Urinary trypsin inhibitor as a therapeutic option for inflammatory disorders

Introduction Urinary trypsin inhibitor, a serine protease inhibitor, has been widely used, particularly in Japan, as a drug for patients with acute inflammatory disorders such as pancreatitis, shock and disseminated intravascular coagulation. Previous in vitro studies have demonstrated that serine protease inhibitors may have anti-inflammatory properties beyond their inhibition of neutrophil elastase at sites of inflammation. However, the therapeutic effects of urinary trypsin inhibitor in vivo remain unclarified. In this review, we introduce the roles of urinary trypsin inhibitor in experimental systemic inflammatory responses induced by both the intraperitoneal and intratracheal administration of lipopolysaccharide using urinary trypsin inhibitor-deficient (–/–) and corresponding wild-type mice. Conclusion Urinary trypsin inhibitor may provide an attractive ‘rescue’ therapeutic option for systemic inflammatory response syndromes such as disseminated intravascular coagulation, acute lung injury and acute liver injury. Introduction Urinary trypsin inhibitor (UTI) is a multivalent Kunitz-type serine protease inhibitor synthesized and released in human urine and blood1. Various serine proteases such as trypsin, chymotrypsin, neutrophil elastase and plasmin are reportedly inhibited by UTI2. Based on the multivalent nature of protease inhibition, UTI appears to prevent organ injury by inhibiting the activity of these proteases3,4. Although the therapeutic effects of UTI on circulatory shock have been recognized, especially in Japan, current understanding regarding the target mechanisms/pathways remains limited. The aim of this review was to discuss UTI as a therapeutic option for inflammatory disorders. Discussion The authors have referenced some of their own studies in this review. These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964), and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed. All human subjects, in these referenced studies, gave informed consent to participate in these studies. General characteristics of UTI UTI is a multivalent Kunitz-type serine protease inhibitor found in human urine and blood. UTI, also referred to as ulinastatin, HI-30, ASPI or bikunin, is an acidic glycoprotein with a molecular weight of 30 kDa by SDS-polyacrylamide gel electrophoresis. It is composed of 143-amino acid residues and its sequence includes two Kunitz-type domains. UTI is produced by hepatocytes as a precursor in which UTI is linked to α1-microgloblin. In hepatocytes, different types of UTI-containing proteins are formed by the assembly of UTI with one or two of the three evolutionarily related heavy chains (HC) 1, HC 2 and HC 3, through a chondroitin sulphate chain7; these proteins comprise inter-α-inhibitor (IαI) family members, including IαI, pre-α-inhibitor (pαI), inter-α-like inhibitor (IαLI) and free UTI. IαI, pαI and IαLI are composed of HC1 + HC2 + UTI, HC3 + UTI and HC2 + UTI, respectively8,9. During inflammation, UTI is cleaved from IαI family proteins through proteolytic cleavage by neutrophil elastase in the peripheral circulation or at the inflammatory site10–13. Therefore, plasma UTI has been considered to be one of the acute-phase reactions, and indeed, the plasma UTI level and its gene expression alter in severe inflammatory conditions11. Further, UTI is rapidly released into urine when infection occurs and is an excellent inflammatory marker, constituting most of the urinary anti-trypsin activity14. Various serine proteases such as trypsin; thrombin; chymotrypsin; kallikrein; plasmin; elastase; cathepsin and factors IXa, Xa, XIa and XlIa are inhibited by UTI2,15. Furthermore, UTI can reportedly suppress urokinase-type plasminogen activator (uPA) expression through the inhibition of protein kinase C16,17. Clinical utility of UTI Although its precise physiological role in normal subjects has not been fully clarified, clinically, UTI is widely used, especially in Japan, to treat acute pancreatitis including post-endoscopic retrograde cholangiopancreatography pancreatitis and septic shock, in which several proteases are considered to play a pathophysiological role18,19. Also, in gynaecological * Corresponding author Email: kinoue@iuhw.ac.jp Center for Medical Science, International University of Health and Welfare, 2600-1, Kitakanemaru, Ohtawara, Tochigi, 324-8501, Japan


Introduction
Urinary trypsin inhibitor (UTI) is a multivalent Kunitz-type serine protease inhibitor synthesized and released in human urine and blood 1 .
Various serine proteases such as trypsin, chymotrypsin, neutrophil elastase and plasmin are reportedly inhibited by UTI 2 .Based on the multivalent nature of protease inhibition, UTI appears to prevent organ injury by inhibiting the activity of these proteases 3,4 .Although the therapeutic effects of UTI on circulatory shock have been recognized, especially in Japan, current understanding regarding the target mechanisms/pathways remains limited.The aim of this review was to discuss UTI as a therapeutic option for inflammatory disorders.

