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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
, IL-18, IL-6, and Neutrophil Infiltration in the KidneyDepartment of Medicine, University of Colorado Health Sciences Center, Denver, Colorado (S.F., E.C.L., L.R., T.S.H., H.S., D.-J.O., L.L., C.L.K., C.A.D., C.L.E.); and Department of Pathology, University Hospital Dubrava, Zagreb, Croatia (D.L.)
Received January 10, 2007; accepted March 29, 2007.
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Abstract |
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, IL-18, IL-6, and neutrophil recruitment. We sought to determine the role of the cytokines IL-1, IL-18, and IL-6 and neutrophil recruitment in cisplatin-induced ARF. We first examined IL-1; renal IL-1 increased nearly 2-fold in cisplatin-induced ARF and was reduced in the caspase-1–/– mice. However, inhibition with IL-1 receptor antagonist (IL-1Ra) did not attenuate cisplatin-induced ARF. Renal IL-18 increased 2.5-fold; however, methods to inhibit IL-18 using IL-18 antiserum and transgenic mice that overproduce IL-18-binding protein (a natural inhibitor of IL-18) did not protect. Renal IL-6 increased 3-fold; however, IL-6-deficient (IL-6–/–) mice still developed cisplatin-induced ARF. We next examined neutrophils; blood neutrophils increased dramatically after cisplatin injection; however, prevention of peripheral neutrophilia and renal neutrophil infiltration with the neutrophil-depleting antibody RB6-8C5 did not protect against cisplatin-induced ARF. In summary, our data demonstrated that cisplatin-induced ARF is associated with increases in the cytokines IL-1, IL-18, and IL-6 and neutrophil infiltration in the kidney. However, inhibition of IL-1, IL-18, and IL-6 or neutrophil infiltration in the kidney is not sufficient to prevent cisplatin-induced ARF.
). A known complication of cisplatin administration is ARF (Safirstein, 2007). The nephrotoxic effect of cisplatin is cumulative and dose-dependent and often necessitates dose reduction or withdrawal. Despite this toxicity, cisplatin remains one of the most commonly used chemotherapy drugs due to its therapeutic efficacy (Schrier, 2002). Therefore, an understanding of the pathogenesis of cisplatin-induced ARF is important for the development of adjunctive therapies to prevent this complication.
Recently, the role of inflammation in ARF has been increasingly appreciated with involvement of leukocytes, adhesion molecules, chemokines, and cytokines (Safirstein, 2007). We have demonstrated that the activity of proinflammatory caspase-1 is increased in the kidney in cisplatin-induced ARF and that caspase-1–/– mice have reduced renal dysfunction, tubular necrosis, and neutrophil infiltration in the kidney (Faubel et al., 2004). Caspase-1 converts the proforms of the proinflammatory cytokines IL-1 and IL-18 to their mature (active) forms (Kuida et al., 1995; Li et al., 1995). Caspase-1–/– mice have decreased IL-6 production (Kuida et al., 1995). Increased IL-1 in the kidney in cisplatin-induced ARF has been documented (Liu et al., 2006). However, it is unknown whether there is increased IL-18 and IL-6 in the kidney in cisplatin-induced ARF. In addition, the effects of IL-1, IL-18, and IL-6 inhibition on cisplatin-induced ARF are also unknown.
Inhibition of proinflammatory mediators in cisplatin-induced ARF is consistently associated with a reduction in renal neutrophils (Kelly et al., 1999; Deng et al., 2001; Ramesh and Reeves, 2002, 2003). A protective effect of neutrophil depletion in cisplatin-induced ARF would demonstrate a cause and effect relationship between neutrophils and cisplatin-induced ARF. However, the effect of neutrophil depletion on cisplatin-induced ARF is unknown. In the present study, we sought to examine the injurious role of the proinflammatory cytokines IL-1, IL-6, and IL-18 and neutrophils in cisplatin-induced ARF.
