PROTEASE-ACTIVATED RECEPTOR 2 BLOCKING PEPTIDE COUNTERACTS ENDOTOXIN-INDUCED INFLAMMATION AND COAGULATION AND AMELIORATES RENAL FIBRIN DEPOSITION IN A RAT MODEL OF ACUTE RENAL FAILURE

Abstract

ABSTRACT 

Glomerular and microvascular thrombosis due to the activation of inflammation and coagulation pathway contribute to the occurrence of acute renal failure in sepsis. The protease-activated receptors (PARs) have been shown to play an important role in the interplay between inflammation and coagulation. We hypothesized that PAR-2 blocking would improve glomerular and vascular thrombosis by attenuating inflammation and coagulation, leading to the prevention of acute renal failure, and assessed the effects of the PAR-2 blocking peptide (PAR-2 BP) in a rat model of LPS-induced acute renal failure. Levels of TNF-α were significantly expressed 1 h after LPS administration, followed by 1) an increase in levels of tissue factor, factor VIIa, factor Xa, thrombin and plasminogen activator inhibitor 1; 2) unchanged levels of tissue factor pathway inhibitor; and 3) subsequent deposition of fibrin in kidney tissues, which led to the elevation of creatinine and blood urea nitrogen. Time-dependent PAR-2 expression was observed at both the gene and protein levels. Immunoreactivities of PAR-2 and fibrin were observed in the glomerulus and small arteries. Protease-activated receptor blocking peptide suppressed TNF-α elevation and attenuated activation of the coagulation, thus leading to a decrease in fibrin formation and its deposition in the glomerulus. However, the levels of creatinine and blood urea nitrogen remained unchanged. These results show that PAR-2 plays a key role in the inflammatory and coagulation process of LPS-induced renal failure; however, PAR-2 inhibition alone does not affect improvement in the renal function.

INTRODUCTION

LPS and proinflammatory cytokines, including TNF-α, increase tissue factor expression in monocytes and endothelial cells, which can activate the extrinsic coagulation pathway. The tissue factor expression, insufficient tissue factor pathway inhibitor (TFPI) not balanced with tissue factor increase, and inhibition of fibrinolysis by plasminogen activator inhibitor 1 (PAI-1) promote disseminated intravascular coagulation, followed by glomerular and microvascular thrombosis, which contribute to the occurrence of acute renal failure in sepsis (^{1, 2}).

Protease-activated receptors (PARs) have been shown to play an important role in the interplay between inflammation and coagulation (^3-5). To date, four distinct PARs, namely, PAR-1, PAR-2, PAR-3, and PAR-4, have been described. In the kidney, PAR-2 expression has been reported in collecting duct cells, mesangial cells, interstitial fibroblasts, vascular endothelial cells, and proximal tubule cells (^{4, 6}). Thrombin activates PAR-1, PAR-3, and PAR-4, whereas tissue factor/factor VIIa (FVIIa) complex and tissue factor/FVIIa/FXa ternary complex are selective ligands for PAR-2 (^{7, 8}). There is growing evidence for the involvement of PAR-2 in both inflammation and the immune response (⁹). Furthermore, in addition to their procoagulant activity, thrombin, tissue factor, FVIIa, and FXa all exert a number of proinflammatory effects that are mediated by the proteolytic activation of the PARs and thus contribute to tissue injury in sepsis (^{4, 5, 10}).

Welty-Wolf et al. (¹¹) and Carraway et al. (¹²) recently showed that the tissue factor/FVIIa complex, which is a selective ligand for PAR-2, contributes to renal failure in Gram-negative sepsis through selective stimulation of proinflammatory cytokine release and fibrin deposition. In their study, the blockade of tissue factor and site-inactivated FVIIa prevented Escherichia coli-induced acute renal failure (^{11, 12}). In addition, inhibition of the downstream coagulation protease thrombin counteracts endotoxin-induced renal dysfunction in the pig (¹³). A combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality in a mouse model of endotoxemia (¹⁴). We have also demonstrated that the blockade of PAR-2 ameliorates liver injury via the normalization of the inflammation, coagulation, and fibrinolytic pathways (¹⁵).

