Immunopharmacology and Inflammation
Mycophenolate mofetil inhibits macrophage infiltration and kidney fibrosis in long-term ischemia–reperfusion injury

https://doi.org/10.1016/j.ejphar.2012.05.001Get rights and content

Abstract

Immunosuppressants have been widely used in renal transplantation, in which ischemia–reperfusion injury is inevitable. Mycophenolate mofetil (MMF) is a relative novel immunosuppressant and also attenuates ischemia–reperfusion injury in the acute phase, but its long-term effects are still obscure. Unilateral renal ischemia–reperfusion injury model was established in Sprague–Dawley rats and 30 mg/kg/day MMF or natural saline was administered a day before the surgery. Renal function was monitored, and histological changes and fibrosis in the kidney were evaluated in both short and long terms. TGF-β1 secretion and MCP-1 expression were determined by immunohistochemistry and real-time PCR respectively. The infiltration of macrophages in renal tissues was also assessed by fluorescence activated cell sorting (FACS). MMF treatment significantly improved renal function in ischemia–reperfusion injury rats in the short and long-term and also effectively prevented interstitial fibrosis. TGF-β1 secretion and MCP-1 expression in the renal tissue of MMF-treated rats were much lower than those in natural saline-treated rats, with much less macrophage infiltration as well. MMF treatment effectively prevented the deterioration of renal function and interstitial fibrosis in ischemia–reperfusion injury rats, which may be associated with decreased TGF-β1, MCP-1 and macrophages. These results provide evidence for the choice of MMF in the renal transplant patients not only for acute renal injury but also for long-term survival of renal allograft.

Introduction

Ischemia–reperfusion injury is a common course in renal transplantation resulting in delayed graft function, acute and chronic rejection, as well as pathological damage (O'Valle et al., 2009, Torras et al., 2002). The progression of ischemia–reperfusion injury is characterized by decreased local oxygen, cellular metabolism impairment with lower level of ATP and glucose, inflammation, free radical production, apoptosis and necrosis (Eickelberg et al., 2002, Gobe et al., 1999, Morimoto, 1998). Depending on the severity of ischemia and the intensity of immune and inflammatory responses, the damage may result in temporary function compromise followed by subsequent recovering; or graft rejection and permanent impairment of renal function due to renal cell death, chronic inflammation and fibrosis (Lieberthal and Levine, 1996, Saikumar et al., 1998, Sutton and Molitoris, 1998).

Inflammatory cells attracted by injured renal endothelial cells play an important role in the pathogenesis of ischemia–reperfusion injury through direct cytotoxicity, secretion of soluble factors and regulation of immune responses in the acute phase, or promoting tissue fibrosis and remodeling by production of growth factors in the long term (Li et al., 2007, Shirali and Goldstein, 2008). It has been reported that TGF-β1 is an important factor involved in the progression of fibrosis (Ahn et al., 2011, Hassane et al., 2010, Yeh et al., 2010) and MCP-1 is essential in the recruitment of macrophages which is associated with glomerulosclerosis and fibrosis (Prasad et al., 2007, Schlondorff et al., 1997). Considering the critical role of immune response in the pathogenesis of renal ischemia–reperfusion injury, immunosuppressants had been employed and shown to be beneficial in many attempts (Lee et al., 2008, Wang et al., 2008, Yeboah et al., 2008). Among these agents, mycophenolate mofetil (MMF), a noncompetitive reversible inhibitor of de novo synthesis of purines, shows excellent results of immunosuppression and ischemia–reperfusion injury attenuation by suppressing the proliferation of T and B lymphocytes and monocytes, and prolonging transplant survival in animal models and humans (Allison and Eugui, 1996, Lui et al., 2001, Ventura et al., 2002). It has also been demonstrated that MMF attenuates inflammatory conditions, such as rheumatoid arthritis, as well as reduces cellular infiltration within the tubulointerstitium with decreased renal damage in the remnant kidney (Sigmon and Beierwaltes, 1998).