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Materials and Methods |
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Mice were maintained on a standard diet, and water was freely available. Mice were housed five per cage under a 12-h light and dark schedule for at least 1 week before cisplatin administration. Six hours before cisplatin administration, food and water were withheld. Cisplatin (Aldrich, Milwaukee, WI) was freshly prepared the day of administration in sterile normal saline at a concentration of 1 mg/ml. Mice were given either 30 mg/kg body weight of cisplatin or vehicle (saline) i.p., after which the mice again had free access to food and water. We have described this model of cisplatin-induced ARF in detail elsewhere (Faubel et al., 2004). In brief, after cisplatin injection, BUN and serum creatinine are normal on day 1 and slightly increased on day 2. On day 3 after cisplatin injection, renal dysfunction, renal neutrophil infiltration, and acute tubular necrosis scores are severe. Under anesthesia with Avertin (2,2,2-tribromoethanol; Aldrich), kidneys were removed and blood samples were collected via cardiac puncture on day 3 after cisplatin administration.
Serum Creatinine Measurement. BUN and serum creatinine were measured using an Astra AutoAnalyzer (Beckman Instruments Inc., Fullerton, CA).
Caspase-1–/– Mice. The caspase-1-deficient mice (Kuida et al., 1995), backcrossed 
8 generations on a C57BL/6 background, were kindly provided by Hiroko Tsutsui of the Department of Immunology and Medical Zoology at Hyogo College of Medicine (Nishinomiya, Japan). Confirmation of caspase-1 deficiency was determined by immunoblotting for the active form of caspase-1 in the spleen and kidneys (Faubel et al., 2004). Age, weight, and gender-matched wild-type C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were used as controls for the caspase-1–/– mice.
IL-18-Binding Protein Transgenic Mice. IL-18-binding protein transgenic (IL-18BP-Tg) mice, which overproduce human IL-18BP, were generated as described previously (Fantuzzi et al., 2003). Genotyping was performed to identify IL-18BP-Tg mice, and wild-type littermates were used as controls. The IL-18BP-Tg mice are well characterized; mRNA for IL-18BP has been identified in the kidney, and the functions of IL-18 in response to endotoxin are effectively inhibited by the high levels of IL-18BP produced in these mice (Fantuzzi et al., 2003). For genotyping, tail DNA was obtained using tissue-DNA-extraction kit XNAT (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. PCR was performed using 2 µl of DNA with 1 µl of 3' primer (20 µM, 5'-TCAGCTGCTCCAGCACCAA-3'), 1 µl of 5' primer (20 µM, 5'-ACACCTGTCTCGCAGACCAC-3'), 10 µl of Red-Extract PCR mix (Sigma-Aldrich), and 5 µl of H2O. Thirty PCR cycles were performed after 3 min at 94°C and included 90 s of denaturation at 94°C, 60 s of annealing at 65°C, and 90 s of extension at 72°C. PCR products were run on 1.5% agarose gel.
IL-6–/– Mice. The IL-6–/– mice in a C57BL/6 background were purchased from Jackson Laboratories. Age, weight, and gender-matched C57BL/6 wild-type mice (Jackson Laboratories) were used as controls for the caspase-1–/– mice.
Cytokine Inhibition and Administration. Recombinant human IL-1Ra was a kind gift of Dr. Daniel Tracey (Upjohn, Kalamazoo, MI). IL-1Ra (40 mg/kg) or vehicle (0.02% azide in PBS) was injected i.p. before cisplatin injection and then every 8 h until the time of sacrifice. This dose was chosen to ensure adequate inhibition of IL-1 in vivo. For example, 10 mg/kg IL-1Ra blocked shock-like hemodynamic parameters and reduced circulating IL-1 and TNF-
levels in a model of Gram-positive sepsis (Aiura et al., 1993), and a similar dose reduced neutrophil infiltration after ischemic ARF in mice (Haq et al., 1998). One microgram of recombinant murine IL-1 (Peprotech, Rocky Hill, NJ) or vehicle (saline) was administered i.p. 24 h before or 24 h after cisplatin injection.
