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NDT Advance Access originally published online on November 3, 2006
Nephrology Dialysis Transplantation 2007 22(2):376-385; doi:10.1093/ndt/gfl608
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

NADPH oxidase subunits (NOX-1, p22phox, Rac-1) and tacrolimus-induced nephrotoxicity in a rat renal transplant model

Ashwani K. Khanna1,2 and Galen M. Pieper3,4

1Division of Cardiology, University of Maryland, Baltimore, MD, 2Transplantation Unit, OncoDetect Inc., Brookfield, WI and 3Surgery (Transplant Surgery) and 4Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI, USA

Correspondence and offprint requests to: Ashwani Khanna, PhD, Division of Cardiology, University of Maryland, 22 S Greene Street S 3B08, Baltimore, MD-21201, USA. Email: akhanna{at}medicine.umaryland.edu



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. TGF-ß and oxidative stress are known mediators of renal injury. However, the precise mechanisms by which TGF-ß and oxidative stress may be involved in the development of nephrotoxicity are not known. We examined whether anti-TGF-ß antibody limits nephrotoxicity produced by tacrolimus (TAC) and whether this altered genes that regulate oxidative stress.

Methods. Renal transplants were performed in Wistar-Furth and Lewis rat strains. Groups included: isograft controls; untreated allografts; allografts treated with 0.25 mg/kg TAC till 90 days with or without 1.0 mg/kg anti-TGF-ß antibody or control antibody. Serum creatinine and BUN levels and renal histology were determined. Real time PCR and western analysis were used to quantify mRNA and protein expression.

Results. BUN and creatinine were elevated in TAC-treated rats. TAC increased expression of TGF-ß (37-fold) and NADPH oxidase subunits, NOX-1 (18-fold), p22phox (31-fold) and Rac-1 mRNA (20-fold), respectively. Contrariwise, expression of antioxidant genes, superoxide dismutase (SOD) and thioredoxin (TRX) was decreased. Anti-TGF-ß antibody but not control antibody reversed the TAC-induced changes in gene expression, renal histology and function.

Conclusions. Our findings suggest a potential for anti-TGF-ß antibody as a novel adjunct therapeutic tool to prevent TAC-induced nephrotoxicity in transplant recipients. The mechanism of protection involves suppression of TGF-ß and the expression of genes that regulate oxidative stress. Moreover, the specific up-regulation of NOX-1, a non-phagocytic NADPH oxidase subunit and its reversal by anti-TGF-ß antibody strongly implicates for the first time the up-regulation of renal parenchymal cell NADPH oxidase in the aetiology of immunosuppression-induced nephrotoxicity.

Keywords: anti-TGF-ß antibody; NADPH oxidase; nephrotoxicity; rat renal transplant model; tacrolimus; TGF-ß



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Acknowledgements
 References
 
The mechanism of immunosuppression-associated nephrotoxicity is not completely understood. Transforming growth factor-ß (TGF-ß) is one of the leading candidates for mediating nephrotoxicity since cyclosporine (CsA), tacrolimus (TAC) and sirolimus (SRL) induce TGF-ß expression [1–7]. Oxidative stress can also stimulate TGF-ß production. Oxidative stress has been considered to be an important mediator of immunosuppression-induced renal injury [8,9]. In fact, there is sufficient evidence showing that TGF-ß alone can increase levels of reactive oxygen in cultured renal cells [10–13]. However, the precise mechanism by which TGF-ß and immunosuppressive drugs increase reactive oxygen species has not been determined. The increased levels of reactive oxygen species might be due to a decrease in antioxidant genes such as the depletion of SOD as has been shown by exposure of rat hepatocytes to TGF-ß [10]. Alternatively, increased levels of reactive oxygen might be increased due to the increased production of reactive oxygen species by certain oxidases.

NAPDH oxidases are prime candidates as sources of reactive oxygen production. Phagocytic NADPH oxidase consists of a catalytic core of two membrane-associated subunits, gp91phox and p22phox. The protein gp91phox is considered to be the subunit responsible for phagocytic Formula production [14]. NADPH oxidase in the resting state becomes activated to produce Formula upon interaction with cytoplasmic components, (p47phox and p67phox), and Rac, a small GTPase. Activation of NADPH oxidase requires translocation of cytoplasmic components to the membrane fraction. Genetic ablation of NADPH oxidase subunits has been shown to inhibit Formula production in inflammatory states [15,16].

