Secondary Logo

Journal Logo

Basic Science Aspects

ROLE OF p38 MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY ON RENAL FAILURE IN THE INFANT RAT AFTER BURN INJURY

Kita, Toshiro; Yamaguchi, Hiroki; Sato, Hiroaki; Kasai, Kentaro; Tanaka, Toshiko; Tanaka, Noriyuki

Author Information
  • Free

Abstract

INTRODUCTION

Many experimental studies support the hypothesis that intestinal barrier damage, leading to enhanced bacterial and/or endotoxin translocation, may play an important role in the development of multiple organ failure after burn injury (1–3). Our group also demonstrated previously that postburn renal failure can be more easily observed in infant rats compared with adult rats because of the weakness of the intestinal barrier of infant versus adult rats (4). A number of investigators have reported the relationship of burn-induced intestinal barrier damage and bacterial translocation (3, 5–8). However, there are no experimental studies that clearly explore the relationship between intestinal barrier damage and remote organ dysfunction. Therefore, we studied the detailed mechanisms found in the development of renal failure after burn-induced intestinal barrier damage.

Donnahoo et al. stated that the p38 MAPK pathway is an important stress-responsive signal molecule pathway in disease states characterized by inflammation and that it is responsible for the production and signal transduction of cytokines (9). Among these families of cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β play an important role in the development of LPS-induced acute renal failure (10–12). MAPK, which controls these cytokines in burn shock, may not only improve intestinal function but also reduce remote renal failure. To elucidate the abovementioned possibilities for MAPK, we examined the effects of FR167653 ] (Fujisawa Pharmaceutical Co. Ltd., Osaka, Japan), a specific inhibitor of p38 MAPK (13–17), in the development of renal failure after burn-induced intestinal barrier damage.

MATERIALS AND METHODS

Animal and animal care

All requests for animals and intended procedures were approved by the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan. Ten-day-old male Wistar pups were obtained from Seiwa Experimental Animal Co. (Oita, Japan) as a dam with a litter of 6–7 pups at 3–5 days of age. The dams and the pups were allowed to acclimate to their new surroundings. The dams were allowed ad libitum intake of water and standard rat chow. The pups were allowed to nurse ad libitum until the 10th day of life. Ten-day-old pups (weight 18–23 g) were used (as infant rats) for the experiment.

Thermal injury procedure

The animals were anesthetized by ether inhalation. The burn group rats were then immersed in a water bath at 95°C for 10 s, resulting in a full-thickness burn of approximately 20% of the total body surface area. FR167653 was diluted with Ringer’s solution (1 mg/mL) and given as a specific inhibitor of p38 MAPK. The animals of the Sham and FR (FR167653 1 mg/100 g s.c.) underwent the same procedure, except for the thermal injury; that is, their backs were immersed in a water bath at 20°C for the same period of time.

All animals were resuscitated with 0.9% saline (5 mL/100 g body weight) solution administered intraperitoneally and received Lepetan (buprenofine 0.2 mg/mL) 0.1 ml/100 g body weight as an analgesic. All animals recovered under an infrared lamp.

Experimental design

Two hundred forty male Wistar infant rats were used. The animals were randomly divided into three sacrificed groups (at 2 h, 6 h, and 24 h after treatment), and each of these groups was divided into the following four subgroups:

  1. 1.
  2. Animals in the sham group (Sham, n = 20) underwent the same treatment but did not receive a thermal injury.
  3. 2.
  4. Animals in the burn group (Burn, n = 20) received a 20% burn wound.
  5. 3.
  6. The burn wound + FR167653 group (Burn + FR, n = 20) received the FR167653 (1 mg/100 g s.c.) treatment before a 30-min burn wound.
  7. 4.
  8. FR167653 group (FR, n = 20) received the FR167653 (1 mg/100 g s.c.) treatment but did not receive a thermal injury.

Tracer experiment

Intestinal barrier damage was estimated by using horseradish peroxidase (HRP) as a tracer. The tracer experiment was assessed 2 h after thermal injury or sham procedure under anesthesia with i.p. pentobarbital (30 mg/kg body weight). The abdominal cavity of the rat was opened, and 2 mg/100 g horseradish peroxidase (HRP; type VI peroxidase; Sigma Chemical Co., St. Louis, MO) was slowly injected into the lumen of the jejunum through the intestinal wall, according to the method of Fujita et al. (18). At 20 min after the HRP injection, the jejunum was isolated, cut into 5-mm-thick slices, and fixed in a mixture of 4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M PBS. The specimens were cut into approximately 30- to 40-μm sections on a microslicer (Dosaka EM Co. Ltd., Osaka, Japan) and inserted into a sample mesh pack (Shiraimatsu & Co. Ltd., Osaka, Japan). Sections were then treated with a solution of 3,3′-diaminobenzidine tetrahydrochloride (DAB) in 0.05 M Tris buffer containing 0.05% H2O2 for 10 min at room temperature, thoroughly washed in 0.05 M Tris buffer, postfixed in 1% osmium tetroxide in 0.1 M PBS for 1 h at 4°C, dehydrated in an ascending ethyl alcohol series, embedded in Quetol 812 on glass slides, and examined by light microscopy (LM).

