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–Neutralizing Antibody in Mouse Cyclosporin A Nephropathy: Morphologic Improvement Associated with Functional Preservation
*Cell Biology, Genzyme Corporation, Framingham, Massachusetts; and Division of Renal Diseases and Hypertension, University of Colorado School of Medicine, Denver, Colorado.
Correspondence to Dr. Steven Ledbetter, Director, Cell Biology, Cell & Protein Therapeutics Division, Genzyme Corporation, One Mountain Road, Framingham, MA 01701. Phone: 508-271-3636; Fax: 508-270-2088;
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is believed to play a central role in the development of Cyclosporin A (CsA)-induced nephropathy. This study investigated the effects of 1D11, a murine pan-specific TGF-–neutralizing monoclonal antibody, in an ICR mouse model of chronic CsA nephropathy. Mice were administered a low-salt diet (0.01% sodium) for 1 wk followed by CsA treatment (30 mg/kg, subcutaneously, daily) for 4 wk. 1D11 was administered (2.5 mg/kg, intraperitoneally, 3 times/wk) beginning immediately after the termination of CsA dosing and continued through 8 wk. CsA caused extensive renal histopathologic alterations, including tubular damage, interstitial infiltrates and fibrosis, deposition of collagen III, and apoptosis of tubular epithelial cells. 1D11 ameliorated the CsA-induced histopathologic alterations, with significant reduction in collagen III expression and deposition. Additionally, elevated levels of mRNA encoding TGF-1 and TGF-2 were significantly reduced. 1D11 also protected tubular epithelial cells from apoptosis by 48% (P < 0.05). In contrast, 13C4 (a control antibody) had no significant effect on any of the endpoints described above. Importantly, the effects of 1D11 on the CsA-induced morphologic alterations were followed by a reduction in serum creatinine level when compared with CsA mice treated with 13C4 (13C4, 0.45 ± 0.09; 1D11, 0.30 ± 0.08; P < 0.05) after 8 wk of treatment. Endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), nitrotyrosine, and tissue hypoxia were examined by immunostaining using specific antibodies. eNOS was significantly reduced in the endothelium of arterioles in the kidneys of mice treated with CsA, whereas iNOS was induced in the cortical tubules. Tissue hypoxia was found in both the arterioles and tubules, whereas nitrotyrosine was localized in the tubules. Administration of 1D11 improved tissue hypoxia and reduced nitrotyrosine formation. Moreover, the reciprocal changes in iNOS and eNOS expression were normalized by 1D11. This study demonstrates that 1D11 administration ameliorated morphologic alterations and preserved renal function in the context of existing chronic CsA nephropathy. E-mail: steve.ledbetter@genzyme.com |
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The mechanisms of chronic CsA nephropathy are yet to be clearly delineated, but they are known to include a role for TGF- (4–5), a group of genetically related, multifunctional cytokines. Three mammalian TGF- isoforms, TGF-1, -2, and -3, have been identified and have regulatory effects on diverse cellular biologic activities through their binding to specific, high-affinity receptors present on most cells (6–7). A number of studies show that elevated expression of TGF-, particularly TGF-1, is associated with tissue fibrosis in a variety of both experimental animal models and human diseases (8–9). Also, there is a significant body of in vitro and in vivo evidence supporting the idea that early expression and persistent activation of TGF- in the kidney is associated with tubulointerstitial fibrosis, one of the most prominent pathologic features of chronic CsA nephropathy (8,10). Recently, it has been shown that patients with chronic CsA nephropathy have an increase in renal TGF- expression (11).
CsA-induced acute injuries, including vasoconstriction and tubular damage, are thought to be related to abnormal expression of nitric oxide (NO) synthase (NOS) (12). Alterations in the expression of both endothelial NOS (eNOS) and inducible NOS (iNOS) in the diseased kidneys results in sustained afferent arteriolar vasoconstriction, resulting in tissue hypoxia and tubular injury via necrosis and apoptosis (12–14). Interestingly, abnormal NOS expression may have an effect on TGF- expression. For example, chronic inhibition of NOS in rats using an L-arginine analogue resulted in increased resistance in afferent arterioles, which was associated with local activation of the renin-angiotensin system (RAS) and induction of TGF-1 expression in the kidneys (15). In addition, Shihab et al. (16) recently reported that chronic CsA nephropathy can be ameliorated by NO enhancement, which is associated with a downregulation in TGF- expression. These observations suggest a complex relationship between the TGF- and NOS systems that collectively contribute to the CsA-induced histopathology associated with renal dysfunction.
