- Email: [email protected]
Comparative Proteomic Analysis of Rapamycin Versus Cyclosporine Combination Treatment in Mouse Podocytes
Comparative Proteomic Analysis of Rapamycin Versus Cyclosporine Combination Treatment in Mouse Podocytes B.S. Kima,b, Y. Chob, H. Leeb, D.J. Joob,c, K.H. Huhb,c, M.S. Kimb,c, and Y.S. Kimb,c,* a Division of Nephrology, Department of Internal Medicine, and cDepartment of Transplantation Surgery, Severance Hospital, Yonsei University Health System, Seoul, Republic of Korea; and bThe Research Institute for Transplantation, Yonsei University College of Medicine, Seoul, Republic of Korea
ABSTRACT Background. The mechanism of podocyte injury observed with the use of rapamycin (RPM) remains unclear. The conversion from calcineurin inhibitors (CNIs) to RPM in kidney transplant recipients has been associated with a higher incidence of proteinuria and renal injury. In this study, we performed proteomic analyses to investigate the alteration of protein expression in mouse podocytes treated with RPM in comparison with CNI/RPM combination. Methods. Immortalized mouse podocytes were treated with 20 nmol/L RPM or 20 nmol/L RPM þ 1 mg/mL cyclosporine. Podocyte proteins were separated by 2-dimensional polyacrylamide gel electrophoresis (2DE) and identified by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and peptide fingerprinting. Selected proteins were analyzed by means of Western blot assay. Results. We identified 36 differently expressed proteins after isolated RPM or CNI/RPM combination treatment in cultured mouse podocytes. There are 3 distinct patterns of protein expression: (1) potentiated down- or upregulation of proteins by CNI/RPM treatment compared with isolated RPM treatment (n ¼ 4); (2) partial offset of downregulation by CNI/RPM in comparison with RPM treatment (n ¼ 25); (3) no difference in down-regulation between RPM and CNI/RPM treatment (n ¼ 5). We found a significant interplay between RPM and CNI on the expression of the selected proteins in mouse podocytes. This might explain the higher incidence of proteinuria by CNI/RPM combination in clinical settings. Conclusions. Further study is required to elucidate the target protein associated with RPM-induced podocyte injury.
P
ODOCYTES play a key role both in maintenance of the glomerular filtration barrier and in glomerular structural integrity. Podocyte injury and loss contribute to proteinuria and progressive sclerosis [1]. Several studies demonstrate an association between mTOR inhibitor (mTORi) and kidney injury after kidney transplantation and suggest that mTORi(s) may alter the behavior and integrity of glomerular podocytes. This effect could be observed to a different extent in diverse settings [2]. In transplant settings, calcineurin inhibitors (CNIs) are often used with mTORi to maintain optimal immunosuppression. However, various adverse events, such as proteinuria, increase in this setting, whereas the mechanisms are still largely unknown. Detailed
analyses of mTOR-associated regulatory events in glomerulopathies and podocytes are required to understand the complex role of this pathway in glomerular disease. This
This work was supported by a faculty research grant of Yonsei University College of Medicine for 2012 (6-2012-0130) and a research grant from Yonsei University Health System IACF (2012-31-0585). *Address correspondence to Yu Seun Kim, Department of Surgery, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Ku, 03722, Seoul, South Korea. E-mail: yukim@ yuhs.ac or [email protected]
ª 2016 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.01.022
Transplantation Proceedings, 48, 1297e1301 (2016)
1297
1298
study focuses on mTORi-associated molecular alteration in podocytes treated with RPM in comparison with CNI/RPM combination. To investigate the issue, we performed proteomic analyses in immortalized mouse podocytes treated with RPM or CNI/RPM combination. METHODS Immortalized Mouse Podocyte Cell Culture Conditionally immortalized mouse podocytes were cultured according to the method described by Mundel et al [3]. Undifferentiated podocytes were cultured in RPMI 1640 medium (GIBCO, NY, United States) with 10% fetal bovine serum (GIBCO) and 10 U/mL recombinant mouse interferon-g (Cell Sciences, Canton, Mass, United States) in an incubator at 33 C in 5% CO2. After differentiation, the podocytes were cultured at 37 C in RPMI 1640 medium without interferon-g.
KIM, CHO, LEE ET AL iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of 0.1 Da. PMF acceptance criteria are probability scoring.
Immunoblot Analysis Protein extracts from the cells were prepared with the use of RIPA buffer. Proteins were separated by use of 10% to 15% SDS-PAGE and blotted onto PVDF membranes. Proteins were detected by use of the following primary antibodies: Gelsolin, Txndc5, PrelaminA/ C, and Vdac2 (Santa Cruz Biotechnology, CA, USA).