In addition to phagocytic NADPH oxidases, there is emerging evidence of the presence of novel protein analogues of these NADPH oxidases that are non-phagocytic in origin. In kidney, NOX-1 is a membrane-bound NADPH oxidase subunit and part of the catalytic core which is a non-phagocytic analogue of the phagocytic subunit gp91phox [17]. The importance of non-phagocytic NAPDH oxidase in renal pathology was demonstrated in studies showing that NOX-1 is increased in rat kidney stressed by infusion with angiotensin II [18].

Since oxidative stress can also stimulate TGF-ß production, oxidative stress is also believed to mediate TGF-ß-induced expression of extracellular matrix proteins. While the latter promotes fibrogenesis, the exact cascade of events initiated by immunosuppressant treatment such as activation of TGF-ß and its downstream signals mediated events leading to the development of nephrotoxicity are not known. The potential role of TGF-ß in mediating CsA-induced nephrotoxicity was demonstrated in one of our recent studies in which treatment with low-dose anti-TGF-ß antibody prolonged graft survival and decreased nephrotoxicity in a rat cardiac transplant model [7].

Despite these new findings, it has not yet been determined if antagonizing the actions of TGF-ß by antibody intervention can uniformly be applied to limit nephrotoxicity mediated by immunosuppressant drugs other than CsA. Contrary to prior dogma, the nephrotoxic effects of TAC have been determined to be similar to CsA [19–23]. Like CsA, we have determined that TAC also induces the expression of TGF-ß [4].

The present study was planned to examine (i) if NADPH oxidase components and TGF-ß mediate the nephrotoxic effects of TAC in a rat model of renal transplantation, (ii) if anti-TGF-ß antibody neutralizes the effects of TGF-ß but also inhibits expression of genes related to oxidative stress and the development of nephrotoxicity. We studied the effect of long-term treatment with TAC on renal function, histology, intragraft mRNA and protein expression of TGF-ß. Since NADPH oxidase subunits components (NOX-1, p22phox and Rac-1) are sources of Formula production whereas SOD decreases Formula and thioredoxin (TRX) decreases redox signalling, we also examined expression of these key genes in modulating oxidative stress. The role of TGF-ß in mediating these changes was determined by a strategy involving treatment with anti-TGF-ß antibody vs control antibody concomitant with TAC on nephrotoxicity. The results demonstrate that the nephrotoxic effects of TAC are mitigated by anti-TGF-ß antibody. Furthermore, our findings show that the mechanism also involves preventing changes in gene expression that would otherwise promote oxidative stress.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Acknowledgements
 References
 
Rat renal transplantation
We used Lewis (LEW, RT11) and Wistar-Furth (WF RT1u) rats, which represent complete genetic disparity at both major and minor histocompatibility loci. LEW or WF donor kidneys were transplanted into LEW recipient rats to represent isogeneic and allogeneic transplantation, respectively. In renal donors, following induction of general anesthesia with 50 mg/kg pentobarbital i.p., a midline laparotomy was performed. The left kidney was mobilized on its vascular pedicle and the ureter was divided. Following heparinization (1000 U, i.p.), the left kidney was removed with a generous cuff of aorta and vena cava. The kidney was then flushed with iced saline, stored on ice and then transplanted. Through a midline laparotomy in the recipient (anaesthetized as above), the infrarenal aorta and vena cava were exposed. Using standard, established microvascular surgical techniques, the left donor kidney was anastomosed in an end-to-side fashion to the recipient vessels and the blood flow restored. The ureter was inserted through the bladder wall and stented with a short segment of PE50 tubing. Both kidneys of the recipients were removed and the only functional kidney in these transplant recipients was the transplanted kidney.

