Adenovirus-mediated antisense ERK2 gene therapy ameliorates chronic allograft nephropathy in a rat model

Abstract

To investigate the effect and underlying mechanism of adenovirus-mediated antisense ERK2 (Adanti-ERK2) gene therapy upon chronic allograft nephropathy (CAN) of rats, male Lewis (LEW, RT11) rats received male Fisher (F344, RT11v1) renal allografts. The recipients were divided into three groups: (1) empty control group; (2) vector control group; (3) gene therapy group. All recipients were sacrificed for the grafts and serum analysis at the 24th week after transplantation. Morphometric analysis was used to determine the fibrosis of grafts. Immunohistochemistry was used to detect the expression of E-Cadherin, Vimentin, TβR I and the infiltration of CD4⁺ T lymphocyte, CD8⁺ T lymphocyte and ED-1⁺ monocytes. Enzyme linked immunosorbent assay (ELISA) was used to detect TGF-β1 in serum. The grafts in the control group and vector control group showed CAN. There was less E-Cadherin in renal tubular epithelial cells in the empty control group but more Vimentin and TβR I. In the gene therapy group, the fibrosis was ameliorated and fewer T lymphocytes and ED-1⁺ monocytes infiltrated in the interstitium. There was no significant difference in the expression of E-Cadherin between the gene therapy group and normal rats. Compared with the empty control group, the expression of TGF-β1 in the gene therapy group was down-regulated. Adanti-ERK2 gene therapy protects the renal allograft and attenuates graft fibrosis, which may be correlated with a decreased renal tubular epithelial mesenchymal transition, a decreased infiltration of CD4⁺ T lymphocyte, CD8⁺ T lymphocytes and ED-1⁺ monocytes in renal interstitium, and the down-regulated TGF-β1 expression.

Keywords

anti-ERK2 / renal transplantation / epithelial mesenchymal transition / chronic allograft nephropathy

Introduction

Chronic allograft nephropathy (CAN) is the most common cause of late graft loss after renal transplantation [1]. Although the processes of CAN are multiple, the main feature of failing renal transplants is tubular atrophy and interstitial fibrosis [2,3]. Extracellular signal-regulated kinase (ERK) is a subfamily of the mitogen-activated protein kinase (MAPK) signaling pathways which is involved in cellular responses including proliferation, differentiation, apoptosis and migration through mediating polypeptide growth factor signaling [4]. Transmission of signals is achieved by a sequential series of phosphorylation reactions wherein ERK is activated by phosphorylation. Activated ERK enters the nucleus and effects on downstream regulatory molecules such as transcription factors, thereby causing changes in gene expression. So far no differential role has been proven in vitro for the two main isoforms of the ERK pathway (ERK1 and ERK2), and they are activated by the same stimuli. However, in vivo invalidation of ERK1 or ERK2 leads to different phenotypes, demonstrating different roles for ERK1 and ERK2 [5]. Indeed, ERK2 is essential for transduction of signals, and ERK1 could instead have an accessory role, possibly enabling a fine tuning of ERK2 activity [6].

In the present study, by using gene transfer technique with adenoviral vectors encoding antisense ERK2 (Adanti-ERK2), we examined the effects of the ERK2 signal transduction pathway in CAN.

Methods

Materials

Dimethyl sulfoxide (DMSO) and propidium iodide (PI) were procured from Sigma (St. Louis, USA). The monoclonal antibodies to rat α-smooth muscle actin (α-SMA), E-cadherin, Vimentin and tissue growth factor β receptor I (TβR I) were purchased from Sigma Chemical Company (USA). TGF-β1 enzyme linked immunosorbent assay (ELISA) kit was from Promega (Madison, USA). The Adeno-XTM expression system kit was from BD Biosciences Clontech (Palo Alto, USA). The plasmid of p3XFLAG-CMV7.1-ERK2 was kindly provided by Dr. Fred L. Robinson (University of Texas, USA).