We hypothesized that PAR-2 blocking would improve glomerular and vascular thrombosis by attenuating inflammation and coagulation, leading to the prevention of acute renal failure. To investigate this hypothesis, we assessed the effects of the PAR-2 blocking peptide (PAR-2 BP) in a rat model of LPS-induced acute renal failure, on the changes in TNF-α, coagulation factors of the tissue factor-dependent pathway, TFPI, and PAI-1, including fibrin deposition within kidney tissue.

MATERIALS AND METHODS

Animals and treatments

In the first series of our study, endotoxemia was induced by administering a single injection of LPS (2 mL, in sterile saline, i.p.) derived from E. coli 055:B5 (15 mg kg⁻¹ body weight) to adult male Wister rats. The animals were killed in groups using pentobarbital (80 mg kg⁻¹ body weight, i.p.) at different time points after LPS administration (1, 3, 6, and 10 h). The control group received an equal volume of sterile saline (2 mL per rat) containing no LPS. Whole kidney tissues were carefully harvested, immediately frozen in liquid nitrogen, and stored at −80°C. For the paraffin section preparations, after the kidney tissues were harvested, they were postfixed in 4% paraformaldehyde overnight and processed routinely for paraffin embedding. All experimental procedures in the present study were performed in accordance with the institutional guidelines of the Hokkaido University Graduate School of Medicine Animal Care and Use Committee.

In the second part of the study, to investigate the specific role of PAR-2 in the LPS-induced fibrin deposition in the kidney, the PAR-2 BP was used to treat the rats in addition to LPS. For this purpose, rats were anesthetized with urethane (35% ethyl carbamate [Wako Pure Chemical Industries, Osaka, Japan] + 4% α-chloralose [Wako] saline wt/vol, 0.4-0.8 g kg⁻¹, i.p.), and the left jugular vein was cannulated for drug administration. All drugs being tested were administered intravenously as a slow bolus injection. Based on our previous study (¹⁵), LPS (15 mg kg⁻¹, i.v.) was administered through the jugular vein at time 0 in different groups of rats, and then the rats were killed at an interval of 1, 3, and 10 h (n = 20 for each time point). However, for the LPS + PAR-2 BP group, 30 min before LPS administration, PAR-2 BP (sc-9278 P; Santa Cruz Biotechnology, Santa Cruz, Calif) was administered as a slow bolus injection (100 μg kg⁻¹; 500 μL kg⁻¹ phosphate-buffered saline, i.v.), and then LPS injection was done, and after LPS administration, PAR-2 BP was continuously infused (20 μ kg⁻¹ h⁻¹; 100 μL h⁻¹ phosphate-buffered saline) through the jugular vein with a pump for 1, 3, and 10 h (n = 20 for each time point). Nontreated rats were used as a control.

Measurements of hemodynamic and biochemical parameters

On the day of the experiment, the rats were anesthetized with pentobarbital sodium (40 mg kg⁻¹ body weight, i.p.), and a microtip pressure transducer catheter (SPC-320; Millar Instruments, Houston, Tex) was inserted into the left carotid artery. The arterial blood pressure and heart rate were monitored with a pressure transducer (model SCK-590; Gould, Rossford, Ohio) and recorded using a polygraph system (amplifier, AP-601G; tachometer, AT-601G; thermal-pen recorder, WT-687G; Nihon Kohden, Tokyo, Japan). Thereafter, plasma and serum samples were prepared. In preparing the plasma samples, the proportion of blood to the sodium citrate dehydrate anticoagulant volume was 9:1. Serum creatinine levels and blood urea nitrogen (BUN) were measured using kits from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Histopathological examination

For the histopathological analysis, the tissue specimens were fixed in a 4% buffered formalin solution, dehydrated through an ethanol series, embedded in paraffin, and sliced into 5-μm-thick sections. After deparaffinization, the sections were stained with hematoxylin and eosin using the standard method.

