interacted with EGFR in vitro, light absorption of IS and PCS in the presence or absence of synthetic extracellular EGFR peptide (amino acids 25–645) was analyzed. The results indicated that the presence of EGFR (10 µM) significantly decreased the OD of IS (50 mg/L) and PCS (50 mg/L) (Figure 1I). The effects of IS and PCS on the EGFR dimerization were tested in vitro by incubating IS, PCS, and synthetic EGFR peptide. Silver staining indicated that EGFR dimer increased as the concentrations of IS and PCS were increased in vitro (Figure 1J). The effects of IS and PCS on the EGFR dimerization were tested in HK2 cells. The Western blot results indicated that IS and PCS treatment increased the dimer formation of EGFR in a dose-dependent manner (Figure 1K). Previous studies suggested that interactions between the extracellular domains of EGFR were important for the stability of EGFR.9,10 It was suggested that IS and PCS might stabilize the active structure of EGFR and facilitate EGFR dimerization.
Western blot analysis indicated that IS and PCS significantly increased EGFR phosphorylation in vitro (Figure 2A). Immunofluorescence staining showed that IS and PCS treatment significantly increased the membrane localization of EGFR in cultured HK2 cells (Figure 2B). In addition, IS and PCS significantly increased the phosphorylation of β-catenin (Figure 2C) and the nuclear localization of β-catenin (Figure 2D). Our results indicated that IS and PCS increased MMP2 and MMP9 expression in a dose-dependent manner in HK2 cells (Figure 2E). We also performed the Western blotting and activity analysis for MMP2 and MMP9 with culture medium of HK2 cells after 4-day IS, PCS, and EGF treatment. The study results showed that the culture medium of cells treated with IS and PCS had significantly increased MMP2 and MMP9 activities (Supplemental Figure 1).
To test whether EGFR activation by IS and PCS was associated with increased expression of MMP2 and MMP9, an in vitro study was performed with EGFR inhibitor (GW2974) treatment. Inhibiting EGFR with GW2974 could significantly suppress EGFR phosphorylation, which was induced by IS and PCS in vitro (Figure 2F). GW2974 treatment decreased Stat1 and Stat3 phosphorylation induced by IS and PCS (Figure 2G). It was also noted that inhibition of EGFR attenuated the effects of IS and PCS on MMP2 and MMP9 expression in vitro (Figure 2H). The in vitro studies also showed that EGFR activation by EGF could be augmented in the presences of IS and PCS. Co-treatment with EGF (1 ng/ml) and IS/PCS significantly increased the MMP2 and MMP9 expression compared with the cells treated with EGF along (Supplemental Figure 2). IS and PCS are the albumin-bound uremic toxins. In vitro experiments in the presence of albumin were performed. The study results showed that the presence of albumin in the culture medium significantly lessened the EGFR activation and attenuated the effects of IS and PCS on MMP2 and MMP9 expression (Supplemental Figure 3).
The animal study results showed that treatment with IS or PCS significantly activated EGFR signaling by increasing the EGFR-p, Erk1/2-p, and Akt-p expression in the kidney of mice (Figure 3A). Immunofluorescence staining for EGFR-p and tubular markers showed that EGFR-p was stained strongly in the tubulointerstitium of mice treated with IS and PCS. In mice treated with IS and PCS, EGFR-p was predominately located in the proximal tubule segments that were positive for Lotus tetragonolobus lectin staining. EGFR-p was also found in tubular cells with positive Dolichosbiflorus agglutinin staining, which was a marker for collecting ducts (Figure 3B). Western blot results revealed that IS or PCS treatment significantly increased the expression of β-catenin-p in the mice (Figure 3C). In addition to the membrane and cytoplasmic localization of β-catenin, immunohistochemistry staining results indicated that IS and PCS significantly increased the nuclear localization of β-catenin in the tubular cells of the mice (Figure 3D). Western blotting results indicated that the study mice treated with IS and PCS significantly increased renal MMP2 and MMP9 expression (Figure 3E). Immunohistochemistry analysis indicated that IS and PCS treatment significantly increased positive tubulointerstitial staining for MMP2 and MMP9 in mice treated with IS and PCS (Figure 3F). The results of EGFR inhibition study indicated that GW2974 treatment significantly attenuated the increase of MMP2 and MMP9 expression by IS and PCS in vivo (Figure 3, G and H).
