Infiltration of CD68+ and CD3+ leukocytes was significantly lower in the 4SC-101–treated rats than in the placebo-treated animals (P<0.05) (Fig. 2B–D), whereas there was no difference in the number of infiltrating CD5+ cells (Fig. 2B–D).
The features of acute rejection, particularly cellular infiltration and tubulitis, were more pronounced 5 days after Tx; however, infiltration of monocytes/macrophages already decreased compared with the animals harvested 3 days after Tx (Fig. 2A,B,D,E).
The treatment group receiving 4SC-101 showed a lower mean acute rejection score compared with the placebo-treated animals (P<0.05) (Fig. 2A,D,E). Infiltration of all examined leukocyte subclasses (CD3+, CD5+, and CD68+) was significantly reduced as compared with the placebo-treated animals (P<0.05) (Fig. 2B,D,E). Furthermore, the expression of IL-2, which is the central mediator of lymphocyte proliferation, was significantly reduced 5 days after Tx in the 4SC-101–treated animals compared with the placebo-treated animals (P<0.05) (Fig. 2C).
IL-17 messenger RNA (mRNA) expression was significantly lower in the 4SC-101–treated animals 5 days after Tx compared with the placebo-treated rats (P<0.05) (Fig. 3A). To detect the source of IL-17 production, immunohistochemistry was performed, showing that, mainly, the infiltrating cells were the source of the IL-17 expression (Fig. 3B).
The Effect of 4SC-101 in 5/6 Nephrectomy
To investigate if 4SC-101 has any effect on kidney function and histopathology in a model of impaired renal function that is relevant for Tx injury, 5/6 nephrectomized rats were treated with 20-mg/kg 4SC-101 per day.
Although 4SC-101 treatment did not change the creatinine clearance compared with the placebo-treated group (P>0.05) (see Table 2, SDC,http://links.lww.com/TP/A662) it significantly decreased proteinuria 12 weeks after 5/6 nephrectomy compared with the placebo-treated animals (P<0.05) (Table 1).
Furthermore, the grade of glomerulosclerosis and tubulointerstitial fibrosis was significantly ameliorated in the 4SC-101–treated group versus the placebo-treated animals (both P<0.05) (data not shown). These changes were paralleled by fewer apoptotic tubular cells demonstrated by terminal deoxynucleotide transferase–mediated dUTP nick-end labeling (see Methods, SDC,http://links.lww.com/TP/A662) (P<0.01 vs. placebo, Fig. 4C) and activated caspase-3 staining (P<0.05 vs. placebo, data not shown).
There was a significant infiltration of CD3+, CD5+, and CD68+ leukocytes in the kidney tissue. Infiltration of CD3+ and CD5+ cells was not influenced, but CD68+ cell infiltration was significantly reduced by 4SC-101 compared with the placebo-treated animals (P<0.05 vs. placebo, data not shown).
IL-17 mRNA expression was significantly lower in the 4SC-101–treated group compared with the controls (P<0.05 vs. placebo) (Fig. 5).
The immunomodulatory effects of the novel DHODH inhibitor 4SC-101 was examined in the prevention of acute kidney rejection after renal Tx. We found that 4SC-101 given as a single agent at a dosage of 20 mg/kg per day significantly prolongs survival of rats after kidney Tx.
The applied doses were chosen based on previously demonstrated pharmacodynamic properties of 4SC-101 in the rat. 4SC-101 at a low dose (4 mg/kg) did not influence the survival of rats after renal Tx. Interestingly, a high-dose (60 mg/kg) 4SC-101 proved to have no protective effect either. Moreover, approximately 60% of animals died within 3 days after Tx, suggesting that higher doses of 4SC-101 might have adverse effects.
In the second setting of our Tx experiments, rats were harvested 3 or 5 days after kidney Tx, and histologic features of the grafts were evaluated. 4SC-101 treatment markedly ameliorated the signs of acute rejection such as interstitial/perivascular infiltration and tubulitis. Furthermore, infiltration of CD3+, CD5+, and CD68+ cells was ameliorated. In line with these observations, the expression of IL-2 that drives the cellular proliferation after Tx was also decreased 3 days after Tx in the 4SC-101–treated group.
4SC-101 is a drug that is effective in inhibiting lymphocyte proliferation, as underlined also by our results, but it was also discovered to block IL-17 release of mononuclear cells (8). Therefore, mRNA expression and the site of IL-17 production were assessed in the kidney. Although tubular cells are also capable of producing IL-17 (17), in our study, IL-17 was mainly detected in infiltrating cells. Overall mRNA expression was significantly lower in the 4SC-101–treated animals 5 days after Tx compared with the placebo-treated animals. These results suggest that 4SC-101 might have two hit points after Tx: (1) decreasing overall proliferation of lymphocytes and (2) decreasing the IL-17 production, leading to a sequential immunosuppression.