Immunohistochemistry

Immunohistochemistry of the kidney tissues was performed as described in our previous study (¹⁶). Briefly, after the tissues were cut (5 μm thick), the sections were deparaffinized and treated for 20 min with a citrate buffer (10 mM citric acid, pH 6.0) in a microwave oven (750 W) before immunostaining. The frozen sections were fixed in acetone and then were air-dried. After blocking nonspecific staining from secondary antibodies, the sections were incubated with primary antibodies overnight at 4°C, followed by incubation with appropriate secondary antibodies coupled to horseradish peroxidase. The immunostaining findings were then viewed by light microscopy with an AEC (3-amino-9-ethylcarbazole) peroxidase substrate solution.

Western blot analysis

Immunoblotting was performed as demonstrated in our previous reports (^{15, 16}). Samples of tissue homogenate were run on a 4% to 15% sodium dodecyl sulfate polyacrylamide gel and then were transferred to a polyvinylidine difluoride filter membrane. The membrane was then blotted with the indicated antibody and was processed via chemiluminescence.

Antibodies

For immunological detection (immunohistochemistry and immunoblot analysis), the following antibodies were used: antihuman PAR-2 goat polyclonal antibody (Santa Cruz Biotechnology); antihuman fibrin mouse monoclonal antibody (Chemicon International, Temecula, Calif), antihuman thrombin goat polyclonal antibody (R&D Systems, Inc., Minneapolis, Minn), antihuman coagulation FVII goat polyclonal antibody (R&D Systems, Inc.), rabbit polyclonal FX (H-120) antibody (Santa Cruz Biotechnology), monoclonal antihuman FXa antibody (R&D Systems, Inc.), rabbit polyclonal TFPI antibody (Santa Cruz Biotechnology), antirabbit tissue factor sheep polyclonal antibody (American Diagnostica, Stamford, Conn), rabbit antirat PAI-1 antibody (American Diagnostica), murine monoclonal antibody against human FVIIa (American Diagnostica), and anti-Xenopus laevis β-actin mouse monoclonal antibody (Abcam, Cambridge, U.K.). In most cases, preliminary experiments were performed to determine the specificity of each antibody by blocking its (primary antibody) immunoreactivity using excess amounts of a competing peptide. In addition, these antibodies were tested for their ability to recognize the target peptide of interest within the rat. The immunoreactivity disappeared when nonimmune immunoglobulin G was used instead of primary antibodies.

RNA preparation and real-time quantitative polymerase chain reaction

All RNA samples were prepared from kidney tissues using the guanidinium thiocyanate-phenol-chloroform single-step extraction method with Isogen (Nippon Gene, Toyama, Japan) used routinely in our laboratory (^{15, 16}). After the RNA was isolated, treated with DNase I and quantified, it was reverse-transcribed to cDNA by omniscript reverse transcriptase using a first-strand cDNA synthesis kit (Qiagen GmbH, Hilden, Germany). The reaction was performed at 37°C for 60 min.

The mRNA expression levels of PAR-2 were analyzed by real-time quantitative polymerase chain reaction with a TaqMan probe using an ABI Prism 7700 Sequence Detector (Perkin-Elmer Applied Biosystems, Foster, Calif), as previously described (^{15, 16}). The gene-specific primers and TaqMan probes were synthesized with the Primer Express v. 1.61 software program (Perkin-Elmer Applied Biosystems, Boston, Mass) according to the published cDNA sequences for each gene. The sequences of the oligonucleotides were as follows: PAR-2 forward: 5′-CCTTGAACATCACCACCTGTCA-3′; PAR-2 reverse: 5′-GGGAGAGGAAGTAACTGAACATGTC-3′; PAR-2 probe: 5′-CCACCAGGACCTCCTC-3′; 18SrRNA forward: 5′-CGCAGCTAGGAATAATGGAATAGGA-3′; 18SrRNA reverse: 5′-GGCCTCAGTTCCGAAAACCA-3′; 18SrRNA probe: 5′-CCGCGGTTCTATTTTGT-3′.

