CN101010001A - NGAL for reduction and amelioration of ischemic and nephrotoxic injuries - Google Patents

Abstract

Use of neutrophil gelatinase-associated lipocalin (NGAL) as a therapeutic and in a method of treating, reducing, or ameliorating an injury selected from an ischemic injury, an ischemic-reperfusion injury, and a toxin-induced injury, to an organ in a patient. The invention includes administering to the patient NGAL in an amount effective to treat, reduce or ameliorate ischemic, ischemic-reperfusion, or toxin-induced injury to the organ, such as the kidney. A siderophore can be co-administered with the NGAL. The invention also relates to administering a sideophore to enhance a response to secretion of NGAL following an ischemic or toxin-induced injury to an organ in a patient.

Description

NGAL is used for reducing and improving ischaemic and nephrotoxicity damage

Invention field

The present invention relates to alleviate or improve the method for ischaemic in the organ (ischemic) and nephrotoxicity damage (nephrotoxic injury).The invention further relates to the patient that treatment suffers from the damage that causes with ischaemic and toxin or injures relevant various disease.The invention further relates to treatment and suffer from patient acute or the chronic renal damage.

Background of invention

The minimizing of oxygen flow is called ischaemic in the organ, causes the incident of a series of complexity, in fact influences each organelle of influenced cell and the 26S Proteasome Structure and Function of subcellular fraction system.Ischemia reperfusion (continuation again of blood flow) damage causes the generation of excessive reactive oxygen species (ROS) and active nitrogen kind (RNS), thereby cause oxidative stress, cause a series of incident, the for example change in the mitochondria oxidisability phosphorylation, the consumption of ATP, the activation of the increase of intracellular Ca2+ and protein kinase, phosphatase, protease, lipase and nuclease, it causes the losing of function/integrality of cell.Shown that it is the great reason that causes tissue and organ injury that ischaemic is poured into the inflammatory response that causes subsequently again.

The ischemia reperfusion damage is serious problem in the organ transplant, because the organ of this collection is removed from health, isolates with blood relationship, and therefore loses oxygen and nutrient in distance.(the acute tubular necrosis of acute tubular necrosis after the problem of a key that will solve in present kidney transplant process is operation, ATN) the delay graft function that causes (delayed graft function, high relatively incidence of disease DGF).Illustrative, DGF influences in many TCs the kidney transplant of about 20-35% and is the most general complication in immediate postoperative period in kidney transplant.Although the incidence of disease of DGF and be defined between the TC differently, this result the most often relates to and prolongs hospital stay, additional invasion procedure and to the extra cost of patient and health care system.Postponing graft function not only influences individual patient, and it also influences the microstructure that obtains organ, and shares as the result who is placed on the consumption (drain) in the available organ supply.DGF also increases the risk of early stage acute rejection outbreak and increases losing from the early stage graft of chronic rejection.

With present store method, preserve the main risk factors that cause DGF after the cold ischemic that causes has been considered to transplant by organ.For kidney, cold ischemia time surpasses 24 hours risks that just obviously increase with DGF.At 20th century middle nineteen sixties, the cold preservation of kidney realizes effectively by using mechanical perfusion and the solution that picks up from CPP.After that, simple cold preserving method is introduced into and comprises and uses cold crystalloid solution.Because to the early stage success that kidney is preserved, preserve the solution that solution develops into synthetic fully clear and definite composition, it is designed to come pre-anti-cold ischemic injuries by suppressing cell expansion with the supply of keeping metabolism.A kind of early stage synthetic solution has been used for pancreas and liver from the solution based on lactobionate (UW) of Wisconsin university.Along with synthetic appearance of preserving preparation with serum-free, quality and duration that feasible organ is preserved have been modified.However, yet, in the kidney about the clinical data of DGF with other relates to the nephrocyte structure and morphologic problem clearly proves the not exclusively success in damage of prevention ischaemic or injury of this solution.

In addition, the acute renal failure (ARF) that is secondary to the damage of ischaemic or renal toxicity also is general and potential huge problem on clinical nephrology always, although tangible progress is arranged on supportive care, has constant high mortality.In the past few decades, many variation and the influences of inflammatory response in the ARF pathogenesis that studies have shown that its lasting vessel retraction, renal tubule obstruction, cell structure and metabolism.To the treatment of ARF with remedy and to be limited with respect to many-sided response of ischemic kidney and the early stage label that lacks ARF.Cell and molecular biological nearest progress in ischaemic and kidney poison injury of kidney have shown that the near-end renal tubular cell has experienced complicated event time sequence, comprise losing of cell polarity, because of Cell Death by Apoptosis and Necrosis, dedifferenting and breeding of vigor cell arranged, and the reconstruction of epithelium phenotype.

Because the result of ischaemic or the infringement of kidney poison, cell can pass through two kinds of various process death.Apoptosis or apoptosis (" cell suicide ") are physiological mechanisms, to remove aging, damage or the unusual cell that influences individual cells.Apoptosis is caused and be it is characterized by dna break by endonuclease and becomes many 180-200 base-pairs.Apoptotic cells do not discharge proteolytic enzyme or poisonous oxygen kind, and this process is not accompanied by inflammation by macrophage or adjacent cells picked-up.On the contrary, downright bad (" cell murder ") is a kind of pathological process, and it influences cell population and causes local organization destruction, inflammation and frequent serious consequence of overall importance.At present, apoptotic cells death demonstrates important effect in increasing kidney disease, comprises ischaemic, ischemia reperfusion, the nephrotoxin, POLYCYSTIC KIDNEY DISEASE, blocks and renal glomerular disease.Therefore the downward modulation of apoptosis provides unique methods of treatment with powerful to improve some acute and chronic renal damages.

If a monomer is considered to suffer from acute renal failure, promptly increase 0.5mg/dL at least, when the baseline serum creatinine levels is lower than 2.0mg/dL when this patient's serum creatinine value or (1); (2) increase 1.5mg/dL at least, when the baseline serum creatinine levels more than or equal to 2.0mg/dL; Or (3) increase 0.5mg/dL at least, no matter the baseline serum creatinine levels, it is the result who is exposed to the radiography agent.

What cDNA microarray technology had allowed that the neutrophil gelatinase is correlated with NGAL (NGAL) is identified in ischaemic and kidney poison damage back in early days as high transcript of inducing in the kidney.The effect of NGAL is not illustrated yet in the kidney.NGAL be protein NGAL family a member and it is characterized by in the human neutrophils particle and to find secretion 25kDa glycoprotein.(people such as Kjeldsen, 1993, J.Biol.Chem.268:10425-10432).NGAL, it can have common three-dimensional β-barrel shaped structure in conjunction with little lipophilic substance, and its function is in conjunction with the lipophilicity part at least some NGALs, for example, steroids, bilin, biostearin or other lipid.Muridae form from the NGAL (homologue) of mouse and rat is known.In mouse, NGAL refers in particular to and is NGAL, 24p3 albumen, SIP24, P25, NGAL 2 and uterocalin.NGAL is considered to NGAL or α 2-microglobulin in rat.The full-length cDNA of coding people NGAL albumen is cloned and is checked order.People NGAL gene, it comprises seven exons and six introns, is also cloned and checks order, and analyzed its expression in different tissues.197 amino acid whose polypeptide of people NGAL gene code, it has 19-or 20-amino acid signal sequence, and ripe NGAL polypeptide comprises 178 amino acid.Motif Gly-X-Trp (the amino acid 48-50 among the ripe people NGAL) and Thr-Asp/Asn-Tyr (the amino acid/11 32-134 among the ripe people NGAL) are present in all known NGALs.On the basis of X-radiocrystallgraphy, hinted that in three grades of β-barrel shaped structure that these motifs share be important between NGAL.Cysteine residues 95 and 194 in the people NGAL sequence is guarded, and has reported the intramolecular disulfide bond bridge of formation.People NGAL comprises single N-glycosylation site (asparagine residue) 84 or 85 positions of NGAL polypeptide (approximately-) in 65 positions of the amino acid sequence of maturation.

A kind ofly can determine that the mechanism of ATN is (mis-localized) iron of location of mistake.Unconjugated iron can catalysis H ₂O ₂Change into OH and OH-(Haber-Weiss reaction), or generate active ferryl or perferryl material.The molecule of the many types of these ion mutagenesis comprises lipid, nucleotide and DNA skeleton.The iron of catalytic discharges into urine or blood from free hemoglobin and myoglobin, and snperoxiaized lipid is put down in writing in the form of many acute renal failures, comprises chemotherapy, ischemia reperfusion, transplanting ischaemic and in the tubular injury of albuminuria-mediation.The animal of load iron is increased the weight of this illness in advance, and siderophore (siderophore) chelating with iron and Deferoxamine or bacterium weakens infringement on the contrary.The infringement of iron-catalysis is considered in the kidney dysfunction one of incident the earliest and may be also very important in other organ, and (referring to people such as Mori: the endocytosis transmission of NGAL-siderophore-iron complexes is from ischemia reperfusion injury protection kidney to comprise heart and liver.J Clin Invest 115：610-621，2005)。

Cell obtains iron by cell surface Railway transportation carrier (for example divalent metal carrier I) from carrier protein (for example transferrins).Intracellular Tie Tong cross the iron response protein (IRP1 for example, IRP2), the effect control of ferritin compound and Heme oxygenase I.Because IRPs is regulated by anoxic, oxidative stress and phosphorylation, the change of their activity can be played important effect by regulating the generation of ferritin compound that the protection cell avoids the infringement of iron mediation in ischemic disease.Ferritin is a kind of iron-phosphorous-albumen composition, comprises about 23% iron, generates in intestinal mucosa.Ferritin is the storage form of iron in tissue such as liver, spleen and the marrow.Haemoglobin in blood and the muscle and myoglobin molecule need to come Catalytic Oxygen to be delivered to cell in conjunction with iron respectively.Yet, know in ischaemic cell or histologic lesion that the Railway transportation that others are seldom arranged is defeated, storage or metabolism, although the iron of catalysis occupies the first place in their pathogenesis in other type.

Although many approach produce ATN, many investigators have been found that the common potential mechanism of near-end renal tubular cell (proximal tubule cell) infringement.This has comprised the release of cell factor.A kind of idea is ischaemic cell and renal tubule toxin generate high concentration partly in the nephron as free myoglobin and haemoglobin a iron.Think that this iron is catalytic activity, and generate oxygen radical.The primary cellular defect of iron catalysis is that the evidence that causes a disease and cause near-end renal tubule dysfunction comprises that the discovery tubular injury is slowed up by the injection of iron chelating agent.What other supported this idea is that iron is the center of the mechanism of organ dysfunction after the ischaemic, it derives from and uses the iron-free that is derived from bacterium, iron chelating agent is called the experiment of siderophore, makes the effect dull of ischemia reperfusion damage in the external model of cardiac ischemia.In these common mechanism each is considered to be in the principle of the pathogenic incident of ATN different times.The infringement of iron catalysis is considered in the kidney dysfunction one of incident the earliest.

Do not know also at present how the near-end renal tubule catches NGAL.In fact still lack clear and definite identification to most of NGAL acceptors.Perhaps being used for the huge albumen that RBP reclaims needs, also is NGAL acceptor (people such as Christensen, 1999, J Am Soc Nephrol.10(4)685-95)。In fact, knock out huge albumen cause urinating in the appearance of NGAL, but these animals also are surprised to find that have higher levels of NGAL information (people such as Hilpert, 2002, Kidney Int.62 (5) 1672-81), the NGAL of hint urine is perhaps synthetic rather than because of catching the failure of filtered load from this locality.Although this ambiguity, NGAL and other NGAL are similar, for example RBP and α-2u globulin NGAL (referring to people such as Borghoff, 1990, Annu Rev Pharmacol Toxicol.30:349-67), it enters cell and is transported to lysosome by huge albumen approach and degrades.These data and the transportation of NGAL in the cell-line of not expressing huge albumen (for example embryonic kidney cells) and wherein this albumen avoid degraded (referring to people such as Yang, 2002, Mol.Cell 10 (5): 1045-56) form to contrast.Similarly, being sent to lysosome by the approach based on huge albumen-cubulin in the near-end renal tubule after, transferrins also degrades (referring to people such as Kozyraki, 2001, Proc.NatlAcad Sci USA 98 (22) 12491-6), yet its usually recycle in cell-line.Therefore be reasonably below proposing, after promptly filtering, NGAL is caught by huge albumen and is degraded by the near-end renal tubule, and does not have recycle.This hypothesis is by following observation support, and the time-delay point after promptly total length NGAL injects in blood no longer occurs.

The cold ischemic injuries of composition and method damage as to(for) the ischaemic that is fit to prevention, reduces or improves in organ such as the kidney still have needs.This composition will be useful in treatment patient's primitive organ, also can use being used for transplanted organ.What need equally is new biomarker, and it can be used for detecting the toxicity damage of pair cell, for example renal toxicity after patient's administration.New with improved treatment with reduce the tissue that causes by organ transplant and the method for organ ischemia reperfusion damage, and treatment and reduce organ cell's structure and the method for metabolism change, practitioner and patient etc. is undoubtedly useful and important.

Summary of the invention

The present invention relates to the relevant NGAL of neutrophil gelatinase (neutrophilgelatinase-associated lipocalin, NGAL) and its application and treatment in composition, alleviate, improve or prevent normally to be selected from the method for illness, damage or the disease of the organ damage that ischaemic, ischemia reperfusion or toxin cause.The NGAL that the present invention also relates to deliver medicine to patient or study subject effective dose treats, alleviates, improves or prevent the method for illness, damage or disease.Described damage can comprise the injury of kidney relevant with illness, processing, treatment or disease, and described illness, processing, treatment or disease easily make patient suffer from ischaemic injury of kidney, renal tubule damage or necrosis/apoptosis.Described damage can comprise acute (including but not limited to shock, apoplexy, pyemia, wound, infection, inflammation) or chronic (including but not limited to hypertension, diabetes, heart failure, lupus, infection, inflammation) kidney disorders.

The present invention also provides the method for the kidney NGAL function reduction that the ischemia reperfusion damage is induced among a kind of patient of improvement, improves the reduction of renal function by the NGAL that gives patient's effective dose.According to this respect, give NGAL and reduce high serum or the plasma creatinine level in ischemia reperfusion damage back.The dosage of NGAL effectively prevents or improves cell death.