Bacterial translocation

The testing for bacterial translocation was assessed at 2 h after treatment. Using aseptic technique, through a midline laparotomy incision, spleens, livers, kidneys, and lungs from six rats per experimental group were taken for bacteriologic cultures. Each collected organ was homogenized in 1.0 mL sterile phosphate-buffered saline, and 0.1 mL of the sample and 1:10 dilution were inoculated onto MacConkey agar plates (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) for isolation of enteric bacteria. Therefore, one colony would represent 1 × 10 and 1 × 102 colony-forming units (CFU) per organ, respectively, for each inoculum size. Limits of detection were 10 organisms per organ. After incubation at 37°C overnight, the bacterial colonies were counted, and then the numbers of CFU per organ were calculated. Species identification was done by biotyping, using an automated Gram Negative Identification (GNI) Card (VITEK, Nippon bioMerieux, Co. Ltd., Tokyo, Japan).

Immunohistochemical experiment

Light microscope—

Antibodies: Rabbit polyclonal anti-rat myeloperoxidase (MPO; antibody as marker for neutrophil: Laboratory Vision Co., CA) was used.

Immunostaining procedures: At 2 h, 6 h, and 24 h after treatment, the rats were sacrificed by bleeding under ether anesthesia, and the jejunum and kidneys were isolated, cut into 2-mm-thick slices, and fixed in 2.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.6. Dewaxed sections were incubated in 3% hydrogen peroxide for 10 min and washed in 0.1 M PBS, pH 7.6. The abovementioned MPO antibody was applied to the sections, which were then incubated at room temperature for 1 h. The products resulting from the immunoreaction were visualized by the peroxidase-conjugated streptavidin-biotin method (Simple-Stain-PO kit; Nichirei Corp., Tokyo, Japan) with 3,3′-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide. The nuclei were counterstained with hematoxylin.

For controls of each immunostaining, Tris buffer or normal sera were substituted for the primary antibodies.

Electron microscopy—

Antibodies: Polyclonal goat anti-rat TNF-α antibody (Genzyme Co., Cambridge, MA) diluted to 1:400 in 0.1 M PBS was used.

Immunostaining procedures: At 2 h, 6 h, and 24 h after treatment, the rats were sacrificed by bleeding under ether anesthesia, and the kidneys were isolated, cut into 2-mm-thick slices, and fixed in 2.5% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate-buffered saline (PBS), pH 7.6. Sections approximately 30–40 μm in thickness were made on a microslicer and inserted into the sample mesh pack. Endogenous peroxidase activity was blocked by incubation in a periodic acid solution (Histofine, Code 415021; Nichirei, Tokyo, Japan) for 45 s, and endogenous avidin and biotin activities were blocked by the endogenous avidin/biotin blocking kit (Histofine, Code 415041; Nichirei, Tokyo, Japan). After treatment with normal rabbit serum at room temperature, sections were incubated for 1 h with the primary antibody at room temperature. The sections were also incubated with a biotinylated anti-goat IgG (Histofine, Code 426021; Nichirei, Tokyo, Japan) for 10 min at room temperature and then with a peroxidase-conjugated streptavidin (Histofine, Code 426061; Nichirei, Tokyo, Japan) for 5 min at room temperature. After incubation, the sections were treated with DAB in 0.05 M Tris buffer containing 0.05% H2O2 at pH 7.6 for 10 min at room temperature. They were thoroughly washed in 0.05 M Tris buffer, postfixed in 1% osmium tetroxide for 1 h at 4°C, dehydrated in an ascending ethyl alcohol series, embedded in Quetol 812, and examined under an electron microscope.

For controls of each immunostaining, Tris buffer or normal serum was substituted for the primary antibodies.

Morphologic experiment

For LM, at 2 h, 6 h, and 24 h after treatment, the rats were sacrificed by bleeding under ether anesthesia, and the kidneys were isolated, cut into 2-mm-thick slices, fixed in 10% buffered formalin, and prepared for LM. Sections were stained with hematoxylin and eosin.

Examinations of organ function

The degree of kidney injury was assessed by measuring the serum urea nitrogen (BUN, mg/dL) in blood collected by a cardiac puncture at 2 h, 6 h, and 24 h after treatment. Results were expressed as a mean ± standard deviation and were analyzed using the analysis of variance. The nonpaired t test was used to determine significance by the Welch t test. Significance was considered to be at the P < 0.01 level.