Animal monitoring, sacrifice, tissue harvesting
Rats housed in metabolic cages were monitored daily for urine output for evidence of graft rejection, which was established by a significant increase in weight gain and a decrease in urine output. Renal function was determined by changes in serum creatinine and BUN levels. At completion of the study period, animals were anaesthetized with sodium pentobarbital (50 mg/kg i.p). Following exsanguination via cardiac puncture, isografts and allografts were obtained for routine histology and immunohistochemistry, gene and protein expression (western analysis, real time RT–PCR).

Experimental groups
The following groups of animals (n = 3 in each group) were used for this study. Group A: isografts kept for 90 days: Group B: 0.25 mg/kg i.m TAC for 90 days; Group C: TAC + anti-TGF-ß antibody (1D11, 1 mg/kg/twice a week); Group D: TAC + control antibody (1 mg/kg/twice a week i.m). Anti-TGF-antibody designated as 1D11 is a murine monoclonal antibody (IgG1), which specifically neutralizes the biological activity of TGF-ß 1, 2 and 3. The antibody recognizes active but not latent TGF-ß. Control antibody (13C4) is also a murine monoclonal antibody (IgG1) that specifically binds shigella toxin. Both antibodies were produced and purified at Genzyme Corporation (Cambridge, MA, USA) and were determined to be free of detectable endotoxin [4]. Based on our experience [7], we designed this study using an antibody dose of 1.0 mg/kg. In contrast to high dose (2.5 mg/kg), this low antibody dose (1.0 mg/kg) limits immunosuppression-induced nephrotoxicity without altering graft survival in the rat [7].

BUN and creatinine levels
Assay of serum levels of BUN and creatinine were performed to assess renal function using specific kits (Wako Chemicals Inc., Richmond, VA and Oxford Biomedical Research, Oxford, MI, USA, respectively).

Allograft histology, immunohistochemistry and quantification of histopathological changes
A portion of transplanted kidneys from each animal was fixed in formalin and paraffin imbedded. Haematoxylin and eosin (H&E) and periodic acid schiff (PAS) staining were used to assess histological changes. Two different individuals graded the histopathological findings using numbered slides that did not give any indication from what group the sample was derived. We quantified renal histological changes by standard procedures. Using light microscopy, we evaluated the severity of the glomerular injury score according to established methods [1,4]. In each field, we counted at least 50–60 glomeruli for each specimen and score the lesions into five grades. In H&E stained renal tissue sections, histopathological grading was defined as: score 0, no proliferation, almost normal histology; score 1, about 25% segmental lesion; score 2, segmental lesion >25 but <50%; score 3, fibrotic lesions with diffuse proliferation; score 4, almost completely fibrotic changes. Using PAS staining, we determined the extent of extracellular matrix protein accumulation; the grading was not performed on matrix deposition. The interlobular arteries in H&E stained slides and the lesions were scored.

Plasma TGF-ß protein and immunochemistry in renal tissues
Plasma levels of TGF-ß protein were measured as previously described [5]. Intragraftprotein expression of TGF- ß was studied using immunohistochemistry [1,2,4]. Formalin-fixed tissues were paraffin-embedded, sliced into fine sections, deparaffinized in xylene and rehydrated in graded ethanol to phosphate-buffered saline (PBS). After blocking endogenous peroxidase activity for 30 min with methanol/H2O2 (18: 1 vol/vol), non-specific binding were blocked for 1 h with 1.5% Avidin/Biotin diluted in PBS supplemented with 10% normal horse serum and 3% BSA. Tissue sections were incubated overnight at 4°C with specific antibodies (50 µg/ml) in above-mentioned PBS. Following extensive washings with PBS, the slides were incubated for 1 h with 1:1000 diluted biotin-labelled anti-mouse IgG horse anti-serum at room temperature, again washed extensively in PBS and then in the ABC solution for 30 min. The slides were then developed for 10 min in diaminobenzidine (DAB) and rinsed with water for 10 min. The slides were counterstained with haematoxylin followed by dehydration in graded ethanol and xylene.

The slides were mounted with Permount for evaluation. Samples from each group were graded for histopathological changes and immunohistochemistry staining. The intensity of immunostaining was graded from 0 (no staining) to 4+ maximum staining, relative to the staining pattern in renal tissues from different groups was recorded as described [1,5,7]. This grading was performed in a blinded fashion and is not quantitative but is relative to each section. The changes in renal histology for each animal were graded blindly by two individuals for interstitial fibrosis, arterial changes and glomerulosclerosis. The number of tubular changes in each field was recorded in kidney sections from animals from different groups. Then, an arbitrary grading from 0 to 4 was assigned for each slide and mean and standard errors were calculated.