Adenovirus construction

Adanti-ERK2 was constructed using the Adeno-XTM expression system, which is based on the procedure developed by Mizuguchi and Kay [7]. The 770-bp length cDNA of ERK2 was obtained by enzyme digesting, and p3XFLAG-CMV7.1-ERK2 was reversely subcloned into the pShuttle vector to create pShuttle-antiERK2. The ERK2 cDNA fragment, obtained by digesting the plasmid of p3XFLAG-CMV7.1-ERK2 with NotI and SalI, was excised by DraI digestion. The resulting 770-bp-length cDNA used for cloning was then separated using agarose gel and reversely ligated into the DraI-NotI site of the pShuttle vector to generate pShuttle-antiERK2. The expression cassette was excised from pShuttle-antiERK2 and inserted into the replication-incompetent (E1/E3-deleted) Ad5 genome via PI-SceI/I-CeuI restriction sites. The recombinant adenoviral vector was packaged in human embryonic kidney (HEK) 293 cells (China Center for Type Culture Collection, Wuhan, China), purified by CsCl density gradient ultracentrifugation, and stored at -80°C. As a vector control of Adanti-ERK2, recombinant adenovirus containing bacterial β-galactosidase gene (Ad-LacZ) was also generated similarly. The titer of the virus was determined by limiting dilution in HEK 293 cells and expressed as plaque forming units (pfu).

Cell culture and treatment

The cultured human proximal tubular epithelial cells (HK-2) were from the China Center for Type Culture Collection (Wuhan, China). HK-2 cells were maintained in dulbecco's modified eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 g/mL streptomycin at 37°C in 5% CO₂. According to the adenovirus infection, the cultured HK-2 cells were divided into three groups: control group (no gene transfer); Ad-LacZ group (transfected with Ad-LacZ, as vector control) and Adanti-ERK2 group (cells transfected with Adanti-ERK2). The medium was changed to DMEM containing 2% FBS and viruses were added to the medium at a multiplicity of infection (MOI) of 100, as we tested previously, when the cells were 60%-70% confluent. Following 18 h infection with adenoviruses, the medium was changed to DMEM containing 10% FBS. The cells in the control group were not transfected, but incubated in DMEM containing 2% FBS for 18 h. The cells were harvested at the 72nd h for further detection.

Western blotting

HK-2 cells and the treated cells were lysed in Triton lysis buffer and 50 μg of total protein extracted from the cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Separated protein was transferred to nitrocellulose membranes and the membranes were blocked in tris-bruffer saline/0.05% tween 20 (TBST) plus 5% fat-free milk powder. The membranes were incubated with primary antibody at 4°C overnight, and then incubated with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. Finally, immunoreactive proteins were visualized by an enhanced chemiluminescense system.

Ex vivo gene transfer and renal transplantation

In renal transplantation, male Fisher (F344, RT11v1) rats were selected as donors and male Lewis (LEW, RT11) rats as recipients [8]. The rats were introduced from the Charles River Laboratory (Boston, MA, USA) via the Vital River Company (Beijing, China). The experimental procedure was approved by the Institutional Animal Care and Use Committee of China. The rats were anaesthetized by intraperitoneal injection of ketamine (80 mg/kg)/xylazine (12 mg/kg) solution (Sigma, St. Louis, USA). Briefly, the renal grafts of F344 rats were isolated and orthotopically perfused by 4°C University of Wisconsin (UW) solution. Ad-LacZ or Adanti-ERK2 (5 × 10⁹ pfu of each) in 500 μL phosphate buffered saline (PBS) was instilled into the renal grafts. Renal grafts were incubated in 4°C UW solution for 45 min and then transplanted into Lewis rats in situ. The recipients were divided into three groups: (1) empty control group (no gene transfer, n = 8); (2) vector control group (allografts transfected with Ad-LacZ, n = 9); (3) gene therapy group (allografts transfected with Adanti-ERK2, n = 6). There was no significant difference in the size of the donor and recipient vessels. All anastomoses were performed using continuous 10-0 prolene suture in an end-to-end technique. The total operative time from removal of the donor graft to reperfusion of the graft in the recipient was approximately 40 min. At the 24th week after transplantation, the grafts were harvested for morphometric and immunohistochemical study, and the serum was collected for cytokine detection.

Immunostaining

Immunohistochemistry was performed on 4-μm thick paraffin-embedded sections of renal tissue. After microwave antigen retrieval in citrate buffer, the sections were incubated overnight at 4°C with mouse anti-rat CD4 (1∶200), mouse anti-rat CD8 (1∶100) and mouse anti-rat ED-1 (1∶100); all reagents were diluted in 20% normal lamb or swine serum. Bound anti-CD8, anti-CD4 or anti-ED-1 was detected using biotinylated rabbit anti-mouse immunoglobulins. Visualization was accomplished by using streptavidin biotin peroxidase to obtain a brown product. For the negative control, the primary antibody was substituted with rabbit IgG or nonimmune serum. Sections in which a biotinylated secondary antibody was used were pretreated with a biotin-blocking system to minimize endogenous biotin activity. Labeled sections were analyzed using Leica Image Analysis and Quality Week in software. Using minor modifications of a method described previously, E-Cadherin (1∶50), Vimentin (1∶100) and TβR I (1∶100) were detected. The marker-positive cells were expressed as \overline{x}\pm s of cells per view-field. Five random view-fields per section were evaluated at 200 power magnification.