Statistics Results are expressed as mean SEM values. One-way ANOVA with Tukey’s post hoc test was used to determine the significance of the differences between means. P values of <.05 were considered statistically significant. The analysis was conducted with the use of the GraphPad Prism software.
Two-Dimensional Polyacrylamide Gel Electrophoresis For the analysis of proteome profiling, 2-dimensional gel electrophoresis (2DE), gel image analyses, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were performed according to the Genomine INC. Protocol-immobilized pH gradient (IPG) dry strips (4e10 nonlinear IPG, 24 cm, Genomine, Korea) were equilibrated for 12 to 16 hours with 7 mol/L urea, 2 mol/L thiourea containing 2% 3-[(3cholamidopropy)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% pharmalyte, respectively, and loaded with 200 mg of samples. Prior to the second dimension, strips were incubated for 10 minutes in equilibration buffer (50 mmol/L Tris-Cl, pH 6.8, containing 6 mol/L urea, 2% SDS, and 30% glycerol), 1% DTT, and 2.5% iodoacetamide, respectively. Equilibrated strips were inserted onto SDS-PAGE gels (20 24 cm, 10% to 16%). The 2D gels were run at 20 C for 1700 Vh. The 2D gels were silver-stained as described by Oakley et al [4]. However, fixing and sensitization steps with glutaraldehyde were omitted.
Image Analysis The quantitative analysis of digitized images was carried out with the use of PDQuest (version 7.0, BioRad) software according to the protocol provided by the manufacturer. The quantity of each spot was normalized by the total intensity of valid spots. Protein spots were selected on the basis of significant expression variation deviated over 2-fold in its expression level compared with control samples.
Peptide Mass Fingerprinting For protein identification by peptide mass fingerprinting (PMF), protein spots were excised, digested with trypsin (Promega, Madison, Wis, United States), mixed with a-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% TFA, and subjected to MALDITOF analyses (Microflex LRF 20, Bruker Daltonics), as described by Fernandez J et al [5]. Spectra were collected from 300 shots per spectrum over m/z range 600 to 3000 and calibrated by means of 2point internal calibration, using trypsin auto-digestion peaks (m/z 842.5099, 2211.1046). A peak list was generated by use of Flex Analysis 3.0. The thresholds used for peak-picking were 500 for minimum resolution of monoisotopic mass and 5 for S/N. The search program MASCOT was used for protein identification by PMF. The following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of 1 missed cleavage,
RESULTS
To investigate molecular alteration in mouse podocytes treated with RPM or CNI/RPM combination, we performed proteomic analyses with the use of 2D-PAGE. Immortalized mouse podocytes were treated with 20 nmol/L RPM or 1 mg/ mL cyclosporine þ 20 nmol/L RPM. Podocyte proteins were separated by use of 2D-PAGE and identified by means of MALDI-TOF mass spectrometry and peptide fingerprinting. Two representative high-resolution, 2-dimensional maps from mouse podocyte cells of 3 groups are shown in Fig 1. In total, 666.5 97.5, 588.5 42.5, and 591.5 29.5 protein spots were separated and visualized, respectively. We analyzed and compared quantitatively 2DE with the use of PDQuest image analyses and 36 differently expressed proteins after isolated RPM or CNI/RPM combination treatment in cultured mouse podocytes (Table 1). Among them, we focused on potentiated down- or up-regulation of proteins by CNI/RPM treatment compared with isolated RPM treatment. The expression of Txndc5, Gelsolin, PrelaminA/C, and VDAC2 exhibited remarkable differences between isolated RPM and CNI/RPM (Fig 2A). Txndc5 was increased by CNI/RPM combination, and Gelsolin, PrelaminA/C, and Vdac2 were significantly decreased by CNI/ RPM treatment compared with isolated RPM treatment (Fig 2B). DISCUSSION
Detailed analyses of mTOR-associated regulatory events in glomerulopathies and podocytes are required to understand the complex role of this pathway in glomerular disease. Although mTORi is well known for inducing severe proteinuria clinically [6,7], only a few reports explain how proteinuria is triggered by mTOR inhibition [7]. Recently, Stallone et al [8] reported that RPM decreased slit diaphragm (SD)-associated molecules dose-dependently; however, they did not show the direct causation between them, and only some patients with the use of mTORi had podocyte injury clinically.
PROTEOMIC ANALYSIS OF RAPAMYCIN TREATED PODOCYTE
1299
Fig 1. Representative 2DE maps (pH 3e10 gradient) in 3 groups: sham operative group (CTRL); rapamycin group (RPM); and CNI/RPM combination group (CNIþRPM).