The study results are summarized in Figure 4. EGFR activation has been shown to contribute to the development and progression of renal fibrosis.11,12 Our results suggested that protein-bound uremic toxins IS and PCS might target EGFR. Hydrophobic interactions between IS/PCS and EGFR might facilitate EGFR dimerization and activate EGFR signaling. The activated EGFR signaling could increase the tubulointerstitial expression of matrix proteases MMP2 and MMP9, which are involved in tissue remodeling. It has been known that organic anion transporters are responsible for transporting IS and PCS into the cells. The intracellular localization of IS and PCS plays a critical role in mediating the toxic effects of IS and PCS. Inhibiting the IS and PCS transportation by organic anion transporters could minimize the cellular toxicity of IS and PCS.13,14 Our data further clarified the molecular mechanism underlying the cellular toxicity of IS and PCS. Our study suggested that the cellular toxicity of IS and PCS could be mediated by an alternative pathway other than the organic anion transporters. Direct targeting and activation of cytokine receptors might be considered as a potential alternative pathologic pathway.
The mechanisms associated with EGFR activation are complicated, and currently are not completely understood. Ligand-driven dimerization and intermolecular allosteric activation of EGFR are the main mechanisms.10,15 It is proposed that monomeric EGFR is in equilibrium with active and inactive receptor oligomers in the cell membrane.15,16 This study suggested that IS and PCS might target the extracellular interdomain pocket of active EGFR, but not inactive EGFR. It has been shown that extracellular domain II is important for the receptor-mediated dimerization of EGFR.8 Intramolecular interactions between the extracellular domains are important for maintaining EGFR active structure. Mutations that disrupt the intramolecular domain II/IV interaction might increase EGF binding affinity.9 It has been reported that the interaction between domains II and III could play a significant part in maintaining EGFR in the extracellular region in an inactive conformation.17 Our data indicated that IS and PCS could bind with the extracellular portion of EGFR. Furthermore, in vitro studies also showed that IS and PCS could increase the dimerization and activation of EGFR. Our data suggest that binding of IS and PCS with extracellular portion of EGFR might stabilize the active EGFR structure, which in turn promotes EGFR dimerization and phosphorylation.
Accumulated evidence indicated that EGFR activation might contributes to development and progression of renal diseases in animal models of CKD. Activation of renal interstitial fibroblasts, induction of tubular atrophy, overproduction of inflammatory factors, and promotion of glomerular injury by EGFR activation have been proposed as the possible pathologic mechanisms for chronic kidney injury.18 Recent studies also indicated that EGFR activation is essential for the upregulation of renal fibrotic genes by TGF-β1.19 Epithelial–mesenchymal transition of renal tubular cells was proposed as a possible mechanism for the renal fibrosis induced by IS and PCS.3 Recent evidence indicated that MMPs are implicated in the initiation and progression of renal fibrosis. MMP2 and MMP9 can induce renal tubular cell epithelial–mesenchymal transition in vitro.20,21 Previous studies had revealed that MMP expression is induced by EGFR signaling.22 Our results showed that IS and PCS could activate signaling of EGFR, Akt, and Erk1/2 in study mice. It was reported that EGFR activation and subsequent tyrosine phosphorylation of β-catenin contribute to the activation and nuclear translocation of β-catenin.23 Nuclear β-catenin could activate β-catenin target genes such as MMP2 by histone H3 and H4 acetylation.24 The results of our study indicated that IS and PCS stimulated β-catenin phosphorylation and increased its nuclear localization in vitro and in vivo. It can be suggested that activation and nuclear translocation of β-catenin by EGFR signaling is an important pathway to facilitate tubulointerstitial MMP expression by IS and PCS.
In conclusion, our study suggested that direct targeting and activation of cytokine receptors by protein-bound uremic toxins might be a potential pathologic pathway other than the injury mediated by organic anion transporters. Activation of EGFR by IS and PCS could contribute to renal tissue remodeling by increasing MMP2 and MMP9 expression.
Molecular docking was performed to investigate the interaction between the EGFR, IS, and PCS by using the SwissDock server and EADock program.25 The structure of extracellular domains of EGFR was adapted by computing the three-dimensional structure of a previous report (1IVO, 1NQL).8,9 The binding modes were generated either in a local docking or in the vicinity of all target cavities. Simultaneously, their Chemistry at HARvard Molecular Mechanics energies were estimated on a grid. The binding modes with the most favorable energies were evaluated with fast analytical continuum treatments of solvation, and clustered. The most favorable clusters were edited using PyMol software.26 Pairwise protein structure superpositions for active and inactive EGFRs (1IVO, 1NQL) were done with a modified quaternion eigenvalue approach in the SuperPose web server by using default parameters.27
Recombinant human EGFR (amino acids 25–645; PeproTech, Rocky Hill, NJ), comprising the extracellular domain of EGFR, was dissolved in PBS at a concentration of 10 µM, and incubated with IS (Sigma-Aldrich, St. Louis, MO) and PCS (Kureha, Tokyo, Japan) at room temperature for 30 minutes in the dark. The experimental conditions and reagent concentrations utilized for the spectrophotometric analysis are shown in the figure legends. Samples were analyzed in the 200- to 400-nm wave length range (SpectraMax M3; Molecular Devices LLC, Sunnyvale, CA).