Assessment of p38 MAPK expression in the kidney

At 2 h, 6 h, and 24 h after treatment, the rats were sacrificed by bleeding under ether anesthesia, and the kidneys were isolated. For western blot analysis, the kidneys were homogenized on ice in extraction buffer, 1 mL/0.1 g tissue (10 mM Tris, pH 8.0, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, and 100 μM sodium vanadate) containing the protease inhibitors leupeptin (1 μg/mL), aprotinin (1 μg/mL), and 4-(2-aminoethylbenzensulfonyl fluoride hydrochloride (2 μg/mL). Insoluble material was removed by centrifugation (18,000 × g, 20 min). The protein content in kidney lysates was measured using a DC protein assay (Bio-Rad Laboratories, Tokyo, Japan). Lysates (20 μg) were resolved by SDS-PAGE and transferred to PVDF membranes (Immobilon, Millipore Corp., Bedford, MA). Nonspecific binding sites were blocked in TBS buffer (10 mmol/L Tris-Cl, pH 7.4, 0.15 mol/L NaCl) containing 5% nonfat dried milk for 1 h at 25°C. The membrane was then washed twice in TBS with 0.1% Tween (TBST). Rabbit polyclonal phospho-specific p38 antibody (1:1000 dilution; New England Biolabs, Berberly, MA) or rabbit polyclonal p38 antibody (1:1000 dilution; New England Biolabs, Berberly, MA) was added to TBST and incubated for 16 h at 4°C. Blots were washed twice and then incubated with goat anti-rabbit IgG peroxidase conjugate (1:1000 dilution; DAKO, Denmark) for 1 h at 25°C. After being washed twice in TBST, membranes were washed once more in TBS, and then the immune complexes were visualized by using enhanced chemiluminescence detection reagents (Amersham Bioscience, Buckinghamshire, United Kingdom). At least three independent experiments were performed with similar results.

The densities of bands that were obtained on the same membrane for the p38 MAPK or phosphorylated p38 MAPK were quantified with Scion Image Beta 4.02 (Scion Corporation, Frederick, MD). We compared the p38 MAPK phosphorylation levels as the ratio to p38 MAPK expression on the same photograph. Because the obtained data were not absolute values, the values are expressed as the ratio to the sham.

Assessment of TNF-α and IL-1β mRNA expression in the kidney

At 2 h, 6 h, and 24 h after treatment, the rats were sacrificed by bleeding under ether anesthesia, and the kidneys were isolated. One microgram of total RNA was reverse-transcribed by using the RNase H-reverse transcriptase and random hexamers (Invitrogen Corp., Carlsbad, CA). The mixture was incubated at 25°C for 10 min and then at 42°C for 50 min, heated to 70°C for 15 min, and cooled to 4°C. The RT sample was then used immediately or stored at −20°C. The sequences of primers for rat TNF-α, IL-1β, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used in this study. The following PCR primers were used: TNF-α sense primer, 5′-TCCCAACAAGGAGGAGAAATT-3′; TNF-α antisense primer, 5′-TCATACCAGGGCTTGAGCTCAG-3′; IL-1β sense primer, 5′-TGAGCTGAAAGCTCTCCACCT-3′; IL-1β antisense primer, 5′-TTGAGAGGTGCTGATGTACCAG-3′; GAPDH sense primer, 5′-GAACGGGAAGCTCACTGGCATGGC-3′; and GAPDH antisense primer, 5′-TGAGGTCCACCACCCTGTTGCTG-3′. To assess the amount of TNF-α and IL-1β mRNA in each sample, we performed a polymerase chain reaction (PCR) for both TNF-α and IL-1β constitutively expressed GAPDH. The primer sequences were chosen from separate exons of the genes so that the RT-PCR product could readily be distinguished from any genomic DNA-induced PCR product. PCR was performed in a total volume of 50 μL consisting of 1.35–5.0 μL of the RT sample (equivalent to 1.0–5.0 μg of total RNA) using the PCR Core Kit (Takara Bio Inc., Tokyo, Japan). The PCR condition was 94°C for 15 min, then 30 cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, followed by a final extension of 5 min at 72°C. A 5-μL aliquot of PCR products that was separated on a 1% agarose gel electrophoresis contained 0.3 μg/mL, was stained with ethidium bromide to visualize DNA bands, and was scanned on FAS mini (Toyobo, Osaka, Japan).

The densities of bands were digitally captured for analysis of intensity with Scion Image Beta 4.02 (Scion Corporation). Levels of TNF-α and IL-1β mRNA were normalized relative to GAPDH mRNA in the same sample. Because the obtained data were not absolute values, the values are expressed as the ratio to the sham.

Statistical analysis

Differences among groups were evaluated using one-way ANOVA and Fisher post hoc test. Statistical significance of difference was set at P < 0.05.