To investigate this issue, we hypothesized that mTORiinduced podocyte injury might be potentiated by commonly accompanying conditions, such as hypoxia, CNI combination, or CNI conversion treatment. Among these, we examined the effect of CNI combination treatment with RPM.
According to the result, there are 3 distinct patterns of protein expression: (1) potentiated down- or up-regulation of proteins by CNIþRPM treatment compared with isolated RPM treatment (n ¼ 4); (2) partial offset of downregulation by CNIþRPM in comparison with RPM (n ¼ 25); (3) no difference in down-regulation between RPM and
Table 1. Differential Expression Proteins Identified by Mass Spectrometry Between the RPM, CNIDRPM, and Control Groups
No.
Sample No.
1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
1701R 2506R 2705R 3701R 3801R 4603R 4702R 4705R 4806R 5802R 5810R 6501R 6504R 6802R 6813R 7006R 7202R 7305R 7605R 7701R 7703R 7707R 7804R 7805R 7808R 8201R 8308R 8309R 8406R 8702R 8801R 8802R 8804R
Protein Name
Chain A, structural basis of Pp2a and Sgo interaction Thioredoxin domainecontaining protein 5 isoform 3 Heat shock protein-8 V-type proton ATPase catalytic subunit-A Stress-70 protein, mitochondrial RuvB-like 2 Coronin-1B Prolyl 4-hydroxylase subunit alpha-1 isoform X1 Alpha-glucosidase 2 alpha neutral subunit, isoform CRA_a Gelsolin isoform 2 Neutral alpha-glucosidase AB isoform 2 precursor Ornithine aminotransferase, mitochondrial precursor NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 Trap1 protein Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 precursor Rassf4 protein mCG13231 3-Mercaptopyruvate sulfurtransferase Aldehyde dehydrogenase mCG2023 Predicted: Dihydropyrimidinase-related protein 2 UPF0317 protein C14orf159 homolog Propionyl-coenzyme A carboxylase, alpha-polypeptide Oxoglutarate dehydrogenase (lipoamide) Predicted: Forkhead-associated domain-containing protein 1 Proteasome subunit-alpha type 6 Voltage-dependent anion-selective channel protein 2 Annexin A2 Chain A, crystal structure of mouse transaldolase WD repeat-containing protein 1 Prelamin-A/C isoform A precursor MICOS complex subunit Mic60 isoform 5 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 isoform 1
Scores
Molecular Weight (Da)
136 147 209 114 241 176 87 167 252 115 132 224 97 210 138 74 81 101 171 103 210 145 238 127 72 88 86 185 141 242 151 205 269
81.0 58.3 95.3 94.5 96.7 31.7 65.0 88.4 137.5 119.2 139.7 58.5 59.8 98.1 120.5 20.9 32.5 40.1 66.3 85.0 85.4 88.2 101.6 138.2 136.3 35.1 41.7 47.3 49.0 93.6 100.1 110.5 127.4
Expression Ratio RPM/CTL CþR/CTL
0.006 3.028 0.127 0.045 0.410 0.284 0.141 0.026 0.076 0.459 0.005 0.543 0.715 0.007 0.007 0.001 0.182 0.005 0.387 0.828 0.579 0.585 0.400 0.006 0.004 0.878 0.505 0.107 0.255 0.062 0.275 0.002 0.274
0.722 3.738 0.330 0.167 0.418 0.452 0.266 0.579 0.392 0.402 0.407 0.656 0.799 0.587 0.144 0.001 0.417 0.005 0.663 0.889 0.799 0.598 0.735 0.482 0.530 0.829 0.454 0.735 0.606 0.459 0.250 0.512 0.320
1300
KIM, CHO, LEE ET AL
proteins and has been shown to have thioreductase activity [9e11]. Reducing ERp46 modulates the expression and/or distribution of the adiponectin receptors and adiponectin signaling [12]. Gelsolin is expressed by most tubular epithelial cells in normal kidney and has also been localized to glomerular endothelium [13], and it is consistent with the expression by all glomerular cell types, including podocytes [14]. Gelsolin was up-regulated in mutant podocytes, whereas the labeling of other glomerular cells was decreased [14]. The mitochondrial voltage-dependent anion channel protein, VDAC2, specifically interacts with the inactive form of Bak and selectively blocks Bak-dependent but not Bax-dependent apoptosis [15,16]. WT1 protein binds to a specific DNA-binding site, which is located in the Bak promoter, and Bak is critical to WT1-mediated apoptosis [16]. PreliminA/C has not been reported in association with mTORi pathway, podocyte damage, or kidney injury yet. Although we found the significant effect of RPM and CNI combination on these molecules, it is still unclear whether these molecules are directly associated with podocyte injury. CONCLUSIONS
We found a significant interplay between RPM and CNI on protein expression in mouse podocytes. This might explain the higher incidence of proteinuria by CNIþRPM combination in clinical settings. Further study is required to elucidate the target protein associated with RPM induced podocyte injury. Fig 2. (A) Comparative close-up views of altered protein spots. Protein spots were zoomed and showed altered expression levels of Ttxndc5, Gelsolin, Vdac2, and LaminA/C. (B) Immunoblot analyses of Txndc5, Gelsolin, Vdac2, and LaminA/C after PRM or CNIþRPM treatment. Mouse podocytes were treated with 1 mg/mL cyclosporine (CsA), 20 nmol/L RPM, and 1 mg/mL CsA þ 20 nmol/L RPM for 24 hours. Total protein lysates were Western-blotted, using the primary antibody of Txndc5, Gelsolin, Vdac2, and LaminA/C (1:1,000).