Detection of mRNA by real time PCR
We performed real time quantitative RT–PCR using a Bio-Rad iCycler system (Bio-Rad, Hercules, CA). RNAs were isolated from renal tissues using a kit from Promega (Madison, USA) and reverse-transcribed into cDNAs by using a cDNA synthesis kit from invitrogen (Carlsbad, CA). The specificity of primers was tested by running a regular PCR for 40 cycles at 95°C for 20 s and 60°C for 1 min, and followed separating in ethidium bromide containing agarose gels. The primer sequences are given in Table 1. The real time PCR was performed using a SYBR supermix kit (Bio-RAD), and running for 40 cycles at 95°C for 20 s and 60°C for 1 min. The PCR efficiency was also examined by serially diluting the template cDNA and the melting curve data was collected to check the PCR specificity and proper negative controls were included in each assay. The mRNA level for each gene for each sample was normalized to ß-actin mRNA and was presented as 2[(Ct/ß-actin ÿ Ct/gene of interest)] as described [24].


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  		Table 1. Primer sequences of genes for real time PCR

 
Western analysis
Frozen tissues were homogenized in ice-cold PBS with 1% Triton X-100, 1 mM phenylmethysulfonyl fluoride, 35 ng/ml pepstatin A and 10 ng/ml leupeptin. After centrifugation, 50 g of protein was electrophoresed by SDS PAGE as described in our laboratory [24]. Blots were probed with appropriate dilutions of a polyclonal anti-p22phox antibody (R5554 a gift Dr Mark Quinn of Montana State University, Bozeman, MT) or anti-NOX antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) and incubated with 1:5000 dilution of horseradish peroxidase-conjugated secondary antibody for visualization by enhanced chemiluminescence.

Statistical analysis
Student's t-test was used to assess differences between the group means for gene expression, plasma levels of TGF-ß protein and histology scores. The analysis was performed using a statistical software programme (GraphPad Inc., San Diego, CA, USA). Results are expressed as the mean ± SEM and two-tailed significance determined at the level of P < 0.05.



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Acknowledgements
 References
 
Renal function
BUN levels (Figure 1A) were elevated in TAC-treated rats after 90 days of treatment vs isograft transplant controls harvested at 90 days post-transplantation (29.9 ± 1.3 vs 17.3 ± 1.3 mg/dl; P < 0.0001). Anti-TGF-ß antibody (21.3 ± 0.9, P < 0.01) but not control antibody (26.6 ± 1.1 mg/dl) decreased BUN levels. Similarly, a significant increase in serum creatinine (0.83 ± 0.06 vs 0.52 ± 0.09 mg/dl; P < 0.04) was observed in TAC-treated recipients compared to time-matched isograft transplant controls. Similar to BUN levels, anti-TGF-ß antibody (0.48 ± 0.08 mg/dl; P < 0.02) but not control antibody (0. 78 ± 0.09) inhibited TAC-induced creatinine levels (Figure 1B).


Figure 1
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  		Fig. 1. Effect of anti-TGF-ß antibody on renal function. (A) BUN levels were elevated in the TAC-treated rats vs isografts (P < 0.0001) and anti-TGF-ß antibody but not control antibody decreased BUN levels (P < 0.01 and not significant, NS, respectively). (B) A significant increase in serum creatinine can be seen in TAC-treated recipients compared to isografts (P < 0.04). Anti-TGF-ß antibody but not control antibody significantly inhibited TAC-induced creatinine levels (P < 0.02 and NS, respectively).

