Methods and compositions for differentiating progressive chronic kidney disease from stable chronic kidney disease

Abstract

The present disclosure provides methods and compositions for detecting and differentiating stable CKD from progressive CKD in a mammal.

Description

[0001] priority

[0002] This application claims the benefit of U.S. Patent Serial No. 63 / 267,181 filed on January 26, 2022 and U.S. Patent Serial No. 63 / 354,878 filed on June 23, 2022, both of which are incorporated by reference in their entirety. Background Art

[0003] Chronic kidney disease (CKD) may be accompanied by increased water consumption, frequent urination, decreased appetite, weight loss and muscle atrophy. Typically, by the time clinical symptoms of CKD appear, irreparable kidney damage has occurred. Early detection allows for earlier treatment, which in turn slows disease progression. Current treatments include, for example, dialysis, addressing any predisposing factors, and a low-phosphorus and low-protein diet. Early detection is essential for improving life expectancy and quality of life.

[0004] In humans, CKD progression is divided into five stages. In companion animals (e.g., canines and felines), CKD progression is divided into four stages described by the International Renal Interest Society. Current methods for detecting CKD in mammals (e.g., canines, felines, and humans) include renal ultrasound, biopsy, measurement of serum creatinine and symmetric dimethylarginine (SDMA), and measurement of urine protein and creatinine. Biopsy is invasive, so its ability as an early screening or diagnostic test is limited. Serum creatinine levels will only increase after a significant decrease in glomerular function, and have poor sensitivity for early, mild, or moderate CKD. Urine and serum creatinine are also affected by muscle mass, diet, and tubular secretion. Currently available methods for detecting and diagnosing CKD cannot distinguish stable CKD from progressive CKD in a timely manner. Summary of the invention

[0005] One embodiment provides a method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD by the following steps: (a) determining the amount of cystatin B polypeptide, clusterin polypeptide, or both in a sample from the subject at a single time point, (b) comparing the amount of cystatin B polypeptide, clusterin polypeptide, or both in the sample with a control sample or a control standard, wherein an increased level of cystatin B polypeptide, clusterin polypeptide, or both in the sample compared to the control sample or the control standard is an indication of progressive CKD.

[0006] In some embodiments, prior to step (a), the subject may be diagnosed with International Kidney Rights Association CKD stage I or CKD with a symmetric dimethylarginine (SDMA) value of up to 20 μg / dl. In some embodiments, the method may also include administering one or more treatments to the subject, the one or more treatments including: surgery for ureteral obstruction, obstructive kidney stones or urolithiasis, dietary management, administration of enteric phosphate binders, antiproteinuric drugs, administration of antihypertensive drugs, fluid therapy for correction of dehydration, acidosis management, administration of diuretics, dialysis, correction of electrolyte abnormalities, administration of antiemetics, administration of antacids, administration of recombinant erythropoietin, overall treatment or a combination thereof. In some embodiments, the method may also include determining the amount of cystatin B polypeptide, clusterin polypeptide or both in a sample from a subject at 2, 3, 4, 5 or more additional time points. In some embodiments, the amount of cystatin B polypeptide can be determined using one or more antibodies or specific binding fragments that specifically bind to cystatin B polypeptide or one or more polypeptides set forth in SEQ ID NO: 1-2, 5-32, fragments thereof or combinations thereof. In some embodiments, clusterin polypeptides can be measured using one or more antibodies or specific binding fragments that specifically bind to clusterin polypeptides or one or more polypeptides set forth in SEQ ID NO:3-4, fragments thereof, or combinations thereof. In some embodiments, measuring the amount of cystatin B polypeptide or clusterin polypeptide in a sample from a subject may include contacting the sample with one or more antibodies or specific binding fragments thereof that specifically bind to cystatin B polypeptide, clusterin polypeptide, or one or more polypeptides set forth in SEQ ID NO:1-32 under conditions suitable for forming a complex of cystatin B polypeptide or clusterin polypeptide with the one or more antibodies or specific binding fragments thereof, and detecting the complex of cystatin B polypeptide or clusterin polypeptide with the one or more antibodies or specific binding fragments thereof. In some embodiments, antibodies or specific binding fragments thereof may be fixed to a solid support. In some embodiments, antibodies or specific binding fragments thereof may be conjugated to one or more markers. In some embodiments, the method may further include contacting a complex of cystatin B polypeptide or clusterin polypeptide with the one or more antibodies or specific binding fragments thereof and an indicator. In some embodiments, the subject may be a non-human mammal. In some embodiments, the non-human mammal may be a canine or a feline. In some embodiments, the sample can be blood, serum, plasma or urine. In some embodiments, the amount of cystatin B or clusterin polypeptide can be determined by immunoassay, competitive immunoassay, sandwich immunoassay, enzyme-linked immunosorbent assay (ELISA), turbidimetric immunoassay, particle enhanced turbidimetric immunoassay, radioimmunoassay (RIA) or Western blot assay.In some embodiments, the amount of cystatin B or clusterin polypeptide can be determined by mass spectrometry, LC-MS, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatography (HPLC) mass balance determination or a combination thereof. In some embodiments, the control sample or control standard can be derived from a normal, healthy subject. In some embodiments, the control sample can be derived from a subject with stable CKD.