ACKNOWLEDGMENTS Conflicts of Interest: The authors disclose the following relevant financial relationships: Y.C. is a research associate supported by Yonsei University Health System IACF (2012-31-0585 and 2013-310834); H.L. is a research associate supported by Yonsei University Health System IACF (2012-31-0604).
REFERENCES
CNIþRPM (n ¼ 5). Among them, we focused on potentiated down- or up-regulation of proteins by CNIþRPM treatment compared with isolated RPM treatment. Although we did not see 2D profile for the isolated CNItreated group, we performed Western blot analysis to evaluate the role of isolated CNI treatment for the proteins, which are down- or up-regulated by CNIþRPM. It is still unclear whether the change of protein expression by interaction between CNI and RPM plays a certain role for podocyte injury. However, to our knowledge, this is the first report to show the effect of CNI and RPM combination on the protein expression in podocyte. Among these proteins, thioredoxin domain containing protein 5 precursor (Txndc5), also called endoprotein disulfide isomerize (EndoPDI), endoplasmic reticulum protein 46 (ERp46), and plasma cell thioredoxin-related protein (PC-TRP), is a member of the thioredoxin family
[1] Fogo AB. The targeted podocyte. J Clin Invest 2011;121: 2142e5. [2] Diekmann F, Andres A, Oppenheimer F. mTOR inhibitorassociated proteinuria in kidney transplant recipients. Transplant Rev 2012;26:27e9. [3] Mundel P, Reiser J, Zuniga Mejia Borja A, et al. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 1997;236:248e58. [4] Oakley BR, Kirsch DR, Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 1980;105:361e3. [5] Fernandez J, Gharahdaghi F, Mische SM. Routine identification of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Electrophoresis 1998;19:1036e45. [6] Jhaveri KD, Schatz JH, Young JW, et al. Sirolimus (rapamycin) induced proteinuria in a patient undergoing allogeneic hematopoietic stem cell transplant. Transplantation 2008;86:180e1.
PROTEOMIC ANALYSIS OF RAPAMYCIN TREATED PODOCYTE [7] Esposito C, Villa L, Grosjean F, et al. Rapamycin reduces proteinuria and renal damage in the rat remnant kidney model. Transplant Proc 2009;41:1370e1. [8] Stallone G, Infante B, Pontrelli P, et al. Sirolimus and proteinuria in renal transplant patients: evidence for a dose-dependent effect on slit diaphragm-associated proteins. Transplantation 2011;91:997e1004. [9] Heiker JT, Wottawah CM, Juhl C, et al. Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling. Cell Signal 2009;21:936e42. [10] Sullivan DC, Huminiecki L, Moore JW, et al. EndoPDI, a novel protein-disulfide isomerase-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor. J Biol Chem 2003;278:47079e88. [11] Wrammert J, Kallberg E, Leanderson T. Identification of a novel thioredoxin-related protein, PC-TRP, which is preferentially expressed in plasma cells. Eur J Immunol 2004;34:137e46.
1301 [12] Charlton HK, Webster J, Kruger S, et al. ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling. Biochem Biophys Res Commun 2010;392:234e9. [13] Lueck A, Brown D, Kwiatkowski DJ. The actin-binding proteins adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine. J Cell Sci 1998;111(Pt 24):3633e43. [14] Harvey SJ, Jarad G, Cunningham J, et al. Podocyte-specific deletion of dicer alters cytoskeletal dynamics and causes glomerular disease. J Am Soc Nephrol 2008;19:2150e8. [15] Cheng EH, Sheiko TV, Fisher JK, et al. VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 2003;301: 513e7. [16] Morrison DJ, English MA, Licht JD. WT1 induces apoptosis through transcriptional regulation of the proapoptotic Bcl-2 family member Bak. Cancer Res 2005;65:8174e82.