An appreciation of the role of TGF- in chronic CsA nephropathy raises the prospect that blocking TGF- bioactivity could prevent or reverse the development of fibrosis that characterizes most chronic renal diseases, including CsA nephropathy. Although the beneficial effects of a TGF-–neutralizing antibody in a rat model of chronic CsA nephropathy have recently been published (17), it is not well documented that blocking TGF- can elicit a therapeutic effect in the context of existing chronic CsA nephropathy accompanied by tubulointerstitial fibrosis. Also, it is not yet known how the neutralization can affect NOS expression, which is believed to play an important role in chronic CsA nephropathy. In the present study, we examined the effects of a murine pan-specific TGF- monoclonal antibody in a mouse model of chronic CsA nephropathy. This study demonstrates that the therapeutic administration of an agent that neutralizes TGF- can effectively reduce fibrosis and preserve renal function in a model of chronic CsA nephropathy.
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isoforms (1, 2, and 3) (18), was produced and purified at Genzyme Corporation (Framingham, MA). This antibody has a circulatory half-life of 34 h in mice when administered by intraperitoneal injection (personal communication, L. Andrews, Genzyme Corp., September 2001). An isotype-matched irrelevant murine IgG1 monoclonal antibody, 13C4, also produced by Genzyme Corporation, directed against Shigela toxin, was used as a control antibody.
Experimental Protocol
Male ICR mice (Taconic Farms, Germantown, NY) weighing 25 to 35 g (6 to 8 wk) were housed in an air-, temperature-, and light-controlled environment, and all protocols were approved by Genzyme’s Institutional Animal Care and Use Committee. All animals were placed on a low-salt diet (0.01%) for 1 wk before CsA (Novartis Pharmaceuticals Corporation, East Hanover, NJ) or PBS administration (for normal control mice). The low-salt diet continued throughout the 4 wk of CsA or PBS administration, as described previously (19). CsA was given subcutaneously at a concentration of 30 mg/kg once a day for 4 wk. Immediately after the termination of CsA dosing, one group (n = 8 for each group) of mice was sacrificed to establish baseline measurements; the remaining mice were randomly divided into the following treatment groups: (1) PBS; (2) 13C4 antibody; and (3) 1D11 antibody. Antibodies were administered intraperitoneally (2.5 mg/kg, 3 times/wk), and the duration of treatment was continued for either 4 or 8 wk, with a total time course for the experiment of 12 wk. Initially, this experiment was conducted as a pilot trial with small numbers of animal, but it was subsequently repeated as outlined above; both experiments provided similar results. At the end of 8 wk of treatment, individual mice were placed in metabolic cages to obtain 24-h urine collection. One hour before sacrifice, all animals received hypoxyprobe-1 (60 mg/kg pimonidazole hydrochloride intravenously; Natural Pharmaceutical, Belmont, MA), to visualize tissue hypoxia by using a specific antibody (see below). At sacrifice, body weight was recorded and blood was taken by eye bleed. The left kidney was perfused with PBS for 5 min and weighed. This kidney was harvested for the analysis of mRNA, and the right kidney was further perfused with 4% PLP solution (4% paraformaldehyde, 5% sucrose, 10 mM sodium periodate, 10 mM lysine) for histopathologic examination and immunofluorescence staining.
Histopathologic Study
PLP-fixed and paraffin-embedded kidney sections (5-µm) were stained with hematoxylin-eosin (H&E) and Masson trichrome using standard techniques. Assessment of CsA-induced histopathologic changes was conducted using commonly accepted semiquantitative scoring as described previously (20). All evaluations were conducted by experienced investigators on coded sections without knowledge of the experimental group to which the mice belonged. Briefly, histopathologic changes were assessed on the basis of tubular damage, interstitial infiltrates, thickening of arterioles, tubulointerstitial expansion, and fibrosis and scored by counting the percentage of the diseased area per kidney section, as follows: 0, none; 1, <10% of diseased area; 2, 11 to 25% of diseased area; 3, 26 to 45% of diseased area; 4, 46 to 75% of diseased area; and 5, >76% of diseased area.