The present invention further provides a kind of after the damage and acute or chronic kidney diseases that ischaemic, ischemia reperfusion or toxin cause, strengthen the method that the kidney epithelium forms (reepithelialization) or renal tubular cell propagation again, strengthen the kidney epithelium by the NGAL that gives patient's effective dose and form or cause renal tubular cell propagation again.

A kind of embodiment of the present invention is included in treatment, alleviates, prevents or improves the application of NGAL in the method for acute renal failure (ARF).Show that NGAL strengthens renal tubular cell propagation and alleviates or improve the renal tubular cell apoptosis.Disclosed method can be improved the reduction of renal function among the patient that damage that ischemia reperfusion or toxin cause induces, and can be further used for treating, alleviate, improving or prevent to be secondary among the patient acute renal failure of ischaemic damage.

The present invention further provides reduce and/or the method for the delay graft function (DGF) of treatment patient transplant organ in the application of NGAL.In some respects, DGF is caused by acute tubular necrosis (ATN).

The present invention also provides transplanting (transplanting) and transplants the application of (grafting) organ NGAL in patient's the method.The application of this method can alleviate or improve organ graft or transplanting is lost or acute cellular rejection, introduce one or more (i) organs or (ii) its donor by NGAL, alleviate or improve organ graft (graft) or transplant losing or acute cellular rejection of (transplant) effective dose.Aspect different, described organ is selected from kidney, liver, heart, brain, lung, stomach, intestines, colon, pancreas, blood vessel, bladder, uterine cervix, skin, or their part or one section.Aspect concrete, described organ is present in organ donor corpse or alive.In this case, NGAL can organ transplant (transplanted) or transplant (grafted) before the patient, among and/or give patient afterwards.Described organ can be the organ of corpse, and wherein this organ is that donor from corpse obtains in the case, the NGAL administration can be given this corpse or organ damage, injury or the failure after preventing to transplant of the organ that taken out.The described organ organ donation product of can be living, organ damage, the injury after the organ that the NGAL administration can be given taking-up in the case prevents to transplant or fail.Described organ can be kidney, liver, heart, brain, lung, stomach, pancreas, blood vessel, bladder, uterine cervix, skin, or their part or one section.The present invention concrete aspect, this organ is a kidney.

The present invention also provides the application of the NGAL relevant with the LD kidney transplant with corpse, and wherein oxidant-mediated Apoptosis is the important factor of facilitating of renal tubular cell death.Except the common complication of acute renal failure (ARF), the ischemia reperfusion damage causes postponing graft function (DGF) in the kidney of known transplanting, and it obviously increases transplants the risk of losing with acute cellular rejection.As described here, NGAL is used for alleviating or the purposes of the method for the ischaemic adverse effect that improvement is relevant with organ transplant being used for making.In one embodiment, NGAL can join in the organ preservation solutions, for example uses in the refrigeration of transplant organ, has the DGF of cadaveric renal transplantation feature with improvement.According to the method, even NGAL administration after local ischemic injury is also effective to small part.The method has advantageously satisfied needs and provides that new ARF is a kind of existing and clinical related adverse events to the ischemic conditions the confirmed treatment means of ARF for example, its common with the bad prognosis of patient is associated.The method that delays or improve DGF relevant with the ischaemic damage in the organ that is implanted among the patient or the graft comprises to be introduced a certain amount of NGAL or with NGAL contact, (i) transplanted organ or graft; The (ii) donor of transplant organ; Or (iii) both (i) and (ii), delay or improve DGF or (transplanted) that transplant or the losing or acute cellular rejection of (grafted) organ of transplanting with effective dose.Illustrative and do not have restricted ground is used for transplanted organ or graft and is selected from kidney, liver, heart, brain, lung, stomach, intestines, colon, pancreas, blood vessel, bladder, uterine cervix, skin, or their part or one section.Aspect concrete, described organ is a kidney.More specifically, the kidney of transplanting is cadaveric donors or LD kidney.And NGAL is the component of organ preservation solutions, and for example, the organ preservation solutions that contains NGAL uses in organ transplant refrigeration.

The present invention further provides the method that a kind of minimizing, improvement, prevention or protection patient avoid injury of kidney, this injury of kidney is with easily to make patient suffer from illness, processing, treatment or the disease of ischaemic injury of kidney relevant.This method comprises that the NGAL that gives patient's effective dose reduces, improves, prevents or protects patient to avoid the injury of kidney relevant with patient's illness, processing, treatment or disease.Illustrative illness, processing or treatment comprise without limitation, developer treatment, Antybody therapy, antibiotic therapy, organ transplant, kidney transplant, cadaveric renal transplantation, cardiac treatment, postoperative cardiac treatment or central nervous system treatment.Comprise infection, bacterial infection, acute nephropathy, chronic kidney disease, ischemia reperfusion damage, shock, wound, pyemia, apoplexy, heart reperfusion injury, cardiopulmonary bypass, OHS and abdominal surgery art without limitation according to illustrative disease in this respect.

The present invention also provides treatment among a kind of patient, alleviates or improve the method for renal tubule damage or necrosis/apoptosis, and it comprises the NGAL that gives the patient effective dose.According in this respect, described patient can suffer from, renal tubule damage, infection, antibiotic therapy, Antybody therapy or the developer treatment of the renal tubule damage of for example, the damage that causes of acute kidney diseases, chronic kidney diseases, ischemia reperfusion damage, organ transplant, toxin, ischaemic, kidney transplant, shock, wound, pyemia, apoplexy, heart reperfusion injury, cardiovascular shunt postoperative, the renal tubule damage behind the OHS, abdominal surgery postoperative.In the method, NGAL can work to strengthen renal tubular cell propagation, because also target kidney near-end renal tubular cell directly of NGAL, causes alleviating or improving the damage or the necrosis/apoptosis of renal tubule.

The present invention also provides a kind of treatment, alleviates or improves the method for patient's organ damage that toxin causes, damage comprises the damage of renal toxicity, treats, alleviates or improve the organ damage that toxin causes by the NGAL that gives the patient effective dose.On the other hand, the method that alleviates or improve the damage that toxin causes comprises co-administered NGAL and the treatment compound poisonous to patient, and NGAL alleviates or improve the toxic action of organ treatment with the effective dose administration.

The present invention also provides treatment, alleviates or improves the method for other acute (including but not limited to shock, apoplexy, pyemia, wound, infection, inflammation) and chronic (including but not limited to hypertension, diabetes, heart failure, lupus, infection, inflammation) injury of kidney.

In the present invention's distinct methods disclosed herein, NGAL can with one or more therapeutic agent administering drug combinations, example for example, vasodilator or oxygenate.In addition, NGAL can be to comprise the physiology acceptable composition administration of carrier, thinner or excipient, and by different methods of administration, it comprises without limitation, intravenous or non-oral approach.And, NGAL can ischaemic or renal toxicity, organ transplant (transplant) transplant (grafting) or renal tubule injury, infringement or damage before, among or administration afterwards, as described herein.

The present invention also provides a kind of method for the treatment of the possibility of inducing renal toxicity of judging, gives test substances to cause the mammal model of nephrotoxicity damage, for example mouse by (a); (b) after giving test substances, measure the existence of the urine or the relevant NGAL (NGAL) of the neutrophil gelatinase in the plasma sample of mammal model, show that with this this material can induce renal damage.Aspect this method concrete, after giving test substances, just in urine or blood plasma, detect NGAL in 3 hours.

The present invention also relates to a kind of composition that is used for the treatment of, alleviates, improves or prevent organ damage in the mammal, comprise NGAL, or derivatives thereof or the analog for the treatment of effective dose.Said composition can further comprise siderophore, typically with 1: 1 mol ratio, comprises the compound of NGAL and siderophore.Said composition can be used for any method disclosed herein.

The present invention also relates to the purposes of siderophore coupling NGAL in composition, with be used for the treatment of, alleviate, improve or prevent the illness that ischaemic or toxin cause and the method for disease, comprise the damage that toxin causes in ischaemic, ischemia reperfusion or the organ.

In one embodiment of the invention, NGAL and the siderophore damage that strengthens treatment, alleviate, improve or prevent ischaemic in the organ, ischemia reperfusion or toxin to cause with the pharmaceutical composition administration of effective dose.

The present invention also relates to comprise the pharmaceutical composition of siderophore, it is used for being administered into patient and strengthens the damage for the treatment of prevention, improve and reducing in the organ by endogenic NGAL.In another embodiment, provide a kind of method of co-administered, typically as the compound compound of a kind of NGAL and siderophore.In another embodiment, siderophore delivers medicine to patient with effective dose and strengthens the propagation that the kidney epithelium forms again or the renal tubular cell that causes is secreted in positive impact by endogenic NGAL.

Particularly, the invention provides a kind of the enhancing and postpone the method that alleviates or improve that graft function (DGF) and organ or graft transplantation repel among the patient, comprise following steps: siderophore is introduced (i) transplanted organ or graft by endogenic NGAL; The (ii) donor of transplant organ; Or (iii) both (i) and (ii), strengthen alleviating of DGF or improve or the losing or acute cellular rejection of (transplanted) that transplant or (grafted) organ of transplanting with effective dose.This method comprises with effective dose administration siderophore treats, alleviates, improves or prevent damage to organ.

In one embodiment of the present invention, NGAL: the siderophore compound can join in the organ preservation solutions, for example uses the DGF that has the cadaveric renal transplantation feature with improvement in transplant organ refrigeration.In another embodiment, the siderophore in buffer solution can join in the organ preservation solutions separately, for example uses in the refrigeration of transplant organ, has the DGF of cadaveric renal transplantation feature with improvement.

The present invention further provides a kind of by giving NGAL or derivatives thereof or analog manipulated cell and method extracellular iron in the organ of ischaemic or toxin infringement.This method also can comprise the organ injury that the iron that gives effective dose is treated, alleviates, improved or further prevent ischaemic or toxin to cause in conjunction with the co-factor or the siderophore of chemicals in conjunction with chemicals, iron.The organ of typically described ischaemic or toxin-infringement is a kidney.

In another embodiment, the invention provides a kind of treatment kit, it comprises first container, and the NGAL of treatment effective dose is housed.This first container can comprise at least one phial, at least one test tube, at least one flask and at least one bottle.This kit also can comprise second container, wherein is placed with at least a composition, and makes device that this container is fixed together to be used for merchandise sales.This medicine box also can comprise the 3rd container, and aseptic, pharmaceutically useful buffer solution or other thinner are housed.This first container can be syringe.The device of fixed container can be plastic containers injection moulding or blowing, is wherein fixing this container.Perhaps, described phial can prepare in such a way, and it allows directly composition to be introduced the intravenous drug transfer system.In operation instruction also typically is included in.

Many-sided, feature and advantage of the present invention will be understood when considering with accompanying drawing after reading detailed description of the present invention better.

Description of drawings

Fig. 1 shows that the Coomassie blue (CB) of purifying NGAL of reorganization of definite amount and the chemiluminescence (ECL uses polyclone NGAL antibody) that strengthens analyze (as shown in the figure).

Fig. 2 show contrast the kidney of non local ischemic animal injection after 1 hour or the ischaemic kidney at injection arbitrary immunofluorescence dyeing after 1 hour or 3 hours, use polyclone NGAL antibody.

Fig. 3 is presented at the detection of NGAL Western blotting in the urine sample that derives from non local ischemic (NI) and ischaemic (I) animal in administration NGAL1 hour.

Fig. 4 showed before contrasting non local ischemic mouse, the pretreated ischaemic mouse of salt solution or ischaemic 1 hour, during or 1 hour after the HE stain of the kidney that obtains of the ischaemic mouse of handling with NGAL cut into slices.

Fig. 5 A, 5B and 5C showed before the pretreated ischaemic mouse of salt solution or ischaemic 1 hour, during or 1 hour after the Histological section of the kidney that obtains of the ischaemic mouse of handling with NGAL mark.This section is analyzed and use is concise and to the point marks 0～4 grade to tubular ectasia (Fig. 5 A), renal tubule tubulose thing (casts) (Fig. 5 B) and renal tubular cell necrosis (Fig. 5 C) scoring.

Fig. 6 shows non local ischemic (Non Isch) control mice, or with (Pre NGAL) before the pretreated mouse of salt solution (PreSal) or the ischaemic damage 1 hour, during the serum creatinine measured in 24 hours after (Dur NGAL) or (PostNGAL) handles with NGAL after 1 hour the mouse ischaemic.

Fig. 7 shows from non local ischemic control mouse, or before the pretreated mouse of salt solution or the ischaemic damage 1 hour, during or 1 hour after the representational section TUNEL coloration result that obtained in 24 hours after the mouse ischaemic handled with NGAL.Arrow refers to the cell nucleus that the dyes aggegation with apoptosis feature, fracture, strong under low and the high-amplification-factor, dyes than proliferating cells nuclear antigen (PCNA).

Fig. 8 shows from non local ischemic control mouse, or before the pretreated mouse of salt solution or the ischaemic damage 1 hour, during or 1 hour after apoptosis (last figure) and breed the quantitative of (middle graph) in the kidney that obtained in 24 hours after the mouse ischaemic handled with NGAL.

Fig. 9 shows from non local ischemic control mouse, or before the pretreated mouse of salt solution or the ischaemic damage 1 hour, during or 1 hour after the propagation that calculates in the kidney that obtained in 24 hours after the mouse ischaemic handled with NGAL: the ratio of apoptosis.

Figure 10 be presented at urine separately, urine and NGAL mixture (urine+Ngal), urine and NGAL: the mixture of siderophore (urine+Ngal:Si), independent blood plasma, independent buffer solution, buffer solution and NGAL mixture (buffer solution+Ngal), buffer solution and NGAL: the mixture of siderophore (buffer solution+Ngal:Sid (1)), second buffer solution and NGAL: the mixture of siderophore (buffer solution+Ngal:Sid (2)) and wherein siderophore by iron saturated the 3rd buffer solution and NGAL: the siderophore mixture (detects in the buffer solution+Ngal:Sid:Fe) ⁵⁵The combination of Fe.

Figure 11 A shows from healthy study subject (Normal), or suffer from the study subject of acute tubular necrosis (ATN) or chronic renal failure (CRF), or from suffering from hepatic cirrhosis, hemochromatosis or cancer of pancreas but lack the Western blotting of NGAL albumen people's urine sample that the study subject (other) of kidney diagnosis obtains.The anti-mouse NGAL of monoclonal anti-human NGAL (Mo) and polyclone (Po) antibody recognition goes out reorganization and natural people NGAL and NGAL standard items.

Figure 11 B shows from healthy study subject (Normal), or suffers from the study subject of acute tubular necrosis (ATN) or chronic renal failure (CRF), or from suffering from hepatic cirrhosis, hemochromatosis or pancreas adenocarcinoma but lack the Western blotting of NGAL albumen the human serum sample that the study subject (other) of kidney diagnosis obtains, than the NGAL standard items.

Figure 11 C show healthy study subject (Normal) suffer from acute tubular necrosis (ATN) or the urine of the study subject of chronic renal failure (CRF) in the Quantitative Comparison of NGAL protein level.ATN further is subdivided into non-pyemia and pyemia group.

Figure 11 D show healthy study subject (Normal) suffer from acute tubular necrosis (ATN) or the serum of the study subject of chronic renal failure (CRF) in the Quantitative Comparison of NGAL protein level.ATN further is subdivided into non-pyemia and pyemia group.

Figure 11 E shows the Western blotting of NGAL in the ATN urine with control mice false processing damage, together with 50,5 and the standard items of 1ng NGAL albumen.

Figure 12 A shows the immunohistochemical staining of NGAL in healthy people's kidney (Normal).

Figure 12 B shows the immunohistochemical staining of NGAL in healthy people's kidney.

Figure 12 C shows the immunohistochemical staining of NGAL in healthy people's kidney.

Figure 12 D shows the immunohistochemical staining of the increase of NGAL in people's kidney of suffering from the ischaemic ATN (ischaemic ATN) that is caused by pyemia.

Figure 12 E shows the immunohistochemical staining of the increase of NGAL in people's kidney of suffering from the ischaemic ATN that causes because of the hypovolemia of vomitting and diarrhoea causes.

Figure 12 F shows the immunohistochemical staining of the increase of NGAL in people's kidney of suffering from the ischaemic ATN that causes because of heart failure.

Figure 12 G shows the immunohistochemical staining of the increase of NGAL in the people's kidney suffer from the toxic ATN (toxic ATN) that the renal toxicity that causes because of diphosphonate (bisphosphonate) compound causes.

Figure 12 H shows the immunohistochemical staining of the increase of NGAL in the people's kidney suffer from the toxic ATN that renal toxicity that excuse spore rhzomorph toxicity causes causes.

Figure 12 I shows the immunohistochemical staining of the increase of NGAL in the people's kidney suffer from the toxic ATN that the renal toxicity that causes because of hemoglobinuria causes.

Figure 12 J shows the immunohistochemical staining of NGAL in people's kidney of suffering from renal glomerular disease, and wherein NGAL faintly expresses in crescent (cresents).

Figure 12 K shows the immunohistochemical staining of NGAL in people's kidney of suffering from renal glomerular disease, and wherein NGAL faintly expresses in the near-end renal tubule that ephrosis becomes.

Figure 13 A shows the Histological section of (ATN+Ngal) kidney of the ATN-damage that false processing (Control), ischemic (ATN) and NGAL handle.Being lost among the ATN of renal tubular cell nuclear observed, but do not observe in the section (last figure) of contrast or ATN+Ngal, the observation of the tube in the same renal tubule that also has cortex (middle figure) and medullary substance (figure below).The NGAL preliminary treatment causes the preservation of cortex renal tubule, except residual cortex-tube of medullary substance.

Figure 13 B shows the Histological section of (ATN+Ngal) kidney of the ATN-damage that ischaemic (ATN) and NGAL handle, wherein PAS dye highlight the chamber tube and protected the cortex renal tubule by the NGAL preliminary treatment.

Figure 13 C shows the Jablonski scoring of (ATN+Ngal) kidney of the ATN-damage that false processing (Sham), ischemic (ATN) and NGAL handle, to confirm that NGAL has protected ischaemic cortex.

N-cadherin dyeing in Figure 14 A demonstration kidney segment is almost abolished (ATN) by ischemia reperfusion, but recovers (ATN+Ngal) again when giving NGAL.

Figure 14 B demonstration total length N-cadherin level is protected by NGAL treatment (ATN+Ngal) with respect to ischaemic (ATN); shown in N-cadherin fragment (arrow) in the animal of ischemia reperfusion and false processing, but it is suppressed in the animal that NGAL handles.GAPDH is the load tester.

Figure 14 C shows that the renal tubule that has TUNEL-positive apoptotic cells (fluorescence) in the ischemia reperfusion mouse (I/R) reduces by NGAL preliminary treatment (I/R+Ngal).Toprol is the nuclear counterstain.

Figure 14 D shows (the quantitative percentage analysis of+Ngal) the renal tubule that comprises apoptotic nucleus of the contrast (false), ischemia reperfusion (I/R) of false processing and the ischemia reperfusion mouse of handling with NGAL.

Figure 14 E shows the Western blotting that Heme oxygenase-1 (HO-1) is expressed in the kidney (ATN+Ngal) that the NGAL of (ATN) of false processing (Sham), ischemia reperfusion or ischemia reperfusion handles.Comprise the HO-1 (HO-1) of reorganization and rat cortex as a comparison.GAPDH is the load tester.

Figure 15 shows the Western blotting that the NGAL albumin removes in the mouse blood and urine behind the intraperitoneal injection 100 μ gNGAL.

Figure 16 A shows that (Alexa568) NGAL of the mark of fluorescence is positioned in the big vesica of near-end renal tubule (figure below) and not in glomerulus or medullary substance (little last figure).The dyestuff of unconjugated fluorescence is the mark kidney not.

Figure 16 B shows that Alexa568-NGAL and FITC-glucan are arranged in the S1 and the S2 district of near-end renal tubule jointly.

Figure 16 C shows ¹²⁵The SDS-PAGE of I-NGAL separates, wherein inject back 1 hour and 5 hours discovery in kidney total length with the NGAL 14kDa fragment.

Figure 16 D shows the intraperitoneal injection load ⁵⁵The radioautogram of 1 hour metanephros of siderophore-NGAL of Fe load.Radioactive decay is found in cortex and is relevant with the apical area of near-end renal tubular cell.

Figure 16 E shows the intraperitoneal injection load ⁵⁵The radioautogram of 1 hour metanephros of siderophore-NGAL of Fe load is not found radioactivity in the medullary substance.

Figure 17 A demonstration stands 30 minutes plasma creatinine in the ischemic mouse.First figure show when local ischemic 15 minutes before or administration in 1 hour after the ischaemic, be derived from the XL-1 bacterium holo-NGAL of (comprising siderophore) (〉=1 μ g) and protected renal function.Yet, when later administration NGAL does not just have effect.Second figure shows that the apo-NGAL minimally that is derived from BL-21 bacterium (no siderophore) is effective, but works as enterochelin in the load, and this albumen just has protective effect.The siderophore (apo-NGAL:Sid:Fe) of iron-free (apo-NGAL:Sid) and load iron all has protective effect.By contrast, the compound of load gallium (apo-NGAL:Sid:Ga) does not have effect, and is the same with the DFO or the free iron carrier (Sid) of single dose.RBP ELISA (RBP) a kind ofly also is filtered and is not had effect by the re-absorbed NGAL of near-end renal tubule.

Figure 17 B shows the immunoprecipitation preparation of NGAL.NGAL:Sid comprises enterochelin, but iron content not.NGAL:Sid:Fe comprises siderophore and iron.

Exist iron in conjunction with co-factor in Figure 18 A demonstration urine.Buffer solution mixes with 55Fe (no albumen), mixes with apo-NGAL, apo-NGAL+ siderophore or apo-NGAL+ siderophore+unlabelled iron.Through a series of cleaning, ⁵⁵Fe is preserved by the apo-NGAL+ siderophore but is not preserved by apo-NGAL or the apo-NGAL that connects the saturated siderophore of iron, proves that unsaturated siderophore is that NGAL preserves ⁵⁵Fe is needed.

Figure 18 B show when urine (＜3,000Da) with ⁵⁵Fe or mix with apo-NGAL by indication, cleans 3 times on the filter of 10KDa then, and apo-NGAL+ urine has been preserved ⁵⁵Fe. ⁵⁵The preservation of Fe is added into excessive ironic citrate (Fe) and blocks.Activity is also blocked by the saturated enterochelin of iron (Sid:Fe).

Figure 19 shows the renal biopsy that carries out in 1 hour from (Fig. 2 and 3) kidney transplant of relevant (figure 0 and 1) of live body or corpse.Section NGAL antibody staining.NGAL is expressed in experience to be increased significantly than the corpse group of lasting arrangement portion ischemic stage.

Figure 20 show from live body relevant (LRD, n=4) or corpse (CAD, n=4) Western blotting of the urine sample that obtains in 2 hours of kidney transplant is sought and visited with NGAL antibody.Do not exist NGAL to express before the operation in the urine.Compare LRD group NGAL in the CAD group and express increase significantly.

Figure 21 is presented among LRD and the CAD quantitative to the NGAL of urine by Western blotting, and it is presented to express significantly among the CAD increases.

Figure 22 shows that CAD transplants the relation of the NGAL and the cold ischemia time of the back urine that obtained in 2 hours.Degree and ischaemic time correlation that the NGAL of urine expresses.

Figure 23 shows that CAD transplants the relation of NGAL with the serum creatinine peak value of operation back measurement in 2-4 days of the back urine that obtained in 2 hours.The degree that the NGAL of urine expresses is relevant with the serum creatinine peak value.

Figure 24 shows the calibration curve of NGAL ELISA, and its linear relation obtains from 10 independent standard curves.

Figure 25 shows the measurement result (n=10) of serum N GAL among a series of patients that suffer from ARF after Surgicenter pulmonary shunt art (CPB).

Figure 26 shows that (square) among patient CBP of a series of ARF of suffering from (n=10) do not have ill (rhombus) (n=30) comparison of value after mean value ± SD of serum N GAL and those experience operations.

Figure 27 shows behind a series of CPB to suffer from the measurement result (n=11) of urinating NGAL among patient CBP of ARF.

Figure 28 shows that (rhombus) among patient CBP of a series of ARF of suffering from (n=11) do not have ill (square) (n=30) comparison of value after urinating the mean value ± SD of NGAL level and those experience operations.

Figure 29 shows behind the CPB 2 hours urine NGAL levels and CPB time relation.

Detailed Description Of The Invention

Definition：

As used herein, the meaning of term " organ " is minute voltinism biological structure that comprises cell and tissue, these cells and be organized in organism for example mammal, comprise among the people performance certain or some function. Representational organ comprises, but is not restricted to, kidney, liver, heart, bone, cartilage, skin, lung, blood vessel, bladder, cervix, stomach, intestines, pancreas, small intestine, colon, pancreas and brain, and their part or one section.

As used herein, term " injury of kidney " or " kidney disease " should comprise acute (including but not limited to ischaemic, ischemia reperfusion, nephrotoxicity, shock, apoplexy, pyemia, wound, infection, inflammation) or chronic (including but not limited to hypertension, diabetes, heart failure, lupus, infection, inflammation) injury of kidney or illness.

Described phrase " pharmaceutically useful ", " acceptable on the pharmacology " and " physiologically acceptable " refer to when delivering medicine to animal, and example is the people for example, does not produce molecular entity disadvantageous, irritated or other unsuitable reaction and composition.

Phrase " pharmaceutically useful carrier " meaning of here using is material, composition or carrier, for example liquid or solid filler, diluent, excipient, solvent or encapsulating material, comprise delivery or transmit compound of the present invention from the part of an organ or health, arrive the part of another organ or health, and do not affect its biological effect. Every kind of carrier should be " acceptable ", and the meaning is compatible with other composition of described composition, and harmless to study subject. Some examples that can be used as the material of pharmaceutically suitable carrier comprise, but be not restricted to: any He all solvents, decentralized medium, dressing, surfactant, antioxidant, anticorrisive agent are (for example, antibacterial agent, antifungal agent), isotonic agent, absorption delay agent, salt, anticorrisive agent, medicine, medicine stabilizing agent, gel, bond, excipient, disintegrant, lubricant, sweetener, flavor enhancement, dyestuff, the material of this class and its combination will be known to those of ordinary skill in the art. Except the carrier of considering any routine and NGAL, siderophore or its compound or other optional activating agent or composition were in inconsistent scope, its application in treatment or pharmaceutical composition also was considered.

Phrase " treatment effective dose " refers to NGAL, siderophore and its mixture or other optional activating agent or the composition of this tittle, effectively produces favourable outcome, especially with regard to described herein the experimenter for example with regard to the treatment among animal or the patient. This tittle at first can be by consulting disclosed document, by carrying out experiment in vitro or judging by carry out metabolism research in the animal used as test of health. Before clinical use, it is useful carrying out deterministic research in animal model, typically in the animal model of accepting extensively of the disease specific that will treat. The typical animal model that is used for definite embodiment is economic rodent and the murine model that uses, and, particularly be accepted widely as the prediction of clinical value because of resulting result.

Ground relevant with NGAL term " derivative ", refer to NGAL compound, material, inhibitor or the stimulant of chemical modification, it still keeps the required effect to characteristics such as ischaemic, renal tubular necrosis, renal toxicity, ischemia reperfusion damages. These derivatives can be included in addition on the parent molecule, elimination or replace one or more chemical groups. These groups can comprise, but are not restricted to, and element is hydrogen for example, halogen, or molecular radical methyl for example. This derivative can be by any method preparation known to those skilled in the art. The characteristic of this analog derivative can be by any to those skilled in the art's method test that describe or known, to test their required characteristics.

Term " analog " comprises the as understood by those skilled in the art suitable thing of structure or dummy.

" patient ", " recipient " or " study subject " mean animal or organism, for example the animal of homoiothermy or organism. Illustrative animal comprises without limitation, mammal, for example, people, non-human primates, pig, cat, dog, rodent, horse, ox, sheep, goat and milk cow. The present invention especially is fit to people patient and study subject.

" inhibitor " means in cell or is derived from the albumen of cell or the translation of activity, function, production, secretion or the mRNA of biologic artifact produces compound, material or the medicament of any reduction of surveying.

(transplanted) and (grafted) organ of transplanting with transplanting used herein is relevant, local ischemic damage or ischemia reperfusion damage " alleviating (reduction) " refers to any measurable minimizing to organ injury, dwindles or reverses, described organ (i) is saved, for example, in preserving liquid or corpse, or (ii) transplanted (transplanted) or transplant (grafted) in patient. Similarly, " alleviate (reducing) " and refer to any measurable to organ injury, damage or injury minimizing or dwindle or suppress fully, this organ is saved, transplants (transplanted) or transplants (grafted) in patient.

Word used herein " a kind of (a) " and " a kind of (an) " refer to " one or more ". More specifically, " comprising ", " having " or the use in the claims of other open language, it requires combination or method to use object, uses expression " one or more objects " in desired method or combination.

The invention provides NGAL or NGAL, in treatment, alleviate or improves that for example the local ischemic damage, ischemia reperfusion of kidney damage and the method for the damage that toxin causes to organ. The present invention also provides the application of NGAL in the method for the treatment of, alleviate or improve acute injury of kidney (including but not limited to shock, wound, apoplexy, pyemia, infection, inflammation, calculus and surgery) and Chronic Renal Impairment (including but not limited to hypertension, diabetes, heart failure, lupus, inflammation, glomerulonephritis and interstitial nephritis). According to the present invention, and do not wish to be subject in the situation of theory constraint, it is dead to have found that the NGAL administration affects renal tubular cell, so that limited the death of the renal tubular cell of apoptosis, that is, and apoptosis, and strengthened epithelium and formed again, that is, the recovery of vigor cell is arranged after the ischaemic in the kidney, it comprises dedifferenting and the reconstruction of propagation and epithelium phenotype of vigor cell after the ischemia reperfusion damage. The NGAL administration also demonstrates the increase that reduces creatinine levels in the serum and plasma behind the local ischemic damage.

Human neutrophil genatinase associated lipocalin is covalently bound to the 25kDa albumen of the gelatinase that is derived from human neutrophils, expresses low-down level in some people's tissue, and these tissues comprise kidney, tracheae, lung, stomach and colon. The expression of NGAL is induced by the epithelial tissue that stimulates significantly and is secreted. For example, NGAL concentration in the phlegm of suffering from asthma or COPD object, and raises in the bronchus fluid from the emphysematose lung in the serum of suffering from the acute bacterial infection patient. NGAL also is one of the highest gene of inducing in the early stage kidney behind the local ischemic damage. These data derive from genetic chip to the analysis of mRNA, the synthetic NGAL of the kidney of hint damage. Other research has shown that NGAL can find to have the level of rising in the serum of suffering from the inflammatory disease patient. Therefore we have estimated the kidney disease incidence that NGAL expresses in people ATN of relative chronic form. NGAL occurs in high expressed and the near-end renal tubule in bioptic people's kidney in clinical definite ATN. Although amount is few, NGAL also expresses in the kidney of the chronic kidney diseases of suffering from several forms. The ATN mouse model that the perfusion of ischaemic/is again induced is also with unusual high level expression NGAL. The NGAL of injection Microgram provides the significant protection of antagonism ATN, and it is measured by plasma creatinine and nephridial tissue. The protective effect of kidney is to be sent to the near-end renal tubule by the NGAL albumen that will comprise Siderophores From Bacteria to realize.

According to the present invention, comprising the pharmaceutical composition of NGAL or the preparation of preparation is known to those skilled in the art, by Remington ' s Pharmaceutical Sciences, and 18th Ed., Mack Printing Company, 1990 as illustration. And, for animal (for example people) administration, should be appreciated that preparation should satisfy required aseptic, the pyrogenicity of FDA Office of Biological Standards, common security and purity rubric.

In the past, the effect of NGAL was unclear; Yet NGAL has been confirmed to be a kind of at the iron transport protein of kidney between the emergence period at present. Although it is to siderophore with to the high-affinity of iron, NGAL can be sent to cytoplasm with iron. The possible mechanism that NGAL transmits iron is endocytosis. The NGAL albumen of fluorescence is by endocytosis, and this transportation is by 4 ℃ temperature retardance. Stop iron to absorb because suppress the medicament of acidifying, it is necessary that the acidifying of these vesicles can discharge the iron of NGAL. And, NGAL is added in the cytoplasm in the cell of the iron indicator of expressing fluorescence, in the indicator change of ironwork of having expressed moderate stimulation, it confirms that NGAL can be used as the iron donor. Significantly, the approach of NGAL endocytosis is different from the approach of full transferrins effect in the cell of cultivating. Under steady shape, full transferrins be transported to rab-5 or rab-7 recirculation vesicles (rab5 and rab7 be respectively early stage and late period endosome sign), and NGAL is transported to the endosome in late period, and fraction is transferred in the lysosome. These data confirm to comprise siderophore as NGAL, and it can be served in iron transmission approach.

NGAL effect in vivo can be different from its pharmacological effect because crucial siderophore external be a kind of bacterial product. Many analyses have shown that endogenic low-molecular-weight co-factor is to the defeated possibility of Railway transportation. This comprises citrate and related compound, is also included within the defeated activity of Railway transportation in the scope that molecular weight is 1000Da. For determining whether this co-factor also can be in kidney, and we will mix with urine sample from the apo-NGAL that the BL21 bacterium obtains. Although urine itself can not catch⁵⁵Fe, and the apo-NGAL that dilutes in the salting liquid can not catch⁵⁵Fe, but the NGAL of dilution allows to keep in the urine⁵⁵Fe. This finds to have co-factor in the hint urine, and it allows the interaction between the NGAL-iron.

In embodiments of the invention, ectogenic administration NGAL can improve the structural damage that the ischemia reperfusion damage causes. Both can both weaken necrosis and apoptosis significantly. Be subject in the situation of theory constraint not wishing, NGAL mechanism of apoptosis inhibit in ischemic conditions comprises and the similar anti-apoptotic effect of Heme oxygenase 1 (HO-1), it can promote the transportation of extracellular iron, thereby the oxidant stress that iron orders about in the restrictive cell inner room (people such as C.D.Ferris, 1999, Nat.Cell Biol., 3:152-157). As any carrier of different siderophores, NGAL also impels and removes excessive intracellular iron, thus the apoptosis of the renal tubular cell death of restriction ischemia reperfusion damage rear oxidation agent mediation. About necrosis, the response of ischemia reperfusion damage metanephros can occur with two stage process, i.e. the initiation of apoptosis then is downright bad cell death. Except the oxidative stress of restriction iron-mediation, the secondary necrosis aspect that can effectively prevent said process by the NGAL apoptosis inhibit. Process by apoptosis inhibit, NGAL can be in multiple kidney disease effectively, it is that apoptosis with increasing is relevant that these kidney diseases are known, and includes but not limited to ischaemic, ischemia reperfusion, the nephrotoxin, POLYCYSTIC KIDNEY DISEASE, blocks, inflammation and renal glomerular disease.

NGAL albumen is comprised of 8 strands of β chains, thereby generates β-barrel-shaped shape thing or calyx shape thing (calyx). This calyx shape thing combination is also transported the low-molecular-weight chemicals, comprises the siderophore of finding in the urine and/or bacterium generates. To the research of the best evidence of the ligand binding characteristic of NGAL from crystallography, it has confirmed the Siderophores From Bacteria (enterochelin) in β-barrel-shaped shape thing. In conjunction with siderophore, and siderophore is with high-affinity (10 with high-affinity (0.4nM) for NGAL^-49M) catch iron. Albumen: siderophore: the stoichiometry of iron is 1: 1: 1, and it is by confirming in conjunction with learning research and x-radiocrystallgraphy. Iron in the siderophore load, NGAL compound are namely donated iron to matter between embryo and clone external, and work as siderophore not iron content, NGAL compound and iron chelating. NGAL is by many cell type endocytosis, and is transported to the late endosome chamber different from the transferrins chamber (late endosomal compartment). The donations of iron occur in the endosome chamber. Because NGAL is first mammalian proteins in conjunction with Siderophores From Bacteria of finding, it is also referred to as siderocalin.

Siderophore (siderophores) is the small protein molecule of removing iron from environment, and it has low-molecular-weight, and scope is from about 500 to about 1000MW. Siderophore can with the ferric iron chelating. The infringement of iron catalysis is considered to the earliest one of event in the damage metanephros dysfunction that ischaemic, ischemia reperfusion or toxin cause, and probably the commitment at other organ injury is important, and these organs comprise heart and liver. The infringement that iron and siderophore chelating can be alleviated these organs.

Siderophore can be the naturally occurring product that synthesizes or collect from Bacteria Culture, and can be purchased. When being mixed together in normally used multiple different buffer solutions under physiological conditions, siderophore is absorbed by NGAL consumingly, and these buffer solutions comprise 10mM Tris or PBS. Typically, siderophore can excessively join in the NGAL albumen of known amount. The NGAL molecule will be in conjunction with the siderophore molecule, thereby each compound will comprise each kind of a part. Clean NGAL: 1: 1 compound that siderophore is removes excessive unconjugated siderophore molecule, then just it further can be processed to implement the present invention. Perhaps, the siderophore of equimolar amounts and NGAL molecule can merge and hatch combination. Consider that the siderophore that is used for external source of the present invention comprises, but be not restricted to enterochelin, carboxyl mycobactin (carboxymycobactin), aminochelin, Deferoxamine, aerobactin, arthrobactin, schizokinen, foroxymithine, pseudobactin, neoenactin, photobactin, ferrichrome, hemin, achromobactin, achromobactin, rhizobactin and other bacterial product, citrate is with the analog that synthesizes and can use that the organic chemistry method prepares and part and other material. Endogenic siderophore also can be compound to NGAL in vivo, and it will be described in the embodiment of using method.

The inventive method provides the advantage of some to patient. The acute renal failure that is secondary to local ischemic damage still is a common problem, and it has confined and not satisfied treatment and selects. Identification suppresses, reduces or antagonism renal tubular cell dead (necrosis/apoptosis) and/or the factor that strengthens convalescence (including dedifferenting and the reconstruction of propagation and epithelium phenotype of vigor renal tubular cell) can be used as new treatment and select. According to the present invention, NGAL, separately and with siderophore, all advantageously demonstrate above-mentioned desirable and characteristic cytoprotection. Verified its renal tubular cell by the restriction apoptosis of the NGAL of external source administration is dead and strengthen the combination that epithelium forms again, has limited morphology and the function result of ischemia reperfusion damage in the mouse model.

Embodiment of the present invention, it is used for the treatment of, alleviates or improves the damage that toxin causes, toxin and/or poisonous medicine can comprise antibiotic, antiinflammatory, antifungal agent, radioactivity developer, medicine, chemotherapeutant, test with medicine (test drug), pharmaceutical substance, or natural, commercial and industrialized chemicals and inorganic matter.The concrete toxin and the nephrotoxin include, but are not limited to, the cancer chemotherapeutics agent, for example cis-platinum, mitomycin, cyclophosphamide, isosfamide and methotrexate, antibiotic comprises gentamicin, vancomycin and tobramycin, antifungal agent, anphotericin for example, antiinflammatory, NSAID for example, immunodepressant, for example cyclosporin and tacrolimus, other medicine, commercial and industrialized chemicals, for example arsenic, mercury, bismuth and lead of hydro carbons, chlorocarbon and fluorocarbon and inorganic matter for example.Other kidney poisons compound can comprise aminoglycoside, phosphine formic acid, pentamidinum, vancomycin, neomycin, nitrous oxide, isoflurane, kanamycin and cyclophosphamide.

According to embodiment of the present invention, as described herein, NGAL can be at ischaemic, ischemia reperfusion damage, organ transplant, ATN, give toxin etc. before, between (with it simultaneously) or administration afterwards.More specifically, NGAL can administration in about 30 minutes to about 90 minutes before organ transplant.Also can consider the scope external administration of said composition at 30 to 90 minutes.

The present invention also comprises a kind of method, and it gives patient about 1 NGAL to about 200mg/kg body weight, more typically from about 1 to about 100mg/kg body weight.The amount that gives patient NGAL can change or exceed the scope of being given.As discussed here, the amount that gives patient NGAL can change.

Composition of the present invention, for example medicine or pharmaceutical composition, the level that can typically comprise NGAL and/or siderophore in composition is at least about 10 micrograms/100 microlitres, and more typically in composition at least about 100 micrograms/100 microlitres.

Composition of the present invention can comprise dissimilar pharmaceutically useful carriers, whether depends on them with solid, liquid or spray form administration, with whether their need as inject be aseptic for this method of administration.The present invention can following mode administration: intravenous, intracutaneous, intra-arterial, in the peritonaeum, in the damage (intralesionally), encephalic, in the joint, in the prostate (intraprostaticaly), in the pleura (intrapleurally), in the tracheae, in the nose, in the vitreum, in the vagina, in the rectum, locality (topically), in the knurl, intramuscular is in the peritonaeum, subcutaneous, under the conjunctiva, in the bladder (intravesicularlly), mucous membrane ground is in the pericardium, in the urethra, intraocular (intraocularally), oral, locality (topically), (locally) partly sucks (for example, spray sucks), injection, transfusion, conduit is passed through in transfusion continuously, lavation, will be in emulsion or lipid composition (for example, liposome) medicine in directly embathes regional perfusion's method of target cell, or by other method or any aforesaid compound mode administration, it will be known to those of ordinary skill in the art.

The present composition give animal patient actual dose can by health with physiological factor for example body weight, by the severity of the illness of being treated, treat the type of disease, treatment intervention before or the while, patient spontaneous disease and method of administration definite.In any case, the doctor who is responsible for administration will determine NGAL, siderophore and its mixture and the concentration of other optional activating agent in composition and the suitable dose that is used for single patient.

In the embodiment of determining, pharmaceutical composition can comprise that for example, the another kind at least about 0.1% is chosen activating agent or composition wantonly.In other embodiments, described activating agent or composition can account for pharmaceutical units about 2% to about 75% between, more typically between about 25% to about 60%, and any scope of drawing thus.In other unrestriced embodiment, activating agent or dose of components can account for from about 1 microgram/kg body weight to about 500 milligrams/kg body weight, more typically from about 5mg/kg body weight to about 100mg/kg body weight.

In some instances, described composition can comprise the oxidation that different antioxidants delays one or more compositions.In addition, the effect of prophylaxis of microbial can produce by for example different antibacteriums of preservative and antifungal agent, include but not limited to parabens (for example, methyl p-hydroxybenzoate, nipasol), anesin, phenol, sorbic acid, thimerosal or its combination.

Described composition can be mixed with composition with free alkali, neutral or salt form.Pharmaceutically useful salt comprises acid-addition salts, and for example, those free amine groups by protein compositions form, or are for example formed by inorganic acid, hydrochloric acid or phosphoric acid, or organic acid such as acetate, oxalic acid, tartaric acid or mandelic acid.The salt that is formed by free carboxy also for example can be derived from inorganic base, sodium hydroxide, potassium hydroxide, ammonium hydroxide, slaked lime or iron hydroxide, or organic base such as isopropylamine, trimethylamine, histidine or procaine.

At composition is in the embodiment of liquid form, carrier can be solvent or dispersion medium comprises, but is not restricted to water, ethanol, polyalcohol (for example glycerine, propane diols, liquid macrogol etc.), and lipid (for example, triglyceride, vegetable oil, liposome) and its combination.Suitable flowability can keep by the following method, for example, by using as the coating of lecithin, by carrier for example in liquid polyol or the lipid dispersion keep required particle size, by use surfactant such as L-HPC, or the combination of these methods.Typically comprise isotonic agent under many circumstances, for example sugar, sodium chloride or its combination.

In other embodiments of the present invention, can use eye drops, nose solution or spray, aerosol or inhalant.These compositions usually are designed to and target tissue type fit.In a non-restrictive example, nose solution is generally the aqueous solution, and it is designed to deliver medicine to nasal meatus with drops or spray.Preparation nose solution makes them similar to nasal discharge aspect a lot, thereby keeps normal ciliary action.Therefore, in embodiment typically, the nose solution of water be generally etc. ooze or slight buffering to keep pH to be about 5.5 to about 6.5.In addition, antimicrobial preservative is similar to and uses in prepared product at eye, the medicine, or suitable medicine stabilizing agent, if desired, can be included in the preparation.For example, the commercial nasal preparation of known difference contains medicine, for example antibiotic or antihistaminic.

In the embodiment of determining, described composition is produced to be used for for example oral medication administration of this approach.In these embodiments, described solid composite for example can comprise, solution, suspension, emulsion, tablet, pill, capsule are (for example, hard or soft shell capsule), sustained release preparation, containing composition, lozenge, elixir, suspension, syrup, wafer (wafers), or its combination.Oral composition can directly mix with food to be taken.The typical carrier of oral administration comprises inert diluent, assimilable edible carrier or its combination.In others of the present invention, Orally administered composition can be prepared as syrup or elixir, and for example can comprise, at least a optional activating agent or composition, sweetener, preservative, fumet, dyestuff, preservative, or its combination.In other embodiment, Orally administered composition can comprise one or more bonds, excipient, disintegrant, lubricant, fumet or its combination.

In the embodiment of determining, composition can comprise one or more following materials: bond, for example, tragacanth, gum Arabic, corn starch, gelatin or its combination, excipient, for example, Dicalcium Phosphate, mannitol, lactose, starch, dolomol, saccharin sodium, cellulose, magnesium carbonate or its combination, disintegrant, for example, corn starch, potato starch, alginic acid or its combination, lubricant, for example, dolomol, sweetener, for example, sucrose, lactose, asccharin or its combination, fumet, for example peppermint, wintergreen, cherry flavor, orange spices etc., or above-mentioned combination.When dosage unit form is a capsule, except the material of the above-mentioned type, it also can comprise carrier, for example liquid-carrier.Other different material can be sugar-coat or other changes the material of dosage unit physical form.For example, tablet, pill or capsule can coat shellac, sugar or both.

Other preparation that is fit to other administering mode comprises suppository.Suppository is the sharp amount form of the solid of Different Weight and shape, mixes medicine usually, is used to insert rectum, vagina or urethra.After the insertion, the suppository deliquescing is melted or is dissolved in the liquid in the chamber.Usually for suppository, traditional carrier can comprise, for example, and poly alkylene glycol, triglyceride or its combination.In the embodiment of determining, suppository can form from mixture, and this mixture for example comprises, about 0.5% activating agent or composition and typically about 1% to about 2% to about 10% scope.

The solution of sterile injectable is by severally above-namedly becoming the reactive compound of aequum phase-splitting mix to prepare, just as required, then be filtration sterilization with other in appropriate solvent.Usually, dispersion prepares by different sterilized reactive compounds is mixed in the aseptic carrier that comprises basic dispersion medium and/or other composition.Prepare at sterile powder under the situation of sterile injectable solution, suspension or emulsion, typical preparation method is vacuum drying or Freeze Drying Technique, and it produces the pulvis of reactive compound and the composition of any additional needs of its liquid medium that is derived from filtration sterilization before.This liquid medium should be suitable buffering if desired, and this liquid diluent etc. is oozed, and injects with sufficient salt or glucose then.Prepare the high concentration composition that is used for directly injection and also be considered, wherein DMSO uses anticipation can cause permeating extremely rapidly as solvent, thereby the reactive compound of transmission high concentration is to the zonule.

Described composition should be stable under the condition of preparation and storage, and avoids the contamination by micro effect, for example bacterium and fungi.Being to be understood that exotoxin pollutes answers bottom line to remain on level of security, for example, is lower than 0.5ng/mg albumen.

Experimental program

The present invention will better be understood by its example of illustrating use and effect.Experimental program described below will be in embodiment subsequently as a reference.

1. the people and the mouse NGAL of expression, purification and radioactive label reorganization:

The NGALcDNA of total length mouse is cloned on the pGEX expression vector, in bacterium, be expressed as the fusion that contains glutathione S-transferase (GST), and use glutathione-agarose column (Amersham) purifying, fibrin ferment cracking then, as in the past by what follows: people such as Bundgaard J, Biochem Biophys Res Commun 202:1468-1475,1994; People such as Yang J, Mol Cell 10:1045-1056,2002; With people such as Del Rio M, J Am Soc Nephrol 15:41-51,2004.The Detoxi-Gel endotoxin that use is recommended by the producer is removed post (Pierce) and is made the NGAL of purifying no endotoxic.Albumen is analyzed by SDS-PAGE, and subsequently NGAL is carried out Coomassie blue stain or carries out Western blotting with polyclonal antibody, as people such as Mishra, J Am SocNephrol 14:2534-2543,2003 is described.Protein concentration uses the Bradford method of testing to determine.Detect the single pure polypeptide of anticipation size, as shown in Figure 1.

2. express and the people and the mouse NGAL of the reorganization of purifying

The people of reorganization and mouse GST-NGAL express in the BL21 of Escherichia coli (Stratagene) bacterial strain or XL1-Blue, use the iron sulfate (50 micromoles, Sigma-Aldrich Co.) that adds.NGAL uses glutathione agarose 4B pearl (beads) (Amersham Biosciences) to separate, by fibrin ferment cracking wash-out (Sigma-Aldrich Co.; St.Louis MO), detects by gel filtration (Superdex75, SMART System, Amersham Biosciences) purification and with coomassie gel (Biorad) then.Not iron content or the saturated enterochelin of iron in the NGAL load in BL-21 source, a kind of siderophore (EMC Microcollections) uses 5 times of molar excess.Unconjugated siderophore (0.7KD) cleans (Microcon YM-10) with PBS and removes.Be the preparation load ⁵⁵The NGAL of Fe or gallium (Ga), we cultivate the enterochelin NGAL of iron-free and wait mole ⁵⁵Fe (18mCi) or Ga are at NaCl (150mM) Hepes (20mM; PH7.4) compound in, and clean this compound 3 times (10K filter).Use iodobeads (Pierce) to utilize ¹²⁵I mark NGAL is not impregnated in ¹²⁵I removes with gel filtration (PD-10 post), then is that (7kDa is by film, Pierce) with antagonism PBS in a large amount of dialysis.According to the producer, Alexa-568 and fluorescein isothiocyanate (Molecular Probes) are attached to NGAL, then dialysis in a large number.Protein concentration is compared definite by the coomassie gel with the bovine serum albumin(BSA) standard.

3.NGAL injection:

The no endotoxic NGAL of purifying is by delivering medicine to mouse in tail vein injection intravenous administration, subcutaneous administration or the peritonaeum.In initial research, NGAL (50,100 or 250 μ g are in 250 μ g/100 μ l solution) the processing animal with 3 kinds of variable concentrations clamped bilateral renal arteries 30 minutes after 1 hour, and adverse current detected after 24 hours.When relatively using equal volume (100 μ l) salt solution pre-treated animal, the group that only gives 250 μ gNGAL demonstrates protection significantly to tubular injury and azotemia.All use the NGAL of 250 μ g dosage to carry out in all researchs subsequently of this report.Compare 5 different animal groups: non local ischemic control group (n=8), use the pretreated ischaemic control group of independent salt solution (n=8), before the arteria renalis clamps 1 hour with NGAL preliminary treatment (n=6), handle (n=6) with NGAL during the arteria renalis clamps, and the arteria renalis clamps 1 hour afterwards with NGAL processing (n=6).

4. human research:

The healthy volunteer suffers from patient's its NGAL protein level of analysis in urine and serum acute or that the chronic renal damage is depleted with being diagnosed as.Acute renal failure (ARF) is by doubling serum creatinine in 5 days and diagnose being less than.The supposition teiology that comprises pyemic ARF, it is by existing two kinds of following standard to determine at least: the evidence of local infection and heating or WBC counting raises in positive blood cultivation or lung, skin or the urinary tract.Some this patients need the blood pressure support.That the teiology of other ARF comprises is hemorrhage or in heart failure, the nephrotoxin or transplant the low blood pressure that the back ischaemic causes.Chronic renal failure (CRF) is defined as serum creatinine and is higher than 2mg/dl, but does not change during at least formerly two months.The teiology that CRF infers comprises obstructive uropathy, arteriosclerotic kidney and diabetes.From the sample that obtains both approvals of Institutional Review Boards and collect blood and urine, use blind method analysis then the patient that Columbia UniversityMedical Center and Kyoto University Hospital determine.

5.NGAL measurement:

Anti-mouse NGAL polyclonal antibody is cultivated in rabbit, goes up purification (referring to following) at the agarose 4 speed stream pearl posts (Amersham Biosciences) in conjunction with recombined small-mouse NGAL then, then at the pH2.5 wash-out.Monoclonal anti-human NGAL (AntibodyShop) also is used for detecting NGAL.People NGAL is discerned by monoclone antibody better and mouse NGAL is only discerned by the polyclone of affinity purification.

The human blood sample is collected in citrate, EDTA or the heparin at first, but because demonstrate similar NGAL immunocompetence by all these prepared products, human serum and mice plasma in example according to collection as described below.This sample see through 100KDa by filter (YM-100, Amicon) centrifugal, and use flow-through centrifugal to be used for Western blotting.In standing the patient of haemodialysis, sample was taked before dialysis immediately.The urine sample that low-speed centrifugal is fresh need not further concentrate then and uses.

6. pathology sample:

The pathology sample comprises ischaemic ATN (10 cases), toxic ATN (11 examples-(5) antibiotic, (2) zoledronate, (1) carbon platinum, (2) non-steroid antiinflammatory, (1) hemoglobinuria), and glomerulopathy (10 examples-comprise the cresentic glomerulonephritis of diabetes, anti-GBM, skeptophylaxis, IgA nephropathy, minimal change disease, focal segmental glomerulosclerosis), also comprise normal kidney (3 example).Formalin fixed, paraffin-embedded tissue are cut into slices (5 μ m) and are used microwave to carry out antigen retrieval (antigen retrieval) 30 minutes in citrate buffer (pH6.0).Endogenic peroxidase 5%H ₂O ₂Stop 30 minutes, then in 10% lowlenthal serum/1%BSA, stop.The anti-mouse NGAL (0.4 μ g/ml) that uses affinity purification spends the night at 4C, then use biotinylated goat anti-rabbit igg (1: 100, Vector) and Avidin-HRP, respectively 30 minutes.Wave carrier piece DAB/0.3%H ₂O ₂2.5 minute develop and use haematoxylin redyeing.Non-immune rabbit igg (0.4 μ g/ml; Vector) with comparing.

7.NGAL transportation

Be to detect the transmission of NGAL to kidney rNGAL (10 or 100 μ g), with Alexa568-NGAL (100 μ g), ¹²⁵I-NGAL (10 μ g, 2 * 10 ⁶Cpm) or load ⁵⁵Enterochelin-NGAL of Fe (10 μ g, 1 * 10 ⁶Cpm) be expelled to peritonaeum, to obtain blood, urine, kidney and liver sample.NGAL detects by Western blotting.Alexa-568 NGAL uses Laser Scanning Confocal Microscope (LSM Meta Detector) to detect, and the defeated light microscopy radioautography by scintillation counter and the kidney by the Epon embedding of the Railway transportation of NGAL mediation detects.Wave carrier piece is exposed to emulsion (Polyscience) week, develops with Microdol then, redyes with Toluidine indigo plant.For detecting the lysosome in the near-end renal tubule, mouse is 24 hours injected fluorescein glucan (46kD before injection Alexa 568 NGAL; 0.5mg; Sigma).LAMP1 (Santa Cruz) detects in the freezing microtome section of the fixing kidney of 4% paraformaldehyde.

8.ATN or the mouse model of ischaemic/reperfusion injury:

Male C57BL/6 mouse (20-25gr; Charles River) anaesthetizes by amobarbital (50mg/kg) in the peritonaeum and be positioned under the heating lamp on the heat pad, to keep 37 ℃ of body temps.Kidney exposes and right kidney or removal or its blood vessel stem of ligation (vascular pedicle) and ureter by laparotomy.The blood vessel stem of left kidney or two kidneys was clamped 30 minutes with microaneurysm folder (Kent Scientific) after the right nephrectomy.Produce reproducible injury of kidney but MIN lethality during the ischaemic.In this process, use PBS (0.5ml) peritonaeum of getting wet.This animal is sewed up with 5-0Nylon.Before the ischaemic 15 minutes or pour into 1-2 hour again after RBP ELISA (RBP), enterochelin or the desferroxamine mesylate (DFO) of salt solution, NGAL, load retinol is expelled to peritonaeum or subcutaneous.

Perfusion again through 6 or 24 hours, obtain heparinize blood plasma, urine and kidney sample, use Western blotting to come measuring N GAL (polyclone 1: 500), Heme oxygenase-1 (Stressgen, 1: 2000), E-cadherin (BD Transduction Labs, 1: 2000), N-cadherin (BD TransductionLabs, 1: 3000) and GAPDH (Chemicon International, 1: 3000).Blood plasma also is used for creatinine and blood urea nitrogen (BUN) colorimetric estimation.The sagittal section of kidney is fixed in 4% formalin, or snap frozen is to be used for mRNA and analysis of protein.(fluorescein-TUNEL, Roche) dyeing is to be used for apoptotic cells nuclear or total cell nucleus (Toprol, Molecular Probes) is analyzed with hematoxylin-eosin or original position kit in paraffin-embedded section (5 μ m).For analysis of cell proliferation, BrdU was expelled to peritonaeum in preceding 1 hour in execution, and according to the producer, freezing microtome section dyes with anti-BrdU (Roche).

For some research, allow mouse in the cage of heating, to recover, and regularly collection obtain urine.After date when difference is poured into is again anaesthetized animal once more, opens the abdominal cavity, and obtains blood by the puncture inferior caval vein, to measure serum creatinine by quantitative colorimetric method.Mouse is put to death, and kidney pours into anchored in place with 4% paraformaldehyde among the PBS, and collects two kidneys.Half snap frozen of each kidney in liquid nitrogen and-70 ℃ of storages up to further processing; Sample is fixed in formalin, paraffin embedding, and section (4 μ m).Paraffin section is with hematoxylin-eosin dyeing and carry out histology.Second half of each kidney is embedded in the OCT compound (Tissue-Tek) and obtains freezing microtome section (4 μ m) and be used for immunohistochemistry.

9. PCR in real time:

According to producer's explanation, use RNeasy mini kit (Qiagen) to extract total RNA from the mouse kidney by DNase digestion on the post.Use the synthetic cDNA template of Omniscript Reverse Transcriptase and oligodeoxythymidylic acid primer (Qiagen).The PCR reaction uses green super mixing of iQ SYBR and the monochromatic PCR in real time detection system of MyiQ (Biorad) to carry out, and it is 2 minutes at 95 ℃ of incubation times, then is the 95 ℃/30s and the 60 ℃/30s of 40 circulations.The specificity of amplification is by curve analysis and agarose gel electrophoresis check.The primer sequence of mouse NGAL mRNA (Genbank NM_008491) be CTCAGAACTTGATCCCTGCC (forward primer, position 93-112) and TCCTTGAGGCCCAGAGACTT (oppositely, 576-557).The sequence of mouse beta-actin mRNA (Genbank X03672) be CTAAGGCCAACCGTGAAAAG (forward, 415-434) and TCTCAGCTGTGGTGGTGAAG (oppositely, 696-677).Every plate comprises the master sample of dilution series, and it is used for determining the amount of mRNA.The content of NGAL mRNA is by beta-actin mRNA standardization.

10. iron is in conjunction with co-factor

The iron that depends on co-factor is in conjunction with NGAL, its use apo-NGAL (10 μ M), ⁵⁵The low molecular weight fraction of Fe (1 μ M) and mouse retention (0-30 μ l) (＜3Kd), in 150mM NaCl-20mMHepes (pH7.4) buffer solution (100 μ l), measure, and incubated at room temperature 70 minutes.(YM-10 and YM-3 Amicon) obtain the urine fraction by 10kDa and 3kDa film successively by making fresh urine.(YM-10 Amicon) goes up cleaning 3 times at the 10kDa film with described mixture then.The positive control that the NGAL of iron-free load enterochelin (not being not in conjunction with the NGAL of siderophore) catches as iron.The enterochelin of ironic citrate (1mM) or load iron (Sid:Fe, 50 μ M) is used for ⁵⁵The competitor of Fe combination.

11.NGAL immunohistochemistry:

Detect for NGAL, freezing kidney segment sealed 1 hour with lowlenthal serum with the 0.2%Triton X-100 infiltrationization processing among the PBS 10 minutes, and cultivated 1 hour with the first antibody (dilution in 1: 500) of NGAL.Wave carrier piece is exposed in the dark 30 minutes second antibody to the Cy5 coupling (Amersham, Arlington Heights, IL) combination, and with the fluorescence microscope that is equipped with the rhodamine light filter (Zeiss Axiophot) video picture then.

12. histopathology scoring:

4 microns kidney segments dye with hematoxylin-eosin, and judge the score of body tubular tissue pathology infringement with blind method, as in the past by people such as Yokota N., Am J Physiol Renal Physiol 285:F319-F325,2003 and people such as Kjeldsen L., Biochim Biophys Acta 1482:272-283,2000 is described.Each parameter is evaluated each average measurement of cutting into slices at endodermis and the outer zone of medullary substance (the most tangible place of tubular injury) with five high power fields (40X).This parameter comprises tubular ectasia, and renal tubule tubulose thing (cast) forms and the renal tubular cell necrosis.Each parameter grade of appraising is 0 to 4, from do not have (0), slight (1), medium (2), serious (3), to very serious/extensive (4).

13. apoptosis is measured:

We utilize ApoAlert DNA Fragmentation Assay Kit (Clontech), detect apoptotic nucleus with the TUNEL determination method.Paraffin section is by zylene and continuous ethanol deparaffnize of reducing, fix 30 minutes with 4% formaldehyde/PBS in 4 ℃, 15 minute thoroughly change processing at room temperature 15 minutes and 0.2%TritonX-100/PBS at 4 ℃ with Proteinase K, and 37 ℃ with the mixtures cultivation of nucleotide and TdT enzyme 60 minutes.This reaction stops with 2X SSC, cleans section with PBS, and be placed on Crystal/count (Biomeda, Foster City, CA) on.The apoptotic nucleus of the TUNEL-positive detects with the fluorescence microscope video picture.Only demonstrate the cell of apoptosis characteristic morphology, comprise the cell nucleus fracture, cell nucleus concentrates and, can be regarded as apoptosis by the intense fluorescence cell nucleus that the TUNEL determination method produces.Only the TUNEL positive cells does not have morphologic criterion, does not think apoptosis.Wave carrier piece detects with blind method, and apoptosis by the quantity of TUNEL positive cell nuclear in per 100 cells of number quantitatively, each on average counting with five high power (40X) visuals field of cutting into slices.

14. proliferation assay:

For detecting proliferative cell, according to producer (ImmunoCruz Staining System, Santa CruzBiotechnology) recommends, to cut into slices and Proliferating Cell Nuclear Antigen (PCNA, dilution in 1: 500, Upstate Biotechnology) monoclone antibody is cultivated, and detects and finish by immunoperoxidase staining.Wave carrier piece detects by blind method, and propagation by the quantity of PCNA positive cell in per 100 cells of counting quantitatively, each on average counting with five high power (40X) visuals field of cutting into slices.

15. statistical analysis:

Use SPSS software (version 8/0) that each continuous variable is produced univariate statistics, comprise mean, standard deviation, distribution, scope and the degree of bias.Detect the normal distribution of data and be evenly distributed.Use unidirectional ANOVA in difference treatment group, to compare the mean ± SD of continuous variable.The data that are used for Non-Gaussian Distribution about the Kruskal-Wallis ANOVA of Ranks.For identifying that one or more groups is different from other group, use multiple comparison procedure (depending on the Tukey test or Dunn ' the s Method of normal distribution).P value＜0.05 is thought statistically evident.People's NGAL level transforms to be used for statistical analysis with log.These data analyze by unidirectional ANOVA and Bonferroni ' s tests the mean value between comparative group afterwards.The Jablonski mark of renal damage is by Kruskal-Wallis test and Dunn ' s test later analysis.

Embodiment

Provide following embodiment more fully to describe the practice of the present invention in its different embodiment.The experimental program that provides above is as the explanation among the embodiment.

Embodiment 1:

The NGAL of body internal jugular vein administration is promptly absorbed by the near-end renal tubular cell.The NGAL of purifying is sent to the action site that it is inferred, i.e. the near-end renal tubule.Mouse is accepted intravenous NGAL (250 μ g are in 100 μ l salt solution) or isopyknic independent salt solution, stands the ischemia reperfusion damage, and detects kidney and urine at different times, as shown in Figure 2.The animal of non local ischemic saline control does not have NGAL (the picture left above), but not the animal that ischaemic NGAL-handles has NGAL (lower-left figure).The animal of pump pickle was not having kidney NGAL (in last figure) in 1 hour.Endogenic NGAL damages at ischemia reperfusion and detected (top right plot) in the animal in saline treatment in back 3 hours.On the contrary, in 1 hour of injection NGAL, mainly in the near-end renal tubule, detect into the cytoplasmic distribution of point-like (middle figure below) easily, and still can see (bottom-right graph) at 3 hours.The identification of near-end renal tubule is based on position and morphology in these sections.This represents the NGAL that has absorbed injection after the ischaemic damage, because NGAL does not detect during 1 hour the adverse current in the animal of pump pickle.In addition, NGAL detects in the urine of injecting in 1 hour, as shown in Figure 3.

Embodiment 2:

Behind administration and local ischemical reperfusion injury, intravenous NGAL promptly occurs in urine.The urine that obtains from the animal of embodiment 1 detects at different times.The animal of pump pickle does not contain NGAL in kidney or the urine during 1 hour adverse current, and NGAL just can detect during 3 hours adverse current, (left figure) as shown in Figure 3.At the endogenic reaction of 3 hours data representation renal tubular cells to local ischemic injuries.On the contrary, also standing simultaneously in the animal of ischemia reperfusion damage with the NGAL injection, NGAL is detecting (right figure) as shown in Figure 3 during 1 hour the adverse current easily in kidney and urine.

Embodiment 3:

NGAL has improved the histopathology infringement to renal tubule that the ischemia reperfusion damage is induced.Before ischaemic during 1 hour, ischaemic or even ischaemic give NGAL after 1 hour and cause significantly minimizing the infringement of body tubular tissue pathology.24 hours representational kidney segments that obtain and that dye with hematoxylin-eosin of adverse current show in Fig. 4.But not ischaemic contrast (non local ischemic figure) demonstrates normal histology, with the independent pre-treated animal of salt solution (salt water pretreatment figure) (100 μ l, diluent volume) demonstrates the feature of acute tubular necrosis widely, comprise that tubular ectasia, renal tubule tubulose thing (cast) form and downright bad cell.On the contrary, the kidney of NGAL processing demonstrates the histopathologic response that weakens.This is with the most obvious (the pretreated figure of NGAL) in the NGAL pre-treated animal, but during the ischaemic damage (NGAL During Isch panel) or even ischaemic damage 1 hour after (NGAL After Isch panel) administration NGAL also obvious.For quantitatively this response,, the histopathology of renal tubule is damaged being used for the blind method kidney segment of appraising.This result illustrates in Fig. 5 A-5C.In 3 parameters of all detections, expansion (Fig. 5 A), tube (Fig. 5 B), and meronecrosis (Fig. 5 C), when with compare, the modes (before the ischaemic, during the ischaemic or after the ischaemic) of all 3 treatment NGAL have caused significantly mark to increase.This species diversity is the most remarkable in the NGAL pre-treated animal, then according to graduate method, finds by during ischaemic or in the animal of handling with NGAL after the ischaemic infringement.Yet this structural defence is also incomplete, promptly uses the NGAL pre-treated animal also to demonstrate the histopathology infringement of some degree really, and this does not exist in the contrast of non local ischemic fully.

Embodiment 4:

NGAL improves the renal function that ischemia reperfusion damage induces and weakens.Before the ischaemic during 1 hour, ischaemic or even ischaemic 1 hour after administration NGAL, measure in 24 hours in adverse current, it causes serum creatinine significantly to reduce, as shown in Figure 6.(0.65 ± 0.13mg/dl), demonstrating serum creatinine with the independent pre-treated animal of salt solution (Pre Sal) (100 μ l, diluent volume) increases (2.6 ± 0.28mg/dl) significantly but not ischaemic contrast (Non Isch) demonstrates serum creatinine.On the contrary, the kidney of NGAL processing demonstrates the function response that weakens.This with the most obvious in the NGAL pre-treated animal (1.25 ± 0.3mg/dl), but administration NGAL during the ischaemic damage (Dur NGAL) (1.5 ± 0.2mg/dl) or even the ischaemic damage after 1 hour (PostNGAL) (1.95 ± 0.2mg/dl) is also obvious.Yet this function and protecting is also incomplete, when comparing with non local ischemic control, promptly uses the NGAL pre-treated animal also to demonstrate the serum creatinine of remarkable increase really.

Embodiment 5:

NGAL improves the renal tubular cell death that ischemia reperfusion damages the apoptosis of inducing.Protection result with the viewed 26S Proteasome Structure and Function of exogenous NGAL administration reduces apoptosis.Test resulting representational kidney segment as shown in Figure 7 adverse current 24 hours and through TUNEL, low multiplication factor (left hurdle) and high-amplification-factor (central hurdle) are arranged.Although non local ischemic control demonstrates minimum apoptosis incidence (each hundred (%) cell detection to 2.2 ± 0.5 cell), but with the independent pre-treated animal of salt solution (100 μ l, diluent volume) demonstrates significantly a large amount of apoptosis renal cells (12.6% ± 2.2), be shown in (left figure) among Fig. 8 as quantitative institute.On the contrary, the kidney of NGAL processing demonstrates the apoptosis response that weakens.This is with the most obvious (6.7% ± 1.6) in the NGAL pre-treated animal, but administration NGAL during the ischaemic damage (7.6% ± 0.8) or even the ischaemic damage after 1 hour (8.5% ± 0.8) also obvious.Yet this apoptotic cell that makes avoids dead protection and incomplete, when comparing with non local ischemic control, promptly uses the NGAL pre-treated animal also to demonstrate the apoptosis infringement of higher degree significantly really.

Embodiment 6:

NGAL strengthens the cell proliferation of ischaemic damage metanephric tubule.Adverse current (reflow) 24 hours and through the resulting representational kidney segment of PCNA antibody staining (right hurdle) as shown in Figure 7.Although non local ischemic control demonstrates minimum cell proliferation incidence (each hundred (%) cell detection to 1.9% ± 0.4 cell), but with the independent pre-treated animal of salt solution (100 μ l, diluent volume) quantitatively demonstrates less but increase (4.4% ± 1.2) significantly at the positive renal cells of PCNA-, be shown in (right figure) among Fig. 8 as quantitative institute.On the contrary, the kidney of NGAL processing demonstrates in cell proliferation significantly and increases.This is with the most obvious (19.1% ± 2.1) in the NGAL pre-treated animal, but administration NGAL during the ischaemic damage (14.9% ± 1.2) or even the ischaemic damage after 1 hour (14.5% ± 1.2) also obvious.

Embodiment 7:

The NGAL balance that tilted makes renal tubular cell turn to survival in ischaemic damage back from death.Unidirectional ANOVA estimation is used in the death of all renal tubular cells of ischaemic damage back, with at the adverse current 24 hours mean ± SD of propagation and apoptosis relatively between the different disposal group.Rate value is 1 can be considered to show that cell survival is identical with dead ratio, and this will expect in the kidney that is in static maturation, as shown in Figure 9.The propagation that the kidney of non local ischemic control shows: the apoptosis ratio is 0.86 ± 0.1, near value 1.Propagation with the independent pre-treated animal of salt solution (100 μ l, diluent volume) demonstration: the apoptosis ratio significantly reduces (0.34% ± 0.05), and this shows that cell death is the principal character of 24 hours adverse current time points.On the contrary, the kidney handled of NGAL demonstrates tangible increase in the ratio to the renal tubular cell of apoptosis of propagation.This is with the most obvious (2.9% ± 0.5) in the NGAL pre-treated animal, but administration NGAL during the ischaemic damage (2.0% ± 0.1) or even the ischaemic damage after 1 hour (1.7% ± 0.1) also obvious.This analysis showed that the NGAL overall balance of renal tubular cell that tilted, and makes it turn to survival in ischaemic damage back from death.

Embodiment 8:

The philtrum that is expressed in acute renal failure of NGAL increases.People's the acute renal failure log value mark of the NGAL protein concentration ascending of serum and urine, as shown in figure 11.The NGAL albumen of urinating in the normal study subject is 22ng/ml (n=10), is 557ng/ml (raising 25 times p＜0.001) in the study subject of suffering from acute renal failure and multiple complications.The NGAL Western blotting of urine shows in Figure 11 A, and as the quantitative diagram of Figure 11 C.GAL is a 21ng/ml (geometric mean with its serum N of normal study subject; N=5) compare, its serum N of the study subject GAL that suffers from acute renal failure and multiple complications has 7.3 times lifting (146ng/ml, p＜0.05).These data are presented among Figure 11 B with Western blotting, and quantitatively diagram in Figure 11 D.Suffer from acute renal failure and easily have the serum (331ng/ml) of highest level and the NGAL of urine (2786ng/ml), but this does not have difference with suffering from acute renal failure on FFI patient statistics with the patient of bacterial infection.For whether the expression of definite NGAL is relevant with the degree of acute injury of kidney, we use simple regression analysis after the NGAL level being done the log conversion.We find serum (r=0.64, n=32) and the NGAL level of urine (r=0.68, n=38) both, and to the urine NGAL of UCre acid anhydride normalization (r=0.67 is n=36) with serum creatinine level height correlation (p＜0.0001).By comparison, the patient who suffers from chronic renal failure serum N GAL (49ng/ml, n=10) and urine NGAL (119ng/ml, n=9) aspect has and lowly promotes significantly, these values are failed relevant with serum creatinine.These data are expressed with the acute kidney infringement NGAL and are associated, and the hint kidney is the main source of serum and urine NGAL.In fact, in several examples of severe renal failure, the drainage mark of NGAL (clearance rate of NGAL, the clearance rate standardization of creatinine relatively) shows that greater than 100% the NGAL of urine is derived from local synthesizing, and just by filtering from blood.By contrast, after the ATN damage is induced, also comprise the lifting of NGAL level significantly in the mouse retention, shown in Figure 11 E.

For make the expression site video picture of NGAL in acute kidney diseases, people's nephridial tissue is cut into slices with the NGAL polyclonal antibody dyeing (Figure 12) of affine purification.The very weak dyeing of demonstration in distal tubule epithelium (mean cortical area 10%) and MCD of normal kidney is shown in Figure 12 A low power and shown in Figure 12 B and the 12C high power.The local dyeing of the wall epithelial cell of glomerulus is rare, rather than the cell of other glomerulus.The near-end renal tubule but is negative fully.On the contrary, always self diagnosis is that the cortex tubule near 50% dyes at NGAL in the kidney segment that suffers the nephrotoxin or ischemic kidney of study subject of following disease: Figure 12 D, the ischaemic ATN that pyemia causes; Figure 12 E, hypovolemia (blood volume is acute to be lost); Figure 12 F, heart failure; Figure 12 G, the renal toxicity that bisphosphonate compound causes; The renal toxicity that Figure 12 H, cephosporin cause; With Figure 12 I, hemoglobinuria.NGAL also expresses in the patient's who suffers from the hyperplasia glomerulopathy near-end renal tubule widely, shown in Figure 12 J and 12K, but with comparing of finding in the ischaemic infringement, degree is less (in the minimal change disease, the percentage of the cortical parenchyma of the NGAL positive is 20%, be 40% in the diabetic nephropathy, among the ANCA be 50% and the anti-GBM disease in be 65%).Renal tubular cell demonstrates the feature of cellular damage, and comprises the repair cell nuclear with outstanding kernel of unification and expansion, and these cells have the denseest dyeing.Renal tubular cell less coloration many with less confusion.These data show that the reactivity of NGAL is newly-generated and is general in the cortex renal tubule of different kidney diseases, and show that it is to the generally response of the epithelium of infringement in people's kidney that NGAL expresses.

Embodiment 9:

The NGAL of external source can protect mouse near-end renal tubule to avoid ATN.For detecting the functional meaning that NGAL expresses in the kidney ischaemic, induce the ATN damage in the mouse.Clamp the arteria renalis 30 minutes and removed the kidney of offside.After pouring into 24 hours again, plasma creatinine rises to 3.16 ± 0.17mg/dl (n=8 from 0.41 ± 0.1mg/dl (n=4); P＜0.001) and NGAL mRNA information and albumen express consumingly.NGAL mRNA level raises about 1000 times, reduce threshold value to be used for detection, its by PCR in real time from false kidney, 17.7 ± 0.87 being recycled to 7.52 ± 0.44 in ischaemic kidney circulation (p＜0.0001, each n=4) (standardization is in β actin mRNA level).NGAL albumen in urine, raise 1000 times (be the 40ng/mls of 40 μ g/ml among the ATN than false operation normal mouse, shown in Figure 11 E), 300 times (being the 100ng/mls of 30 μ g/ml than false operation mouse among the ATN) raise in blood, and in the kidney extract, raise nearly 100 times (average 73 μ g/g in false operation kidney＜1 μ g/g kidney weight in wet base, n=3, p＜0.05).The amount of NGAL albumen is very relevant with the duration of cross-clamp in the kidney.

For determining whether NGAL has protective effect in the ischaemic model of ATN, we will or unclamp general ground administration NGAL (by subcutaneous or endoperitoneal injection 1-300 μ g) within an hour before unclamping vessel clamp.Before clamping, inject 100 μ g NGAL 15 minutes the time and stoped rising (1.18 ± 0.18mg/dl, the n=7 that pours into the plasma creatinine that detects after 24 hours again; In the animal of being untreated 3.16 ± 0.17mg/dl).Similar data is obtained from the dosage range of NGAL 10-300 μ g, but 1 μ gNGAL do not have protective effect (creatinine=3.09 ± 0.11mg/dl, n=3).1 hour injection NGAL has also stoped azotemia (creatinine=1.60 ± 0.28, n=3, p＜0.001) after the perfusion again, but to compare degree less with the NGAL preliminary treatment.These data confirm (data not shown) by measuring blood urea nitrogen (BUN).

The activity of NGAL also confirms by histological finding, the reservation in the S1 of near-end renal tubule and S2 district of normal epithelial morphology but not downright bad renal tubule and chamber fragment, as shown in FIG. 13A, in the kidney (ATN+NGAL) of contrast and NGAL processing, compare with the ATN kidney.S3 district outside medullary substance is outer field in the striped is less to be protected by injection NGAL, but renal tubule tubulose thing is more not obvious, shown in Figure 13 B.These observed results are marked by the Jablonski to section and are supported, shown in Figure 13 C.On the contrary, handled with NGAL in 2 hours after the ischaemic and do not have protective effect (creatinine=3.12 ± 0.35mg/dl, n=3).

Embodiment 10:

The correlative of ischaemic perfusion damage.Because the transportation of cadherin and metabolism are promptly influenced by ischaemic, and because NGAL serves as the derivant of E-cadherin in the matter between the rat embryonic metanephros, so NGAL has protected the expression of cadherin in ischemic kidney.For detecting this hypothesis, we at first confirm can detect in mouse near-end renal tubule by immunofluorescence when the E-cadherin, and the N-cadherin just all exists in all parts of near-end renal tubule, shown in Figure 14 A, and is shown as its main cadherin.Known N-cadherin produces 30-40Kd cytoplasm fragment by Guang winter protease, beta-secretase and matrix metalloproteinase processing, and these fragments are signaling molecules of potential important adjusting CREB signal transduction.The N-cadherin is degraded to the 30Kd fragment behind the ischemia reperfusion, is confirmed by Western blotting among Figure 14 B, hints the activation of one or more approach.The degraded of albumen can detect pouring in 6 hours again in some animals, and time 24 hours, N-cadherin immunofluorescence and full-length proteins have nearly all been abolished.On the contrary, when perfusion again 6 hours (in some animals) or when monitoring in 24 hours, N-cadherin immunofluorescence has been preserved in the NGAL preliminary treatment, strengthens the expression of total length N-cadherin and reduces the appearance of its fragment.Therefore, the preservation of near-end renal tubule mark N-cadherin is active relevant with NGAL, and is the sensitivity label thing of NGAL activity.The E-cadherin of high expressed in distal tubule and collecting pipe, the influence that handled by ischaemic and NGAL is very little.Similarly, the kidney of metal inducement poison ATN causes the N-cadherin but not the degraded of E-cadherin.Therefore, a kind of effect of NGAL is the signal transduction that suppresses N-cadherin fragment.

Because breaking of near-end cell causes apoptotic cells death, the effect of NGAL pair cell vigor uses the TUNEL determination method of apoptosis induction to determine.Poured into back 24 hours, we calculate the percentage of the renal tubule of the renal tubular cell with at least one TUNEL-positive, shown in Fig. 1 4C again.Ischaemic kidney (I/R) shows that the cortex renal tubule that comprises the TUNEL-positive cell is 11.5% ± 0.6 (SEM, n=4 animal), and with after NGAL (I/R+Ngal) processing, the percentage of positive renal tubule is reduced to 2.9% ± 0.9 (SEM, n=7; P＜0.001).By contrast, 0.5% ± 0.3 cortex renal tubule has the TUNEL-positive cell in the false kidney, as the quantitative diagram of Figure 14 D.

BrDU absorbs and is decided to be a kind of method of measuring cell proliferation, and this method is to measure the percentage (unlisted) of the cortex renal tubule with the positive renal tubular cell of at least one BrDU-in the nephridial tissue section.Ischaemic cortex renal tubule comprises rare BrDU-positive cell (1.9% ± 0.3; N=3), have little but growth (3.9% ± 0.5 significantly with pretreated its positive cell of ischaemic kidney of NGAL; N=4; P＜0.05), measure in the damage after 24 hours.By contrast, 3.7% ± 0.7 cortex renal tubule has the BrdU-positive cell in the false kidney.Therefore, the protective effect of NGAL has reduced the apoptosis of cortex cell, and or the propagation of boost compensator renal tubular cell or protection cell avoid damage.

Because the expression of NGAL is relevant with the ischaemic infringement, has measured the endogenic NGAL that handles afterwards with ectogenic NGAL albumen and expressed.The growth that the ischaemic animal that 100mg NGAL handles has reduced endogenous NGAL RNA reaches 72% ± 16 (p＜0.01; N=5), PCR in real time is measured in pouring into back 24 hours again.The processing of the 100mg NGAL that Western blotting is measured makes the appearance of endogenous NGAL albumen in kidney reduce by 2.5 times of (ischaemic 73 ± 7 μ g/g; The ischaemic that NGAL handles, 29 ± 7 μ g/g; Each n=3; P＜0.01), it has preserved the renal tubular cell with proliferation potential.

Embodiment 11:

NGAL raises the Heme oxygenase 1 among the ATN.The conditioning agent of the key of near-end renal tubule in the kidney ischaemic has been regarded Heme oxygenase 1 (HO-1) as in many researchs.HO-1 recovers necessary from ATN and its expression directly damages relevant with protective tissue.Shown in Figure 14 E, ischemia reperfusion (ATN swimming lane) strengthens the expression of HO-1, but when the mouse of ATN-damage is handled with 10-100 μ gNGAL (ATN Ngal swimming lane), this enzyme further raised 5-10 doubly after the perfusion in 24 hours again.For determining whether NGAL induces HO-1 separately, the NGAL of healthy mice (false swimming lane) injection increasing gradually dosage, and measurement HO-1 protein level.Yet the kidney of the ATN-damage that the relative NGAL of the expression of HO-1 handles after the NGAL injection is a lot of less, and this shows that NGAL and other activating agent act synergistically and raises the HO-1 during the ischemia reperfusion and protect kidney to avoid the infringement that iron mediates.

Embodiment 12:

Mechanism from ATN rescue: NGAL the near-end renal tubule as target.In the peritonaeum or determine after the hypodermic injection that the distribution of exogenous NGAL sets up the mechanism that NGAL protection near-end renal tubule avoids the ischaemic infringement.In urine, found NGAL in injection within NGAL10 minute of 100 μ g external sources, hinted that this albumen removed by kidney rapidly, as shown in figure 15.Same time point is also observed (not shown) behind injection 10 μ g NGAL.Yet only the NGAL of 0.1-0.2% injection reclaims in urine in the 1st hour.For spike transportation better, give the fluorescence conjugate of NGAL.Two kinds of fluoresceins-and the NGAL of Alexa-mark with (S1 of the nephron and S2 district) in the big vesica that concentrated on the nearly top zone of cortex near-end renal tubule in 1 hour, but not in other of renal tubule distinguished, shown in Figure 16 A.Should be pointed out that albumen transportation itself also can not be the mechanism of kidney protection, since second NGAL, the RBP of load biostearin, it is also caught by the near-end renal tubule and degrades in lysosome, and it is invalid (Fig. 6 A).For determining whether these organelles are lysosome, we are giving Alexa-568NGAL usefulness the previous day fluorescein-glucan (43kDa) mark near-end renal tubule lysosome.Inject after NGAL1 hour, 33% NGAL vesicles also comprises glucan, shown in Figure 16 B.In addition, many these vesicles are by lysosomal label L AMP1 dye altogether (data is not shown).The injection [ ¹²⁵I]-observing similar result behind the NGAL, Figure 16 C shows 1 hour time point full-length proteins and promptly removes from blood and be gathered in the kidney.In fact, kidney contain than liver/mg albumen Duo 13 times [ ¹²⁵I]-NGAL.Reported the almost same data about people NGAL in the past, people NGAL promptly removes circulation (t _1/2=10 minutes) and to accumulate in the kidney than the level that liver/mg albumen is high 12 times.The albumen that concentrates on kidney is (70%) of TCA deposition, and is made up of total length NGAL and specific 14kDa catabolite.These materials continue to exist, and only slowly disappear after back 5 hours in injection.On the contrary, blood plasma, especially urine comprise low-molecular-weight mostly, TCA soluble [ ¹²⁵I]-fragment, (35% and 20%TCA deposition, respectively).

These data show total lengths NGAL is promptly removed by the near-end renal tubule, is transported to lysosome and degrades to the fragment of 14kDa at near-end renal tubule NGAL.Endogenic probably albumen (low serum N GAL level) transportation in a similar fashion, because in normal mouse or people's urine, contain the NGAL of considerably less urine, although the fact is NGAL freely to be filtered in circulation (people: filtered load=20ng/mlX GFR, and urinate NGAL=22ng/ml; Mouse: filtered load=100ng/ml X GFR, and urine NGAL=40ng/ml).

Embodiment 13:

The near-end renal tubule is saved from ATN needed Fe: siderophore.For determining whether NGAL can transmit iron to the near-end renal tubule, with 1: 1 stoichiometry ( ⁵⁵Fe:Sid:NGAL) cultivate iron-free enterochelin-NGAL (Sid:NGAL) with ⁵⁵Fe makes NGAL by the radionuclide iron substance ⁵⁵Fe is saturated.Inject this radiolabeled ⁵⁵Back 1 hour of Fe:Sid:NGAL compound (10 μ g are endoperitoneal), great majority ⁵⁵Fe reclaims (55%) in kidney, and has only trace to be found in blood plasma (4.3%), urine (0.6%), liver (2.4%) and spleen (0.2%).For determining ⁵⁵The histologic section radioautography is carried out in the position of Fe in kidney. ⁵⁵Fe is gathered in the near-end renal tubule, especially along top end surface, and under brush border (brush border), shown in Figure 16 E, in the table 1, X wherein ²=21.2 and p=0.0017.

Table 1

The position	Silver granuel a(% total amount)	Area b(% point counting)	Relative SA c	X 2
					Tube chamber	26.46	18.49	1.43	3.43
Top MB	12.91	5.84	2.21	8.54
					Cytosol	48.98	48.81	1	0.00062
Karyon	4.02	7.17	0.56	1.39
					Substrate MB	3.81	6.41	0.59	1.06
Between matter	3.71	12.2	0.3	5.91
					The bead bundle	0.13	1.16	0.11	0.91

Annotate: ^aTotal silver granuel=2601 ^bTotal some counting=999 ^c% particle/% point counting.

On the contrary, ⁵⁵Fe does not find in medullary substance, shown in Figure 16 D.These data show when the injection external source ⁵⁵The Fe:Sid:NGAL compound, NGAL albumen and its part iron can both be caught by the near-end renal tubule.Should be understood that ⁵⁵The distribution of Fe:Sid:NGAL is with no protein bound ⁵⁵The distribution of Fe citrate differs widely, and wherein has only 1.5% iron to reclaim (not shown) in kidney.

Embodiment 14:

For determining that iron is transmitted in the effect in the kidney protection, we relatively use two kinds of not NGAL of the escherichia coli cloning of homology.The NGAL that clones in the XL-1Blue bacterium comprises enterochelin and load iron, and the NGAL that clones in the BL-21 bacterium does not comprise enterochelin.10 μ gXL-1Blue-clone's NGAL (holo-NGAL 10 μ g, left figure) compares with the kidney (ATN) of untreated ATN-damage with (Sham) of false processing, has protected kidney, shown in Figure 17 A.Yet, reduce with its protection level of NGAL (apo-NGAL 10 μ g, the figure of central authorities) of the 10 μ gBL-21-clone who lacks enterochelin.Therefore, the enterochelin (apo-NGAL:Sid:Fe) that BL-21-clone's NGAL and iron-free (apo-NGAL:Sid) and iron are saturated is reformulated.Load enterochelin (contain or not iron content) strengthens the kidney protective effect and weakens the rising of serum creatinine.The existence of iron reduces protective effect a little, because the saturated siderophore of iron has weakened the ability of its chelated iron in kidney.Because two kinds of NGAL:Sid of the form of load iron and iron-free form have protective effect, may be siderophore itself, but not iron work.Be the further effect of test iron, gallium: Sid:NGAL:Sid: gallium (apo-NGAL:Sid:Ga) compound is also tested.

Because gallium is the metal that occupies the iron binding site with high-affinity, (gallium is with the degree prevention same with unlabelled iron to comprise the enterochelin siderophore ⁵⁵Fe is in conjunction with enterochelin), and because it can not experience the redox reaction of typical iron, gallium combines siderophore with the iron competition.Opposite with iron complexes, ischaemic was handled mouse with gallium complexes in 15 minutes does not before have protective effect (creatinine=3.17 ± 0.1; N=4).In addition, the free siderophore (Sid) of single dose, deferoxamine mesylate (DFO) or fail protection antagonism ATN in conjunction with the albumen (RBP) of retinol.These data acknowledgements NGAL: the siderophore compound provides the protection activity of NGAL, and by the defeated NGAL that depends on of the Railway transportation of siderophore.The iron content (Fe) or the NGAL of iron content (Fe) not: the Western blotting of siderophore compound is shown in Figure 17 B.

Embodiment 15:

The confirmation of urine siderophore.The effect of endogenic NGAL may be different with the pharmacological action of the NGAL of external source in the body, because the siderophore of the key relevant with the NGAL of external source is a bacterial product.Yet the existence of the endogenous low molecular weight factor of transportation iron is hinted by multiple research institute.These molecules can comprise citrate and related compound, but also comprise the defeated activating agent of the Railway transportation that has in the molecular weight 1Kd scope.For determining whether the NGAL co-factor is present in the urine, and the apo-NGAL that is derived from the BL21 bacterium mixes with the urine sample that derives from healthy mice.Although the lower-molecular-weight component of urine (＜3Kd) fail to catch by filter being higher than 10Kd ⁵⁵Fe, and the apo-NGAL that is diluted in Tris or phosphate buffer fails to catch ⁵⁵Fe, but with NGAL and urine (＜3,000Da) cultivate and make ⁵⁵Fe allows to keep, shown in Figure 18 A.NGAL suppresses catching by 1000 times of unlabelled iron citrates of iron, and is suppressed by the saturated enterochelin of the iron of 50 times of concentration stronglyer, shown in Figure 18 B.Catching of iron is saturable, and when using 30 μ l mouse retentions, about 20% NGAL molecule is in conjunction with iron.These find that the hint mouse retention comprises the low-molecular-weight co-factor, and it allows the mutual effect of NGAL-iron phase.Because the endogenic factor is competed mutually with the bacterium siderophore, in conjunction with calyx, appear the bacterium and the mammal factor all to occupy the same binding pocket of NGAL with high-affinity (0.4nM).

Embodiment 16:

NGAL among the patient of experience kidney transplant expresses.The people of assessment experience kidney transplant determines that NGAL is in convalescent expression.Renal biopsy from the relevant donor of living (LRD, n=10) or corpse (CAD n=12) carries out in 1 of kidney transplant hour.Biopsy sample is cut into slices and with the histochemical stain of NGAL antibody mediated immunity.NGAL is expressed in the CAD group to be increased significantly, as shown in figure 19.Because in the longer time of external preservation, the degree of ischaemic damage is usually bigger than the typical kidney that obtains from the relevant donor of living for the kidney of corpse, therefore express relevant with NGAL clearly.

Carry out Western blotting before transplanting and at the urine sample that in LRD (n=4) or CAD (n=4) kidney transplant 2 hours, obtains, as shown in figure 20.The operation that is expressed in of NGAL does not exist before in the urine.Compare being expressed in the CAD group of NGAL increases significantly with the LRD group.The NGAL of the urine by the Western blotting method quantitative measurement, LRD to CAD in, demonstrate the expression of remarkable increase among the CAD, as shown in figure 21.The quantitative assay of ELISA has confirmed similar (not shown) as a result.This discovery makes the ischaemic long period relevant with the CAD kidney transplant again.CAD transplants the correlation of the NGAL and the cold ischemia time of the back urine that obtained in 2 hours, as shown in figure 22.

Serum creatinine level, it reached peak value in 2-4 days after transfer operation, and also the NGAL with urine is relevant.The correlation that CAD transplants the NGAL of the back urine that obtained in 2 hours and serum creatinine peak value as shown in figure 23.

Embodiment 17:

Use the diagnostic tool of NGAL measurement as acute renal failure.A kind of unfortunate result of cardiopulmonary bypass in the OHS (CPB) is the generation of acute renal failure (ARF).It can be to being in the patient's that ARF danger takes place height prediction that serum N GAL measures.The calibration curve of NGAL ELISA as shown in figure 24, linear relation is obtained from 10 independent standard curves.A series of serum N GAL suffer from the measurement (n=10) in the patient's sample of ARF behind CPB, as shown in figure 25.NGAL raises significantly in the sample of collecting after operation, and keeps raising at least 4 days.On the contrary, the patient who does not suffer from ARF did not increase serum N GAL level in back 4 days in initial operation.Figure 26 shows the comparison that does not have ill (n=30) value after the mean value ± SD of (n=10) serum N GAL among the patient of a series of ARF of suffering from and those operations.The measurement result (n=11) of a series of urine NGAL of suffering from the patient of ARF behind the CPB has also raise, as shown in figure 27, but than the easier variation of serum N GAL.Yet urine NGAL level is the omen of ARF, as shown in figure 28, does not have the comparison of ill (n=30) value among the patient of a series of ARF of suffering from of its analysis after mean value ± SD of (n=11) urine NGAL and those operations.The urine NGAL level of performing the operation back 2 hours is relevant with the length of intra-operative CBP time, as shown in figure 29.

Although the present invention is illustrated by the description of its embodiment and embodiment, although and this embodiment and embodiment be described in considerable detail, be not to limit or by any way the scope of the additional claim of constraint among these details.The advantage and the modification that add for those skilled in the art will be conspicuous.The present invention it be not limited to aspect wider concrete details, representational method and structure and shown in and the embodiment that illustrates that describes.Therefore, the place different with these details can be arranged as long as without prejudice to scope of the present invention or spirit.

Claims (10)

1. the relevant NGAL (NGAL) of neutrophil gelatinase is used for the treatment of, alleviates, improves in preparation or prevent among the patient the purposes in the medicine of organ damage, and these damages are selected from the damage that ischaemic is damaged, ischemia reperfusion damages and toxin causes.

2. according to the purposes of claim 1, wherein organ is selected from kidney, liver, heart, brain, lung, stomach, intestines, colon, pancreas, blood vessel, bladder, uterine cervix, skin and a part thereof, and preferably wherein organ transplant is gone among the patient, and the NGAL administration is given organ donor corpse or that live before patient is gone in organ transplant.

3. according to the purposes of claim 1, wherein damage comprises and easily makes patient suffer from the illness of ischaemic injury of kidney, handle, treatment or the relevant injury of kidney of disease, and preferably wherein injury of kidney by illness, handle or treat and cause, these illnesss, processing or treatment are selected from the developer treatment, Antybody therapy, antibiotic therapy, organ transplant, kidney transplant, the kidney transplant of corpse, the treatment of heart, the treatment of operation back heart and central nervous system treatment, or be selected from following disease: infect, bacterial infection, the kidney disease, the ischemia reperfusion damage, the heart reperfusion injury, cardiopulmonary bypass, OHS and abdominal surgery art.

4. according to the purposes of claim 1, wherein said medicine further comprises siderophore, preferably with the form of the complex chemical compound that forms with NGAL.

5. the relevant NGAL (NGAL) of neutrophil gelatinase is used for alleviating or improve purposes in the medicine of repulsion of the delay graft function (DGF) of the donor that is implanted into patient or transplant organ or graft or organ or graft in preparation.

6. according to the purposes of claim 5, wherein transplanted organ or graft are selected from kidney, liver, heart, brain, lung, stomach, intestines, colon, pancreas, blood vessel, bladder, uterine cervix, skin and a part thereof, and preferably wherein transplanted organ or graft are kidney corpse or LD.

7. according to the purposes of claim 6, wherein said medicine further comprises siderophore, preferably with the form of the complex chemical compound that forms with NGAL.

8. a composition that is used for the treatment of, alleviates, improves or prevent organ damage in the mammal comprises neutrophil gelatinase relevant NGAL (NGAL), or derivatives thereof or analog.

9. composition according to Claim 8, per 100 microlitre compositions comprise at least 10 microgram NGAL.

10. composition according to Claim 8 further comprises siderophore, preferably the complex chemical compound that forms with NGAL.

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