ABSTRACT Background. The mechanism of podocyte injury observed with the use of rapamycin (RPM) remains unclear. The conversion from calcineurin inhibitors (CNIs) to RPM in kidney transplant recipients has been associated with a higher incidence of proteinuria and renal injury. In this study, we performed proteomic analyses to investigate the alteration of protein expression in mouse podocytes treated with RPM in comparison with CNI/RPM combination. Methods. Immortalized mouse podocytes were treated with 20 nmol/L RPM or 20 nmol/L RPM þ 1 mg/mL cyclosporine. Podocyte proteins were separated by 2-dimensional polyacrylamide gel electrophoresis (2DE) and identified by matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry and peptide fingerprinting. Selected proteins were analyzed by means of Western blot assay. Results. We identified 36 differently expressed proteins after isolated RPM or CNI/RPM combination treatment in cultured mouse podocytes. There are 3 distinct patterns of protein expression: (1) potentiated down- or upregulation of proteins by CNI/RPM treatment compared with isolated RPM treatment (n ¼ 4); (2) partial offset of downregulation by CNI/RPM in comparison with RPM treatment (n ¼ 25); (3) no difference in down-regulation between RPM and CNI/RPM treatment (n ¼ 5). We found a significant interplay between RPM and CNI on the expression of the selected proteins in mouse podocytes. This might explain the higher incidence of proteinuria by CNI/RPM combination in clinical settings. Conclusions. Further study is required to elucidate the target protein associated with RPM-induced podocyte injury.
P
ODOCYTES play a key role both in maintenance of the glomerular filtration barrier and in glomerular structural integrity. Podocyte injury and loss contribute to proteinuria and progressive sclerosis [1]. Several studies demonstrate an association between mTOR inhibitor (mTORi) and kidney injury after kidney transplantation and suggest that mTORi(s) may alter the behavior and integrity of glomerular podocytes. This effect could be observed to a different extent in diverse settings [2]. In transplant settings, calcineurin inhibitors (CNIs) are often used with mTORi to maintain optimal immunosuppression. However, various adverse events, such as proteinuria, increase in this setting, whereas the mechanisms are still largely unknown. Detailed
analyses of mTOR-associated regulatory events in glomerulopathies and podocytes are required to understand the complex role of this pathway in glomerular disease. This
This work was supported by a faculty research grant of Yonsei University College of Medicine for 2012 (6-2012-0130) and a research grant from Yonsei University Health System IACF (2012-31-0585). *Address correspondence to Yu Seun Kim, Department of Surgery, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-Ku, 03722, Seoul, South Korea. E-mail: yukim@ yuhs.ac or [email protected]
ª 2016 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/16 http://dx.doi.org/10.1016/j.transproceed.2016.01.022
Transplantation Proceedings, 48, 1297e1301 (2016)
1297
1298
study focuses on mTORi-associated molecular alteration in podocytes treated with RPM in comparison with CNI/RPM combination. To investigate the issue, we performed proteomic analyses in immortalized mouse podocytes treated with RPM or CNI/RPM combination. METHODS Immortalized Mouse Podocyte Cell Culture Conditionally immortalized mouse podocytes were cultured according to the method described by Mundel et al [3]. Undifferentiated podocytes were cultured in RPMI 1640 medium (GIBCO, NY, United States) with 10% fetal bovine serum (GIBCO) and 10 U/mL recombinant mouse interferon-g (Cell Sciences, Canton, Mass, United States) in an incubator at 33 C in 5% CO2. After differentiation, the podocytes were cultured at 37 C in RPMI 1640 medium without interferon-g.
KIM, CHO, LEE ET AL iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of 0.1 Da. PMF acceptance criteria are probability scoring.
Immunoblot Analysis Protein extracts from the cells were prepared with the use of RIPA buffer. Proteins were separated by use of 10% to 15% SDS-PAGE and blotted onto PVDF membranes. Proteins were detected by use of the following primary antibodies: Gelsolin, Txndc5, PrelaminA/ C, and Vdac2 (Santa Cruz Biotechnology, CA, USA).
Statistics Results are expressed as mean SEM values. One-way ANOVA with Tukey’s post hoc test was used to determine the significance of the differences between means. P values of <.05 were considered statistically significant. The analysis was conducted with the use of the GraphPad Prism software.