RESULTS

Tracer experiment

Figure 1shows the result of the tracer experiment on the jejunum. In the Burn group, HRP reaction products were observed in the capillary net of the intestinal villi, blood vessels of lamina propria mucosae, and the vasucular bed of tunica submucosa (Fig. 1A). In the Burn+FR group (Fig. 1B), HRP reaction products were partly observed in the capillary net of the intestinal villi. In the other groups, HRP reaction products were observed only in the coated vesicle.

Fig. 1.
Fig. 1.:
Light micrograph of tracer experiments showing the jejunum of infant rats. (A) In the Burn group, HRP reaction products were observed in the capillary net of the intestinal villi, blood vessels of lamina propria mucosae, and vascular bed of the tunica submucosa. (B) In the Burn+FR group, HRP reaction products were partly observed in the capillary net of intestinal villi. (Bar = 120 μm)

Bacterial translocation

The results of the quantitative bacterial culture of tissue samples are depicted in Table 1. In the Burn group, 67% of the animals showed bacterial translocation to the liver, 33% to the spleen and kidney, and 17% to the lung. Bacteria isolated from the organs were Escherichia coli and Morganella spp. In the other groups, positive tissue cultures for enteric bacteria were not observed.

Table 1
Table 1:
Bacterial translocation of enteric organisms after thermal injurya

Immunohistochemical experiment

The immunohistochemical neutrophil staining in the tissue of the jejunum and kidneys was observed. In the Burn group, a clustered appearance of reddish brown precipitates seen in the jejunum indicated the presence of neutrophils (Fig. 2A). In the Sham, FR, and Burn+FR (Fig. 2B) groups, such neutrophil immunoreactions were not observed. In the kidneys, the neutrophil immunoreactions were observed in the glomeruli of the Burn group (Fig. 3A). In the Sham, FR, and Burn+FR (Fig. 3B) groups, such immunoreactions were not observed.

Fig. 2.
Fig. 2.:
Light micrograph of immunohistochemical experiments for the stain of MPO showing the jejunum of infant rats. (A) In the Burn group, a clustered appearance of reddish brown precipitates seen in the intestinal villi indicates the presence of neutrophils. (B) In the Burn+FR group, no neutrophil immunoreactions were observed. (Bar = 25 μm)
Fig. 3.
Fig. 3.:
Light micrograph of immunohistochemical experiments for the stain of MPO showing the kidney of infant rats. (A) In the Burn group, a clustered appearance of reddish brown precipitates seen in the glomeruli indicates the presence of neutrophils. (B) In the Burn+FR group, the neutrophil immunoreactions were not observed. (Bar = 25 μm)

In the Burn group, TNF-α immunoreactions were localized in the lysosomes of neutrophils (Fig. 4A), macrophages, and mesangial cells. In addition to this, these immunoreactions were localized in the lysosomes of renal proximal tubular cells, which show edematous degenerative changes at 24 h after burn (Fig. 4B). In the other groups, TNF-α immunoreactions were not observed.

Fig. 4.
Fig. 4.:
Electron micrograph of immunohistologic experiments for the stain of TNF-α showing the kidney of infant rats. (A) In the Burn group, TNF-α immunoreactions were localized in the lysosomes of neutrophils at 2 h after burn. (Bar = 2 μm) (B) In the Burn group, TNF-α immunoreactions were localized in the lysosomes of renal proximal tubular cells, which show edematous degenerative changes at 24 h after burn. (Bar = 2 μm)

Morphologic experiment

LM findings show that some neutrophils, which often existed in the smaller vessels such as glomerular and vacuolar, were observed in the renal proximal tubular cell in the kidney of the Burn group at 2 h after treatment. The frequency of the infiltrations of inflammatory cells and the edematous degenerations of renal proximal tubular cells increased at 24 h after treatment. Abnormal histologic renal changes were not seen in the Sham, FR, or Burn+FR groups.

Examinations of organ function

Figure 5 shows a significant increase of serum BUN values in the Burn group compared with the Sham group at 2 h, 6 h, and 24 h after treatment. The BUN values in the FR group were significantly decreased compared with the Sham group at 2 h and 6 h after treatment, and the Burn+FR group did not increase compared with the Sham group at 2 h, 6 h, and 24 h after treatment.

Fig. 5.
Fig. 5.:
Evaluation of kidney function of infant rats after burn injury with FR167653 (1 mg/100 g) treatment. The degree of kidney injury was assessed by measuring BUN (mg/dL). Data were expressed as the mean ± SD for 5 rats. **P < 0.01 versus Sham

Assessment of p38 MAPK expression in the kidney

Figure 6 shows the relative p38 MAPK phosphorylation levels that were expressed relative to sham levels after normalization with p38 MAPK. At 2 h, 6 h, and 24 h after treatment, the ratio levels of p38 MAPK phosphorylation in the Burn group were significantly increased compared with those in the Sham group. At 2 h and 6 h after treatment, the administration of FR167653 significantly decreased the phosphorylation levels of p38 MAPK in the Burn+FR and FR groups compared with the Sham group. At 24 h after treatment, there were no significant differences in the Burn+FR and FR groups compared with the Sham group.