 
Anti-TGF-ß antibody and TAC-induced expression of intragraft TGF-ß, NOX-1, p22phox and Rac-1 mRNA
We performed a real time PCR analysis for intragraft expression of TGF-, NOX-1 and p22phox mRNA in renal tissues from isografts, TAC, TAC + anti-TGF-antibody and TAC + control antibody-treated animals at 90 days post-transplantation. The results are presented with respect to isograft transplant controls. Treatment with TAC increased mRNA expression of TGF-ß (by 37-fold), NOX-1 (by 18-fold), and p22phox (by 31-fold) and Rac-1 (by 20-fold). Increases in gene expression were inhibited by anti-TGF-ß (P < 0.01; P < 0.008, P < 0.04 and P < 0.03, respectively) but were not inhibited by control antibody (Figure 2A). The mRNA expression of gp91phox was undetectable in all the groups and the levels of ß-actin mRNA (in terms of Ct value) were similar in each group.


Figure 2
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  		Fig. 2. (A) Effect of anti-TGF-ß antibody on intragraft expression of TGF-ß, NOX-1, p22phox and RAC-1 mRNA. Anti-TGF-ß antibody but not the control antibody inhibits TAC-induced intragraft TGF-ß, NOX-1, p22phox and RAC-1 mRNA expression. (B) Effect of TAC on TGF-ß, SOD and TRX mRNA expression in rat renal transplants. TGF-ß vs SOD and TRX mRNA are reciprocally expressed in TAC-treated rat transplant recipients. Anti-TGF-ß antibody inhibited intragraft TGF-ß but partially restored SOD and TRX mRNA expression. (C) Western blot analysis for p22phox and NOX-1 protein expression. TAC treatment induced intragraft p22phox and NOX-1 protein in allografts (lanes 5, 6) compared with untreated allografts (lanes 3, 4) or isograft transplant controls (lanes 1, 2), and anti-TGF-ß decreased TAC-induced expression of p22phox and NOX-1 protein (lanes 7, 8).

 
Differential intragraft mRNA expression of TGF-ß, SOD and TRX
Since we observed that treatment of renal transplant recipients with TAC resulted in increased intragraft expression of TGF-ß and NADPH oxidase subunit components, the next step was to determine the effect of prolonged treatment with TAC on antioxidant genes. We examined intragraft mRNA expression of SOD and TRX and compared it with TGF-ß mRNA expression. The data demonstrate that SOD and TRX mRNA decreased whereas TGF-ß increased in TAC-treated rats compared to isograft transplant controls. SOD mRNA was partially reversed whereas TRX mRNA was completely reversed in TAC + anti-TGF-ß antibody-treated rats. We calculated the fold-increase in expression of TGF-ß, SOD and TRX mRNA. Treatment with TAC resulted in a 37-fold increase in TGF-ß whereas mRNA expression of SOD and TRX decreased 27- and 80-fold, respectively. These changes were reversed by anti-TGF-ß antibody (Figure 2B).

Intragraft expression of p22phox and NOX-1 protein
We studied the expression of p22phox and NOX-1 protein in kidney tissues from isografts, and treated with TAC. The protein lysates (10 µg) were electrophoresed, transferred to nitrocellulose paper and probed with anti-p22phox and anti NOX-1 antibodies. The results (Figure 2C) show that treatment with TAC resulted in increased intragraft p22phox and NOX-1 protein. More significantly, anti-TGF-ß treatment decreased TAC induced increased intragraft p22phox and NOX-1 protein expression.

Treatment with TAC results in the transplant-nephrotoxicity specific renal histological changes
We examined the effect of TAC, TAC + a control antibody and TAC+ anti-TGF-ß antibody treatment on morphological changes by histopathological examination in H&E- and PAS-stained thin kidney sections. We scored the morphological changes. The light microscopic findings of kidneys of transplant control rats from isografts (panels 5 and 6) showed normal glomeruli, afferent arterioles and tubule cells. In sharp contrast, the renal tissues of rats treated with TAC showed marked histological changes including, severe to moderate epical blebbing, hyalinization, glomerular basement thickening, a pattern of tubulointerstitial fibrosis including arteriolopathy of afferent arteriole (panel 1) and terminal portions of the interlobular arteries. These changes were not observed in co-administration of TAC and anti-TGF-ß antibody (panels 3 and 4). However, similar changes can be seen in animals treated with TAC + a control antibody.