IL-18 antiserum was obtained from a New Zealand rabbit immunized by intradermal injection of murine recombinant IL-18 in the presence of Hunter's Titermax adjuvant (Fantuzzi et al., 1999). Three hundred microliters of IL-18 antiserum or vehicle (normal rabbit serum; Santa Cruz Biotechnology, Santa Cruz, CA) was administered i.p. before cisplatin injection. IL-18 antiserum has been used in mice in vivo to block endogenous IL-18 (Fantuzzi et al., 1999), and this dose has been shown to be effective in ischemic ARF (Melnikov et al., 2001, 2002).
Neutrophil Depletion. Rat IgG2b monoclonal antibody RB6-8C5 was used to deplete neutrophils. This antibody is commonly used to study the effects of neutrophil depletion (Wipke and Allen, 2001). Mice were injected i.p. with 0.2 mg of RB6-8C5 (BD PharMingen, San Diego, CA) or vehicle (normal rat IgG; Sigma-Aldrich) on days –1, 0, +1, and +2 relative to cisplatin injection (day 0 being the day of cisplatin injection). This dose has been shown to be effective at depleting both peripheral and renal neutrophils in ischemic ARF (Melnikov et al., 2002).
Peripheral Blood Neutrophil Determination. The percentage of peripheral neutrophils were determined at baseline and in cisplatin-treated and vehicle-treated mice on days 1, 2, and 3 after injection. Peripheral blood smears were immediately prepared from one to two drops of whole blood obtained from the tail vein. Smears were air-dried for approximately 10 min at room temperature and fixed with 100% methanol. Smears were stained with Wright-Giemsa using an automated slide stainer. Slides were read by experienced clinic laboratory technicians in a blinded fashion and reported as percentage of total leukocytes. A sufficient number of fields were examined so that at least 100 leukocytes were counted.
Histological Examination. Paraformaldehyde (4%)-fixed and paraffin-embedded kidneys were sectioned at 4 µm and stained with periodic acid-Schiff (PAS) by standard methods. All histological examinations were performed by the renal pathologist in a blinded fashion. Histological changes due to ATN were evaluated in the outer stripe of the outer medulla on PAS-stained tissue and were quantified by counting the percentage of tubules that displayed cell necrosis, loss of brush border, cast formation, and tubule dilatation as follows: 0 = none, 1 =<10%, 2 = 10–25%, 3 = 26–45%, 4 = 46–75%, and 5 =>75%. At least 10 fields (200x) were reviewed for each slide. Neutrophil infiltration was quantitatively assessed on PAS-stained tissue by the renal pathologist by counting the number of neutrophils per high powered field (400x). At least 10 fields were counted in the outer stripe of the outer medulla for each slide.
Electrochemilumenescence Assay for IL-1, IL-18, and IL-1. The electrochemiluminescence (ECL) assay for IL-1, IL-18, and IL-1 in whole-kidney homogenates was performed as described previously in detail (Fantuzzi et al., 1999; Wang et al., 2005). The ECL assay detects both pro- and mature forms of IL-1, IL-18, and IL-1.
IL-6, IL-18, and IFN-
ELISA. The ELISA assay for IL-6 in whole-kidney homogenates was performed using a Quantikine ELISA kit for murine IL-6 (catalog number M6000B; R&D Systems, Minneapolis, MN). The detection limit for this assay was 1.6 pg/ml. IL-18 was measured in serum using a mouse IL-18 ELISA kit (Medical and Biological Laboratories, Nagoya, Japan) according to manufacturer's directions. The detection limit for this assay is 25 pg/ml. IFN- was measured in whole-kidney homogenates using a mouse ELISA set (BD Biosciences, San Diego, CA) according to manufacturer's directions.
Statistical Analysis. All values are expressed as mean ± S.E. For single comparisons, normally distributed data were evaluated using unpaired, two-tailed Student's t tests, and non-normally distributed data were analyzed by the nonparametric unpaired Mann-Whitney test. Multiple group comparisons were performed using analysis of variance with post test according to Newman-Keuls. A P value of <0.05 was considered statistically significant.
