Transplantation/Immunology
The Experimental Agent Pirfenidone Reduces Pro-Fibrotic Gene Expression in a Model of Tacrolimus-Induced Nephrotoxicity

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Background

Tacrolimus nephrotoxicity is thought to contribute to renal allograft dysfunction and subsequent failure, a process that is underpinned by alterations in mRNA expression of genes involved in matrix metabolism. The new anti-fibrotic pirfenidone was tested for its potential to reverse markers of renal dysfunction.

Materials and methods

Rats were salt-depleted before tacrolimus and pirfenidone treatment. Serum creatinine, urinary protein/creatinine ratio, extracellular matrix deposition (ECM), and mRNA expression of genes involved in matrix turnover were assessed.

Results

Tacrolimus reduced TGF-β mRNA expression below control levels and treatment with pirfenidone at all doses did not alter this effect. Likewise, TIMP-1 mRNA expression was depressed by the addition of tacrolimus and pirfenidone caused a further decrease in expression. Collagen III, MMP-2, and MMP-9 expression was unchanged by tacrolimus, but pirfenidone reduced collagen III below control levels. ECM was slight (1–4%) and not significantly different between groups.

Conclusions

These findings suggest that pirfenidone can attenuate the limited fibrotic potential of tacrolimus.

Introduction

The use of calcineurin-inhibitor based immunosuppressants is a contributory factor in chronic renal allograft dysfunction (CRAD), the pathophysiological entity that is the leading cause of allograft loss after the first post-transplant year [1]. Evidence for the nephrotoxicity of cyclosporine is now well established [2], and a number of short- and long-term animal and human studies in the setting of organ allografting have demonstrated the nephrotoxic potential of tacrolimus [3, 4, 5]. The histopathological changes in renal allograft biopsies, consisting of tubular vacuolisation, striped interstitial fibrosis and arteriolar hyalinosis, are associated with both long-term cyclosporine and tacrolimus therapy [6].

The rat salt-depleted model [7] has become a paradigm for the study of chronic calcineurin-inhibitor nephrotoxicity, because it allows examination of the underlying mechanisms without the influence of multiple confounding factors that can confuse the findings of clinical studies. It has elucidated some of the structural, functional [5, 8] and molecular mechanisms [3] of tacrolimus nephrotoxicity, which include alterations in the levels of profibrotic cytokines and changes in extracellular matrix metabolism.

To date, no pharmacological agents have proved effective in the clinical setting of CRAD. Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone; Marnac Inc., Dallas, TX) is a novel anti-fibrotic agent that has demonstrated potential in a number of animal models of fibrotic disease. It can prevent or reverse ECM accumulation in several organs [9, 10, 11], by decreasing the expression of profibrotic cytokines such as TNF-α and TGF-β. The aim of this study was to examine the effects of pirfenidone on physiological and histological markers of tacrolimus-induced renal injury in the rat salt-depleted model. Further, we appraised the underlying molecular mechanisms, looking specifically at the mRNA expression of genes involved in extracellular matrix turnover and fibrosis. We hypothesized that pirfenidone reduces the renal functional, histological, and molecular markers of tacrolimus-induced fibrosis.

Section snippets

Treatment schedule

Male Sprague-Dawley rats (350–500 g), obtained from Harlan (Cambridge, U.K.) were housed and cared for in accordance with the Animals (Scientific Procedures) Act 1986, in cages of three animals in a temperature and light controlled environment, with water ad libitum. The rats were acclimatized for 7 days on a 12:12 h light:dark cycle. They were fed a salt-depleted diet for 7 days (0.05% sodium, Special Diets Services, Witham, Essex, U.K.), before the introduction of a regimen of tacrolimus

Animal weight

Animals in all groups gained weight at an approximately equal rate, and there were no significant differences in weight across groups (data not shown). This suggests that neither tacrolimus nor pirfenidone treatment displayed systemic toxicity.

Serum creatinine

Tacrolimus treatment caused a rise in serum creatinine compared to the low salt-diet treated controls at 21 days (75 versus 55 μmol/l, P = 0.001) and 28 days (76 versus 61 μmol/l, P = 0.01) (see Table 2). Treatment with tacrolimus plus pirfenidone (at all

Discussion

This study has demonstrated that pirfenidone attenuates the creatinine rise seen in a model of chronic tacrolimus toxicity. It has also shown a reduction in both TGF-β and TIMP-1 expression by tacrolimus. Although pirfenidone had no effect on TGF-β, TIMP-1 levels were further reduced by its addition, and collagen III expression was reduced below control levels by high-dose pirfenidone. Neither tacrolimus nor pirfenidone altered the expression of the matrix metalloproteinases 2 and 9. Renal

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    Nicholas Brook is partly funded by The Royal College of Surgeons of Edinburgh Robertson Trust Fellowship.

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