Measurement of TGF-β1 concentration

The concentration of TGF-β1 in the recipients’ serum was measured by specific ELISA assay in accordance with the manufacturer’s protocol. The absorbance (A) value was measured in an ELISA microplate reader at 492 nm. The concentration of TGF-β1 was obtained according to the standard curve constructed by plotting the mean A for each standard against the concentration.

Statistical analysis

Data were expressed as \overline{x}\pm s. Differences in mean values were compared using SPSS 11.0 by one-way ANOVA and S-N-K test. Statistical significance was defined as P <0.05.

Results

Adanti-ERK2 down-regulated ERK2 expression in vitro

To prove the efficiency of Adanti-ERK2 gene therapy, the ERK2 protein was measured using Western blotting with anti-ERK2 antibody. As shown in Fig. 1, the expression of ERK2 in HK-2 cells was down-regulated following the treatment of Adanti-ERK2 for 72 h, but the cells in Ad-LacZ group were at a normal level as the control.

Adanti-ERK2 modulated CD4⁺T, CD8⁺T lymphocyte and macrophage infiltration

By 24 weeks, the grafts in the empty control group and vector control group developed graft interstitial fibrosis and infiltration of CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and ED-1⁺ monocytes in the renal interstitium. Meanwhile, moderate fibrosis and less T lymphocyte and ED-1⁺ monocyte infiltrating in the interstitium of the gene therapy group was observed, indicating that CAN was ameliorated (Figs. 2, 3, Table 1).

Adanti-ERK2 attenuated tubular epithelial-myofibroblast transition in transplants after renal transplantation in rats

To further demonstrate the role of the ERK2 signal transduction pathway in renal tubular epithelial-myofibroblast transition in transplant nephropathy, we tested the effect of Adanti-ERK2 on a rat chronic allograft nephropathy model. At the 24th week after transplantation, the renal tubular epithelial cells of grafts in the control group and vector control group expressed less E-Cadherin but the interstitium expressed more Vimentin and α-SMA in comparison with the gene therapy group, indicating that the Adanti-ERK2 inhibited the process of EMT in the allografts (Fig. 4).

Adanti-ERK2 down-regulated the expression of TGF-β1 and suppressed TβR I up-regulation in CAN in rat model

At the 24th week after transplantation, the serum was collected and then subjected to ELISA, and paraformaldehyde-fixed and paraffin-embedded sections of the grafts were stained with immunohistochemistry to measure TβR I with mouse anti-rat TβR I antibody (1∶100). Compared with the empty control groups, the serum expression of TGF-β1 in the gene therapy group was down-regulated. In the control group and vector control group, the renal tubular epithelial cells expressing TβR I were more than those in the gene therapy group (Fig. 5, Table 2).

Discussion

CAN is the chief cause of kidney transplant failure and is characterized by progressive sclerosis of the renal interstitium, glomeruli and vessels [9]. Whatever the original insult occurring on the graft, the end-result is fibrogenesis, i.e. the activation of myofibroblasts leading to the accumulation of extracellular matrix. The origin and mechanism of activation of these fibroblasts are controversial, but increasing evidence indicates that a relevant proportion of these extracellular matrix producing cells arise from the phenotypic conversion of tubular epithelial cells (TECs) into mesenchymal cells through EMT [10].

Intracellular signal pathways that mediate EMT are multiple. Different cytokines that have been implicated in mediating EMT may be in different or similar way. Our previous study has also suggested that MAPK family members increased expression and delivered extracellular signals to nucleus through Ras-MAPK pathway in C Response [11,12]. And antisense ERK1/2 gene therapy can attenuate graft arteriosclerosis [13]. The ERK signal pathway was the first characterized MAP kinase cascade, being a vital mediator of a number of cellular processes, including growth, proliferation, and survival [14,15]. In this article, we mainly focused our discussion of the intracellular signal transduction on EMT in the setting of CAN mainly related to the ERK signal transduction pathway.