Immunoassays

The TNF-α levels in the plasma and kidney tissue specimens were detected using a rat TNF-α enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Inc.). A rat PAI-1 activity ELISA kit (ZYMUTEST Rat-PAI-1-Activity, Hyphen Biomed, 95000 Neuville-sur-Oise, France) was used to detect PAI-1 in the rat serum and kidney tissues. Plasma thrombin-antithrombin complex (TAT) was measured using ELISA (SRL, Tokyo, Japan).

Statistical analysis

The results are expressed as the mean ± SEM, and the sample numbers are equivalent to the number of animals in each group. Means were compared by one-factor ANOVA, followed by the Scheffé test for multiple comparisons. Differences were considered to be significant at a value of P < 0.05.

RESULTS

Part 1

The effects of LPS on different parameters in plasma and TNF-α induction

Table 1 summarizes the values for biochemical markers in both the control and LPS-treated rats. The serum creatinine level increased at all of the time points after the LPS administration, but the serum BUN level began to increase significantly from 3 h after the LPS administration. The plasma TAT and PAI-1 levels significantly increased after the LPS administration in comparison to the control rats. The plasma and renal levels of TNF-α were elevated after the administration of LPS and peaked at 1 h (92- and 15-fold in comparison to the control, respectively) but declined thereafter.

TABLE 1: Time courses for the hemodynamic and biochemical markers

Histopathology after LPS administration

Although the histopathological evaluation showed only minimal tubular degenerative changes, they are consistent with a septic kidney. That is, focal tubular dilatation with mild swelling or thinning of the epithelial cells was observed beginning at 1 h, reached a peak at 3 h, then returned to almost normal findings at 6 to 10 h after the LPS administration (data not shown).

The expression levels of the coagulation and fibrinolytic markers in the kidney

A Western blot analysis for tissue factor (a single band migrating at 45 kDa) in rat kidney tissues showed elevated levels in the LPS-treated group in comparison to the control group (Fig. 1A). Tissue factor pathway inhibitor (a single band migrating at 35 kDa), however, remained almost unchanged between the control and the LPS-treated rats (Fig. 1B). In addition, the relative level of α-thrombin (target molecular weight ∼37 kDa) was significantly increased at different hours after the LPS administration (Fig. 1C). Figure 1D shows the statistically high levels of PAI-1 in the kidney tissues after the LPS administration. The relative amounts of immunodetectable fibrinogen decreased and fibrin significantly increased after LPS administration (Fig. 1, E and F). The expression level of FVII in the kidney tissue (target molecular weight, 50 kDa) decreased from 1 h after the LPS administration, whereas the expression of FVIIa was significantly increased in kidney tissues after LPS administration (Fig. 2, A and B). Consistent with the findings of FVII, the expression of FX in the kidney tissue (target molecular weight, 56 kDa) decreased after the LPS administration (Fig. 2C), whereas that of FXa progressively increased after LPS administration (Fig. 2D).

Fig. 1: An immunoblot analysis of tissue factor (A); an immunoblot analysis of TFPI (B); an immunoblot analysis of α-thrombin (C); ELISA of PAI-1 (D); and immunoblot analyses of fibrinogen and fibrin in kidney tissues (E and F). All of the experiments were done in the control rats and at different time points after LPS administration (n = 17). In each of the immunoblot experiments, the band obtained in the control was normalized to 1.0. *P < 0.01 in comparison to the control.

Fig. 2: Immunoblot analyses of kidney tissues FVII (A), FVIIa (B), FX (C), and FXa (D). All of the experiments were done in the control rats and at different time points after LPS administration (n = 17). In each of the immunoblot experiments, the band obtained in the control was normalized to 1.0. *P < 0.01 in comparison to the control.

Expression of PAR-2 in the kidney

The PAR-2 mRNA expression increased in the kidney tissues after LPS administration with a peak at 3 h in comparison to the control rat (Fig. 3A). The protein levels of PAR-2 were detected as a band of approximately 55 kDa, which was consistent with the manufacturer's information. The band increased over time, peaking at 3 h post-LPS treatment (2.5-fold; Fig. 3B), which was consistent with the mRNA expression.