Cell Culture and Treatment
HK2 cells were cultured as previously reported.28 HK2 cell cultures at 70% confluence were synchronized under serum-free conditions for 4 hours. The HK2 cells under the serum-free condition were then treated with IS or PCS at concentrations of 0, 1, 5, and 50 mg/L for 24 hours. For EGFR inhibition experiments, GW2974 (Sigma-Aldrich), a potent and selective dual inhibitor of EGFR and ErbB-2 receptor tyrosine kinase,29 at concentrations of 0, 0.4, 2, and 10 µM were added to HK2 cells 2 hours before IS and PCS treatment.
In Vitro Analyses of EGFR Dimerization
The recombinant human EGFR was incubated with IS and PCS at concentrations of 0, 1, 5, and 50 mg/L in PBS at room temperature for 12 hours in the dark. When the incubation was completed, the reaction mixtures (100 µl) were treated with 5 µl of 2.3% freshly prepared solution of glutaraldehyde (Sigma-Aldrich) for 5 minutes at 37°C. The reaction was terminated by the addition of 10 µl of 1 M Tris-HCl (Sigma-Aldrich), pH 8.0. The protein samples were analyzed by electrophoresis in 12% polyacrylamide gels, and visualized with silver staining. Cultured HK2 cells were extracted in cell lysis buffer without Triton after treatment with IS and PCS at concentrations of 0, 1, 5, and 50 mg/L for 24 hours. The cell lysates were then treated with glutaraldehyde (Sigma-Aldrich) to facilitate protein cross-linking and were subjected to electrophoresis.
The procedure for animal study was according to a previous report, with some modifications.3,30 In brief, 10-week-old B-6 mice subjected to half nephrectomy were used in this study. The experimental mice received intraperitoneal injection with IS (n=8) or PCS (n=8) at a dosage of 100 mg/kg per day for 1 week. The control mice (n=8) were administered daily with PBS injection for 1 week at similar volumes. For GW2974 treatment, the IS- and PCS-injected mice simultaneously administered GW2974 by the oral route at a dosage of 10 mg/kg per day for 1 week (n=8 for each group). At the end of the study, the renal cortex was microdissected for further analysis. All animal experiments were approved by the experimental animal ethics committee of the Chang Gung Memorial Hospital.
Western Blotting Analyses
Total protein was extracted using a commercial kit following the manufacturer’s instructions (Protein Extraction Kit; EMD Millipore, Billerica, MA). Thirty milligrams of protein from each sample was mixed with sample-loading buffer and loaded onto separate lanes in 12% SDS-polyacrylamide gel. Proteins were electrotransferred onto polyvinylidene fluoride membranes (0.2 mm, Immun-Blot; Bio-Rad), and then immunoblotted with antibodies against EGFR (Cell Signaling Technology, Denver, MA), EGFR-p (Tyr-1068; Cell Signaling), MMP2 (Abcam, Inc., Cambridge, MA), MMP9 (Abcam, Inc.), Stat1-p (Abcam, Inc.), Stat3-p (Abcam, Inc.), Erk1/2-p (Thr202/Tyr204; Cell Signaling Technology), Akt-p (Ser473; Cell Signaling Technology), β-catenin (Abcam, Inc.), β-catenin-p (Ser33/Ser37; Cell Signaling Technology), and β-actin (Abcam, Inc.). The intensity of each band was quantified using National Institutes of Health Image software (Bethesda, MD), and the densitometric intensity corresponding to each band was normalized with β-actin expression.