IL-17 was mostly produced by the infiltrating mononuclear cells in our study. Previous studies have demonstrated that locally produced IL-17 amplifies intragraft inflammation early after Tx and promotes tissue injury by facilitating T-cell recruitment into the graft. Targeting the IL-17 signaling network may decrease injury and improve graft survival. For example, in the heart, IL-17 antagonism significantly improved cardiac allograft survival (18), and IL-17 deficiency resulted in delayed T-cell recruitment into cardiac allografts (19) because of attenuated neutrophil and macrophage infiltration. In the kidney, according to the study of Loong et al. (20), IL-17 was associated with acute rejection, which correlated well with renal IL-17 mRNA expression.
In summary, in line with previous reports on DHODH inhibition and IL-17, 4SC-101 reduced the infiltration of T lymphocytes and macrophages and caused a significant reduction in the intragraft IL-17 levels. These findings indicate that these mechanisms were probably responsible for ameliorated acute rejection after kidney Tx.
In the next step, the impact of 4SC-101 on renal function to elucidate potential nephrotoxic effects was examined in a remnant kidney model. Many of the immunosuppressive drugs have nephrotoxic adverse effects, making them unfavorable after kidney Tx (21). We tested only the most effective dose (20 mg/kg) of 4SC-101 in which it ameliorated acute rejection to exclude potential nephrotoxic effects. 4SC-101 significantly reduced proteinuria and the grade of glomerulosclerosis, IF, and apoptosis of tubular cells, suggesting that 4SC-101 might have protective effects even in chronic renal insufficiency. This is in line with previous observations of our group, where we have shown that, for example, the immunosuppressant calcineurin inhibitor tacrolimus markedly increased the efficacy of angiotensin-converting enzyme inhibitors in reducing proteinuria after 5/6 nephrectomy (22).
4SC-101 decreased the mRNA expression of transforming growth factor (TGF)-β, which is a key fibrogenic cytokine, and its role has been described in several fibrosis-related conditions such as IF and glomerular mesangial matrix deposition (23, 24). By Masson trichrome staining, marked deposition of collagen was demonstrated, which was reduced by 4SC-101 treatment. These results are in well accordance of another previous study of ours showing that the leflunomide derivate FK778 reduced TGF-β mRNA expression in the kidney during chronic allograft injury (25). Moreover, the lymphocyte-specific inhibitor FTY720 ameliorated cyclosporine nephropathy in rats, paralleled by reduced fibrosis and TGF-β expression in the tissue (26).
Various cytokines and chemokines secreted by leukocytes are well-known regulators of fibrosis. Therefore, it is plausible that immunosuppressive agents targeting different effector mechanisms of the immune system might influence the development of tissue fibrosis.
B cells are, for example, implicated in fibrosis, either by producing autoantibodies or by secreting IL-6, a well-known fibroblast growth factor (17). T helper (Th)2-type cytokines are critically involved in the progression of fibrosis; on the other hand, Th1-associated cytokines might have potent antifibrotic activities (27). There is much less known about the role of IL-17 and its major source, the Th17 cells in fibrosis. However, several lines of evidence indicate a profibrotic IL-17 effect. For example, IL-17A–deficient mice have reduced interstitial myocardial fibrosis (28), and the Th17 response is associated with cardiac fibrosis after Tx (29). Moreover, genetic deletion of IL-17 receptor or treatment with the receptor antagonist reduced collagen deposition in experimental models of myocardial and lung fibrosis (30, 31). Because 4SC-101 is a known inhibitor of IL-17 secretion, we hypothesized that, in addition, the antifibrotic effects of 4SC-101 might be associated with a reduced IL-17 levels. Therefore, IL-17 mRNA expression was examined and was found to be significantly lower in the 4SC-101–treated animals. This was paralleled by lower infiltration of CD68+ cells; however, there was no difference in CD3+ and CD5+ cell infiltration when compared with the controls.
These results suggest that IL-17–mediated mechanism might be a part of 4SC-101 protective effects in chronic kidney disease, particularly in renal fibrosis. The fact that we could not detect differences in lymphocyte infiltration might be because of the examined time point, and we speculate that the differences might be more prominent in a later point in time.
For future clinical relevance, it is also important to discuss that, in the high-dose range (60 mg/kg), there were signs of putative 4SC-101 toxicity. Although dose and toxicity ranges can be very different in rodents and humans, normally, these results might possibly indicate that the same might be present in humans as well. However, 4SC-101 (vidofludimus) has already been tested in humans, and after the completion of single- and multiple-dose safety and tolerability phase I studies, 4SC-101 at once-a-day dosages was also tested in three subsequent phase II studies in patients with rheumatoid arthritis and inflammatory bowel diseases. Multiple-dose application showed no toxicity, and both tested clinical doses have been demonstrated to be safe and well tolerated. These clinical studies have a high relevance in the context of our results because they indicate that 4SC-101 could already be safely administered in humans, showing also therapeutic benefits.