Immunofluorescence Staining
The expression and localization of type III collagen, eNOS, iNOS nitrotyrosine and tissue hypoxia were visualized by immunofluorescence staining. Briefly, transverse sections (5-µm) of kidneys were prepared, deparaffinized in xylene, and hydrated in graded ethanol to H2O and PBS. All incubations were performed at room temperature. Sections were first treated with an unmasking solution (1%; Vector Laboratories, Burlingame, CA) and then incubated with 0.25% trypsin for antigen retrieval. To block potential endogenous peroxidase, sections were quenched with 0.3% H2O2 plus 0.3% horse serum in PBS. To block nonspecific binding, sections were incubated for 30 min with 1% BSA in PBS. For the mouse monoclonal antibody used to detect tissue hypoxia, a Vector M.O.M.–specific blocking reagent was employed. Sections were incubated for 1 h with specific rabbit polyclonal antibodies against eNOS, iNOS (Santa Cruz Biotechnology, Santa Cruz, CA), nitrotyrosine (Upstate Biotech, Lake Placid, NY), or a mouse monoclonal antibody recognizing reduced, protein-bound pimonidazole, which is reductively activated at low oxygen concentrations and binds to cellular macromolecules (21). All antibodies were used at 2.5 µg/ml in PBS, except for the latter, which was diluted 1:1. After three washes with PBS, sections were incubated for 30 min with either anti-rabbit or anti-mouse IgG linked to horseradish peroxidase (HRP, Amersham, Piscataway, NJ). Fluorescein (FITC)-labeled tyramide, an HRP-catalyzed signal amplification reagent (NEN Life Products, Boston, MA), was then applied in a dilution of 1:100 for 10 min according to manufacturer’s instruction. After thorough washing with PBS, specimens were counterstained with 0.04% Evan blue (Sigma, St. Louis, MO) to better visualize the stained tissue under explicit background of renal architecture. Sections were mounted with anti-fade mounting medium (Vector). The immunofluorescence detection of type III collagen using a specific goat polyclonal antibody (Southern Biotechnology Associates, Birmingham, AL) was followed by 45 min of incubation with FITC-labeled donkey anti-goat IgG (1:100 dilution; Jackson ImmunoResearch, West Grove, PA). To assess the specificity of the immunostaining, adjacent sections were incubated with nonimmune rabbit, mouse, or goat IgG in place of the primary antibodies and then processed under identical conditions. Microscopic observation and photography were performed with a Nikon Eclipse E-800 fluorescence microscope (Nikon, Melville, NY).
Ribonuclease Protection Assay (RPA)
Total cellular RNA from whole kidney tissue was obtained using the RNAqueous kit (Ambion, Austin, TX). To prepare the RPA probe template, primers complementary to the cDNA sequence of TGF-1, TGF-2, and type III collagen (22) were included in the reverse transcription PCR (RT-PCR), which was performed on total kidney RNA. Linearized cDNA was transcribed in vitro using the Maxiscript kit (Ambion) according to the manufacturer’s instructions. T7 polymerase and P32CTP (3000 Ci/mmol; Dupont-NEN, Boston, MA) were included in the reaction mixture to generate P32-labeled riboprobe. The reaction mixture was incubated for 60 min at 37°C, and the DNA template was removed by digestion with 0.5 U of RNAase-free DNAase. Full-length probes were purified from the transcription reaction by electrophoresis on 6% polyacrylamide/TBE gel (Novex, San Diego, CA), followed by autoradiography, excision of the gel band, and passive diffusion into probe elution buffer (Maxiscript kit, Novex) overnight at 37°C. The activity of the probe was quantified by scintillation counting. RNAase protection assay was performed using the Hybspeed RPA kit (Ambion) according to the manufacturer’s instructions. Briefly, radiolabeled antisense RNA probe for type III collagen, TGF-1, and TGF-2 were combined and hybridized with 10 µg of total cellular RNA from the kidney samples. A probe for 18S RNA (Ambion) was also included in each hybridization mixture to normalize total RNA in individual samples. Yeast RNA was used as a negative control. Digestion with RNAaseA/RNAaseT1 mix was performed to degrade unhybridized RNAs. Hybridized RNA, protected from digestion, were resolved by electrophoresis and visualized with a Fujifilm LAS-1000 phosphoimager (Fuji, Elmsford, NY). Bands on the phosphoimager representing mRNA encoding type III collagen, TGF-1, and TGF-2 were quantified using Mac BAS Version 2.4 software. For all samples, the expression of each gene was corrected by dividing probe-specific signal by that obtained for 18S RNA.