Fig. 3: A, A real-time polymerase chain reaction analysis showing the gene expression for PAR-2 in kidney tissues from the control and LPS-treated rats. Values represent the amount of mRNA relative to that of 18SrRNA (n = 15). *P < 0.01 in comparison to the control. B, An immunoblot analysis of the PAR-2 protein in the kidney tissues of the control rats and at different time points after LPS administration (n = 15). In each of the immunoblot experiments, the band obtained in the control was normalized to 1.0. *P < 0.01 in comparison to the control.

Immunohistochemistry of fibrin and PAR2

At baseline, immunohistochemistry revealed negative to faint fibrin immunoreactivity in the small arteries, glomerulus, and interstitial areas (Fig. 4A). Three hours after the LPS injection, the immunoreactivity of fibrin was moderate in the glomeruli and moderate to strong in the tunica intima and tunica media of the small arteries (Fig. 4B).

Fig. 4: Immunohistochemistry of fibrin and PAR-2 in the kidney tissues from the LPS-treated and control rats. A, At baseline, immunohistochemistry revealed negative to, in part, mild fibrin immunoreactivity (reddish-orange) in the small arteries, glomerulus, and interstitial areas. B, Immunoreactivity of fibrin was moderate in the glomerular mesangial areas and, in part, glomerular capillary loops and moderate to strong in the endothelium of the small arteries 3 h after LPS injection (arrow). C, Immunohistochemistry at the base line revealed negative to minimal PAR-2 immunoreactivity (reddish-orange) in the glomeruli (asterisk), small arteries (arrow), and mild in the interstitium (arrowhead). D and E, Immunoreactivity ofPAR-2 was moderate in glomeruli (asterisk), tunica intima of small arteries(arrow), veins (long arrow), and moderate to strong in the interstitium (arrowhead) 3 h after LPS injection. Magnification, ×200 (A, B) and ×100 (C-E).

An immunohistochemical analysis revealed that immunoreactivity of PAR2 at 3 h (Fig. 4, D and E) had clearly increased in the glomeruli, tunica intima of the small arteries and veins, and in the interstitium in comparison to the control kidneys (Fig. 4C).

Part 2

To explore the differences in the PAR-2 expression in relation to the time-kinetics and the patterns of expressions in the LPS-treated rats, PAR-2 BP was administered as a bolus injection, followed by continuous infusion for 1, 3, and 10 h. From our detailed investigation, we found that the blockage of PAR-2 at 3 h was the most beneficial in reversing the different parameters related to renal fibrin deposition, proinflammatory cytokine, and coagulation and fibrinolytic factors. The PAR-2 BP dosage used in the present study was selected based on the findings of preliminary research using different doses of PAR-2 BP. Moreover, the trends demonstrated in this study for the alterations of different parameters responsible for the LPS-induced renal fibrin deposition were almost identical, irrespective of either the intraperitoneal or intravenous administration of LPS.

Changes in the different plasma parameters after the blockade of PAR-2

The elevated serum levels of both creatinine and BUN in LPS-treated rats at 3 h were almost unchanged after the administration of PAR-2 BP to the LPS-treated rats. The competitive blockade of PAR-2 for 3 h was found to completely normalize the increased plasma levels of TAT in the LPS-treated rats, whereas the elevated serum PAI-1 level remained unchanged. PAR-2 blockade for 3 h significantly suppressed the increased plasma TNF level in the LPS-administered rats. The results are shown in Table 2.

TABLE 2: Effects of PAR-2 BP on the biochemical markers

The effects of PAR-2 BP on the expression levels of different target molecules in the kidney

The renal level of TNF-α increased at 3 h in the LPS-treated rats in comparison to the control group and was significantly prevented by the administration of PAR-2 BP for 3 h in the LPS-treated rats (Fig. 5A). The elevated tissue factor level in the kidney tissue was significantly reversed in the LPS-treated rats after the PAR-2 blockade for 3 h (Fig. 5B); in contrast, the renal PAI-1 level was almost unaltered with the treatment of PAR-2 BP (Fig. 5C).