Discussion
The authors have referenced some of their own studies in this review.These referenced studies have been conducted in accordance with the Declaration of Helsinki (1964), and the protocols of these studies have been approved by the relevant ethics committees related to the institution in which they were performed.All human subjects, in these referenced studies, gave informed consent to participate in these studies.

General characteristics of UTI
UTI is a multivalent Kunitz-type serine protease inhibitor found in human urine and blood.UTI, also referred to as ulinastatin, HI-30, ASPI or bikunin, is an acidic glycoprotein with a molecular weight of 30 kDa by SDS-polyacrylamide gel electrophoresis.It is composed of 143-amino acid residues and its sequence includes two Kunitz-type domains.UTI is produced by hepatocytes as a precursor in which UTI is linked to α 1 -microgloblin 5,6 .In hepatocytes, different types of UTI-containing proteins are formed by the assembly of UTI with one or two of the three evolutionarily related heavy chains (HC) 1, HC 2 and HC 3, through a chondroitin sulphate chain 7 ; these proteins comprise inter-α-inhibitor (IαI) family members, including IαI, pre-α-inhibitor (pαI), inter-α-like inhibitor (IαLI) and free UTI.IαI, pαI and IαLI are composed of HC1 + HC2 + UTI, HC3 + UTI and HC2 + UTI, respectively 8,9 .During inflammation, UTI is cleaved from IαI family proteins through proteolytic cleavage by neutrophil elastase in the peripheral circulation or at the inflammatory site [10][11][12][13] .Therefore, plasma UTI has been considered to be one of the acute-phase reactions, and indeed, the plasma UTI level and its gene expression alter in severe inflammatory conditions 11 .Further, UTI is rapidly released into urine when infection occurs and is an excellent inflammatory marker, constituting most of the urinary anti-trypsin activity 14 .Various serine proteases such as trypsin; thrombin; chymotrypsin; kallikrein; plasmin; elastase; cathepsin and factors IXa, Xa, XIa and XlIa are inhibited by UTI 2,15 .Furthermore, UTI can reportedly suppress urokinase-type plasminogen activator (uPA) expression through the inhibition of protein kinase C 16,17 .

Clinical utility of UTI
Although its precise physiological role in normal subjects has not been fully clarified, clinically, UTI is widely used, especially in Japan, to treat acute pancreatitis including post-endoscopic retrograde cholangiopancreatography pancreatitis and septic shock, in which several proteases are considered to play a pathophysiological role 18,19  Competing interests: none declared.Conflict of interests: none declared.
All authors contributed to conception and design, manuscript preparation, read and approved the final manuscript.