However, most of these studies were carried out using specific inhibitors of the ERK pathway such as the mitogen-activated protein kinase (MEK) inhibitors (PD98059 or U0126) or antisense oligonucleotides. Notably, these approaches do not discriminate between ERK1 and ERK2. In vivo and in vitro evidence has shown that ERK2 is crucial for transduction of signals [5]. The technique of the graft transfected directly was used in gene therapy widely [16]. Therefore, in this study, by using Adanti-ERK2 for specifically inhibiting endogenous ERK2, we examined the biologic role of ERK2 in HK-2 cells in vivo. The expression of ERK2 in HK-2 cells was down-regulated following the treatment of Adanti-ERK2, but the cells in the Ad-LacZ groups were at a normal level as the control. We further explored the effects of Adanti-ERK2 gene therapy on CAN in vivo. First, we employed a rat CAN model, which had been developed by Tilney and Tullius [17,18]. At the 24th week post transplantation, the typical features of CAN, including glomerulopathy, arteriopathy, fibrosis and tubular atrophy, were observed. In the specimens from the empty control and vector control, tubular EMT was proven by monitoring the specific epithelial (E-cadherin) and mesenchymal (α-SMA and Vimentin) markers. However, in the grafts receiving Adanti-ERK2 gene therapy, phenotype monitoring showed that EMT was also ameliorated, and the therapy exerted a protective effect on the transplants.

EMT is regulated by numerous growth factors, cytokines, hormones, and extracellular cues in different ways. The extraordinary ability of TGF-β1 underscores that induction of EMT may be a major pathway that leads to interstitial fibrosis under pathologic conditions [19]. Even more notable is the recent work by Robertson and colleagues documenting the association of S100A4, a calcium binding protein and a marker of EMT, with the presence of CD8⁺ T-cells within allografts with CAN [20]. This study suggests that infiltrating T-cells may directly induce epithelial cells to transform and migrate into the interstitium. Recently, some experiments have illustrated that the ERK signal correlates with inflammation [21]. In this study, transfection with Adanti-ERK2, through reducing the expression of TβR I, could attenuate renal tubular EMT. Meanwhile, we found that Adanti-ERK2 therapy effectively decreased infiltration of CD4⁺ T lymphocytes, CD8⁺ T lymphocytes and ED-1⁺ monocytes in the renal interstitium after renal transplantation. This treatment was inserted at the time of transplant, based on the concern that the process of CAN is initiated from the very beginning of transplant, as intimal activation is triggered immediately by immunologic and nonimmunologic injuries [20].

Although this Adanti-ERK2 therapy aims at transfection of the therapeutic gene into the graft itself, it is also capable of stabilizing the inflammation in the graft so as to lessen the innate immunity through downregulation of cytokines and growth factors; for example, TGF-β1 in serum was down-regulated in our study. Furthermore, the virus-soaked transplant also could release some vectors, which then infect T cells, correlating with inhibition of immune response, as ERK activation is an important event of T cell activation, differentiation and function [22]. So ex vivo Adanti-ERK2 gene transfer before transplantation can ameliorate the microenvironment of tubular epithelial cells, attenuating tubular EMT. However, Adanti-ERK2 gene therapy did not totally inhibit the onset of CAN in our study. One reason may be the limited duration of the therapeutic gene expression, failing to elicit a complete long-term inhibition after transplant. Alternatively, it is also possible that blockade of the ERK pathway in vivo is insufficient to prevent the activation of myofibroblasts because of the multi-factorial and complicated mechanism for transplant CAN. Therefore, vector improvement and therapeutic gene modification should be addressed in the future study.

In conclusion, EMT should be considered as a critical process that may be initiated by both antigen dependent and independent insults in transplant. Our results suggest antisense ERK2 gene therapy attenuates tubular EMT in transplant. Furthermore, knowledge about ERK pathway involved in CAN via different molecular mechanisms may contribute to promising novel efficient therapeutic targets for treating CAN.