Fig. 5: Kidney tissue expressions for level of TNF-α (A, ELISA); tissue factor (B, immunoblotting); PAI-1 (C, ELISA); α-thrombin (D, immunoblotting); and fibrin (E, immunoblotting) from the control, 3-h LPS-treated rats, and 3-h PAR-2 BP-treated LPS rats (n = 15). In each of the immunoblot experiments, the band obtained in the control was normalized to 1.0. *P < 0.01 vs. control, †P < 0.01 vs. 3-h LPS-treated rats. F, The immunohistochemical findings of fibrin (reddish-orange) shown in the glomerular area in 3-h LPS-injected rats with or without the treatment of PAR-2 BP (arrows). Magnification, ×200.

The elevated level of α-thrombin in the kidney tissue specimens was significantly reversed after the 3-h blockade of PAR-2 in the LPS-treated rats, as demonstrated by the immunoblotting findings (Fig. 5D). The expression of fibrin in the kidney tissues of the LPS-treated rats was also significantly prevented by PAR-2 BP for 3 h (Fig. 5E). The immunoreactivity of fibrin clearly decreased in the glomerulus (Fig. 5F) but remained almost unchanged or decreased in part in the small arteries after PAR-2 BP treatment for 3 h in the LPS-induced septic rats.

The treatment of PAR-2 BP for 3 h in the LPS-treated rats restored the FVII expression in the kidney tissues (Fig. 6A) and normalized the elevated renal expression of FVIIa (Fig. 6B). Consistent with the renal expression of FVII, the treatment with PAR-2 BP for 3 h in the LPS-treated rats also recovered the FX expression in the kidney tissues (Fig. 6C). In addition, the up-regulated kidney tissue expression of FXa significantly diminished after the blockade of PAR-2 at 3 h in the LPS-treated rats (Fig. 6D).

Fig. 6: An immunoblot analysis of kidney tissues. FVII (A), FVIIa (B), FX (C), and FXa (D) from the control group, 3-h LPS-treated rats, and 3-h PAR-2 BP-treated LPS rats (n = 15). In each of the immunoblot experiments, the band obtained in the control was normalized to 1.0. *P < 0.01 vs. control; †P < 0.01 vs. 3-h LPS-treated rats.

DISCUSSION

In the present study, the LPS administration induced TNF-α expression and activated the tissue factor-dependent pathway, leading to an increase in the levels of FVIIa, FXa, and thrombin, followed by renal fibrin deposition in the failed kidney. This is the first study to show PAR-2 expression at both the mRNA and protein levels in rat injured kidney tissue and the removal of glomerular fibrin by the inhibition of both inflammation and activation of coagulation after PAR-2 blocking.

TNF-α produced by activated monocytes induces tissue factor synthesis by cultured umbilical vein endothelial cells, and it is likely that the same holds true for glomerular endothelial cells. In addition, rat mesangial cells are induced to synthesize increased amounts of tissue factor by LPS and TNF-α (¹⁷). After marked increases in the levels of TNF-α both in the plasma and kidney tissues 1 h after LPS administration, the significant expression of tissue factor, FVIIa, FXa, thrombin, and elevated TAT levels were observed in the present study. However, the levels of TFPI remained unchanged. All of these changes suggest the formation of tissue factor/FVIIa and tissue factor/FVIIa/FXa ternary complexes in the kidney, which are selective ligands for PAR-2 (^{7, 8}).

The elevation of PAI-1 and the activation of extrinsic coagulation pathway led to a decrease in fibrinogen and gave rise to fibrin protein expression in the kidney tissues and fibrin deposition in the glomerulus and arteries, including endothelial layer. The immunoreactivity of fibrin was observed in the glomerular mesangial areas and glomerular capillary loops. These localizations are consistent with LPS and TNF-α-induced intravascular microthrombosis and local fibrin formation by mesangial cells (^{17, 18}). Glomerular and microvascular fibrin thrombosis compromise glomerular capillary flow, leading to focal ischemia and necrosis, which is considered to be the main pathogenesis of LPS-induced acute renal failure (^{2, 18}). As a result, we observed elevated levels of serum creatinine and BUN in our study.