Fig. 6.
Fig. 6.:
Effects of FR167653 on p38 MAPK activation (assessed by Western blotting) in an infant rat kidney. (A) Representative Western blotting bands for phosphorylated p38 MAPK and p38 MAPK in the kidney. (B) Relative p38 MAPK phosphorylation levels were expressed relative to Sham levels after normalization with p38 MAPK. Data were expressed as mean ± SE for four rats. *P < 0.05 versus Sham

Assessment of TNF-α and IL-1β mRNA expression in the kidney

Figure 7 shows the TNF-α mRNA expression levels that were expressed relative to the Sham levels after normalization with GAPDH. The ratio levels of TNF-α mRNA expression in the Burn group significantly increased compared with the Sham group at 2 h, 6 h, and 24 h after treatment. The TNF-α mRNA expression in the FR group significantly decreased versus the Sham group at 2 h and 6 h after treatment, and the TNF-α mRNA expression in the Burn+FR group significantly increased compared with the Sham group at 6 h and 24 h after treatment.

Fig. 7.
Fig. 7.:
Effects of FR167653 on TNF-α mRNA (assessed by RT-PCR method) expression in the infant rat kidney after burn injury. (A) Representative PCR bands for TNF-α and GAPDH in the kidney. (B) Relative TNF-α mRNA expression levels were expressed relative to Sham levels after normalization with GAPDH. Data were expressed as the mean ± SE for five rats. *P < 0.05 versus Sham

Figure 8 shows the IL-1β mRNA levels that were expressed relative to the sham levels after normalization with GAPDH. The ratio levels of IL-1β mRNA expression in the Burn group significantly increased compared with the Sham group at 2 h, 6 h, and 24 h after treatment. The IL-1β mRNA expression in the Burn+FR and FR group significantly decreased versus the Sham group at 2 h after treatment, and the IL-1β mRNA expression in the Burn+FR group significantly increased compared with the Sham group at 6 h and 24 h after treatment.

Fig. 8.
Fig. 8.:
Effects of FR167653 on IL-1β mRNA (assessed by RT-PCR method) expression in the infant rat kidney after burn injury. (A) Representative PCR bands for IL-1β and GAPDH in the kidney. (B) Relative IL-1β mRNA expression levels were expressed relative to Sham levels after normalization with GAPDH. Data were expressed as the mean ± SE for five rats. *P < 0.05 versus Sham

DISCUSSION

Many studies have reported that thermal injury increases intestinal mucosal permeability, and these studies also support the concept that the increase of mucosal permeability is produced during the accumulation of neutrophils (19–22). Our observations are similar to their data; that is, our tracer experiment showed the burn-induced intestinal barrier damage and the immunohistochemical data indicating the accumulation of neutrophils in the intestine after burn. Previous studies have clearly demonstrated that the bacterial LPS activated the p38 MAPK pathway and that p38 MAPK is important for the nuclear factor-κB (NF-κB) activation and subsequent TNF-α and IL-1β production (9, 15, 23). Spies et al. suggested that TNF-α plays an important role in gut mucosal damage after burn (24). Therefore, we examined the effects of FR167653, a specific inhibitor of p38 MAPK, in the development of renal failure after the burn-induced intestinal barrier damage. FR167653 prevented the accumulation of neutrophils in the intestine and blocked the intestinal barrier damage. Eaves-Pyles et al. have demonstrated that bacterial translocation occurred within only 5 min postburn, and viable bacteria reached the remote organ within 30 min postburn (25). Our quantitative bacterial culture data demonstrated that viable bacteria reached the remote organ at 2 h after burn. Our present study demonstrated that FR167653 blocked the burn-induced intestinal barrier damage and also blocked the viable bacteria invading the kidney, and this fact indicates that FR167653 prevents the bacterial LPS from reaching the remote kidney. Western blotting identified the increased phosphorylation of p38 MAPK in the kidneys after thermal injury, which may indicate the possibility that bacterial LPS enhances the activation of the p38 MAPK pathway.