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick 3' End Labeling (TUNEL)
TUNEL assay, combined with the identification of condensed or fragmented nuclei, was performed to evaluate apoptosis in CsA-treated mice. Sections (5-µm) of paraformaldyhyde-fixed, paraffin-embedded kidney tissue were deparaffinized, rehydrated, and washed in PBS. After washing, sections were incubated with proteinase K (20 µg/ml) for 20 min at room temperature. The sections were then incubated with 80 µl of a mixed solution, including 25 U/µl terminal deoxynucleotidyl transferase (TdT), 50 µM FITC-labeled 12-dUTP, 100 µM Tris-HCl (pH 7.6), and 1 mM EDTA for 10 min at 37°C in a humidified chamber. After thorough washing, the sections were counterstained with 0.3 µg/ml propidium iodide (Sigma) in PBS for 1 min at room temperature, to visualize non-FITC stained nuclei. A Nikon fluorescence microscope was used to count apoptotic cells. A minimum of four microscopic fields (magnification, x200) were examined to identify a combination of FITC-positive staining and condensed or fragmented nuclei.
Renal Function Analysis
Serum creatinine (SCr) was measured using an Astra Autoanalyzer (Beckman Instruments, Fullerton, CA). Albumin excretion in 24-h urine was measured by fluorescence assay using albumin blue 580 (Sigma) and mouse albumin as a standard.
Statistical Analyses
Values are expressed as means ± SEM. Group comparisons were done using Student t test or ANOVA when appropriate. P < 0.05 was considered statistically significant.
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1 (threefold) and TGF-2 (fourfold) mRNA levels in the kidneys, and these levels remained elevated through the end of the experiment (Figure 4B). Treatment with 1D11 for 4 or 8 wk inhibited the CsA-mediated upregulation of both genes, whereas treatment with 13C4 had no significant effect. These results support a central role of both isoforms of TGF- in the development of CsA nephropathy.
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neutralization would affect tubular cell apoptosis determined by TUNEL assay. We noted apoptotic cell death mainly in cortical tubular epithelial cells in mice treated with CsA, but this was rarely seen in the kidneys of normal animals (data not shown). Apoptotic cell numbers were determined on samples taken from mice that received treatment for 4 wk after CsA dosing (incidence of apoptosis was highest at this time point). As shown in Figure 7, mice treated with 1D11 showed fewer (47%) apoptotic tubular epithelial cells by comparison with mice treated with 13C4 (n = 8; P < 0.05).
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–neutralizing antibody, 1D11, on further disease progression and also examined tissues for evidence of regression from existing pathology. As was expected from earlier reports that defined this model (19), 4 wk of CsA administration induced substantial histopathology, including tubular damage, a robust inflammatory response, arteriolopathy, and striped tubulointerstitial expansion. We also noted early signs of damage associated with vasculature in the cortical-medullary junction and outer medullary capillary rays, consistent with earlier reports of CsA-mediated medullary ray injury (23–24). With the cessation of CsA treatment, further deterioration of renal structure, mostly in the form of tubular damage, and vascular and tubulointerstitial fibrosis was seen. Tissue fibrosis, as judged by histologic and immunostaining findings, developed slowly, beginning in the cortical-medullary area and progressing to include the cortical interstitium 8 wk after CsA dosing was discontinued.