Stimulation with TNF-α and IL-1, as well as LPS, has been reported to result in a marked elevation of PAR-2 gene expression in human umbilical vein endothelial cells (¹⁹). A recent study showed that primary cultures of human proximal tubular cells express PAR-2 and produce proinflammatory monocyte chemoattractant protein 1, which can induce the expression of IL-6 and IL-8, in response to stimulation with a PAR-2-activating peptide (²⁰). These studies suggest that PAR-2 may be involved in inflammatory responses, such as inflammatory cell infiltration and inflammatory cytokine production, in the kidney (⁶). Furthermore, another study showed that FXa induces mesangial cell proliferation in a PAR-2-dependent manner, and that a specific FXa inhibitor inhibited this effect and fibrin deposition on the mesangial cells (²¹). These results suggest that PAR-2 may be further involved in the development of renal injury via a coagulation-dependent mechanism.

Collectively, the significant increases in PAR-2 at both the mRNA and protein levels in the kidney observed in the present study suggest the role of PAR-2 in LPS-induced renal failure via inflammation and a coagulation-dependent manner. Moreover, we also revealed the distribution of PAR-2 in the glomerulus and arterial endothelium, which was consistent with the localization of fibrin in the kidney. We discovered results that prove the importance of PAR-2 in LPS-induced renal failure in which PAR-2 blockade significantly prevents the elevation of TNF-α in both the plasma and kidney tissues. The marked decrease in TNF-α levels may explain the inhibition of the activation of the tissue factor-dependent coagulation pathway, which was associated with decreased fibrin formation in the kidney tissues. In addition, PAR-2 BP prevented fibrin deposition in the glomerulus, as observed in the immunohistochemistry findings.

Contrary to our expectations, however, we were not able to show an improvement in the renal function as measured by the serum creatinine and BUN levels using a PAR-2 blockade for 10 h. Although a human study has documented the role of TNF-α in the induction of PAI-1 levels, a recent study using LPS-infused rabbits suggests the PAI-1 increase observed in severe endotoxemia may be independent of TNF-α (^{22, 23}). TNF-α-independent PAI-1 induction may be one of the reasons for the failure in the improvement of renal function in the present study. In our study, PAR-2 BP showed a significant but partial blockade of the inflammatory, coagulation, and fibrinolytic markers. Despite the blockade of fibrin deposition in the glomerulus, fibrin in the small arteries remained unchanged or decreased in part after PAR-2 BP treatment. These results may be another reason for the failed improvement of renal function. Welty-Wolf et al. (¹¹) and Carraway et al. (¹²) found a significant decrease in the creatinine levels using direct blockade of tissue factor and FVIIa in sepsis-induced renal failure. In a mouse model of endotoxemia, deficiency of PAR-2 alone did not affect survival; however, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality (¹⁴). In addition, PAR-4 antagonist dose-dependently diminished the severity of endotoxemia and preserved kidney function in a murine model of systemic inflammation and disseminated intravascular coagulation (²⁴). These studies and the present results suggest that several mechanisms, as well as PAR-2, may be involved in the pathogenesis of LPS-induced acute renal failure.

In summary, we herein demonstrated that the increase in the expression of TNF-α after LPS administration in rats, which is followed by the activation of the tissue factor-dependent coagulation pathway, insufficient suppression of the coagulation, and the inhibition of fibrinolysis, gives rise to renal fibrin deposition, which leads to acute renal failure. A time-dependent induction of PAR-2 at both the gene and protein levels was also observed, which was predominantly localized in the glomerulus, arterial endothelium, and interstitium. Protease-activated receptor 2 blocking peptide counteracts endotoxin-induced inflammation and coagulation and ameliorates glomerular fibrin deposition, but it did not improve the renal function.

We conclude that PAR-2 plays a key role in the inflammatory and coagulation process of LPS-induced renal failure. However, PAR-2 inhibition alone does not affect the improvement in the renal function, and thus, some other undefined mechanisms may also be involved in the pathogenesis of this disease.