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in Cisplatin-Induced ARF. Renal IL-1 was determined on day 3 after cisplatin-administration in vehicle-treated wild-type, cisplatin-treated wild-type, and cisplatin-treated caspase-1–/– mice. IL-1 was significantly increased in cisplatin-treated wild type compared with vehicle-treated wild type and was significantly reduced in the cisplatin-treated caspase-1–/– mice (Fig. 1A). We have previously reported that renal IL-1 increases on day 3 after cisplatin injection and is reduced in the caspase-1–/– mice (Faubel et al., 2004).
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in cisplatin-induced ARF, IL-1 receptor antagonist IL-1Ra (40 mg/kg) or vehicle (0.02% azide in PBS) was administered every 8 h i.p. beginning at the time of cisplatin injection. IL-1Ra did not protect against cisplatin-induced ARF, as determined by serum creatinine (Fig. 1B) and BUN (Fig. 1C). IL-1Ra did not protect against cisplatin-induced ARF, as determined by ATN score. The ATN score was 3.2 ± 0.7 in cisplatin-treated mice administered vehicle compared with 3.0 ± 0.3 in cisplatin-treated mice administered IL-1Ra (P > 0.05, n = 6). To determine that the IL-1Ra was having an in vivo effect, we measured IL-6 in vehicle-treated and IL-1Ra-treated mice. IL-6 was significantly decreased after IL-1Ra treatment. IL-6 (picogram/milligram) was 10.3 ± 1.5 in cisplatin-treated mice administered vehicle compared with 5.7 ± 0.9 in cisplatin-treated mice administered IL-1Ra (P = 0.03, n = 5).
To further examine the role of IL-1 in cisplatin-induced ARF, 1 µg of recombinant murine IL-1 or vehicle was administered i.p. either 24 h before cisplatin injection or 24 h after cisplatin injection. Administration of IL-1 did not exacerbate cisplatin-induced ARF. Serum creatinine on day 3 was 1.9 ± 0.2 in mice administered vehicle and 1.7 ± 0.2 in mice administered IL-1 24 h before cisplatin injection (P > 0.05, n = 15). Serum creatinine on day 3 was 1.8 ± 0.2 in mice administered vehicle and 1.7 ± 0.3 in mice administered IL-1 24 h after cisplatin injection (P > 0.05, n = 10).
Role of IL-18 in Cisplatin-Induced ARF. Renal IL-18 was determined on day 3 in vehicle-treated wild-type, cisplatin-treated wild-type, and cisplatin-treated caspase-1–/– mice. IL-18 was significantly increased on day 3 in cisplatin-treated wild type compared with vehicle-treated wild type and was significantly reduced in the cisplatin-treated caspase-1–/– mice (Fig. 2A).
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To examine whether IL-18 was adequately inhibited by a single dose of IL-18 antiserum, we determined serum levels of IL-18. Serum IL-18 significantly increased on day 3 after cisplatin injection. Treatment with IL-18 antiserum before cisplatin administration resulted in undetectable levels of serum IL-18 on day 3 after cisplatin administration. Serum IL-18 (picogram/milliliter) was 150 ± 20 in controls (administered rabbit serum and saline), 295 ± 25 in cisplatin-treated mice administered vehicle, and undetectable in cisplatin-treated mice administered IL-18 antiserum (n = 4 per group). Although IL-18 antiserum was given as a one-time dose, these data indicated that IL-18 was inhibited until the time of sacrifice.
To confirm data obtained with the use of IL-18 antiserum, mice that are transgenic for human IL-18BP were studied. IL-18BP is a naturally occurring inhibitor of IL-18. Transgenic status was determined by PCR for all mice studied. These mice have been well characterized; IL-18BP mRNA has been identified in the kidneys of these mice (Fantuzzi et al., 2003). IL-18BP-Tg mice were not protected against cisplatin-induced ARF (Fig. 2D).
To confirm the suppression of IL-18 activity in the kidney of the IL-18BP-Tg mice, IFN- (picogram/milligram) was determined in whole-kidney homogenates of wild-type and IL-18BP-Tg mice. IFN- was examined because IL-18, formerly known as IFN--inducing factor, stimulates IFN- production (Gu et al., 1997). IFN- was 780 ± 69 in wild type and 55 ± 20 in IL-18BP-Tg kidneys (P < 0.01, n = 5–6).