Renal failure increased with time after burn by BUN assay, but the assessment of TNF-α and IL-1β mRNA expression in the kidney demonstrated that the peak of these genetic cytokine levels were observed at 2 h after burn. Some investigators have reported that these cytokines play an important role in the first phase of the development of organ failure after burn and suggested that these inflammatory cytokines act in concert to produce burn-mediated organ failure (26, 27). Our present data are similar to their data and showed the possibility that these genetic activations of cytokines were induced by the first attack of p38 MAPK phosphorylation, which was enhanced by the bacterial LPS. Furuichi et al. reported that the plasma concentration of FR167653 reached a peak 2 h after subcutaneous administration (16), and this fact supported our data, which showed that FR167653 dramatically decreased p38 MAPK levels at 2 h after burn. At 24 h after burn, the p38 MAPK levels in the Burn+FR group returned to Sham levels. It seems that they were correlated with the plasma concentration of FR167653, which decreased time-dependently. Our data showed that FR167653 decreased the BUN levels in the Burn+FR group as compared with the Burn group at 2 h after burn, and the BUN levels continued at the same levels as the Sham group at 24 h after burn, although the plasma concentration of FR167653 decreased time-dependently. The inhibitory effects of FR167653 on renal failure extended up to 24 h after burn even after FR167653 diminished in the animal, which agreed with the extinction of subsequent outbreak of inflammatory vicious circuit induced by burn injury.

Although the p38 MAPK activity in the Burn+FR group decreased markedly, the BUN levels did not decrease as compared with the Sham group at 2 h after burn. The p38 MAPK pathway is activated primarily only in abnormal conditions, such as thermal injury, and induced the additional rise of BUN levels. It seems that BUN levels are not controlled by p38 MAPK pathway unless phosphorylated p38 MAPK reaches a certain level. There are many different factors related to the changes of BUN values, so only p38 MAPK could not explain the movement of BUN values. In contrast to the other p38 MAPK inhibitor, SB203580, FR167653 has no effect on cyclooxygenase (COX)-1 or COX-2 activity (16), so the BUN levels in the Burn+FR group at 2 h after burn may be influenced by COX activity. From these reasons mentioned above, we concluded that FR167653 inhibited the burn-induced BUN increases through the suppression of p38 MAPK as well as TNF-α and IL-1β, what we call block of the first attack. The activation levels of p38 MAPK phosphorylation were decreased at 6 h and increased again at 24 h combined with the levels of TNF-α mRNA. Our morphologic data demonstrated that the frequency of inflammatory cell infiltrations and edematous degenerations of renal proximal tubular cells increased at 24 h after burn, and our immunohistochemical data showed that TNF-α immunoreactions were localized in the lysosomes of neutrophils and renal edematous proximal tubular cells. The abovementioned data indicate the possibility that the activated cytokines, which may be induced by neutrophils, play an important role in the burn-induced renal failure.

Stambe et al. demonstrated that the p38 MAPK pathway is activated within endothelial cells in acute inflammatory renal injury, and blockade using an inhibitor of p38 MAPK resulted in reduced neutrophils and platelet accumulation (28). Our observations are similar to their study; that is, our morphologic and immunohistochemical data showed that FR167653 prevents the accumulation of neutrophils in the glomerulus after burn and that BUN assay indicated that FR167653 blockaded the burn-induced renal failure. Recent studies have demonstrated that FR167653 attenuates renal damage by reducing the production of inflammatory mediators, and have also suggested that the p38 MAPK pathway has an important role in the recruitment and activation of neutrophils (11–14). Among them, TNF-α and IL-1β are well known to play an important role in the development of LPS-induced acute renal failure. For example, TNF-α promotes renal dysfunction via direct cytotoxicity, vasoconstriction, and decreased renal blood flow and by the recruitment of neutrophils and monocytes (9, 29, 30). Our present data also showed that FR167653 especially decreased the phosphorylation levels of p38 MAPK in the kidneys, and immunoreactions of TNF-α were not observed in the kidney and TNF-α mRNA and IL-1β mRNA were decreased through the p38 MAPK pathway. Thus, it is reasonable to say that the p38 MAPK pathway plays an important role in the pathogenesis of renal failure in infant rats after thermal injury.

In addition, our previous data showed that thermal injury did not induce renal failure in the adult rat, but using carrageenan (CAR) and nonlethal LPS together induced renal failure in the burned adult rat (4). CAR is known to destroy macrophages (31, 32) and increase TNF-α mRNA in neutrophils (CAR by itself did not induce TNF-α production) (33). The activated neutrophils and CAR pretreatment may have increased susceptibility to the relatively low doses of bacterial LPS, and nonlethal LPS assisted the bacterial LPS. Our abovementioned data indicate a causal relationship between the burn-induced renal failure and the bacterial LPS. Our present data demonstrated that thermal injury induced the invading bacteria of kidney and the accumulation of neutrophils in the glomeruli and increased the expression of TNF-α mRNA and IL-1β mRNA in the kidney. Therefore, the burn-induced renal failure in the infant rat may be induced by the bacterial LPS-activated neutrophils. However, the p38 MAPK pathway can be activated not only by bacterial LPS but also by ischemia. Ramzy et al. demonstrated that the gut epithelial apoptosis increased significantly in the burn group compared with the mechanical ischemia perfusion group and suggested that this damage is primarily related to burn-induced proinflamatory mediators (34). Hansbrough et al. demonstrated that bactericidal/permeability-increasing protein increased the infiltrating neutrophils into kidneys after burn (35). With these facts taken into consideration, the present data suggest that the bacterial LPS-activated p38 MAPK pathway plays an important role in the pathogenesis of renal failure in infant rats after thermal injury.