Immunohistochemistry and Immunofluorescence Staining
For immunohistochemistry analysis, paraffin tissue sections were cut, mounted, deparaffinized, rehydrated, and stained with hematoxylin and eosin by using standard histologic techniques. The Ventana BenchMark automated staining system and Ventana reagents were used (Ventana Medical Systems, Tucson, AZ), and primary antibodies against MMP2/MMP9 (1:100 dilution; Abcam, Inc.) and β-catenin-p (Ser33/Ser37, 1:100 dilution; Cell Signaling Technology) at 4°C were used overnight. For immunofluorescence staining, cultured HK2 cells and serial cryostat sections of renal cortex were incubated with a primary rabbit antibody against EGFR (1:100 dilution; Cell Signaling Technology), EGFR-p (Tyr1068, 1:100 dilution; Cell Signaling Technology), and β-catenin-p (Ser33/Ser37, 1:100 dilution; Cell Signaling Technology). For renal tubule segment identification, frozen kidney samples were incubated with primary antibodies including FITC-conjugated anti-Lotus tetragonolobus lectin (1:500 dilution; Vector Laboratories, Burlingame, CA) and FITC-conjugated anti-Dolichosbiflorus agglutinin (1:500 dilution; Vector Laboratories).31 The sections were counterstained with 4′,6-diamidino-2-phenylindole (dilution 1:500; Sigma-Aldrich) to identify cellular nuclei.
All data are expressed as the mean±SE. Data from different groups were compared using the Wilcoxon–Mann–Whitney test. P<0.05 was considered statistically significant.
The authors thank the staff of the Medical Research Institute of Keelung Chang Gung Memorial Hospital for the experimental assistance.
This study was supported by a grant from the Taiwan National Science Council (NMRPG2B6032).
A portion of this work was presented at the 2013 Annual Meeting of the American Society of Nephrology, held November 5–10, 2013, in Atlanta, Georgia.
Published online ahead of print. Publication date available at www.jasn.org.
This article contains supplemental material online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2014010021/-/DCSupplemental.
1. Raff AC, Meyer TW, Hostetter TH: New insights into uremic toxicity. Curr Opin Nephrol Hypertens 17: 560–565, 2008
2. Watanabe H, Miyamoto Y, Honda D, Tanaka H, Wu Q, Endo M, Noguchi T, Kadowaki D, Ishima Y, Kotani S, Nakajima M, Kataoka K, Kim-Mitsuyama S, Tanaka M, Fukagawa M, Otagiri M, Maruyama T: p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase. Kidney Int 83: 582–592, 2013
3. Sun CY, Chang SC, Wu MS: Uremic toxins induce kidney fibrosis by activating intrarenal renin-angiotensin-aldosterone system associated epithelial-to-mesenchymal transition. PLoS ONE 7: e34026, 2012
4. Klein T, Bischoff R: Physiology and pathophysiology of matrix metalloproteases. Amino Acids 41: 271–290, 2011
5. Hudson LG, Moss NM, Stack MS: EGF
-receptor regulation of matrix metalloproteinases
in epithelial ovarian carcinoma. Future Oncol 5: 323–338, 2009
6. Matrisian LM, Hogan BL: Growth factor-regulated proteases and extracellular matrix remodeling during mammalian development. Curr Top Dev Biol 24: 219–259, 1990
7. Bolati D, Shimizu H, Higashiyama Y, Nishijima F, Niwa T: Indoxyl sulfate induces epithelial-to-mesenchymal transition in rat kidneys and human proximal tubular cells. Am J Nephrol 34: 318–323, 2011
8. Ogiso H, Ishitani R, Nureki O, Fukai S, Yamanaka M, Kim JH, Saito K, Sakamoto A, Inoue M, Shirouzu M, Yokoyama S: Crystal structure of the complex of human epidermal growth factor and receptor extracellular domains. Cell 110: 775–787, 2002
9. Ferguson KM, Berger MB, Mendrola JM, Cho HS, Leahy DJ, Lemmon MA: EGF
activates its receptor by removing interactions that autoinhibit ectodomain dimerization. Mol Cell 11: 507–517, 2003
10. Ferguson KM: Structure-based view of epidermal growth factor receptor regulation. Annu Rev Biophys 37: 353–373, 2008
11. Terzi F, Burtin M, Hekmati M, Federici P, Grimber G, Briand P, Friedlander G: Targeted expression of a dominant-negative EGF
-R in the kidney reduces tubulo-interstitial lesions after renal injury. J Clin Invest 106: 225–234, 2000