[0007] Specific embodiments will become apparent from the following more detailed description of certain preferred embodiments and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The uCysB values ​​for each dog grouped by disease state are shown. Control and stable CKD dogs appear to have similar uCysB values. Progressive CKD dogs tend to have greater uCysB values ​​than control and stable CKD dogs. Serial measurements for a given dog do not appear to be independent.

[0009] Figure 2 The Y-intercept calculated from the linear regression of each dog with urinary cystatin B as the dependent variable and follow-up time as the independent variable is shown. While the y-intercepts were relatively comparable for control dogs and dogs with stable CKD, they were consistently higher for dogs with progressive CKD. Kruskal-Wallis ANOVA was used to detect differences in the y-intercepts between groups (p<10 -4 ).

[0010] Figure 3 Shown are the slopes calculated from linear regression of each dog with log(uCysB) as dependent variable and follow-up time as independent variable. Differences between the slopes of control, stable CKD and progressive CKD dogs were detected on Kruskal-Wallis ANOVA (p=0.006).

[0011] Figure 4 The first uCysB measurement values ​​for the CKD group are shown. Although only eight dogs had progressive IRIS stage 1 CKD at the first visit, six (75%) had first uCysB values ​​above the reference range (RR) 50, of which four (67%) progressed to beyond IRIS stage 1 CKD.

[0012] Figure 5 The use of cystatin B levels to differentiate stable CKD from progressive CKD in canine International Kidney Advocate stage I CKD patients was shown.

[0013] Figure 6 The use of cystatin B levels in canine CKD patients with SDMA values ​​up to 20 was shown to differentiate stable CKD from progressive CKD.

[0014] Figure 7 The use of clusterin levels to differentiate stable CKD from progressive CKD in canine International Kidney Advocate stage I CKD patients was shown.

[0015] Figure 8 The use of clusterin levels in canine CKD patients with SDMA values ​​up to 20 was shown to differentiate stable CKD from progressive CKD.

[0016] Fig. 9 The creatinine reciprocal slope cutoff is shown to differentiate stable renal function in healthy dogs (HD) from renal dysfunction in progressive CKD caused by X-linked hereditary nephropathy (XLHN). The slope cutoff is shown as a gray dashed line.

[0017] Fig.10 The SDMA inverse slope cutoff is shown to differentiate stable renal function in healthy dogs (HD) from renal dysfunction in progressive CKD caused by XLHN. The slope cutoff is shown as a grey dashed line.

[0018] Fig.11 Shown is how to determine the normal biomarker range for reciprocal creatinine.

[0019] Fig.12 Shown is how to determine the normal biomarker range for the inverse of SDMA.

[0020] Fig.13 Shown is how to determine the minimum change in the reciprocal creatinine slope from normal to abnormal.

[0021] Fig.14 It shows how to determine the minimum change in the SDMA reciprocal slope from normal to abnormal.