In conclusion, our present data provide evidence for the hypothesis that the p38 MAPK pathway controls inflammatory mediators and not only improves intestinal function but also reduces remote renal failure after burn. We identified the pathophysiologic role of the p38 MAPK pathway in the development of renal failure after burn.

ACKNOWLEDGMENTS

The authors thank Miss Ogawa and Mr. Miyamoto for their excellent microbiological advice; and Miss Matsumura and Mr. Fusasaki for their skillful technical assistance.

REFERENCES

1. Bahrami S, Redl H, Yao YM, Schlag G: Involvement of bacteria/endotoxin translocation in the development of multiple organ failure. Curr Top Microbiol Immunol 216:239–258, 1996.
2. Deitch EA, Rutan R, Waymack JP: Trauma, shock, and gut translocation. New Horiz 4:289–299, 1996.
3. Fang WH, Yao YM, Shi ZG, Yu Y, Wu Y, Lu LR, Sheng ZY: Effect of recombinant bactericidal/permeability-increasing protein on endotoxin translocation and lipopolysaccharide-binding protein/CD14 expression in rats after thermal injury. Crit Care Med 29:1452–1459, 2001.
4. Yamaguchi H, Kita T, Sato H, Tanaka N: Escherichia coli endotoxin enhances acute renal failure in rats after thermal injury. Burns 29:133–138, 2003.
5. Tokyay R, Zeigler ST, Traber DL, Stothert JC Jr, Loick HM, Heggers JP, Herndon DN: Postburn gastrointestinal vasoconstriction increases bacterial and endotoxin translocation. J Appl Physiol 74:1521–1527, 1993.
6. Khorram-Sefat R, Goldmann C, Radke A, Lennartz A, Mottaghy K, Afify M, Kupper W, Klosterhalfen B: The therapeutic effect of C1-inhibitor on gut-derived bacterial translocation after thermal injury. Shock 9:101–108, 1998.
7. Choudhry M, Fazal N, Namak S, Haque F, Ravindranath T, Sayeed M: PGE2 suppresses intestinal T cell function in thermal injury: a cause of enhanced bacterial translocation. Shock 16:183–188, 2001.
8. Eaves-Pyles T, Alexander J: Comparison of translocation of different types of microorganisms from the intestinal tract of burned mice. Shock 16:148–152, 2001.
9. Donnahoo KK, Shames BD, Harken AH, Meldrum DR: Review article: the role of tumor necrosis factor in renal ischemia-reperfusion injury. J Urol 162:196–203, 1999.
10. Kita T, Tanaka N, Nagano T: The immunocytochemical localization of tumour necrosis factor and leukotriene in the rat kidney after treatment with lipopolysaccharide. Int J Exp Pathol 74:471–479, 1993.
11. Granger R, Hughes T, Ramji D: Stimulus- and cell-type-specific regulation of CCAAT-enhancer binding protein isoforms in glomerular mesangial cells by lipopolysaccharide and cytokines. Biochim Biophys Acta 1501:171–179, 2000.
12. Jo SK, Cha DR, Cho WY, Kim HK, Chang KH, Yun SY, Won NH: Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells. Nephron 91:406–415, 2002.
13. Wada T, Furuichi K, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Kobayashi K, Mukaida N, Matsushima K, Yokoyama H: A new anti-inflammatory compound, FR167653, ameliorates crescentic glomerulonephritis in Wistar-Kyoto rats. J Am Soc Nephrol 11:1534–1541, 2000.
14. Wada T, Furuichi K, Sakai N, Hisada Y, Kobayashi K, Mukaida N, Tomosugi N, Matsushima K, Yokoyama H: Involvement of p38 mitogen-activated protein kinase followed by chemokine expression in crescentic glomerulonephritis. Am J Kidney Dis 38:1169–1177, 2001.
15. Yoshinari D, Takeyoshi I, Koibuchi Y, Matsumoto K, Kawashima Y, Koyama T, Ohwada S, Morishita Y: Effects of a dual inhibitor of tumor necrosis factor-alpha and interleukin-1 on lipopolysaccharide-induced lung injury in rats: involvement of the p38 mitogen-activated protein kinase pathway. Crit Care Med 29:628–634, 2001.
16. Furuichi K, Wada T, Iwata Y, Sakai N, Yoshimoto K, Kobayashi Ki K, Mukaida N, Matsushima K, Yokoyama H: Administration of FR167653, a new anti-inflammatory compound, prevents renal ischaemia/reperfusion injury in mice. Nephrol Dial Transplant 17:399–407, 2002.