Cyclosporin-mediated morphologic alterations correlated with the expression of TGF- mRNA in the kidneys; both TGF-1 and TGF-2 were significantly upregulated after 4 wk of CsA administration (fourfold and fivefold increases, respectively) and remained high in the mice that did not receive 1D11. The upregulation of mRNA encoding TGF-2 was unexpected, as TGF-1 has been more frequently associated with pathologic states, particularly fibrosis. The role of TGF-2 in this context is presently unclear. It is well established that there is an axis of TGF- autoregulation existing in most mammalian cells. When the proteins are activated, they stimulate not only their targeted molecules, such as connective tissue production, but they also enhance their own transcription and thus maintain an elevated transcriptional activity and constant production (6–7,25). It was therefore not surprising that 1D11 disrupted TGF- autoregulation loop by neutralizing these molecules and subsequent reduction in transcriptional activity. What remains unclear is the degree of coordinate transcriptional regulation of individual isoforms of TGF-, i.e., does a reduction in TGF-1 expression also reduce TGF-2 expression in the kidney?
Our study demonstrated that 1D11 treatment for 8 wk offered beneficial effects in ameliorating CsA-induced pathology. Histologically, we saw a reduction in overall tubular damage, attenuated inflammatory response, and a decreased tubulointerstitial expansion and fibrosis. These changes were accompanied with better preservation of histologic architecture in both cortical and medullary regions. Marked reduction in type III collagen expression and production provided further support that the histopathology was substantially improved in the animals treated with 1D11 for 8 wk but not in those dosed with a control antibody. Significant preservation of renal function was also achieved upon dosing with 1D11, as seen in terminal SCr levels. These results therefore provide direct evidence that TGF- plays an important role in this model of chronic CsA nephropathy. Indeed, numerous experimental and clinical studies have previously implicated a role for TGF- in mediating chronic CsA nephropathy and chronic allograft rejection, which is at least partially caused by CsA treatment (11,16,23). Recent studies by Islam et al. (17) showed that TGF-–neutralizing antibody reduced SCr but did not affect CsA-mediated interstitial lesions in a rat model of chronic CsA nephropathy, and Khanna et al. (26) demonstrated reduction in connective tissue gene expression in response to 1D11 administered to mice dosed with CsA. These studies and other studies of TGF-–neutralizing reagents administered to renal disease models (27–29) have made important observations on the role of TGF- in disease progression, but in all cases these agents have been administered simultaneously with the induction of disease or at a very early stage of disease when renal fibrosis was not yet developed. These studies do not address the efficacy of TGF- neutralization in a setting of established chronic nephropathy, which is most relevant to human disease. Our studies showed that 1D11 not only prevented further deterioration of renal architecture and development of tubulointerstitial fibrosis, but it also apparently reversed the progression of disease by improving the histologic appearance to a level better than that observed at the end of CsA administration. Our findings underscore a remarkable capacity of the kidney to repair itself and to apparently reverse the accumulation of fibrotic deposits. It has long been held that pathologic fibrosis is irreversible, though recent studies in the human diabetic kidney by Fioretto et al. (30) provide evidence that challenge this belief, and our data provide further preliminary evidence of the potential for reversing existing fibrosis with renal function preservation. Clearly, further work needs to be performed to explore the machinery of extracellular protein turnover upon TGF- neutralization.
CsA is known to cause both alterations in production of, or vascular reactivity to, several vasoactive factors as well as tubular cytotoxicity (31). Recently, increasing evidence has identified endothelial injury/dysfunction and tubular cytotoxicity as mechanisms that are central to CsA-induced nephropathy. These data show that enhancing NO production by administering the NOS substrate, L-arginine, improved CsA-induced impairment in endothelium-dependent relaxation of afferent arteries and chronic tissue hypoxia (12,16,32). On the other hand, there is also evidence that inadequate NOS expression is closely associated with CsA-induced nephropathy (31–32). However, none of these studies has uncovered a unifying mechanism concerning the role of NOS expression in CsA-induced renal pathology. Our results showed reciprocal changes in the expression of eNOS, a potent vasodilator that is constitutively expressed, and iNOS, the cytotoxic activities of which can be induced by a number of stimuli (such as cytokines and hypoxia), in the kidneys of the animals