12. Chen J, Chen JK, Nagai K, Plieth D, Tan M, Lee TC, Threadgill DW, Neilson EG, Harris RC: EGFR signaling
promotes TGFβ-dependent renal fibrosis. J Am Soc Nephrol 23: 215–224, 2012
13. Enomoto A, Niwa T: Roles of organic anion transporters in the progression of chronic renal failure. Ther Apher Dial 11[Suppl 1]: S27–S31, 2007
14. Miyamoto Y, Watanabe H, Noguchi T, Kotani S, Nakajima M, Kadowaki D, Otagiri M, Maruyama T: Organic anion transporters play an important role in the uptake of p-cresyl sulfate, a uremic toxin, in the kidney. Nephrol Dial Transplant 26: 2498–2502, 2011
15. Yarden Y, Schlessinger J: Self-phosphorylation of epidermal growth factor receptor: Evidence for a model of intermolecular allosteric activation. Biochemistry 26: 1434–1442, 1987
16. Endres NF, Das R, Smith AW, Arkhipov A, Kovacs E, Huang Y, Pelton JG, Shan Y, Shaw DE, Wemmer DE, Groves JT, Kuriyan J: Conformational coupling across the plasma membrane in activation of the EGF
receptor. Cell 152: 543–556, 2013
17. Katz WS, Lesa GM, Yannoukakos D, Clandinin TR, Schlessinger J, Sternberg PW: A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog. Mol Cell Biol 16: 529–537, 1996
18. Tang J, Liu N, Zhuang S: Role of epidermal growth factor receptor in acute and chronic kidney injury. Kidney Int 83: 804–810, 2013
19. Samarakoon R, Dobberfuhl AD, Cooley C, Overstreet JM, Patel S, Goldschmeding R, Meldrum KK, Higgins PJ: Induction of renal fibrotic genes by TGF-β1 requires EGFR activation, p53 and reactive oxygen species. Cell Signal 25: 2198–2209, 2013
20. Cheng S, Lovett DH: Gelatinase A (MMP-2) is necessary and sufficient for renal tubular cell epithelial-mesenchymal transformation. Am J Pathol 162: 1937–1949, 2003
21. Tan TK, Zheng G, Hsu TT, Wang Y, Lee VW, Tian X, Wang Y, Cao Q, Wang Y, Harris DC: Macrophage matrix metalloproteinase-9 mediates epithelial-mesenchymal transition in vitro in murine renal tubular cells. Am J Pathol 176: 1256–1270, 2010
22. Kodali R, Hajjou M, Berman AB, Bansal MB, Zhang S, Pan JJ, Schecter AD: Chemokines induce matrix metalloproteinase-2 through activation of epidermal growth factor receptor in arterial smooth muscle cells. Cardiovasc Res 69: 706–715, 2006
23. Rosanò L, Cianfrocca R, Masi S, Spinella F, Di Castro V, Biroccio A, Salvati E, Nicotra MR, Natali PG, Bagnato A: Beta-arrestin links endothelin A receptor to beta-catenin signaling
to induce ovarian cancer cell invasion and metastasis. Proc Natl Acad Sci U S A 106: 2806–2811, 2009
24. Rosanò L, Cianfrocca R, Tocci P, Spinella F, Di Castro V, Spadaro F, Salvati E, Biroccio AM, Natali PG, Bagnato A: β-arrestin-1 is a nuclear transcriptional regulator of endothelin-1-induced β-catenin signaling
. Oncogene 32: 5066–5077, 2013
25. Grosdidier A, Zoete V, Michielin O: Fast docking using the CHARMM force field with EADock DSS. J Comput Chem 32: 2149–2159, 2011
26. Seeliger D, de Groot BL: Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des 24: 417–422, 2010
27. Maiti R, Van Domselaar GH, Zhang H, Wishart DS: SuperPose: A simple server for sophisticated structural superposition. Nucleic Acids Res 32: W590–W594, 2004
28. Nightingale J, Patel S, Suzuki N, Buxton R, Takagi KI, Suzuki J, Sumi Y, Imaizumi A, Mason RM, Zhang Z., Oncostatin M: A cytokine released by activated mononuclear cells, induces epithelial cell-myofibroblast transdifferentiation via Jak/Stat pathway activation. J Am Soc Nephrol 15: 21–32, 2004
29. Busch H, Camacho-Trullio D, Rogon Z, Breuhahn K, Angel P, Eils R, Szabowski A: Gene network dynamics controlling keratinocyte migration. Mol Syst Biol 4: 199, 2008
30. Sun CY, Chang SC, Wu MS: Suppression of Klotho expression by protein-bound uremic toxins is associated with increased DNA methyltransferase expression and DNA hypermethylation. Kidney Int 81: 640–650, 2012
31. Kusaba T, Lalli M, Kramann R, Kobayashi A, Humphreys BD: Differentiated kidney epithelial cells repair injured proximal tubule. Proc Natl Acad Sci U S A 111: 1527–1532, 2014