Histology in TAC + control antibody-treated animals was not different than TAC alone (not shown). Renal toxicity specific to proximal tubular epithelial cells specific changes specific to renal toxicity can be seen in TAC and TAC + control antibody-treated rat transplant recipients and not in isografts or TAC + anti-TGF-ß antibody-treated rats (Figure 3B). TAC-induced tubular changes (tubular atrophy/vacuolization, hyalinization are also shown in Figure 3B. These changes were seen only in TAC and TAC + control antibody-treated renal transplant recipients but not in isografts and TAC + anti-TGF-ß antibody-treated transplant recipients. The quantitative analysis (Figure 3C) demonstrated a statistically significant (P < 0.036) score in TAC-treated recipients compared to the control isografts which were not different than TAC + anti-TGF-ß antibody-treated allografts.


Figure 3
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  		Fig. 3. Effect of TGF-ß antibody on renal histology. (A) The light microscopic findings of kidneys of control rats (isografts) showed normal glomeruli, afferent arterioles and tubule cells (panels 5 and 6). In sharp contrast, the renal tissues of rats, which received TAC showed marked histological changes including, severe to moderate epical blebbing, hyalinization, glomerular basement thickening, a pattern of tubulo-interstitial fibrosis and arteriolopathy of afferent arteriole (panel 1, black arrow) and terminal portions of the interlobular arteries. These changes were not observed in co-administration of TAC and anti-TGF-ß antibody (panels 3 and 4). Tubular thickening, remodelling and arteriolopathy tubulo-interstitial fibrosis and glomerular basement membrane thickening can be seen in TAC-treated recipients (panels 1 and 2) but not in TAC + anti-TGF-ß antibody-treated recipients (panels 3 and 4). B: Tubular changes such as dilation, swelling, thickening, remodelling and hyalinization are seen in TAC and TAC + control antibody-treated recipients but not in the isografts or TAC + anti-TGF-ß antibody-treated recipients, magnification 400x. (C) Statistically significant semi-quantitative changes (*P < 0.036) in kidneys from TAC-treated rat renal transplant recipients compared to TAC+ anti-TGF-ß antibody are shown. There were no differences in TAC alone vs TAC + control antibody and isografts vs TAC+ anti-TGF-ß antibody-treated animals.

 
Tacrolimus increases intragraft TGF-ß protein expression
We have also examined intragraft expression of TGF-ß protein in kidney tissues from recipients of renal transplants treated with TAC, TAC + anti-TGF-ß antibody, TAC + control antibody-treated or untreated allografts. Renal sections were stained with anti-TGF-ß antibody as earlier described by our group [7]. Beside hyalinization and arteriolopathy tubulo-interstitial fibrosis and glomerular basement membrane thickening, a positive staining for TGF-ß protein can be seen in TAC and TAC + control antibody-treated recipients but not in TAC + anti-TGF-ß antibody-treated recipients (Figure 4A). The intragraft expression of TGF-ß protein in TAC + anti-TGF-ß antibody-treated recipients was slightly higher compared to the isografts, based on our experience; this suggests that the anti-TGF-ß antibody only neutralized the induced but not the constitutive TGF-ß protein expression.


Figure 4
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  		Fig. 4. Intragraft expression of TGF-ß protein. Representative slides of immuno-histochemical staining for TGF-ß in renal tissues from isografts, TAC, TAC + anti-TGF-ß and TAC + control antibody-treated animals are shown. The results demonstrate a significantly higher staining of intragraft TGF-ß protein expression in TAC-treated recipients which was not different in control antibody + TAC-treated recipients. TGF-ß protein staining in animals treated with TAC + anti-TGF-ß antibody (1.0 mg/kg) was abolished, however, was slightly higher than isografts (A). (B) Circulating levels of TGF-ß protein. Circulating levels of TGF- protein in plasma samples were quantified by ELISA. A statistically significant increased expression in TAC-treated recipients can be seen compared to isograft transplant controls. A statistically significant decrease of TGF-ß levels can be seen in TAC + anti-TGF-ß antibody compared to TAC-treated animals; whereas, no difference with TAC + control antibody-treated recipients can be seen. *P < 0.01; **P < 0.002.