Two-Dimensional Polyacrylamide Gel Electrophoresis For the analysis of proteome profiling, 2-dimensional gel electrophoresis (2DE), gel image analyses, and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were performed according to the Genomine INC. Protocol-immobilized pH gradient (IPG) dry strips (4e10 nonlinear IPG, 24 cm, Genomine, Korea) were equilibrated for 12 to 16 hours with 7 mol/L urea, 2 mol/L thiourea containing 2% 3-[(3cholamidopropy)dimethylammonio]-1-propanesulfonate (CHAPS), 1% dithiothreitol (DTT), and 1% pharmalyte, respectively, and loaded with 200 mg of samples. Prior to the second dimension, strips were incubated for 10 minutes in equilibration buffer (50 mmol/L Tris-Cl, pH 6.8, containing 6 mol/L urea, 2% SDS, and 30% glycerol), 1% DTT, and 2.5% iodoacetamide, respectively. Equilibrated strips were inserted onto SDS-PAGE gels (20 24 cm, 10% to 16%). The 2D gels were run at 20 C for 1700 Vh. The 2D gels were silver-stained as described by Oakley et al [4]. However, fixing and sensitization steps with glutaraldehyde were omitted.
Image Analysis The quantitative analysis of digitized images was carried out with the use of PDQuest (version 7.0, BioRad) software according to the protocol provided by the manufacturer. The quantity of each spot was normalized by the total intensity of valid spots. Protein spots were selected on the basis of significant expression variation deviated over 2-fold in its expression level compared with control samples.
Peptide Mass Fingerprinting For protein identification by peptide mass fingerprinting (PMF), protein spots were excised, digested with trypsin (Promega, Madison, Wis, United States), mixed with a-cyano-4-hydroxycinnamic acid in 50% acetonitrile/0.1% TFA, and subjected to MALDITOF analyses (Microflex LRF 20, Bruker Daltonics), as described by Fernandez J et al [5]. Spectra were collected from 300 shots per spectrum over m/z range 600 to 3000 and calibrated by means of 2point internal calibration, using trypsin auto-digestion peaks (m/z 842.5099, 2211.1046). A peak list was generated by use of Flex Analysis 3.0. The thresholds used for peak-picking were 500 for minimum resolution of monoisotopic mass and 5 for S/N. The search program MASCOT was used for protein identification by PMF. The following parameters were used for the database search: trypsin as the cleaving enzyme, a maximum of 1 missed cleavage,
RESULTS
To investigate molecular alteration in mouse podocytes treated with RPM or CNI/RPM combination, we performed proteomic analyses with the use of 2D-PAGE. Immortalized mouse podocytes were treated with 20 nmol/L RPM or 1 mg/ mL cyclosporine þ 20 nmol/L RPM. Podocyte proteins were separated by use of 2D-PAGE and identified by means of MALDI-TOF mass spectrometry and peptide fingerprinting. Two representative high-resolution, 2-dimensional maps from mouse podocyte cells of 3 groups are shown in Fig 1. In total, 666.5 97.5, 588.5 42.5, and 591.5 29.5 protein spots were separated and visualized, respectively. We analyzed and compared quantitatively 2DE with the use of PDQuest image analyses and 36 differently expressed proteins after isolated RPM or CNI/RPM combination treatment in cultured mouse podocytes (Table 1). Among them, we focused on potentiated down- or up-regulation of proteins by CNI/RPM treatment compared with isolated RPM treatment. The expression of Txndc5, Gelsolin, PrelaminA/C, and VDAC2 exhibited remarkable differences between isolated RPM and CNI/RPM (Fig 2A). Txndc5 was increased by CNI/RPM combination, and Gelsolin, PrelaminA/C, and Vdac2 were significantly decreased by CNI/ RPM treatment compared with isolated RPM treatment (Fig 2B). DISCUSSION
Detailed analyses of mTOR-associated regulatory events in glomerulopathies and podocytes are required to understand the complex role of this pathway in glomerular disease. Although mTORi is well known for inducing severe proteinuria clinically [6,7], only a few reports explain how proteinuria is triggered by mTOR inhibition [7]. Recently, Stallone et al [8] reported that RPM decreased slit diaphragm (SD)-associated molecules dose-dependently; however, they did not show the direct causation between them, and only some patients with the use of mTORi had podocyte injury clinically.
PROTEOMIC ANALYSIS OF RAPAMYCIN TREATED PODOCYTE
1299
Fig 1. Representative 2DE maps (pH 3e10 gradient) in 3 groups: sham operative group (CTRL); rapamycin group (RPM); and CNI/RPM combination group (CNIþRPM).
To investigate this issue, we hypothesized that mTORiinduced podocyte injury might be potentiated by commonly accompanying conditions, such as hypoxia, CNI combination, or CNI conversion treatment. Among these, we examined the effect of CNI combination treatment with RPM.