All authors abide by the Association for Medical Ethics (AME) ethical rules of disclosure.
In other studies, UTI reportedly had preventive effects on drug/metal-induced nephrotoxicity by gentamycin, mercuric chloride and cisplatin [47][48][49][50][51] .The proposed mechanisms for this renal protection include reductions of lysosomal fragility and proximal tubule damage 50,51 .Koizumi and colleagues have shown that UTI prevents experimental crescentic glomerulonephritis in rats, at least in part, by inhibiting the intraglomerular infiltration of inflammatory cells 52 .On the other hand, Huang et al., employing both clinical and in vivo tests, showed that UTI reverses myocardial damage accompanied by severe burn via modulation of the inflammatory status with lipid peroxidation with cardiac apoptosis 53 .Interestingly, Tsujimura and colleagues reported a case of infectious interstitial pneumonia associated with mixed connective tissue disease, in whom the bolus infusion of UTI improved the pathology 54 .Also, Komori et al. illustrated that UTI improves peripheral microcirculation and relives bronchospasm associated with systemic anaphylaxis in rabbits 55 .Further, UTI reportedly ameliorates refractory skin diseases such as Stevens-Johnson syndrome and toxic epidermal necrolysis 56 .Thus, these in vivo findings may pave the way for alternative therapeutic strategies for disorders other than sepsis and postsurgical haemodynamic abnormality, such as nephrotoxicity, nephritis, burn-associated tissue damage, interstitial pneumonitis, anaphylactic shock and/or refractory skin diseases in the future.
Alternatively, UTI may be applicable in combination with other drugs.For example, UTI with amphotericin B can ameliorate invasive pulmonary aspergillosis 57 .On the other hand, a combined therapeutic strategy with low-dose dopamine, gabexate mesilate and UTI improves the water balance and reduces the incidence of pulmonary complications in thoracic esophagectomy patients 58 .sion 18,[31][32][33] through modulating TNF-α production via the inhibition of early growth response factor (Egr)-1 in monocytes and pulmonary induction of inducible nitric oxide synthase 31 and reduces mortality caused by sepsis 34 .Also, UTI can alleviate coagulatory disturbance accompanied by sepsis such as an increase in the serum level of fibrinogen and fibrinogen degradation products 35 .Another group showed that UTI protects against haemorrhagic shock by preserving the myocardial mitochondrial function 36 .Likewise, UTI has a protective effect against ischemia-reperfusion injury in the liver 37 , kidney 38 , heart 39 and lung 40 in vivo via the actions of its radical scavenging elements 41 .In addition, UTI reduces C-X-C chemokine production during liver ischemia/ reperfusion in vivo 42 .Intravenous pretreatment with UTI was also suggested to have a neuroprotective action in a transient cerebral ischemia model 43 .
In humans, pre-pump administration (5,000 U/kg) of UTI reportedly improves cardiopulmonary bypassinduced haemodynamic instability and pulmonary dysfunction through the attenuation of IL-6 and IL-8 release in humans 44 .Furthermore, UTI can inhibit coagulatory activation accompanied by severe inflammation such as tissue factor expression on monocytes in vitro and in vivo 35 as well as coagulation and fibrinolysis during surgery in humans 45 .On the other hand, Kurosawa et al. clinically showed that (intravenous) pretreatment with 300,000 IU of UTI prevents an increase in pulmonary artery pressure and shunting in patients after aortic unclamping during abdominal aortic aneurysmectomy 46 .Taken together, it is likely that UTI itself other than IαI/PαI also regulates hemodynamic stability during surgical stress.However, the detailed mechanisms and concise methodology for UTI treatment remain to be determined in the future.Also, further evaluation are needed using each gene-depleted mice for IαI and/or PαI.area, for instance, UTI therapy is reportedly effective in patients at risk for premature labour, with a moderately developed bulging membrane during the second trimester 20 , and can reduce meconium-induced chemical peritonitis and thereby facilitate intrauterine remission of foetal ascites 21 .Further, recently, it is used for the prevention of imminent abortion via vaginal administration.

Anti-inflammatory property of UTI
Beyond its inhibition of inflammatory proteases mentioned above, UTI exhibits anti-inflammatory activity and suppresses the infiltration of neutrophils and release of elastase and chemical mediators from them 13,22,23 .As well, UTI reportedly inhibits the production of tumour necrosis factor (TNF)-α 24,25 and interleukin (IL)-1 25 in lipopolysaccharide (LPS)-stimulated human monocytes and LPS-or neutrophil elastase-stimulated IL-8 gene expression in HL60 cells 26 or bronchial epithelial cells 27 in vitro.Matsuzaki et al. demonstrated that UTI inhibits LPS-induced TNF-α and subsequent IL-1β and IL-6 induction by macrophages, at least partly, through the suppression of mitogenactivated protein kinase (MAPK) signalling pathways such as ERK1/2, JNK and p38 in vitro 28 .Nakatani and colleagues demonstrated that UTI inhibits neutrophil-mediated endothelial cell injury in vitro, suggesting that UTI can act directly on neutrophils and suppress the production and secretion of activated elastase from them 23 .Further, UTI downregulates stimulated arachidonic acid metabolism such as thromboxane B2 production in vitro 29 , which plays a role in the pathogenesis of sepsis syndrome 30 .
On the other hand, a large number of in vivo reports have provided evidence that UTI protects against pathological traits related to septic shock induced by gram-negative bacteria: UTI reduces LPS-induced hypoten-using UTI (-/-) mice via the regulation of systemic (serum) levels of cytokines such as IL-6 and IL-10 and chemokines such as MCP-1 and MIP-1α 73 .