 
Effect of treatment with TAC on circulating levels of TGF-ß protein
These results (Figure 4B) demonstrate that compared to isografts, long-term treatment of TAC resulted in a significant increase in circulating levels of TGF-ß protein (9.8 ± 1.7 vs 57 ± 6 ng/mL; p <0. 01). There was no difference in TGF-ß levels among isografts and TAC + control antibody-treated recipients (57 ± 6 vs 52 ± 6 ng/ml); however, a statistically significant difference (9 ± 1.3 vs 57 ± 6 ng/ml; P < 0.01) was observed between TAC and TAC + anti-TGF-ß antibody-treated recipients (Figure 4B).



   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Acknowledgements
 References
 
Immunosuppression-induced nephrotoxicity remains an unavoidable and significant problem in organ transplantation. Furthermore, there is a lack of sufficient understanding of its pathogenesis and mechanism(s) involved. The results of the present study for the first time demonstrate that besides TGF-ß, oxidative stress also contributes significantly to this process. This action occurs through two separate mechanisms. The first mechanism is by down-regulation of antioxidant defense genes which detoxify reactive oxygen such as SOD and TRX, which detoxify reactive oxygen. The second mechanism is by up-regulation of NADPH oxidase subunit genes which increase reactive oxygen production.

We used a rat renal transplantation model with long-term treatment with TAC that results in renal dysfunction as determined by increases in BUN and creatinine and by changes in renal histopathology. Treatment with TAC also increased intragraft expression of TGF-ß mRNA and protein expression as well as the circulating levels of TGF-ß protein. This is similar to our previous findings in a rat cardiac transplant model treated with CsA [7]. In that model using anti-TGF-ß antibody treatment, we were able to document the important role of CsA-induced up-regulation of TGF-ß in nephrotoxicity using an anti-TGF-ß antibody strategy. In the present study, we zfound that treatment with anti-TGF-ß antibody inhibited TAC-associated renal dysfunction. This conclusion is based upon the documented by inhibition of changes in BUN, creatinine levels and renal histopathology. These results suggest that anti-TGF-ß antibody treatment may protect against nephrotoxicity in renal transplant recipients by potentially neutralizing the induced TGF-ß protein and gene expression.

Immunosuppressive agents such as CsA and TAC have been shown to promote generation of reactive oxygen species in a number of cell types [25–29]. Despite this, the exact mechanism of how reactive oxygen levels are up-regulated by these agents remains unresolved. One theory is that reactive oxygen species are by-products of the metabolism of immunosuppressive drugs [27]. Accordingly, the normal Formula due to CsA induced inhibition of NADPH cytochrome P-450 reductase might have resulted in Formula , increased NADPH activity and lipid peroxidation [30–32]. Currently, there is no evidence if a similar mechanism of drug metabolism also plays a role in the TAC-induced increased Formula . Reactive oxygen species as well as the end-products of lipid peroxidation like 4-hydroxy-2, 3-nonenal induce expression of TGF-ß in renal tissue 1 h after TAC administration in rats [33]. These data suggest that TAC-induced nephrotoxicity may be related to increased oxidative stress and lipid peroxidation.

It has also been determined that reactive oxygen species enhance TGF-ß release in vitro and in vivo [34]. Thus, increased reactive oxygen may be both a promoter of TGF-ß expression as well as a potential mediator of downstream actions of TGF-ß. Regarding the latter, we have previously shown that TGF-ß increased albumin permeability in isolated glomeruli and that this injury was antagonized by SOD or dimethylthiourea [35]. This finding suggests a potential role of O2 and hydroxyl radicals as downstream mediators of the nephrotoxic actions of TGF-ß. The results of the present study demonstrate that not only intragraft mRNA and protein expression of TGF-ß are significantly increased in rat renal transplant recipients treated with TAC. In addition, our study shows for the first time that the marked up-regulation of NADPH oxidase components (p22phox, NOX-1, Rac-1) are significant sources for increased Formula production in TAC-induced nephrotoxicity. This suggests that interventions to limit actions of TGF-ß such as in this study using anti-TGF-ß antibody might also have additional benefits to limit downstream actions of oxidative stress and nephrotoxicity associated with long-term treatment of transplant recipients with TAC.