According to the result, there are 3 distinct patterns of protein expression: (1) potentiated down- or up-regulation of proteins by CNIþRPM treatment compared with isolated RPM treatment (n ¼ 4); (2) partial offset of downregulation by CNIþRPM in comparison with RPM (n ¼ 25); (3) no difference in down-regulation between RPM and
Table 1. Differential Expression Proteins Identified by Mass Spectrometry Between the RPM, CNIDRPM, and Control Groups
No.
Sample No.
1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
1701R 2506R 2705R 3701R 3801R 4603R 4702R 4705R 4806R 5802R 5810R 6501R 6504R 6802R 6813R 7006R 7202R 7305R 7605R 7701R 7703R 7707R 7804R 7805R 7808R 8201R 8308R 8309R 8406R 8702R 8801R 8802R 8804R
Protein Name
Chain A, structural basis of Pp2a and Sgo interaction Thioredoxin domainecontaining protein 5 isoform 3 Heat shock protein-8 V-type proton ATPase catalytic subunit-A Stress-70 protein, mitochondrial RuvB-like 2 Coronin-1B Prolyl 4-hydroxylase subunit alpha-1 isoform X1 Alpha-glucosidase 2 alpha neutral subunit, isoform CRA_a Gelsolin isoform 2 Neutral alpha-glucosidase AB isoform 2 precursor Ornithine aminotransferase, mitochondrial precursor NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 Trap1 protein Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 precursor Rassf4 protein mCG13231 3-Mercaptopyruvate sulfurtransferase Aldehyde dehydrogenase mCG2023 Predicted: Dihydropyrimidinase-related protein 2 UPF0317 protein C14orf159 homolog Propionyl-coenzyme A carboxylase, alpha-polypeptide Oxoglutarate dehydrogenase (lipoamide) Predicted: Forkhead-associated domain-containing protein 1 Proteasome subunit-alpha type 6 Voltage-dependent anion-selective channel protein 2 Annexin A2 Chain A, crystal structure of mouse transaldolase WD repeat-containing protein 1 Prelamin-A/C isoform A precursor MICOS complex subunit Mic60 isoform 5 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 isoform 1
Scores
Molecular Weight (Da)
136 147 209 114 241 176 87 167 252 115 132 224 97 210 138 74 81 101 171 103 210 145 238 127 72 88 86 185 141 242 151 205 269
81.0 58.3 95.3 94.5 96.7 31.7 65.0 88.4 137.5 119.2 139.7 58.5 59.8 98.1 120.5 20.9 32.5 40.1 66.3 85.0 85.4 88.2 101.6 138.2 136.3 35.1 41.7 47.3 49.0 93.6 100.1 110.5 127.4
Expression Ratio RPM/CTL CþR/CTL
0.006 3.028 0.127 0.045 0.410 0.284 0.141 0.026 0.076 0.459 0.005 0.543 0.715 0.007 0.007 0.001 0.182 0.005 0.387 0.828 0.579 0.585 0.400 0.006 0.004 0.878 0.505 0.107 0.255 0.062 0.275 0.002 0.274
0.722 3.738 0.330 0.167 0.418 0.452 0.266 0.579 0.392 0.402 0.407 0.656 0.799 0.587 0.144 0.001 0.417 0.005 0.663 0.889 0.799 0.598 0.735 0.482 0.530 0.829 0.454 0.735 0.606 0.459 0.250 0.512 0.320
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KIM, CHO, LEE ET AL
proteins and has been shown to have thioreductase activity [9e11]. Reducing ERp46 modulates the expression and/or distribution of the adiponectin receptors and adiponectin signaling [12]. Gelsolin is expressed by most tubular epithelial cells in normal kidney and has also been localized to glomerular endothelium [13], and it is consistent with the expression by all glomerular cell types, including podocytes [14]. Gelsolin was up-regulated in mutant podocytes, whereas the labeling of other glomerular cells was decreased [14]. The mitochondrial voltage-dependent anion channel protein, VDAC2, specifically interacts with the inactive form of Bak and selectively blocks Bak-dependent but not Bax-dependent apoptosis [15,16]. WT1 protein binds to a specific DNA-binding site, which is located in the Bak promoter, and Bak is critical to WT1-mediated apoptosis [16]. PreliminA/C has not been reported in association with mTORi pathway, podocyte damage, or kidney injury yet. Although we found the significant effect of RPM and CNI combination on these molecules, it is still unclear whether these molecules are directly associated with podocyte injury. CONCLUSIONS
We found a significant interplay between RPM and CNI on protein expression in mouse podocytes. This might explain the higher incidence of proteinuria by CNIþRPM combination in clinical settings. Further study is required to elucidate the target protein associated with RPM induced podocyte injury. Fig 2. (A) Comparative close-up views of altered protein spots. Protein spots were zoomed and showed altered expression levels of Ttxndc5, Gelsolin, Vdac2, and LaminA/C. (B) Immunoblot analyses of Txndc5, Gelsolin, Vdac2, and LaminA/C after PRM or CNIþRPM treatment. Mouse podocytes were treated with 1 mg/mL cyclosporine (CsA), 20 nmol/L RPM, and 1 mg/mL CsA þ 20 nmol/L RPM for 24 hours. Total protein lysates were Western-blotted, using the primary antibody of Txndc5, Gelsolin, Vdac2, and LaminA/C (1:1,000).