Effect of Combination Therapy with IL-1Ra- and IL-18-Neutralizing Antiserum in Cisplatin-Induced ARF. Combination therapy with both IL-1Ra (40 mg/kg i.p. every 8 h after cisplatin administration) and IL-18 antiserum (300 µl on day 2 after cisplatin administration) or the vehicles (0.02% azide in PBS and normal rabbit serum) was administered. Combination therapy did not significantly protect against cisplatin-induced ARF. Serum creatinine (milligram/deciliter) was 2.2 ± 0.3 in cisplatin-treated mice administered vehicle compared with 2.2 ± 0.2 in cisplatin-treated mice administered combination therapy of IL-1Ra and IL-18 antiserum (P > 0.05, n = 6). BUN (milligram/deciliter) was 214 ± 16 in cisplatin-treated mice administered vehicle compared with 180 ± 26 in cisplatin-treated mice administered combination therapy of IL-1Ra and IL-18 antiserum (P > 0.05, n = 6).
Role of IL-6 in Cisplatin-Induced ARF. Renal IL-6 was determined on day 3 in vehicle-treated wild-type, cisplatin-treated wild-type, and cisplatin-treated caspase-1–/– mice. IL-6 was significantly increased on day 3 in cisplatin-treated wild type compared with vehicle-treated wild type and was significantly reduced in the cisplatin-treated caspase-1–/– mice (Fig. 3A). There was no significant difference in IL-6 levels in the kidney between control wild-type and control caspase-1–/– mice. IL-6 (picogram/milligram) in kidney homogenates was 2.5 ± 0.3 in wild-type mice and 3.4 ± 0.2 in caspase-1–/– mice (P > 0.05, n = 5).
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To examine the role of IL-6 in cisplatin-induced ARF, IL-6–/– mice were studied. IL-6–/– mice were not protected against cisplatin-induced ARF as determined by serum creatinine (Fig. 3B) and BUN (Fig. 3C). IL-6–/– mice were not protected against cisplatin-induced ARF as determined by the ATN score. The ATN score was 3.4 ± 0.2 in cisplatin-treated wild-type mice compared with 4.0 ± 0.1 in cisplatin-treated IL-6–/– mice (P > 0.05, n = 5).
Role of Neutrophils in Cisplatin-Induced ARF. To examine the role of neutrophils on the development of cisplatin-induced ARF, 0.2 mg of the neutrophil-depleting antibody RB6-8C5 or vehicle was administered i.p. on days –1, 0, +1, and +2 relative to cisplatin injection. The effect of the anti-neutrophil antibody on neutrophils was determined by examining peripheral blood and renal neutrophils.
Peripheral blood smears were obtained at baseline and in vehicle-treated and cisplatin-treated mice. Neutrophils in the blood were increased significantly in cisplatin-treated mice, and treatment with the anti-neutrophil antibody prevented the increase in peripheral neutrophils. (Fig. 4A). Neutrophils (percentage of total peripheral leukocytes) were 13 ± 1 at baseline before cisplatin administration, 22 ± 4 on day 3 in the vehicle-treated controls, 80 ± 4 on day 3 after cisplatin administration (P < 0.001 versus baseline, vehicle-treated, and cisplatin plus anti-neutrophil antibody treatment, n = 5), and 25 ± 2 on day 3 after cisplatin administration in anti-neutrophil antibody-treated mice.
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IL-1 in Cisplatin-Induced ARF. Caspase-1–/– mice are known to be deficient in IL-1 via an unknown mechanism (Kuida et al., 1995; Li et al., 1995). Renal IL-1 was determined on day 3 in vehicle-treated wild-type, cisplatin-treated wild-type, and cisplatin-treated caspase-1–/– mice. IL-1 was markedly increased on day 3 in cisplatin-treated wild-type mice compared with vehicle-treated wild type. In addition, IL- was significantly reduced in the cisplatin-treated caspase-1–/– mice compared with cisplatin-treated wild type on day 3 (Fig. 5).