treated with CsA. Specifically, decreased expression of eNOS in the arterioles and increased expression of iNOS in the damaged tubules was observed. These findings, when considered in context with the CsA-mediated morphologic changes, suggest that the induction of iNOS in the tubules caused extensive tubular damage while the depression of eNOS likely contributed to vasoconstriction, leading to hypoxia in tissues. Accordingly, using a specific antibody that recognized protein-bound pimonidazole adduct, we demonstrated increased tissue hypoxia in the renal arterioles and tubules. The localization of tissue hypoxia in the diseased tubules was similar to the staining pattern of iNOS. Considering that the iNOS gene promoter contains a hypoxia-responsive domain (33), the data may indicate that depressed eNOS production may have led to an induction of iNOS via hypoxia. It is conceivable to believe that the tissue hypoxia per se is caused by both arteriolopathy and interstitial scaring, although it could be due to CsA-mediated vasoconstriction at the early stage of disease (13). In a further attempt to implicate the cytotoxicity of iNOS, we examined the localization of nitrotyrosine by immunostaining. The study revealed an increase in nitrotyrosine production in the kidneys of the CsA-treated mice with localization to the damaged tubules. Nitrotyrosine is a footprint of peroxynitrite formation, a highly toxic compound resulting from the reaction between superoxide and NO generated from iNOS induction; our results therefore suggest that iNOS is cytotoxic to the tubules. Further investigation showed that the administration of 1D11 improved tissue oxygenation, normalized the expression of eNOS and iNOS, and reduced the amount of nitrotyrosine in the diseased kidneys. Our observations could begin to reconcile the two divergent views of the effect of NO in chronic CsA nephropathy, providing insight into distinct roles of eNOS and iNOS in this model.
Excessive apoptotic cell death has recently been incriminated in the pathogenic, "silent," loss of cells seen in progressive renal scarring during the evolution of focal glomerular sclerosis and chronic CsA nephropathy (34–35). However, the role of TGF- in this pathway remains unknown. Using the TUNEL assay, an increase in apoptotic cells was found in the kidneys of CsA-treated mice while 1D11 protected tubular epithelial cells from apoptosis. Although a number of factors such as hypoxia or free oxygen radicals could be involved in inducing tubular epithelial cell apoptosis in CsA nephropathy, increasing evidence has reported that TGF- triggers apoptosis in a variety of cells, including kidney cells (36–38). As documented by Schiffer et al. (38), induction of apoptosis in renal podocytes was observed in TGF-1 transgenic mice. Also, tubular epithelial cell apoptosis was associated with an increase in TGF- expression in both diabetic and obstructive rats, which was prevented by the agents that diminish TGF- levels (39–40), suggesting an involvement of TGF- in apoptosis induction. Similarly, TGF- neutralizing antibody protected tubular epithelial cells from apoptosis in both cultured kidney NRK-52E cells and a rat model of unilateral ureteral obstruction, in which apoptosis was accompanied by an increase in TGF- (41). TGF- was hence very likely directly implicated in the CsA-induced apoptosis in the present study. However, mechanisms by which TGF- exerts its apoptotic effect are still poorly understood. For example, it is also possible that reduced apoptosis after treatment with 1D11 was partially or largely an indirect response to reduction in vascular wall fibrosis and improved tissue oxygenation. For future studies, it will be important to define the basis of cytoprotection afforded by TGF- neutralization and the apoptotic pathway induced by TGF- and to ascertain which isoform(s) of TGF- cause apoptosis in the evolution of renal fibrosis.
In summary, a murine monoclonal antibody, 1D11, that neutralizes all isoforms of TGF- has been shown to provide therapeutic benefit, as measured by functional and histologic endpoints, in a mouse model of chronic CsA nephropathy. Specifically, the administration of this antibody can ameliorate the progression of CsA-induced tubular damage and tubulointerstitial fibrosis. The beneficial effects are attributed largely to a reduction of the overexpressed TGF-1 and TGF-2 production as well as the prevention of tubular apoptosis. In addition, 1D11 restores the balance between eNOS and iNOS and subsequently improves tissue oxygenation in the kidneys.
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 Y. Motomura, H. Kanbayashi, W. I. Khan, Y. Deng, P. A. Blennerhassett, P. J. Margetts, J. Gauldie, K. Egashira, and S. M. Collins The gene transfer of soluble VEGF type I receptor (Flt-1) attenuates peritoneal fibrosis formation in mice but not soluble TGF-{beta} type II receptor gene transfer Am J Physiol Gastrointest Liver Physiol, January 1, 2005; 288(1): G143 - G150. [Abstract] [Full Text] [PDF]
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