Critical involvement of Th1-related cytokines in renal injuries induced by ischemia and reperfusion

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Abstract

Renal ischemia and reperfusion injury (IRI) is considered an inflammatory syndrome. To move forward in its pathogenesis, we exploited the role of several cytokines on renal damages triggered by IRI. Specifically to evaluate the role of Th1 immune profile in this system, IL-12, IFN-γ, and IFN-γ/IL-12 deficient (KO) mice on C57BL/6 background and their controls were subjected to IRI. In each group, blood and kidney samples were harvested. Renal function was evaluated by serum creatinine and renal morphometric analyses. Gene expression of IL-6 and HO-1 were also investigated by Q-PCR. IFN-γ KO animals presented the highest impairment in renal function compared to controls. Conversely, IL-12 KO animals were absolutely protected and, in a lesser extent, IFN-γ/IL-12 KO double knockout was also protected from IRI. Gene expression analyses showed higher expression of HO-1, a cytoprotective gene, and IL-6, a pro-inflammatory cytokine, in IFN-γ deficient animals subjected to IRI. Our results confirm that Th1 related cytokines such as IL-12 and IFN-γ are critically involved in renal ischemia and reperfusion injury.

Introduction

Acute renal failure (ARF) is an inflammatory consequence of a sudden and transient interruption of blood flow in the kidneys [1], [2] and remains as a common issue in the clinical practice, lacking satisfactory therapeutic options. Despite diagnostic and treatment advances, the mortality and morbidity rates remain unchanged for the last decades [3]. Ischemia reperfusion injury (IRI) has an important role in its pathogenesis, in native organs and in transplanted grafts [4]. ARF pathogenesis is multifaceted, as the sudden interruption of blood flow to the kidneys unleashes inflammation and innate immune responses such as production of reactive oxygen species, complement and neutrophils activation, up-regulation of adhesion molecules and tubular cell death. Nevertheless it is known that more specific immune response plays a crucial role in this process [5].

In the last years, leucocytes have been shown to have a role in the pathogenesis of IRI, and at this time, many studies focused on neutrophils infiltration [6]. The role of lymphocytes emerged in IRI after the observation that treatment with anti-ICAM-1 elicits protection in renal graft human receptors. It was observed that delayed graft function (DGF) rates were smaller than those from untreated patients receiving the contra-lateral organ [7]. In an experimental study, mice that received anti-ICAM-1 at the moment of renal ischemia presented a protection, with decreased functional damage of the organ [8], showing that both leukocytes and adhesion molecules play critical roles in ARF. From these studies to now, many groups have reported the participation of TCD4+ and TCD8+ lymphocytes in renal and other organs subjected to IRI [9], [10], [11]. One of these reports focused on nu/nu mice that had been adoptively transferred with an enriched CD4+ T cell population obtained from mice that were deficient in either CD28 or IFN-γ. They found that both CD28 and IFN-γ are important components of CD4+ T cell-mediated renal injury following IRI, as adoptively transferred mice had no increase in serum creatinine levels compared with nontransferred nu/nu mice [11].

Experiments with B cell deficient mice reported the involvement of these cells on the pathogenesis of IRI, since they presented an improvement of renal function and less tubular injury when subjected to IRI, compared to wild type [12]. However, it was observed that RAG-1 deficient mice, which lack B and T cells were not protected from IRI [13]. On the other hand, Yokota-Ikeda et al. tested the effect of depletion of T cells in an experimental IRI model using thymectomized mice or T cell-depleting monoclonal antibodies (GK1.5, 2.43, 30.H12). Animals receiving GK1.5 and 2.43 showed marked depletion but no renal function protection, measured by serum creatinine, after IRI. However, when treated with all antibodies, animals presented significant protection [14]. So, the possible mechanisms for the participation of lymphocytes on IRI, leading to tissue injury remain unknown, once they seem to be dependent not only on the cell types involved but also on the interactions between cells and cytokines into the lesion site.

Classically, CD4+ T cells can be functionally differentiated into subsets of helper T cells, distinguishable by their patterns of cytokine production: the T helper 1 (Th1) phenotype is best characterized by the production of IFN-γ and tumor necrosis factor β (TNF-β), and the Th2 phenotype by the production of interleukin-4 (IL-4), IL-5 and IL-13 [15]. Th1 cell differentiation is regulated by the cytokines and by transcription factors such as IL-12, IL-23, IL-27, IFN-γ, STAT4 and t-bet, whereas IL-4, IL-21, STAT6, Gata-3, c-maf and NFATs (nuclear factors of activated T cells) regulate Th 2 cell segregation [16], [17]. Recently, it was demonstrated that the Th1/Th2 balance plays an important role in the pathogenesis of renal IRI once STAT6-deficient mice, with a defective Th2 phenotype, have presented enhanced renal dysfunction after IRI, whereas STAT4-deficient mice, with a defective Th1 phenotype, have mildly improved renal function in an experimental model of renal ischemia using Balb/c mice. [18].