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(Li et al., 1995) and IL-18 (Gu et al., 1997) and have decreased production of IL-6 after stimulation with lipopolysaccharide (Kuida et al., 1995). Caspase-1–/– mice have been a useful tool to study a variety of proinflammatory conditions, such as lethal endotoxemia (Li et al., 1995, 1997), ischemic brain injury (Liu et al., 1999), chronic colitis (Siegmund et al., 2001), and pneumococcal meningitis (Koedel et al., 2002). In all of these conditions, caspase-1–/– mice have been protected. Caspase-1–/– mice have also been studied in a variety of models of ARF. We have demonstrated that caspase-1–/– mice are protected against ischemic ARF (Melnikov et al., 2001), endotoxemic ARF (Wang et al., 2005), and cisplatin-induced ARF (Faubel et al., 2004). The proinflammatory nature of cisplatin-induced ARF has been well documented (Kelly et al., 1999; Deng et al., 2001; Ramesh and Reeves, 2002). In the present study, we sought to determine the mechanism of protection against cisplatin-induced ARF seen in the caspase-1–/– mice, with regard to the proinflammatory cytokines IL-1, IL-18, and IL-6.
Caspase-1, formerly known as interleukin-1-converting enzyme or ICE, is a potent inflammatory agent via its activation of IL-1. IL-1 has been shown to be a proximal mediator of the inflammatory events associated with infection, sepsis, and ischemia. Therefore, we first examined the role of IL-1 in cisplatin-induced ARF and found that inhibition with IL-1Ra was not protective.
In addition to activation of IL-1, caspase-1 is known to activate the proinflammatory cytokine IL-18 (Gu et al., 1997). IL-18, formerly known as IFN--inducing factor, is involved in a diverse array of functions, including inflammation (Akira, 2000), ischemic tissue injury (Daemen et al., 1999), and T-cell-mediated immunity (Nakanishi et al., 2001; Kinjo et al., 2002). Although not protective against endotoxemic ARF (Wang et al., 2005), we have previously demonstrated that inhibition of IL-18 is protective against ischemic ARF (Melnikov et al., 2001, 2002). However, inhibition of IL-18 using both IL-18 antiserum and IL-18BP-Tg mice did not result in protection against cisplatin-induced ARF.
IL-6 is a well known proinflammatory cytokine. Cisplatin has been demonstrated to increase IL-1, IL-18, and IL-6 levels in peripheral blood mononuclear cells derived from patients with head and neck tumors (Okamoto et al., 2000). Monocytes from caspase-1–/– mice do not produce IL-6 after stimulation with lipopolysaccharide (Kuida et al., 1995). IL-6 was increased in the kidney in cisplatin-induced ARF and decreased in caspase-1–/– mice. However, IL-6–/– mice were not protected against cisplatin-induced ARF.
We next examined neutrophils in cisplatin-induced ARF. Reduced renal neutrophil infiltration has been observed with other anti-inflammatory agents, such as anti-TNF- antibodies, IL-10, and anti-intercellular adhesion molecule-1 antibodies, which protect against cisplatin-induced ARF (Kelly et al., 1999; Deng et al., 2001; Ramesh and Reeves, 2002, 2003). However, the pathogenic role of infiltrating neutrophils in cisplatin-induced ARF has not been specifically addressed. In the present study, renal neutrophils were absent in mice treated with the neutrophil-depleting antibody RB6-8C5, yet renal function and tubular necrosis were not improved. Our data suggest that, although neutrophil recruitment is associated with cisplatin-induced ARF, infiltrating neutrophils are not essential for tubular necrosis and renal failure to occur.