17. Iwata Y, Wada T, Furuichi K, Sakai N, Matsushima K, Yokoyama H, Kobayashi K: p38 mitogen-activated protein kinase contributes to autoimmune renal injury in MRL-Fas(lpr) mice. J Am Soc Nephrol 14:57–67, 2003.
18. Fujita M, Baba R, Oshikawa T, Miyoshi M: Relationship between endocytic pathway and cytoskeleton in absorptive cells of the small intestine of the sucking rat. Dig Absorp 19:86–90, 1996.
19. Gianotti L, Braga M, Vaiani R, Almondo F, Di-Carlo V: Experimental gut-derived endotoxaemia and bacteraemia are reduced by systemic administration of monoclonal anti-LPS antibodies. Burns 22:120–124, 1996.
20. Konaka A, Kato S, Tanaka A, Kunikata T, Korolkiewicz R, Takeuchi K: Roles of enterobacteria, nitric oxide and neutrophil in pathogenesis of indomethacin-induced small intestinal lesions in rats. Pharmacol Res 40:517–524, 1999.
21. Sir O, Fazal N, Choudhry MA, Goris RJ, Gamelli RL, Sayeed MM: Role of neutrophils in burn-induced microvascular injury in the intestine. Shock 14:113–117, 2000.
22. Sir O, Fazal N, Choudhry MA, Gamelli RL, Sayeed MM: Neutrophil depletion prevents intestinal mucosal permeability alterations in burn-injured rats. Am J Physiol Regul Integr Comp Physiol 278:R1224–R1231, 2000.
23. Ono K, Han J: The p38 signal transduction pathway: activation and function. Cell Signal 12:1–13, 2000.
24. Spies M, Chappell VL, Dasu MR, Herndon DN, Thompson JC, Wolf SE: Role of TNF-alpha in gut mucosal changes after severe burn. Am J Physiol Gastrointest Liver Physiol 283:G703–G708, 2002.
25. Eaves-Pyles T, Alexander JW: Rapid and prolonged impairment of gut barrier function after thermal injury in mice. Shock 9:95–100, 1998.
26. Chen J, Zhou YP, Rong XZ: An experimental study on systemic inflammatory response syndrome induced by subeschar tissue fluid. Burns 26:149–155, 2000.
27. Maass DL, Hybki DP, White J, Horton JW: The time course of cardiac NF-kappaB activation and TNF-alpha secretion by cardiac myocytes after burn injury: contribution to burn-related cardiac contractile dysfunction. Shock 17:293–299, 2002.
28. Stambe C, Atkins R, Tesch G, Kapoun A, Hill P, Schreiner G, Nikolic-Paterson D: Blockade of p38alpha MAPK ameliorates acute inflammatory renal injury in rat anti-GBM glomerulonephritis. J Am Soc Nephrol 14:338–351, 2003.
29. Cunningham PN, Dyanov HM, Park P, Wang J, Newell KA, Quigg RJ: acute renal failure in endotoxemia is caused by TNF acting directly on TNF receptor-1 in kidney. J Immunol 168:5817–5823, 2002.
30. Niimi R, Nakamura A, Yanagawa Y: Suppression of endotoxin-induced renal tumor necrosis factor-alpha and interleukin-6 mRNA by renin-angiotensin system inhibitors. Jpn J Pharmacol 88:139–145, 2002.
31. Catanzaro PJ, Schwartz HJ, Graham RC Jr: Spectrum and possible mechanism of carrageenan cytotoxicity. Am J Pathol 64:387–404, 1971.
32. Sawicki JE, Catanzaro PJ: Selective macrophage cytotoxicity of carrageenan in vivo. Int Arch Allergy Appl Immunol 49:709–714, 1975.
33. Ogata M, Matsui T, Kita T, Shigematsu A: Carrageenan primes leukocytes to enhance lipopolysaccharide-induced tumor necrosis factor alpha production. Infect Immun 67:3284–3289, 1999.
34. Ramzy PI, Wolf SE, Irtun O, Hart DW, Thompson JC, Herndon DN: Gut epithelial apoptosis after severe burn: effects of gut hypoperfusion. J Am Coll Surg 190:281–287, 2000.
35. Hansbrough J, Tenenhaus M, Wikstrom T, Braide M, Rennekampff OH, Kiessig V, Bjursten LM: Effects of recombinant bactericidal/permeability-increasing protein (rBPI23) on neutrophil activity in burned rats. J Trauma 40:886–892, 1996.
Keywords:

KEYWORDS; Burn injury; renal failure; p38 MAPK pathway; TNF-α; IL-1β; bacterial translocation

©2004The Shock Society