Although the therapeutic role of MMF on acute renal dysfunction caused by ischemia–reperfusion injury has been established, its effect on renal function in the long-term of ischemia–reperfusion injury is still obscure. It has been reported that MMF can reduce compensatory hypertrophy, cellular proliferation, myofibroblast infiltration and collagen III deposition in rat remnant kidneys (Badid et al., 2000). This leads to a hypothesis to test whether MMF can suppress chronic inflammation and fibrosis in the long-term of renal ischemia–reperfusion injury. In the present study, MMF was delivered to rats with unilateral renal ischemia–reperfusion injury and renal function was determined in both acute and late phases. Tissue fibrosis was analyzed, and chemokine expression and lymphocyte infiltration were further determined to disclose the possible mechanism underlying the effect of MMF on renal function in the long-term post ischemia–reperfusion injury.

Section snippets

Renal ischemia–reperfusion injury model

All experimental protocols for animal studies were approved by the Animal Ethics Committee of Fudan University. Renal ischemia–reperfusion injury model was established in Sprague–Dawley rats. Briefly, male rats (200–220 g) were anesthetized with ketamine (100 mg/kg, intraperitoneally) and underwent midline abdominal incision, the left renal pedicle was occluded for 45 min, followed by immediate contralateral nephrectomy. MMF was prepared in natural saline and was given by intragastric

Dose response of MMF on renal function

As shown in Fig. 1, the level of blood urea nitrogen in 30 and 40 mg/kg MMF treated groups was similar, but statistically significantly lower than that in the natural saline group, whereas there was no significant decrease in the group treated with 10 or 20 mg/kg MMF. In addition, 10 mg/kg MMF significantly prevented the elevation of serum creatinine and UA, and a dose dependent manner was shown in a range of 10 to 30 mg/kg MMF. However, there was no further change in serum creatinine and UA in the

Discussion

Ischemia reperfusion injury is inevitable in renal transplantation and is one of key risk factors for the development of acute and chronic allograft dysfunction. Ischemia–reperfusion injury initiates immune and inflammatory responses that associated with cytokine production and leukocyte infiltration, and subsequently cause damage to the renal allograft (Rabb et al., 2000, Sutton and Molitoris, 1998). This study using a rat renal ischemia–reperfusion injury model demonstrated completed dose and

Conclusion

In conclusion, MMF treatment effectively prevented the deterioration of renal function and the formation of fibrosis in ischemia–reperfusion injury rats, which may be contributed by decreased TGF-β1 and MCP-1 expression as well as macrophage infiltration. These results demonstrated that MMF was a promising immunosuppressant for improving renal function not only at the acute phase, but also in the long-term, which will be eventually beneficial to the survival of transplant organs.

References (36)

  • M.M. Yeboah et al.

    Cholinergic agonists attenuate renal ischemia–reperfusion injury in rats

    Kidney Int.

    (2008)
  • Y.C. Yeh et al.

    Transforming growth factor-{beta}1 induces Smad3-dependent {beta}1 integrin gene expression in epithelial-to-mesenchymal transition during chronic tubulointerstitial fibrosis

    Am. J. Pathol.

    (2010)
  • J.Y. Ahn et al.

    The inhibitory effect of ginsan on TGF-beta mediated fibrotic process

    J. Cell. Physiol.

    (2011)
  • A.C. Allison et al.

    Purine metabolism and immunosuppressive effects of mycophenolate mofetil (MMF)

    Clin. Transplant.

    (1996)
  • O. Eickelberg et al.

    Functional activation of heat shock factor and hypoxia-inducible factor in the kidney

    J. Am. Soc. Nephrol. JASN

    (2002)
  • S. Hassane et al.

    Elevated TGFbeta-Smad signalling in experimental Pkd1 models and human patients with polycystic kidney disease

    J. Pathol.

    (2010)
  • D.A. Hesselink et al.

    The pharmacogenetics of calcineurin inhibitor-related nephrotoxicity

    Ther. Drug Monit.

    (2010)
  • S. Jain et al.

    Tacrolimus has less fibrogenic potential than cyclosporin A in a model of renal ischaemia–reperfusion injury

    Br. J. Surg.

    (2000)
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    There is no conflict of interest for each author.

    1

    The authors contribute equally to the article.

    2

    Research design and performing.

    3

    Research performing.

    4

    Research design and supervising.

    5

    Animal experiment.

    6

    Experiment supervising.

    6

    Immunohistochemistry.

    8

    Animal experiment.

    9

    Animal experiment.

    7

    Pathology.

    10

    Research supervising.

    12

    Pathology, research supervising and manuscript revising.

    13

    Research and statistic supervising.

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