The role of inflammation in ARF has been increasingly appreciated with involvement of leukocytes, adhesion molecules, chemokines, and cytokines (Safirstein, 2007). However, our study demonstrates that the well known proinflammatory cytokines IL-1, IL-18, IL-6, and neutrophils do not play a pathophysiologic role in cisplatin-induced ARF. Thus, there are alternate proinflammatory mechanisms involved in the pathophysiology of cisplatin-induced ARF. In this regard, nonspecific anti-inflammatory strategies have afforded protection against cisplatin-induced ARF. For example, IL-10 inhibited cisplatin-induced increases in mRNA for TNF- and intercellular adhesion molecule-1 in the kidney (Deng et al., 2001). In addition, WY-14,643, a fibrate class of peroxisome proliferator-activated receptor- ligands, suppressed cisplatin-induced up-regulation of cytokines and chemokines, including TNF-, IL-6, IFN-, and monocyte chemoattractant protein-1, prevented neutrophil infiltration, and ameliorated renal dysfunction (Li et al., 2005). Recently, it has been demonstrated that specific inhibition of TNF- using specific antibodies and anti-TNF--deficient mice ameliorates cisplatin-induced renal dysfunction and structural damage, suggesting that TNF- plays a central role in the pathogenesis of cisplatin-induced ARF (Ramesh and Reeves, 2002, 2003).
Inhibition of anti-ICAM-1 protects against cisplatin-induced ARF, suggesting a role of inflammatory cell-endothelial cell interactions in cisplatin-ARF (Kelly et al., 1999). Our data suggest that neutrophils are not mediators of cisplatin-induced ARF. In this regard, a pathophysiologic role for T lymphocytes in murine acute cisplatin nephrotoxicity has recently been demonstrated (Liu et al., 2006). In this study, CD4 T-cell and, to a lesser degree, CD8 T-cell-deficient mice were protected against cisplatin-induced ARF.
IL-1 is synergistic with cisplatin in its ability to kill cells (Nakamura et al., 1991). The original articles that described the caspase-1–/– mice noted that these mice were also deficient in IL-1 (Kuida et al., 1995; Li et al., 1995). Pro-IL-1 is cleaved to its active form by the calcium-dependent cysteine protease, calpain. The reason that caspase-1-deficient mice have less IL-1 remains unknown. IL-1, like IL-1, may be characterized as a proinflammatory cytokine, and it initiates many of the same biological effects as IL-1. However, IL-1 is unique in that it is not secreted from the cell and is rarely found in the extracellular compartment, even after activation. IL-1 exerts its proinflammatory actions intracellularly or at the membrane (Werman et al., 2004). The intracellular location of IL-1 is relevant in regard to cisplatin toxicity because cisplatin is preferentially taken up by the proximal tubule, which is responsible for its preferential renal toxicity. In the present study, IL-1 was found to be markedly increased in the kidney after cisplatin injection and was notably reduced in the caspase-1–/– mice. However, at present, a specific inhibitor for IL-1 is unavailable.
In summary, we sought to determine the mechanism of protection against cisplatin-induced ARF seen in caspase-1–/– mice, with regard to IL-1, IL-18, IL-6, and neutrophils. Our data demonstrated that renal IL-1, IL-18, and IL-6 and neutrophils increase in cisplatin-induced ARF. However, inhibition of IL-1, IL-6, IL-18, or neutrophil infiltration in the kidney is not sufficient to prevent cisplatin-induced ARF.
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ARF, acute renal failure; IL, interleukin; BUN, blood urea nitrogen; IL-1Ra, IL-1 receptor antagonist; IL-18BP, IL-18-binding protein; Tg, transgenic; ELISA, enzyme-linked immunosorbent assay; ATN, acute tubular necrosis; PAS, periodic acid-Schiff; ANAb, anti-neutrophil antibody; PBS, phosphate-buffered saline; caspase-1–/–, caspase-1-deficient; AS, antiserum; ECL, electrochemiluminescence; TNF-, tumor necrosis factor-; IFN-, interferon-; WY-14,643, [4-chloro-6(2,3-xylidino)-2-pyrimidinylthio] acetic acid.
Address correspondence to: Dr. Charles L. Edelstein, Division of Renal Diseases and Hypertension, University of Colorado School of Medicine, Box C281, 4200 E. 9th Ave, Denver, CO 80262. E-mail: charles.edelstein{at}uchsc.edu
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