ACKNOWLEDGMENTS Conflicts of Interest: The authors disclose the following relevant financial relationships: Y.C. is a research associate supported by Yonsei University Health System IACF (2012-31-0585 and 2013-310834); H.L. is a research associate supported by Yonsei University Health System IACF (2012-31-0604).
REFERENCES
CNIþRPM (n ¼ 5). Among them, we focused on potentiated down- or up-regulation of proteins by CNIþRPM treatment compared with isolated RPM treatment. Although we did not see 2D profile for the isolated CNItreated group, we performed Western blot analysis to evaluate the role of isolated CNI treatment for the proteins, which are down- or up-regulated by CNIþRPM. It is still unclear whether the change of protein expression by interaction between CNI and RPM plays a certain role for podocyte injury. However, to our knowledge, this is the first report to show the effect of CNI and RPM combination on the protein expression in podocyte. Among these proteins, thioredoxin domain containing protein 5 precursor (Txndc5), also called endoprotein disulfide isomerize (EndoPDI), endoplasmic reticulum protein 46 (ERp46), and plasma cell thioredoxin-related protein (PC-TRP), is a member of the thioredoxin family
[1] Fogo AB. The targeted podocyte. J Clin Invest 2011;121: 2142e5. [2] Diekmann F, Andres A, Oppenheimer F. mTOR inhibitorassociated proteinuria in kidney transplant recipients. Transplant Rev 2012;26:27e9. [3] Mundel P, Reiser J, Zuniga Mejia Borja A, et al. Rearrangements of the cytoskeleton and cell contacts induce process formation during differentiation of conditionally immortalized mouse podocyte cell lines. Exp Cell Res 1997;236:248e58. [4] Oakley BR, Kirsch DR, Morris NR. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem 1980;105:361e3. [5] Fernandez J, Gharahdaghi F, Mische SM. Routine identification of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels or polyvinyl difluoride membranes using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Electrophoresis 1998;19:1036e45. [6] Jhaveri KD, Schatz JH, Young JW, et al. Sirolimus (rapamycin) induced proteinuria in a patient undergoing allogeneic hematopoietic stem cell transplant. Transplantation 2008;86:180e1.
PROTEOMIC ANALYSIS OF RAPAMYCIN TREATED PODOCYTE [7] Esposito C, Villa L, Grosjean F, et al. Rapamycin reduces proteinuria and renal damage in the rat remnant kidney model. Transplant Proc 2009;41:1370e1. [8] Stallone G, Infante B, Pontrelli P, et al. Sirolimus and proteinuria in renal transplant patients: evidence for a dose-dependent effect on slit diaphragm-associated proteins. Transplantation 2011;91:997e1004. [9] Heiker JT, Wottawah CM, Juhl C, et al. Protein kinase CK2 interacts with adiponectin receptor 1 and participates in adiponectin signaling. Cell Signal 2009;21:936e42. [10] Sullivan DC, Huminiecki L, Moore JW, et al. EndoPDI, a novel protein-disulfide isomerase-like protein that is preferentially expressed in endothelial cells acts as a stress survival factor. J Biol Chem 2003;278:47079e88. [11] Wrammert J, Kallberg E, Leanderson T. Identification of a novel thioredoxin-related protein, PC-TRP, which is preferentially expressed in plasma cells. Eur J Immunol 2004;34:137e46.
1301 [12] Charlton HK, Webster J, Kruger S, et al. ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling. Biochem Biophys Res Commun 2010;392:234e9. [13] Lueck A, Brown D, Kwiatkowski DJ. The actin-binding proteins adseverin and gelsolin are both highly expressed but differentially localized in kidney and intestine. J Cell Sci 1998;111(Pt 24):3633e43. [14] Harvey SJ, Jarad G, Cunningham J, et al. Podocyte-specific deletion of dicer alters cytoskeletal dynamics and causes glomerular disease. J Am Soc Nephrol 2008;19:2150e8. [15] Cheng EH, Sheiko TV, Fisher JK, et al. VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 2003;301: 513e7. [16] Morrison DJ, English MA, Licht JD. WT1 induces apoptosis through transcriptional regulation of the proapoptotic Bcl-2 family member Bak. Cancer Res 2005;65:8174e82.