In conclusion, the novel DHODH inhibitor 4SC-101 was able to prolong the mean survival time and ameliorate histologic features of acute rejection after kidney Tx in an experimental rat model. Furthermore, 4SC-101 decreased the progression of chronic renal injury by marked inhibition of tissue fibrosis; therefore, it seems to be a promising immunosuppressive drug in Tx medicine. Because 4SC-101 was shown to be safe in human clinical trials, it might be a relevant drug for further investigations in the clinical settings.
MATERIALS AND METHODS
Male Brown-Norway and Lewis rats (weighing 250 to 300 g; Charles River, Sulzfeld, Germany) with free access to standard rat chow and tap water were used. Tx was performed as described previously (10). Kidney Tx was performed in two independent settings. In the first setting, the animals were allowed to survive after Tx. The rats that died within 72 hr after surgery were excluded from the analysis as surgical failure. The animals were studied for 30 days, and at the end of day 30, the surviving rats were killed.
In the second setting of kidney Tx, the rats were harvested 3 or 5 days after Tx, when kidney graft was rapidly removed for further analysis.
The principles of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the German Law on the Protection of Animals were followed.
5/6 nephrectomy was performed in Lewis rats weighing between 250 and 300 g. All operations were carried out under isoflurane anesthesia. A left-flank incision was made, and the left kidney was exposed. The renal artery was temporarily occluded, and the upper and lower thirds of the kidney were ligated and excised. Thus, one third of the mass of the left kidney remained. The muscle and skin incisions were sutured with polypropylene suture. The animals were returned to the vivarium to recover. One week later, a right-flank incision was made, the renal vessels and ureter were tied, and the right kidney was excised. The animals were returned to the cages to recover. The animals were studied for 12 weeks, and at the end of the 12th week, they were killed.
4SC-101 Treatment and Experimental Groups
In the first setting of Tx experiments, rats (n=8 per group) were treated with 4-, 20-, or 60-mg/kg 4SC-101 per day. 4SC-101 and the respective vehicle (placebo) were obtained from 4SC AG (Planegg-Martinsried, Germany). The drug was given to the recipients 2 days before Tx and was administered orally each day. The control rats were treated with placebo. If the rats survived for 30 days, they were killed.
In the second setting of Tx experiments, the following animal groups were studied:
- Placebo 3d (n=7) was treated with vehicle and harvested 3 days after Tx.
- 4SC-101 20 mg/kg 3d (n=5) was treated with 20-mg/kg 4SC-101 and harvested 3 days after Tx.
- Placebo 5d (n=6) was treated with vehicle and harvested 5 days after Tx.
- 4SC-101 20 mg/kg 5d (n=7) was treated with 20-mg/kg 4SC-101 and harvested 5 days after Tx.
Placebo treatment served as control.
In the 5/6 nephrectomy experiments, the rats (n=6 per group) were given 20-mg/kg 4SC-101 everyday after the operation through oral gavage until harvesting. The control rats were treated with placebo.
Paraformaldehyde-fixed, paraffin-embedded tissue sections were stained with periodic acid–Schiff reagent. Histologic analysis was performed using the Banff criteria for acute rejection in a blind fashion (32). To investigate acute rejection, the intensity of perivascular and diffuse interstitial inflammation and tubulitis of samples was scored from 0 to 3 according to the Banff classification (9), and then the scores were added.
To investigate glomerulosclerosis, periodic acid–Schiff stainings were used, and the following scoring was applied: grade 0, no signs; grade 1, mild (affecting 5%–15% of the glomeruli); grade 2, moderate (affecting 16%–50% of the glomeruli); and grade 3 (affecting >50% of the glomeruli).
To investigate collagen deposition, we analyzed Masson trichrome stainings using the following scoring: grade 0, no signs; grade 1, mild (affecting 5%–15% of section); grade 2, moderate (affecting 16%–50% of section); and grade 3 (affecting >50% of section). The number of animals was then presented for each grade for both scorings. Histopathologic analysis was performed in a blind review.
Data are expressed as mean±SEM. Data were tested using the chi-square or Mann-Whitney U test. A global P value of less than 0.05 was considered significant. All tests were performed two tailed. Data were analyzed using SPSS statistical software package (version 13.0; SPSS GmbH, Munich, Germany).
The authors thank Sandra Haderer and Marcel Konhaeuser for their technical assistance.
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Kidney; Acute rejection; Novel dihydroorotate dehydrogenase inhibitor
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