One of the tissue reactions to aggressive stimuli is an increase of the expression of molecules with cytoprotective properties such as the anti-apoptotic and anti-oxidant proteins or enzymes like the Heme oxygenases (HO) [19]. HO-1 and HO-2 genes encode functional stress-responsive enzymes that act during inflammatory reactions as the rate-limiting step in the catabolism of heme, yielding equimolar amounts of iron (Fe), biliverdin, and the gas carbon monoxide (CO) [20]. HO-2 is constitutively expressed in most cell types while HO-1 is not. Expression of HO-1 regulates inflammatory and immune responses, such as those involved in the rejection of transplanted organs. In at least one of the studies involving transplanted organs, induction of HO-1 in the organ donor was shown to be sufficient to prevent chronic allograft dysfunction that develops months after transplantation [21]. Besides, illustrating the importance of IRI in the pathogenesis of chronic graft dysfunction, this observation also suggests that inhibition of IRI by induction of HO-1 expression can prevent chronic graft dysfunction [21]. The mechanisms by which HO-1 affords this protective effect are not clear and remain the subject of intensive research.

Previous work of our group showed that IL-4 deficient mice on C57BL/6 background presented significant increase in serum urea levels and in tubular injury and impairment in cell regeneration post IRI, compared to control animals. The IRI in IL-4 deficient mice was accompanied by higher levels of HO-1, t-bet and later up-regulation of MCP-1 in kidneys These data suggest strongly both functional and morphologic renal damage in mice lacking Th2 response, while the IL-12-deficient mice were not affected, which suggests a major role for pro-inflammatory Th1 cytokines in the immune response after ischemia and reperfusion injury [22]. Here, we hypothesize that a polarized Th1 activation response may negatively influence the renal IRI through an IL-12/IFN-γ pathway balance. To address this question, IL-12, IFN-γ, and IFN-γ/IL-12 deficient mice, in a C57/BL6 background representing a defective Th1 response were subjected to renal IRI.

Section snippets

Animals

Isogenic male IL-12, IFN-γ deficient and IFN-γ/IL-12 double deficient C57BL/6 mice (H2-IA b), aged 5–9 weeks (25–28 g), were obtained from the University of São Paulo (USP) Animal Facility, São Paulo, Brazil. All animals were housed in individual and standard cages and had free access to water and food. All procedures were previously reviewed and approved by the internal Ethical Committee of the Institution. Wild type C57BL/6 mice, aged and sex-matched, were used as control animals and were

Specific cytokines of the Th1 pathway are involved in the renal ischemia and reperfusion injury

The T cell effectors' mechanisms involved in tissue damage after renal IRI are poorly understood. As shown in a previous work, IL-4 knockout mice had the worse renal outcome after IRI [22]. So, we hypothesize that a polarized Th1 activation response may negatively influence the renal IRI through an IL-12/IFN-γ pathway balance.

Indeed, IL-12 and IFN-γ/Il-12 deficient mice presented better renal function after renal ischemia and reperfusion injury compared to control wild type mice. IL-12

Discussion

In the last years, leucocytes have been studied as mediators of ischemic injury, with focus on neutrophils infiltration [6] and on lymphocytes after the observation that their depletion or the inhibition of cell adhesion conferred protection to renal ischemia [7], [8]. The role of TCD4+ and TCD8+ cells [9], [10], [11] and B cells [12] in IRI is substantial in literature. However, the effector mechanisms are elusive.

Although the Th1/Th2 functional subsets of lymphocytes theory are under strong

Acknowledgements

The authors thank to Paulo Albe Bernardo for preparing the histology slides and Dr. Silvia Maria Gomes Massironi for genotyping the knockout animals. The authors are thankful for FAPESP and CNPq financial support.

References (34)

  • H. Rabb et al.

    Pathophysiological role of T lymphocytes in renal ischemia reperfusion injury in mice

    Am J Physiol

    (2000)
  • R.M. Zwacka et al.

    CD4(+) T-lymphocytes mediate ischemia/reperfusion-induced inflammatory responses in mouse liver

    J Clin Invest

    (1997)
  • M.J. Burne et al.

    Identification of the CD4+ T cell as a major pathogenic factor in ischemic acute renal failure

    Clin Invest

    (2001)
  • M.J. Burne-Taney et al.

    B Cell deficiency confers protection from renal ischemia reperfusion injury

    J Immunol

    (2003)
  • N. Yokota et al.

    Protective effect of T cell depletion in murine renal ischemia–reperfusion injury

    Transplantation

    (2002)
  • A.K. Abbas et al.

    Functional diversity of helper T lymphocytes

    Nature

    (1996)
  • D. Agnello et al.

    Cytokines and transcription factors that regulate T helper cell differentiation: new players and new insights

    J Clin Immunol

    (2003)
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