References

[1] PascualM, TheruvathT, KawaiT, Tolkoff-RubinN, CosimiA B. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med, 2002, 346(8): 580–590

[2] BurnsW C, KantharidisP, ThomasM C. The role of tubular epithelial-mesenchymal transition in progressive kidney disease. Cells Tissues Organs, 2007, 185(1-3): 222–31

[3] NankivellB J, BorrowsR J, FungC L, O'ConnellP J, AllenR D, Chapman J R. The natural history of chronic allograft nephropathy. N Engl J Med, 2003, 349(24): 2326–2333

[4] KyriakisJ M, AvruchJ. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev, 2001, 81(2): 807–869

[5] BostF, AouadiM, CaronL, EvenP, BelmonteN, ProtM, DaniC, HofmanP, PagèsG, PouysségurJ, Le Marchand-BrustelY, BinétruyB. The extracellular signal–regulated kinase isoform ERK1 is specifically required for in vitro and in vivo adipogenesis. Diabetes, 2005, 54(2): 402–411

[6] VantaggiatoC, FormentiniI, BondanzaA, BoniniC, NaldiniL, BrambillaR. ERK1 and ERK2 mitogen-activated protein kinases affect Ras-dependent cell signaling differentially. J Biol, 2006, 5(5): 14

[7] MizuguchiH, KayM A. A simple method for constructing E1- and E1/E4-deleted recombinant adenoviral vectors. Hum Gene Ther, 1999, 10(12): 2013–2017

[8] LeeS. An improved technique of renal transplantation in the rat. Surgery, 1967, 61(5): 771–773

[9] PascualM, TheruvathT, KawaiT, Tolkoff-RubinN, CosimiA B. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med, 2002, 346(8): 580–590

[10] IwanoM, PliethD, DanoffT M, XueC, OkadaH, NeilsonE G. Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest, 2002, 110(3): 341–350

[11] GongN, ZhangW, LiG, GuoH, YeQ. MAPK cascade pathway and anti-stress response of hepatocyte after live transplantation. Chin J Organ Transplant, 2002, 23(5): 288–290 (in Chinese)

[12] LiG, GongN, YeQ, GuoH. Different effects of several signal pathways after liver transplantation. Zhonghua Ganzangbing Zazhi, 2003, 11(12): 742–745 (in Chinese).

[13] DongC, GongN, ChenZ, ChenX, XuQ, GuoH, ZengZ, MingC, ChenZ K. Antisense ERK1/2 oligodeoxynucleotide gene therapy attenuates graft arteriosclerosis of aortic transplant in a rat model. Transplant Proc, 2006, 38(10): 3304–3306

[14] WhitehurstA W, RobinsonF L, MooreM S, CobbM H. The death effector domain protein PEA-15 prevents nuclear entry of ERK2 by inhibiting required interactions. J Biol Chem, 2004, 279(13): 12840–12847

[15] ChenR H, AbateC, BlenisJ. Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase. Proc Natl Acad Sci USA, 1993, 90(23): 10952–10956

[16] GongN, PleyerU, VogtK, AnegonI, FlügelA, VolkH D, RitterT. Local overexpression of nerve growth factor in rat corneal transplants improves allograft survival. Invest Ophthalmol Vis Sci, 2007, 48(3): 1043–1052

[17] TilneyN L, WhitleyW D, TulliusS G, HeemannU W, WasowskaB, BaldwinW M 3rd, HancockW W. Serial analysis of cytokines, adhesion molecule expression, and humoral responses during development of chronic kidney allograft rejection in a new rat model. Transplant Proc, 1993, 25(1 Pt 2): 861–862

[18] TulliusS G, HancockW W, HeemannU, AzumaH, TilneyN L. Reversibility of chronic renal allograft rejection. Critical effect of time after transplantation suggests both host immune dependent and independent phases of progressive injury. Transplantation, 1994, 58(1): 93–99

[19] Meindl-BeinkerN M, DooleyS. Transforming growth factor-beta and hepatocyte transdifferentiation in liver fibrogenesis. J Gastroenterol Hepatol, 2008, 23(Suppl 1): S122–S127

[20] RobertsonH, AliS, McDonnellB J, BurtA D, KirbyJ A. Chronic renal allograft dysfunction: The role of T cell-mediated tubular epithelial to mesenchymal cell transition. J Am Soc Nephrol, 2004, 15(2): 390–397

[21] ReedR C, BerwinB, BakerJ P, NicchittaC V. GRP94/gp96 elicits ERK activation in murine macrophages. A role for endotoxin contamination in NF-kappa B activation and nitric oxide production. J Biol Chem, 2003, 278(34): 31853–31860

[22] ZhangY L, DongC. MAP kinases in immune responses. Cell Mol Immunol, 2005, 2(1): 20–27

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg