Nephrotic protein biomarker and its application

Abstract

The present invention relates to biomarkers, methods and assay kits for identifying and screening renal disease, particularly diabetic nephropathy, in individuals in need thereof, or for predicting diabetic nephropathy or early progressive renal function decline (ERFD) in diabetic patients.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 688,142, filed on June 21, 2018, under 35 U.S.C. §119, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates to biomarkers, methods, and assay kits for identifying and screening renal disease, particularly diabetic nephropathy, in individuals in need thereof, or for predicting diabetic nephropathy or early progressive renal function decline (ERFD) in diabetic patients. Background Art

[0004] Kidney damage, also known as nephropathy, can be caused by, for example, drug toxicity, inflammation, high blood pressure, and diabetes. Kidney disease is usually a progressive disease, meaning that damage to the kidneys is often permanent and irreversible. Therefore, it is important to detect kidney disease early, before damage occurs. If kidney disease is detected in the early stages, it can be treated very effectively. Treatment of chronic kidney disease focuses on slowing the progression of kidney damage, usually by controlling the underlying cause. Chronic kidney disease can progress to end-stage renal failure, which is fatal without artificial filtration (dialysis) or a kidney transplant. In particular, diabetic nephropathy (DN) is one of the most common complications among patients with diabetes. Nephropathy develops in approximately 20-40% of patients with type 2 diabetes (T2D) [1]. In addition, diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD). Microalbuminuria (urinary albumin excretion 30 to 300 mg / 24 hours) is the first sign of renal insufficiency as it progresses to macroalbuminuria (>300 mg / 24 hours) and subsequently to renal failure [2,3].

[0005] Typically, nephropathy is diagnosed by analyzing the amount of protein in urine (e.g., the amount of albumin in urine) or by checking the glomerular filtration rate (GFR). Other relevant parameters include, for example, systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), and hemoglobin A1c (HbA1c). However, these methods lack sufficient sensitivity and / or selectivity, especially for detecting early nephropathy when no obvious symptoms occur. Although nephropathy can also be detected by renal biopsy, this invasive procedure is not ideal because most patients are unwilling to do so and may result in a late diagnosis until clinical features become apparent or disease progression has already developed. Renal biopsy may also result in the risk of serious bleeding complications.

[0006] There is a need to develop a method for detecting renal lesions, particularly for general screening, early detection, and non-invasive methods. Summary of the invention

[0007] In the present invention, it was unexpectedly found that Klebsiella pneumoniae (CC16) was specifically and highly expressed in patients with nephropathy compared to control individuals without nephropathy. It was also found that CC16 was highly correlated with the development of diabetic nephropathy or early progressive renal function decline (ERFD) in diabetic patients. Therefore, Klebsiella pneumoniae (CC16) can be used as a specific biomarker for diagnosing nephropathy, especially for early detection; and also for predicting diabetic nephropathy or ERFD in diabetic patients.

[0008] In one aspect, the present invention provides a method for detecting renal lesions in an individual, the method comprising:

[0009] (i) providing a biological sample obtained from the patient; and

[0010] (ii) detecting a biomarker in the biological sample to obtain a detection level, comparing the detection level with a reference level of the biomarker to obtain a comparison result, and assessing whether the individual suffers from renal disease or has a risk of developing renal disease based on the comparison result, wherein the biomarker comprises CC16, and an increase in the detection level compared to the reference level indicates that the individual suffers from renal disease or has a risk of developing renal disease.

[0011] In some embodiments, the individual is a diabetic patient.

[0012] In some embodiments, the renal disease is diabetic nephropathy.

[0013] In some embodiments, if the individual is determined to have renal disease, the individual is subjected to a method for treating the renal disease.

[0014] In another aspect, the present invention provides a method for predicting diabetic nephropathy or early progressive renal function decline (ERFD) in a diabetic patient, the method comprising:

[0015] (i) providing a biological sample obtained from the diabetic patient; and

[0016] (ii) detecting a biomarker in the biological sample to obtain a detection level, comparing the detection level with a reference level of the biomarker to obtain a comparison result, and assessing whether the individual suffers from renal disease or has a risk of developing renal disease or ERFD based on the comparison result, wherein the biomarker comprises CC16, and an increase in the detection level compared to the reference level indicates a higher risk of developing diabetic nephropathy or ERFD.

[0017] In some embodiments, if a diabetic patient is determined to have a higher risk of developing diabetic nephropathy or ERFD, a method for preventing diabetic nephropathy or ERFD is performed on the individual.

[0018] In some specific embodiments of the invention, the detecting is performed by mass spectrometry.

[0019] In some specific embodiments of the present invention, the biological sample is a urine sample.

[0020] In some specific embodiments of the present invention, one or more additional biomarkers or parameters may be further detected to improve the accuracy of the detection. In some examples, the biomarkers to be detected according to the present invention also include β2-microglobulin (β2-microglobulin, B2M).

[0021] In some embodiments of the present invention, the renal disease is chronic kidney disease (CKD).

[0022] In some specific embodiments of the present invention, the chronic kidney disease (CKD) is early CKD, specifically stage 1 or stage 2, or the CKD is stage 3 or stage 4 chronic kidney disease (CKD).

[0023] In another aspect, the invention provides a kit for practicing the methods described herein and instructions for using the kit to detect the presence or amount of a biomarker as described herein.

[0024] Also provided is the use of a reagent that specifically recognizes a biomarker as described herein for diagnosing renal disease in an individual in need thereof, or for predicting diabetic nephropathy or ERFD in a diabetic patient, or for preparing a kit or composition for diagnosing renal disease in an individual in need thereof, or for predicting diabetic nephropathy or ERFD in a diabetic patient.

[0025] The details of one or more specific embodiments of the present invention are described in the following description. Other features or advantages of the present invention will become apparent from the detailed description of the following specific embodiments and the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to illustrate the present invention, specific embodiments are described below. However, it should be understood that the present invention is not limited to the preferred specific embodiments shown.

[0027] In the attached figure:

[0028] Figure 1 Shown are representative MALDI-TOF mass spectra of urine samples from healthy individuals and patients with WDM-NPs, DM-WNPs, and DM-NPs.

[0029] Figure 2A-2B Shown are the excretion levels of (2A) 11.7 kDa and (2B) 15.8 kDa proteins in urine samples from 39 healthy, 44 WDM-NP, 85 DM-WNP, and 51 DM-NP individuals. Relative intensity is presented as a box plot, represented as a medium with quartile values ​​(25%, 75%). Error bars represent minimum and maximum values. *p<0.05, ***p≤0.001.

[0030] Figure 3 Shown is the corresponding peptide identified as the m / z 647.91 peak by nanoLC-M / MS.

[0031] Figures 4A-4B Shown are urine samples from 39 healthy, 44 WDM-NP, 85 DM-WNP, and 51 DM-NP individuals ( Figure 4A )β2-microglobulin (B2M) and ( Figure 4B ) Excretion content of Klebsiella pneumoniae protein (CC16). Relative intensity is presented as a box plot, expressed as medium with quartile values ​​(25%, 75%). Error bars represent minimum and maximum values. *p<0.05, **p<0.01, ***p≤0.001. DETAILED DESCRIPTION

[0032] In order to provide a clear and quick understanding of the present invention, some terms are first defined. Additional definitions are set forth throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs.

[0033] As used herein, the articles "a" and "an" refer to one or more than one (ie, at least one) of the grammatical object of the article. For example, "a component" means one component or more than one component.

[0034] As used herein, the words "about" or "approximately" refer to acceptable degrees of deviation that would be understood by a person of ordinary skill in the art, which may vary depending on the context in which it is used. Generally, "about" or "approximately" can indicate a value with a ±10% range around the quoted value.

[0035] As used herein, the words "comprise (verb)" or "include (gerund)" are generally used in the sense of include (verb) / including (gerund), which represents that one or more features, ingredients or components are allowed to be present. The words "comprise (verb)" or "include (gerund)" include the words "consist of (verb)" or "consist of (gerund)".

[0036] As used herein, the terms "subject", "individual" and "patient" refer to any mammalian individual, particularly humans, who need diagnosis, prognosis, treatment or medical treatment. Other individuals may include cows, dogs, cats, guinea pigs, rabbits, rats, mice, horses, etc.

[0037] As used herein, the term "diagnosis" as used herein generally includes determining whether an individual is likely to be affected by a target disease, condition, or dysfunction. Those skilled in the art generally diagnose based on one or more diagnostic indicators (i.e., markers), diagnosing the presence, absence, or amount of the marker that indicates the presence or absence of a disease, condition, or dysfunction. Those skilled in the art will understand that diagnosis does not mean determining the presence or absence of a particular disease with 100% accuracy, but rather an increased likelihood of the presence of a disease in an individual.

[0038] As used herein, the term "antibody" refers to an immunoglobulin capable of binding to an antigen. Antibodies used herein are meant to include intact antibodies and any antibody fragments (e.g., F(ab')2, Fab', Fab, Fv) capable of binding to an antigenic determinant, an antigen, or a target antigen fragment. The antibodies of the present invention are immunoreactive or immunospecific, and therefore specifically and selectively bind to a target protein, such as Klebsiella pneumoniae protein (CC16) or B2-microglobulin (B2M) protein. Antibodies for target proteins are preferably immunospecific, that is, they have essentially no cross-reaction with related materials, although they can recognize their homologs across species. The term "antibody" includes all types of antibodies (e.g., monoclonal antibodies and polyclonal antibodies).

[0039] As used herein, the term "treatment" refers to the application or administration of one or more active agents to an individual affected by a disease, a symptom or condition of the disease, or the progression of the disease, for the purpose of curing, healing, alleviating, alleviating, altering, remedying, improving, promoting, or affecting the disease, a symptom or condition of the disease, a disability caused by the disease, or the progression of the disease.

[0040] As used herein, the term "prevention" refers to measures to prevent or avoid a disease or a symptom or condition of a disease, including, but not limited to, applying or administering one or more active agents to an individual who has not yet been diagnosed with the disease or a symptom or condition of the disease, but who may be affected by or susceptible to the disease. The purpose of preventive measures is to avoid, prevent, or delay the occurrence of the disease or a symptom or condition of the disease.

[0041] As used herein, the term "normal individual" may be used to refer to an individual who is essentially in a healthy state without a specific disease (eg, renal disease), and may refer to a single normal / healthy individual or a group of normal / healthy individuals.

[0042] As used herein, the term "control individual" may be used to refer to an individual who does not suffer from a target disease (e.g., nephropathy), and may refer to a single control individual or a group of control individuals. In some specific embodiments, the control individual may refer to a normal / healthy individual. In some specific embodiments, the control individual may refer to an individual who does not suffer from nephropathy (or a diabetic patient).

[0043] As used herein, "abnormal amount" refers to the amount of an increased indicator compared to an individual or reference or control amount not suffering from the target disease (e.g., nephropathy). Specifically, for example, the abnormal amount can be 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more higher than the reference. The reference may refer to the amount measured in a control sample (e.g., a tissue or cell or any biological sample that does not contain the target disease). In the art, a range of normal values ​​can be obtained by analyzing the detected amounts of markers in samples from a normal population of individuals using conventional detection and statistical methods.

[0044] As used herein, "low expression" and "high expression" of a biomarker as used herein refer to relative terms relative to the amount of the biomarker found in a sample. In some embodiments, low expression and high expression can be determined by comparing the expression level of the biomarker in a control group or a non-diseased sample, wherein low expression can refer to a lower or equivalent expression level relative to the expression level in the control or non-diseased sample, and high expression can refer to a higher expression level relative to the expression level in the control or non-diseased sample.

[0045] As used herein, a biological marker (biomarker) is an objectively measured and evaluated characteristic (e.g., protein, amino acid, metabolite, gene or genetic expression) as an indicator of normal or abnormal biological processes, diseases, pathogenic processes, or response to treatment or therapeutic intervention. A biomarker may include the presence or absence of a characteristic or pattern or collection of characteristics that indicates a specific biological process. A biomarker measurement may increase or decrease to indicate a certain biological event or process. Markers are primarily used for diagnostic and prognostic purposes. However, they can be used for treatment, monitoring, drug screening, and other purposes described herein, including evaluating the effectiveness of therapeutic agents.

[0046] As used herein, the biological sample to be analyzed by any of the methods described herein can be any type of sample obtained from an individual to be diagnosed. In some embodiments, the biological sample can be a body fluid sample, for example, a blood sample, a urine sample, or an ascites sample. Typically, the biological sample is a urine sample. In other embodiments, the blood sample can be whole blood or a portion thereof, such as serum or plasma, heparinized or EDTA treated to prevent blood coagulation. Alternatively, the biological sample can be a tissue sample or a biopsy sample obtained from a kidney.

[0047] As used herein, the term "physiological parameter" as used herein generally refers to any parameter that can be monitored to determine one or more quantitative physiological aspects and / or activities associated with a patient. Examples of physiological parameters include, but are not limited to, age, sex, systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBG), hemoglobin A1c (HbA1c), duration of diabetes, creatinine, estimated glomerular filtration rate (eGFR), albuminuria, albumin to creatinine ratio (ACR), and any combination thereof. In certain specific embodiments, the physiological parameter includes fasting blood glucose (FBG) and / or diastolic blood pressure (DBP).

[0048] As used herein, the term "nephropathy" refers to a physiological condition in which damage to the kidney occurs, which specifically disrupts its ability to properly regulate the concentration of solutes in the blood and urine. Nephropathy can be characterized by one or more pathological changes: glomerular size, tufted fibrosis, Bowman's capsule fibrosis, dilatation, microvascular narrowing, basement membrane thickening, pleocytosis (interannular membrane or endothelium), leukocyte infiltration, microvascular thrombosis, tubular atrophy, necrosis, vacuolar and hyaline droplet changes, basement membrane thickening, dilatation, inflammatory cells and casts within the tubular lumen, interstitial fibrosis, edema, acute and chronic leukocyte infiltration, arteriolar fibrosis, thrombosis, hyaline changes and narrowing. Generally speaking, in the early stages of nephropathy, the kidneys can still filter waste products from the blood well; in the middle stages, the kidneys may need to work harder to get rid of waste products; in the late stages, the kidneys may stop working. Usually and routinely, nephropathy can be assessed by urine protein concentration. Early clinical features of nephropathy may be low but abnormal albumin concentration in urine (albumin excretion rate (AER): 30-300 mg / 24 hours; or albumin to creatinine ratio (ACR): 30-300 mg / g), called microalbuminuria, and the patient has initial nephropathy (primary nephropathy); if not properly treated, such patients will develop persistent microalbuminuria and turn into severe nephropathy (overt nephropathy), also called macroalbuminuria (albumin excretion rate (AER)>300 mg / 24 hours or albumin to creatinine ratio (ACR)>300 mg / g), and finally progress to end-stage renal disease (ERSD). Estimated glomerular filtration rate (eGFR) can also be used as an indicator of nephropathy. Chronic kidney disease (CKD) can be defined as an estimated glomerular filtration rate (eGFR) of less than 60 ml / min for more than 3 months in patients with or without proteinuria, or proteinuria for more than 3 months despite low or high eGFR. Nephropathy can also be assessed based on, for example, serum creatinine concentration, urine protein concentration, urine protein to creatinine ratio, or by using tracer compounds such as phthalates.

[0049] In some embodiments, chronic kidney disease (CKD) can be considered to include five (5) stages of kidney damage, ranging from very mild damage in stage 1 to complete kidney failure in stage 5. See Table A.

[0050] Table A. Different stages of chronic kidney disease (CKD).

[0051]

[0052] In particular, the early stages of chronic kidney disease (CKD) as described herein may include Stage 1 and Stage 2 as shown above, where patients may have a relatively high (normal) estimated glomerular filtration rate (eGFR) but have at least one sign of kidney damage, such as microalbumin.

[0053] As used herein, the term "diabetic nephropathy" refers to kidney disease caused by diabetes. In certain embodiments, the diabetes is type 2 diabetes. Many diabetic patients experience early progressive renal failure (ERFD) before the onset of microalbuminuria, although they may still have normal renal function. Once the decline process begins, without appropriate treatment, it will progress and may lead to impaired renal function. Specifically, when the estimated glomerular filtration rate (eGFR) is above 1.73 m / s, the kidney function will be reduced. 2 When the decrease in blood pressure exceeds 3.3 mL / min, it can be determined as early progressive renal function decline (ERFD).

[0054] The present application is based (at least in part) on the identification of CC16 as a new and reliable biomarker for nephropathy. As demonstrated in some of the following examples, increased CC16 levels were found in urine samples of individuals suffering from nephropathy. Therefore, the nephropathy detection method described herein can be used to identify whether an individual suffers from, is suspected of suffering from, or has a risk of developing nephropathy. The detection method described herein can be applied to any individual, especially as an initial, routine, and regular (or early) screening method to identify those patients suffering from nephropathy or having a risk of progressive nephropathy. As further demonstrated in other examples below, the presence of CC16 in diabetic patients is associated with the later development of early progressive renal function decline (ERFD). Therefore, when applied to diabetic patients, the detection method described herein can be used to predict the risk of ERFD or diabetic nephropathy.

[0055] In some specific embodiments, B2M is further performed to improve the accuracy of the detection.

[0056] As used herein, Klebsiella pneumoniae protein (CC16) is a 15.8-kDa isomeric binary protein that is secreted in large quantities in the airways by non-ciliated bronchiolar Klebsiella pneumoniae cells. CC16 has been shown to regulate the production and / or activity of various mediators of inflammatory responses, including PLA2, interferon-γ, and tumor necrosis factor-α (Clinical & Experimental Allergy 30(4):469-75, May 2000). β2-microglobulin (B2M) is a subunit of the major histocompatibility complex (MHC) class I molecule. The amino acid sequences of these protein biomarkers are well known in the art, for example, CC16: P11684, and B2M: P61769.

[0057] The presence and amount of the biomarkers described herein in a biological sample can be determined by conventional techniques. In some embodiments, the presence and / or amount of the biomarkers described herein can be determined by mass spectrometry, which allows for direct measurement of analytes with high sensitivity and reproducibility. There are many mass spectrometry methods to choose from. Examples of mass spectrometry include, but are not limited to, matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI-TOF), surface-enhanced laser desorption ionization / time of flight mass spectrometry (SELDI-TOF), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography tandem mass spectrometry (LC-MS-MS), and electrospray ionization mass spectrometry (ESI-MS). One example of such a method is tandem mass spectrometry (MS / MS), which involves multiple steps of mass selection or analysis, usually by some form of fragmentation.

[0058] In other embodiments, the presence and / or amount of the biomarker can be determined by immunoassay. Examples of immunoassays include, but are not limited to, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunoprecipitation assay (RIPA), immunofluorescence assay (IFA), enzyme-linked fluorescent immunoassay (ELFA), electrochemiluminescence (ECL), and capillary gel electrophoresis (CGE). In some examples, the presence and / or amount of the biomarker can be determined using a reagent that specifically recognizes the biomarker, such as an antibody that specifically binds to the biomarker.

[0059] As used herein, the antibody may be a polyclonal antibody or a monoclonal antibody. Polyclonal antibodies against a specific protein are prepared by injecting an effective amount of a peptide or antigen component into a suitable experimental animal, collecting serum from the animal, and isolating the specific serum by any known immunoadsorption technique. Animals that can be easily used to produce polyclonal antibodies used in the present invention include chickens, mice, rabbits, rats, goats, horses, etc.

[0060] To perform the methods described herein, the levels of the biomarkers described herein are detected or measured in a biological sample taken from an individual in need thereof (e.g., a human patient without any symptoms of renal disease, or a human patient suffering from, suspected of suffering from, or at risk of suffering from renal disease) by any method known in the art, such as those described herein, such as mass spectrometry. Typically, the biological sample is a urine sample.

[0061] In some embodiments, the amount of a biomarker in a sample from a candidate individual can be compared to a standard value. A higher amount of a biomarker as described herein can indicate a positive result, i.e., the individual has nephropathy or is at risk of developing nephropathy, or when the individual is a diabetic, he / she has a higher risk of developing diabetic nephropathy or early progressive renal function decline (ERFD). The standard value represents the amount of a biomarker as described herein in the control sample. The control sample can be taken from an individual who does not have nephropathy. In addition, the control sample can be a mixture of samples from a group of such individuals. Alternatively, the control individual is matched to the candidate individual in, for example, age, sex, and / or ethnic background. Preferably, the control sample and the biological sample of the candidate individual are samples of the same species.

[0062] In some instances, the amount of marker in the control sample is undetectable in the control sample (i.e., the reference value is 0), and the presence of the marker (detectable marker) detected in a biological sample from an individual using conventional analytical methods, such as mass spectrometry and immunoassays, and using the same analytical methods can indicate a positive result.

[0063] In some specific embodiments, CC16 is detected as a first biomarker according to the present invention. A higher level of the first biomarker compared to a (first) control level of the biomarker may indicate a first positive result. In some other specific embodiments, B2M is further detected as a second biomarker according to the present invention. A higher level of the second biomarker compared to a (second) control level of the biomarker may indicate a second positive result with increased accuracy. In further specific embodiments, one or more physiological parameters may be additionally measured. For example, such physiological parameters may be selected from the group consisting of estimated glomerular filtration rate (eGFR), albuminuria, urine albumin to creatinine ratio (ACR), and any combination thereof.

[0064] When an individual, such as a human patient, is diagnosed as having, suspected of having, or at risk for developing renal disease, the individual may undergo further testing (e.g., conventional physical testing, including surgical biopsies or imaging methods, such as X-ray imaging, magnetic resonance imaging (MRI), or ultrasound) to confirm the presence of the disease and / or determine the stage and type of renal disease.

[0065] In some embodiments, the methods described herein may further include treating the patient with nephropathy to at least alleviate symptoms associated with the disease. Treatment may be performed by administering conventional drugs for nephropathy. Examples of such drugs include, but are not limited to, (i) drugs for reducing albuminuria, such as phosphodiesterase inhibitors, for example, dipyridamole and pentoxifylline; (ii) antihypertensive drugs, such as angiotensin converting enzyme (ACE) inhibitors, for example, imidapril, and angiotensin receptor blockers ( (iii) phosphate binders, such as sevelamer carbonate, lanthanum carbonate, and Al(OH)3 hexitol complex; (iv) calcium supplements, such as calcium carbonate, calcium citrate, and vitamin D; (v) anti-anemic drugs, such as erythropoietin (EPO) and iron supplements; (vi) drugs for lowering blood lipids, such as statins, such as simvastatin, pravastatin, and atorvastatin; (vii) drugs for lowering uric acid, such as isopurinol, febuxostat, and benzbromarone; (viii) other drugs, such as corticosteroids, such as prednisolone, non-steriodal anti-inflammatory drugs (NSAIDs), and N-acetylcysteine ​​(for prevention of contrast-induced nephropathy (CIN)). These drugs can be administered to a subject in need thereof in an effective amount. Treatment of nephropathy may also include a diet regimen with a low protein and / or low salt diet.

[0066] As used herein, "effective amount" refers to the amount of each active substance that can be administered to the individual alone or in combination with one or more other active substances to confer a therapeutic effect on the individual. The effective amount may vary and must be determined by a person skilled in the art, depending on the specific circumstances at the time of administration, the severity of the condition, various parameters of the patient, including sex, age, weight, height, physical condition, treatment regimen, the nature of parallel treatments (if any), the specific route of administration, and other possible factors determined by the knowledge and professional judgment of the medical staff. These factors are well known to those of ordinary skill in the art and can be introduced without further routine experiments.

[0067] The present invention also provides a kit or composition for implementing the method, which includes a reagent (e.g., an antibody or a labeling reagent) that specifically recognizes a biomarker as described herein. The kit may further include instructions for using the kit to detect the presence or amount of the biomarker described herein, thereby detecting renal lesions in individuals in need thereof, or for predicting diabetic nephropathy or early progressive renal function decline (ERFD) in diabetic patients. The components comprising the detection reagents as described herein can be packaged together in the form of a kit. For example, the detection reagents can be packaged in separate containers, such as antibodies (bound to a solid matrix or packaged separately from reagents for binding them to the matrix), control reagents (positive and / or negative) and / or detectable labels, instructions for performing the analysis (e.g., written, tape, VCR, CD-ROM, etc.) can also be included in the kit. For example, the assay format of the kit can be a chip or an enzyme-linked immunosorbent assay (ELISA). Further provided is the use of such reagents for implementing the methods described herein. Such reagents include reagents that specifically recognize biomarkers. In some specific embodiments, such reagents include (i) molecules that specifically recognize CC16, optionally (ii) molecules that specifically recognize B2M, or (iii) a combination of (i) and (ii). The reagent can be mixed with a carrier, such as a pharmaceutically acceptable carrier, to form a composition for detection or diagnosis purposes. Examples of such carriers include injectable saline, injectable distilled water, injectable buffer solutions, and the like.

[0068] Without further elaboration, it is believed that those skilled in the art will be able to apply the present invention to the greatest extent based on the above description. Therefore, the purpose of the following specific examples is to illustrate, but not to limit the scope of application of the present invention in any way. All documents cited herein are incorporated herein by reference.

[0069] Example

[0070] In this study, C 18The urinary protein profiles of 238 individuals from the following 4 groups were compared by using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS): patients with type 2 diabetes (T2D) with microalbuminuria, patients with diabetes (DM) without micro- or macroalbuminuria, patients with micro- or macroalbuminuria caused by non-diabetes, and healthy controls. It was found that β2-microglobulin (B2M) and Klebsiella pneumoniae protein (CC16) were highly released in the urine of patients with proteinuria caused by non-diabetes or diabetes. The sensitivity and specificity of the combined B2M and CC16 markers were 77.3% and 91.8%, respectively, in distinguishing between nephrotic and healthy individuals. The sensitivity and specificity of the combined markers were 66% and 73%, respectively, in distinguishing between patients with type 2 diabetes (T2D) with microalbuminuria and patients with type 2 diabetes (T2D). The predictive ability of B2M and CC16 for early progressive renal failure (ERFD) was verified in 125 patients with type 2 diabetes (T2D) during the follow-up period. The odds ratio (OR) of the combined marker of B2M and CC16 for the occurrence of ERFD was 7.59 (95% CI: 1.97-29.24). 18 The disc-MALDI-TOF MS method for detecting B2M as well as CC16 may be an attractive practical test for rapid diagnosis of nephropathy in non-diabetic / diabetic patients and as a predictor of early progressive renal function decline (ERFD) in patients with type 2 diabetes (T2D) who do not show overt renal disease at baseline.

[0071] Abbreviations: DM = diabetes mellitus; DN = diabetic nephropathy; WDM-NP = patients with micro-or macroalbuminuria due to nondiabetic disease; DM-WNP = patients with type 2 diabetes mellitus without micro-or macroalbuminuria; DM-NP = patients with type 2 diabetes mellitus with microalbuminuria; MALDI-TOF-MS = matrix-assisted laser desorption / ionization time-of-flight mass spectrometry; SELDI-TOF-MS = surface-enhanced laser desorption / ionization time-of-flight mass spectrometry; SA = Sinapic ionization mass spectrometry. acid) = sinapinic acid; CC16 = Klebsiella pneumoniae protein; B2M = β2-microglobulin.

[0072] 1. Materials and Methods

[0073] 1.1 Chemicals

[0074] Polydimethylsiloxane (PDMS) prepolymer was purchased from Dow Corning (Sylgard 184 Midland County, Michigan, USA). Acetonitrile (ACN) and trifluoroacetic acid (TFA) were purchased from JTBaker (Phillipsburg, New Jersey, USA). Dithiothreitol (DTT), iodoacetamide (IAA), and formic acid (FA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Octadecyl-coated silica particles (C 18 ,3μm, Develosil was purchased from Nomura Chemical Co., Ltd. (Seto, Japan). Sinapic acid (SA) was purchased from Bruker Daltonics (Germany). Trypsin (modified, sequenced grade) was purchased from Promega (Madison, Wisconsin, USA). Urea was purchased from Bio Basic (Toronto, Canada).

[0075] 1.2 Study population and samples

[0076] A cross-sectional study design was used to discover and validate protein markers by C18 disk / MALDI-TOF MS. The study protocol, including sample collection, preparation, and analysis, was approved by the local ethics committee of China Medical University, Taichung, Taiwan, and was conducted in accordance with the principles of the Declaration of Helsinki. All individuals (n=238) had provided their informed consent prior to the study. The following 4 groups were defined according to the clinical course and urinary albumin excretion content: diabetic patients with microalbuminuria (DM-NP; n = 53, 30 < albumin to creatinine ratio (ACR) < 300 mg / g), diabetic patients without micro- or macroalbuminuria (DM-WNP; n = 87, albumin to creatinine ratio (ACR) < 30 mg / g), patients with micro- or macroalbuminuria not caused by diabetes (WDM-NP; n = 48, 30 mg / g < albumin to creatinine ratio (ACR)), and healthy controls (n = 50; albumin to creatinine ratio (ACR) < 30 mg / g). The clinical characteristics of the 4 groups are shown in Table 1.

[0077] Table 1. Clinical and biochemical parameters of healthy, WDM-NP, DM-WNP, and DM-NP individuals.

[0078]

[0079]

[0080] Values ​​are expressed as mean ± standard deviation. WDM-NP group, patients with micro- or macroalbuminuria due to non-diabetes; DM-WNP group, patients with diabetes (DM) without micro- or macroalbuminuria; DM-NP, patients with diabetes with microalbuminuria; BMI, body mass index; eGFR, estimated glomerular filtration rate; ACR, albumin to creatinine ratio.

[0081] 1.3 Follow-up verification of the study population

[0082] A total of 125 individuals with type 2 diabetes (T2D), including 56 with early progressive renal function decline (ERFD) (cases) and 69 without early progressive renal function decline (ERFD) (controls) were included in this nested case-control study. All patients had normal renal function (estimated glomerular filtration rate [eGFR]>60mL / min / 1.73m2) at enrollment. 2 , albumin to creatinine ratio (ACR) <300 mg / g). Of these participants, 56 had early progressive renal function decline (ERFD) and 69 did not have early progressive renal function decline (ERFD) during the follow-up period. Early progressive renal function decline (ERFD) was defined as a decrease in estimated glomerular filtration rate (eGFR) per 1.73 m / day per year. 2 A decrease of more than 3.3 mL / min

[12] . Glomerular filtration rate (GFR) was estimated using the Modified Diet in Renal Disease (MDRD) equation

[13] .

[0083] 1.4 Preparation of urine samples for protein analysis

[0084] The midstream urine was collected in a 15 mL centrifuge tube for protein sampling. To reduce the effects of protein degradation, 500 μL of a protease inhibitor cocktail solution (1 protease inhibitor tablet dissolved in 10 mL of double distilled water (ddH2O)) was added to 10 mL of each collected urine sample. The urine samples were centrifuged at 3000 g and 4 ° C for 20 minutes. After eliminating the precipitate, the supernatant was collected immediately or stored at -80 ° C.

[0085] 1.5 by C 18 Protein desalting and protein analysis by MALDI-TOF MS

[0086] According to our previous study

[11] , C 18 First, wash the C with 100% methanol solution. 18 The urine samples (20 μL) were loaded directly onto the C 18 The spots were then washed with double distilled water (ddH2O) to remove salts. 3 μL of 80% acetonitrile (ACN) / 0.1% TFA solution was used to remove the salts from the C 18The desalted protein was eluted. For MALDI-TOF MS analysis, the eluted protein was mixed with 2 μL of sinapinic acid (SA) solution (saturated sinapinic acid (SA) in 30% acetonitrile (ACN) / 0.1% TFA) on a MALDI target. After sinapinic acid (SA) / protein co-crystallization, the MALDI-target was analyzed using a MALDI-TOF / TOF MS system (Ultraflex III TOF / TOF; Bruker Daltonics) equipped with a Smartbeam laser system in linear mode.

[0087] 1.6 Purification of protein marker peaks

[0088] The LC column (XBridge Protein BEH C4 column, The protein was purified using a liquid chromatography (LC) pump system (Ultimate 3000; Dionex) with a flow rate of 3.5 μm, 2.1 mm x 150 mm; Waters). The mobile phases were solvent A (5% acetonitrile (ACN) and 0.1% formic acid (FA)) and solvent B (100% acetonitrile (ACN) and 0.1% formic acid (FA)). The gradient flow was set at a flow rate of 250 μL / min as follows: 1% solvent B for 1.5 minutes, 1% to 30% solvent B over 1 minute, 30% to 80% solvent B over 16 minutes, 80% solvent B for 2 minutes, and 80% solvent B reduced to 1% solvent B in 5 minutes. The eluate was monitored at wavelengths of 220 and 280 nm with a UV detector (VWD-3400RS; Dionex). The eluate was collected at intervals of 60 seconds. Each fraction was analyzed by MALDI-TOF MS to confirm successful purification of the marker peak at m / z ∼ 15860. The purified protein subfraction with the marker peak m / z 15860 and its adjacent subfraction (as a control subfraction) were dried in a centrifugal concentrator (miVac Duo Concentrator; Genevac, NY, USA) and then subjected to in-solution digestion and identification by nanoLC-MS / MS analysis.

[0089] 1.7 In-solution digestion

[0090] The purified protein marker peak at m / z 15800 was redissolved in 4 M urea and reduced with 10 mM DTT at 37°C for 45 min. Then, 55 mM IAA was added and the mixture was incubated at 25°C in the dark for 60 min. Ammonium bicarbonate buffer (10 mM) was added to the protein solution to reduce the urea concentration to below 1 M. Trypsin was then added to the protein solution at an enzyme-substrate ratio of 1:25 (w / w) at 37°C for 16 h. The peptide solution was heated to 40 °C for 24 h. 18 Z-tips were desalted, dried in a centrifugal concentrator, and then reconstituted with 10 μL of 0.1% formic acid (FA) for nanoLC-MS / MS analysis.

[0091] 1.8 NanoLC-MS / MS analysis

[0092] NanoLC-MS / MS was performed using a nanoflow ultra-high performance liquid chromatography system (UltiMate 3000 RSLCnano system; Dionex) coupled to a hybrid quadrupole time-of-flight (Q-TOF) mass spectrometer (maXis Impact; Bruker). After sample loading, the peptides were eluted from the capture column to the analytical column (Acclaim PepMap C 18 , 2μm, 75μm x 250mm; Thermo Scientific) and coupled to the nano-electrospray ionization source on the Q-TOF mass spectrometer. A gradient flow of 8% acetonitrile (ACN) (0.1% formic acid (FA)) to 40% acetonitrile (ACN) (0.1% formic acid (FA)) in 36 minutes was used for tryptic peptide separation at a flow rate of 300nL / min. The 8 charge precursors +2, +3, and +4 for each TOF MS scan were dynamically selected and separated for MS / MS fragment ion scanning. MS and MS / MS accumulation were set to 1 and 10Hz, respectively.

[0093] 1.9 Protein database search

[0094] The spectra acquired by nanoLC-MS / MS were converted to xml files using DataAnalysis (version 4.1; Bruker) and searched against the Swissprot (version 51.0) database using MASCOT (version 2.2.07). The MASCOT search parameters for precursor ion and fragment ion tolerance were 80 ppm and 0.07 Da, respectively. The following search parameters were selected: Taxonomy, human; missed cleavages, 1; enzyme, trypsin; fixed modifications, carbamidomethyl (C); and variable modifications, oxidation (M) and deamidation (NQ). Peptides were considered "identified" if their individual MASCOT ion scores were higher than 25 (p < 0.01).

[0095] 1.10 ELISA measurement of B2M and Klebsiella pneumoniae proteins

[0096] According to the manufacturer's instructions, commercial kits (Cloud-Clone) were used to measure urine β2-microglobulin (B2M) and Klebsiella pneumoniae protein (CC16) concentrations by enzyme-linked immunosorbent assay (ELISA). All samples were processed using the same equipment and the same laboratory technicians, who were blinded to all clinical data. The Mann-Whitney test was used to compare the differences in median values, which were expressed as median values ​​with quartile values ​​(25%, 75%).

[0097] 1.11 Statistical analysis

[0098] Continuous data were expressed as mean and standard deviation or median and interquartile range, and categorical data were expressed as proportions. Two independent sample T tests were used to compare the means of continuous variables, and the chi-square test was used to compare the frequencies of categorical variables between groups. Logistic regression models were used to estimate the association between potential urinary biomarkers and early progressive renal function decline (ERFD), and the odds ratio (OR) and 95% confidence intervals (CIs) were calculated. Receiver operating characteristic (ROC) curves were constructed to determine the sensitivity and specificity of potential biomarkers. Statistical analysis was performed using SigmaPlot 11.1 (Systat Software, California, USA) and SPSS statistical software 22.0 (IBM Corporation, New York, USA). p values ​​less than 0.05 (bilateral) were considered significant.

[0099] 2. Results

[0100] 2.1 C 18 1. Disk Preparation of Urine Samples

[0101] High salt content in urine can interfere with MALDI crystallization and result in poor mass spectrometer signals. To evaluate the effect of salt on urine protein analysis, 20 μL of urine was directly applied to MALDI-TOF MS analysis without desalting. To avoid salt interference effects, we used hydrophobic C 18 The disk removes salt but retains protein in the urine sample. The desalted urine sample is applied to the C 18 After the hour, the protein signal was greatly improved.

[0102] To evaluate the minimum amount of protein required to obtain protein profiles in this study, different amounts of urine protein were tested. Similar protein profiles were obtained when 0.6-2.38 μg was administered. The protein concentrations measured by Bradford protein assay in urine samples of the healthy, WDM-NP, DM-WNP, and DM-NP groups (expressed as median values ​​with quartile values ​​(25%, 75%)) were 0.06 μg / μL (0.03–0.09 μg / μL), 0.34 μg / μL (0.13–1.11 μg / μL), 0.05 μg / μL (0.03–0.08 μg / μL), and 0.13 μg / μL (0.10–0.19 μg / μL), respectively. Therefore, the amount of protein in a 20 μL urine sample was sufficient to provide informative protein profiles in this study.

[0103] 2.2 Normal and pathological protein excretion patterns in urine

[0104] Desalinuric protein samples from 87 DM-WNP patients, 53 DM-NP patients, 48 ​​WDM-NP patients, and 50 healthy controls were analyzed by MALDI-TOF MS. Representative MALDI-TOF mass spectra from healthy and WDM-NP individuals are shown in Figure 1 The significant peaks of m / z 11732±2 (or oxidized form m / z 11748±2) and m / z 15840±3 (or oxidized form m / z~15856±3) were found to be highly expressed in WDM-NP and DM-NP individuals; their representative pseudogel and spectral analysis were also performed (data not shown). The 9.7 kDa peak (sphingolipid B) was used as an internal standard to evaluate the diagnostic value of the two protein marker peaks of 11.7 kDa and 15.8 kDa. Figure 2A As shown, the peak ratio of WDM-NP and DM-NP was 11.7 / 9.7 kDa, which was significantly higher than that of the healthy group (p<0.001). The peak ratio of WDM-NP and DM-NP was 15.8 / 9.7 kDa, which was significantly higher than that of the healthy group (p<0.001) and the DM-WNP group (p<0.001) ( Figure 2B ).

[0105] To differentiate between WDM-NP patients and healthy individuals, the area under the curve (AUC) of the receiver operating characteristic (ROC) plot was studied. The area under the curve (AUC) was 0.75 for the 11.7 kDa peak and 0.74 for the 15.8 kDa peak. Since both peaks can be detected simultaneously in a single MALDI-TOF mass spectrum, both can be used as diagnostic markers. The sensitivity and specificity of these two peaks were 77.3% and 91.8%, respectively, with an improved area under the curve (AUC) of 0.8, thus serving as markers for differentiating between WDM-NP (nephropathy) and healthy individuals. For the differentiation between DM-NP (diabetic nephropathy) and DM-WNP patients, the area under the curve (AUC) was 0.6 for the 11.7 kDa peak and 0.67 for the 15.8 kDa peak. In this case, the combined signature of these two peaks had a sensitivity and specificity of 66% and 73%, with an area under the curve (AUC) of 0.62.

[0106] 2.3 Purification and identification of differentially expressed proteins

[0107] The 11.7 kDa peak has been reported to be β2-microglobulin (B2M)

[14] and was also identified in our previous study

[15] . To confirm the identity of the 15.8 kDa peak, urine samples were fractionated by C4 reverse phase chromatography as described in the Materials and Methods section. LC-UV chromatograms of urine protein from DM-NP individuals were performed (data not shown), and the 15.8 kDa peak was purified from subfraction 10 and identified as Klebsiella pneumoniae protein (CC16) based on MS / MS sequencing of a doubly charged tryptic peptide peak at m / z 647.91 (Mascot identification score of 88), showing a complete y- and b-ion series corresponding to the sequence KLVDTLPQKPR ( Figure 3 ).

[0108] 2.4 ELISA evaluation of B2M and CC16

[0109] Since enzyme-linked immunosorbent assay (ELISA) is commonly used in clinical laboratories to quantify protein marker abundance for diagnostic purposes, ELISA was performed for 11.7 kDa B2M and 15.8 kDa CC16 to assess the relative abundance of the two markers in urine. (Figure 4) The expression of B2M in the WDM-NP, DM-NP, and DM-WNP groups was significantly higher than that in the healthy control group (p<0.001). However, the expression of CC16 in the DM-NP group was significantly higher only relative to the DM-WNP group (p<0.05) and the healthy control group (p<0.001). In order to distinguish between WDM-NP patients and healthy individuals, the area under the curve (AUC) of the receiver operating characteristic (ROC) plot was evaluated and found to be 0.87 for B2M, 0.67 for CC16, and 0.74 for the combination of B2M and CC16. In the case of distinguishing DM-NP from DM-WNP, the area under the curve (AUC) of B2M was 0.59, the area under the curve (AUC) of CC16 was 0.60, and the area under the curve (AUC) of the combination was 0.60.

[0110] 2.5 Validation of protein signatures in T2D patients who have developed ERFD

[0111] A nested case-control study design was used to investigate the predictive ability of B2M and CC1 in the development of renal disease in patients with type 2 diabetes (T2D). T2D individuals with (n=56) and without (n=69) early progressive renal function decline (ERFD) as the primary endpoint had similar demographics (age and sex), duration of diabetes, BMI, systolic blood pressure (SBP), diastolic blood pressure (DBP), HbA1c, creatinine, and albumin to creatinine ratio (ACR) at baseline examination. Estimated glomerular filtration rate (eGFR) was higher in patients with ERFD than in those without ERFD (mean ± standard deviation: 114.13 ± 25.86 vs. 98.86 ± 23.36 [p value = 0.001]). Albumin to creatinine ratio (ACR) was not a significant marker at baseline for predicting ERF) (p value of chi-square test was 0.196 (Table 2)). For both potential urine markers, 16 of 56 T2D patients with ERFD (28.6%) and 9 of 69 T2D patients without ERFD (13.0%) had detectable CC16 markers (CC16 / SAP ratio>0) at baseline (p-value for chi-square test was 0.031 (Table 2)). No significant difference in the presence of B2M was observed between the two groups (39.3% vs. 31.9% in ERFD and non-ERFD groups, p=0.389). The data showed that the presence of CC16 at baseline was associated with the later development of ERFD.

[0112] Table 2. Demographic and clinical characteristics of patients stratified by estimated glomerular filtration rate (eGFR) decline ≥ 3.3% per year (ERFD)

[0113]

[0114]

[0115] Abbreviations: ACR, albumin-to-creatinine ratio; SBP, systolic blood pressure; DBP, diastolic blood pressure; eGFR, estimated glomerular filtration rate;

[0116] BMI, body mass index; ERFD, early stage renal failure; DM, diabetes mellitus.

[0117] Group 1: B2M / SAP_MALDI ratio = 0, and CC16 / SAP MALDI ratio = 0

[0118] Group 2: B2M / SAP_MALDI ratio = 0, and CC16 / SAP MALDI ratio > 0,

[0119] B2M / SAP_MALDI ratio>0, and CC16 / SAP MALDI ratio=0

[0120] Group 3: B2M / SAP_MALDI ratio>0 and CC16 / SAP MALDI ratio>0

[0121] Logistic regression was used to further examine the effects of the potential biomarkers B2M and CC1 on ERFD independently or in combination (Table 3). When comparing individuals with the markers to those without the markers, the odds ratios (ORs) for ERFD were 2.01 (95% CI: 0.90-4.52) and 4.87 (95% CI: 1.77-13.44) for the β2-microglobulin (B2M) marker and the Klebsiella pneumoniae (CC16) marker, respectively, after adjusting for follow-up time. In addition, the combination of CC16 and B2M had a significant additive effect in increasing the risk of ERFD (p-value for the interaction term = 0.003). When comparing individuals with both markers to those without any markers, the odds ratio (OR) for ERFD was 7.59 (95% CI: 1.97-29.24).

[0122] Table 3. Logistic regression model

[0123]

[0124] a P values ​​for logistic regression models, unadjusted

[0125] b P values ​​of logistic regression models adjusted for follow-up time

[0126] Group 1: B2M / SAP_MALDI ratio = 0, and CC16 / SAP MALDI ratio = 0

[0127] Group 2: B2M / SAP_MALDI ratio = 0, and CC16 / SAP MALDI ratio > 0,

[0128] B2M / SAP_MALDI ratio>0, and CC16 / SAP MALDI ratio=0

[0129] Group 3: B2M / SAP_MALDI ratio>0 and CC16 / SAP MALDI ratio>0

[0130] 3. Discussion and Conclusion

[0131] Recently, MALDI-TOF MS has been successfully approved as an in vitro diagnostic device for routine bacterial identification in hospitals

[16] . Therefore, disease markers detected by MALDI-TOF MS have become practical for clinical use. Since salts in urine can interfere with MALDI-TOF mass spectrometry signals, in this study, C 18 The disk quickly removes salts from clinical samples and retains urine protein in urine samples for MALDI-TOF MS analysis. Our results showed that WDM-NP and DM-NP groups had higher expression of WDM-NP protein (CC16) than healthy group (p<0.001) and DM-WNP group (p<0.001). However, in the results of enzyme-linked immunosorbent assay (ELISA), the expression of WDM-NP protein (CC16) in DM-NP group was only slightly higher than that in DM-WNP group (p=0.05). These results also indicate that MALDI-TOF MS can identify WDM-NP protein (CC16) more specifically than ELISA analysis.

[0132] β2-microglobulin (B2M) is a low molecular weight protein that is filtered by the glomerulus and degraded in the proximal tubule

[17] . Some studies have shown that B2M increases in tubular damage, suggesting that B2M is an early diagnostic marker for tubular damage [18,19]. In type 2 diabetes, urinary B2M excretion is associated with macrovascular disease

[20] and nephropathy [1,21,22].

[0133] The 15.8-kDa Klebsiella cell protein (CC16) (also known as CC10, uteroglobin, or urine protein 1) is rapidly eliminated by glomerular filtration and is reabsorbed and catabolized in the renal proximal tubular cells. Dysfunction of the proximal tubular cells results in decreased reabsorption of CC16 and increased levels in the urine. CC16 has been reported as a marker for proximal tubular dysfunction in adults

[23] and children

[24] . This protein marker is sensitive to very subtle defects in proximal tubular dysfunction that may not be detected by classical urinary low-molecular-weight proteins

[25] . To our knowledge, our study is the first to identify CC16 in the urine of patients with renal disease and diabetic nephropathy (DN).

[0134] Through long-term follow-up studies, it was found that the odds ratio (OR) of B2M and CC16 for early progressive renal function decline (ERFD) was 7.59, which was an independent predictor of ERFD in patients with type 2 diabetes (T2D) who did not show obvious kidney disease at baseline. It was also shown that the protein peaks of B2M and CC16 detected by C18 disk / MALDI-TOF can improve the sensitivity of predicting renal disease before the appearance of urinary albumin.

[0135] Through the analysis of a large sample size, we discovered and validated 2 protein peaks, B2M (11.7 kDa) and CC16 (15.8 kDa), as biomarkers associated with nephropathy and verified their discriminatory ability in 238 individuals including diabetic and non-diabetic patients. The odds ratio (OR) of the combined B2M and CC16 markers for developing ERFD was 7.59 (95% CI: 1.97-29.24). This is the first report of CC16 as a urinary marker for nephropathy and diabetic nephropathy (DN). Therefore, we used C 18 The method of detecting B2M and CC16 by plate-MALDI-TOF MS can provide rapid diagnosis and prediction of nephropathy in patients with type 2 diabetes.

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Claims

1. Use of a reagent comprising a molecule that specifically recognizes Klebsiella pneumoniae protein, for preparing a kit or composition for predicting the risk of early progressive renal function decline in diabetic patients.

2. The use according to claim 1, wherein the reagent further comprises a molecule that specifically recognizes β2-microglobulin.

3. The use according to claim 1 or 2, wherein the agent is an antibody.

4. The use according to claim 1 or 2, wherein the diabetic patient is a type 2 diabetic patient.

Images