Conclusion
Our experiments employing genetic approach suggest that UTI can protect against the systemic inflammatory response and subsequent organ injury induced by LPS, at least partly, through the inhibition of proinflammatory cytokine and chemokine expression, which provide important in vivo evidence and understanding about a protective role of UTI in inflammatory conditions.UTI may therefore provide an attractive 'rescue' therapeutic option for systemic inflammatory response syndromes such as disseminated intravascular coagulation, acute lung injury and acute liver injury.
The protein expression levels of proinflammatory molecules such as macrophage chemoattractant protein (MCP)-1 in the lungs; MCP-1 and keratinocyte-derived chemoattractant (KC) in the kidneys and IL-1β, macrophage inflammatory protein (MIP)-2, MCP-1 and KC in the livers were significantly greater in UTI (-/-) than in WT mice after LPS challenge.These results indicate that UTI protects against systemic inflammation induced by the i.p. administration of LPS, at least partly, through the inhibition of proinflammatory cytokine production/release 66 , suggesting that UTI may be therapeutic against sepsis in humans.
A previous study showed that UTI improves acute lung injury in vivo 67 ; however, no evidence has been reported using a genetic approach.In another series of studies 68,69 , therefore, we also showed that that UTI also protects against acute lung inflammation induced by the intratracheal administration of LPS, at least in part, via the local suppression of proinflammatory cytokines 68 and antioxidation 69 , suggesting that UTI may be a useful therapeutic tool for acute lung injury.
One study has shown that plasma UTI levels increase in patients with acute hepatitis and markedly decrease in those with fulminant hepatitis, suggesting that the plasma UTI level is closely linked to the severity of liver damage 70 .Further, the plasma UTI level is reportedly correlated with the degree of liver damage in patients with chronic liver diseases such as liver cirrhosis and hepatocellular carcinoma 71 .Using UTI (-/-) mice, we showed that UTI protects against the local inflammatory response accompanied by severe liver injury, which supports its antiinflammatory properties in vivo 72 , implicating a therapeutic potential of UTI in fulminant hepatitis in humans.In this regard, Nobuoka and colleagues have recently implicated UTI in normal liver regeneration Subsequent studies indicate that UTI can inhibit the invasiveness of tumour cells of various histologic origins [59][60][61][62] .Moreover, UTI has been shown to downregulate the expression of the cancer metastasisassociated molecules uPA and uPA receptor possibly through MAPKdependent signalling cascades in vitro and in vivo 63,64 .In addition, UTI has anti-inflammatory effects against several forms of malignancy in vitro 60,65 .These studies suggest that UTI is a candidate anti-cancer drug, although further studies are required in the future.
In order to confirm its anti-inflammatory potential in vivo, we conducted a series of studies on the role of UTI in the inflammation related to bacterial endotoxin (LPS) using UTI (-/-) mice.In one study 66 , both UTI (-/-) and wild-type (C57/BL6: WT) mice were injected intraperitoneally with vehicle or LPS at a dose of 1 mg/kg body weight.Evaluation of the coagulatory and fibrinolytic parameters and white blood cell (WBC) counts at 72 h after intraperitoneal (i.p.) challenge showed that fibrinogen levels were significantly greater in LPS-than in vehicle-challenged mice with the same genotypes.In the presence of LPS, however, they were also significantly higher in UTI (-/-) than in WT mice.WBC counts significantly decreased after LPS challenge in UTI (-/-) mice.LPS appeared to shorten prothrombin time (PT) when compared to vehicle treatment in UTI (-/-) mice, although this difference did not reach significance.In the presence of LPS, the PT was significantly shorter in UTI (-/-) than in WT mice.Further, histopathological changes in the lung, kidney and liver of both genotypes after LPS challenge revealed severe neutrophilic inflammation in UTI (-/-) lungs challenged with LPS, whereas little neutrophilic infiltration was found in LPS-treated WT mice.The overall trend was similar regarding findings in the kidney and liver.
. Also, in gynaecological Licensee OA Publishing London 2013.Creative Commons Attribution License (CC-BY)For citation purposes: Inoue KI, Takano H. Urinary trypsin inhibitor as a therapeutic option for inflammatory disorders.