In our study, we were able to show for the first time that expression of major antioxidant genes, SOD and TRX, are down-regulated in renal transplants treated with TAC. These proteins are primary enzymes important in detoxifying reactive oxygen species in cells. We found that co-administration with anti-TGF-ß antibody inhibited the down-regulation in SOD and TRX gene expression. While TGF-ß has previously been shown to decrease MnSOD in cultured hepatocytes [10], it has not; however, been determined that this action may occur in other cell types or tissues including renal cells/tissues.

In our renal transplant model, the antagonizing of the down-regulation of SOD and TRX by anti-TGF-ß gives evidence of a novel potential pathway by which TAC may induce nephrotoxicity in transplant recipients. Our study describes this novel finding that TAC also alters the redox state of cells by down-regulating TRX expression. To the best of our knowledge, TRX expression has not been previously determined for any immunosuppressant agent. Furthermore, our findings suggest a new source contributing to enhanced oxidative stress in this condition.

This conclusion arises from our other novel findings that anti-TGF-ß antibody but not control antibody prevents TAC-induced up-regulation of NADPH oxidase gene and protein expression. Taken together with our findings with SOD and TRX, we find that TAC has potentially two mechanisms of increasing oxidative stress; one by up-regulating genes that increase Formula production and a second by down-regulating genes that remove or scavenge Formula . Our findings on the collective role of TGF-ß and Formula are also supported by studies on other models of renal injury such as salt-sensitive Dahl rats [36,37] and an angiotensin-induced hypertension model [38]. In these studies, the improvement in the renal injury was associated with a simultaneous inhibition of TGF-ß and oxidative stress.

The findings of the increased mRNA expression of NADPH oxidase components (p22phox, NOX-1 and Rac-1) by TAC distinguishes it from studies showing renal dysfunction due to salt loading in the Dahl S rat in which p22phox and Rac-1 mRNA were not induced but rather gp91phox and p47phox [39]. This suggests that up-regulation of specific NADPH oxidases may mediate renal pathology under different conditions.

In addition to phagocytic NADPH oxidases, there is emerging evidence that non-phagocytic NADPH oxidases are present in many cells and could be important sources for Formula production in inflammatory states. In kidney, NOX-1 is the non-phagocytic membrane analogue of the phagocytic subunit gp91phox. Despite this knowledge, the detailed analysis of these regulatory components in the events leading to increased Formula production and initiation of events leading to nephrotoxicity have not been explored.

Our findings with gp91phox do not support a significant role of phagocytic NADPH oxidase from infiltrating inflammatory cells in this model of TAC-induced nephrotoxicity. Thus, this distinguishes it from the role of phagocytic NADPH oxidases in both salt loading-induced and angiotensin-induced nephropathy. In contrast, our additional findings of up-regulation in NOX-1 and its reversal by TGF-ß antibody for the first time implicate non-phagocytic NADPH oxidase as a major role in TAC-induced nephrotoxicity as well. We suggest that this may be a direct action on the non-phagocytic NADPH oxidase unit within renal cells per se. Indeed, supporting this concept are new data from our laboratory showing that TGF-ß has the direct capacity to increase NADPH oxidase gene expression in proximal epithelial cells in culture and that this increase is blocked by TGF-ß siRNA (unpublished observations). In summary, the results from this study demonstrated that the expression of TGF-ß and NADPH oxidase components is increased while that of antioxidant genes SOD and TRX is decreased in TAC-treated rat renal transplant recipients. These effects were reversed in recipients treated with TAC + anti-TGF-ß antibody but not the control antibody. This suggests that TGF-ß may be a direct mediator of both the oxidative stress and nephrotoxicity in TAC-treated transplant recipients.

Collectively, our studies demonstrate that TGF-ß and Formula participate in the events leading to renal damage. Therefore, a strategy to inhibit TGF-ß and/or Formula would assist in the prolongation of graft survival without unwanted side effects of TAC for transplant recipients.



   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors are grateful to Matthew Plummer and Gail Hilton for their excellent technical assistance and to Dr Mark Quinn for anti-p22phox antibody. This work was supported, in part, by NIH (National Institutes of Health) grants HL-6637 (to G.M.P).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 24. 6.06
Accepted in revised form: 20. 9.06


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