[0022] Fig.15 Representative serial creatinine observations for 30 dogs randomly selected from a cohort of 113 healthy dogs are shown.

[0023] Fig.16 Shown are representative reciprocal serial changes in creatinine in 30 dogs randomly selected from a cohort of 113 healthy dogs.

[0024] Fig.17 The inverse slope plot of creatinine for the HD cohort is shown.

[0025] Fig.18 Representative sSDMA serial observations of 30 dogs randomly selected from a cohort of 113 healthy dogs are shown.

[0026] Fig.19Shown are the sequential changes and slopes of the SDMA reciprocal in 30 dogs randomly selected from a cohort of 113 healthy dogs.

[0027] Fig. 20 The SDMA inverse slope plot for the HD cohort is shown.

[0028] Fig.21 Shown are the sequential observations of creatinine in the XLHN cohort.

[0029] Fig. 22 Shown are the temporal changes in s-creatinine in the XLHN cohort.

[0030] Fig.23 Serial changes in reciprocal creatinine in the XLHN cohort are shown.

[0031] Fig.24 The inverse creatinine slope plot for the XLHN cohort is shown.

[0032] Fig.25 Shown are sequential observations of sSDMA in the XLHN cohort.

[0033] Fig.26 Shown are the temporal changes in sSDMA in the XLHN cohort.

[0034] Fig. 27 Serial changes in the inverse of SDMA in the XLHN cohort are shown.

[0035] Fig.28 Shown is a plot of the SDMA inverse slope for the XLHN cohort.

[0036] Fig.29 Creatinine reciprocal slope cutoff values ​​are shown to differentiate stable renal function in healthy dogs (HD) from renal dysfunction in progressive CKD caused by X-linked hereditary nephropathy (XLHN).

[0037] Fig.30 SDMA reciprocal slope cutoff values ​​are shown to differentiate stable renal function in healthy dogs (HD) from renal dysfunction in progressive CKD caused by X-linked hereditary nephropathy (XLHN).

[0038] Fig.31 Shown are box plots demonstrating reciprocal creatinine (left panel) and reciprocal SDMA (right panel) slope cutoffs (dotted lines) to distinguish stable renal function in healthy dogs (HD) from renal dysfunction in progressive CKD caused by X-linked hereditary nephropathy (XLHN).

[0039] These and other objects and features will be better understood from the following detailed description taken in conjunction with the accompanying drawings. DETAILED DESCRIPTION

[0040] The terms used in this specification in the context of the compositions and methods described herein and in the specific context in which each term is used generally have their ordinary meaning in the art. Some terms are more specifically defined below to provide additional guidance to the practitioner regarding the description of the compositions and methods.

[0041] Overview

[0042] Chronic kidney disease (CKD) is defined as the presence of structural or functional abnormalities in the kidney for more than 3 months. Changes such as fibrosis and renal parenchymal degeneration dominate. CKD is irreversible and is often progressive in nature. CKD is usually diagnosed based on the change of functional markers such as serum creatinine and symmetric dimethylarginine (SDMA) concentration, and these functional markers are also used to monitor disease progression. However, due to the nonlinear relationship with glomerular filtration rate (GFR) and because the reference range of creatinine and SDMA is relatively wide, these functional markers may not be sensitive in early CKD. In some dogs with CKD, active persistent damage (acute kidney injury; AKI) markers may also increase, which shows that even if the disease seems stable based on functional markers, active persistent damage is also present in dogs with CKD. Therefore, CKD and AKI may not be completely independent processes, and may have some common features.

[0043] Although CKD is often progressive in all dogs, the degree of progress varies greatly between dogs and dogs. Such progress differences can be observed in the rate of change or slope of functional kidney biomarkers (e.g., creatinine, SDMA). When assessing the progress of CKD, the functional kidney biomarker value can be converted to its reciprocal (" biomarker reciprocal "), from which the slope (" slope reciprocal ") can be calculated. Several risk factors for CKD progress have been identified, including the renal proteinuria degree, hypertension and hyperphosphatemia reflected by urine protein: creatinine (UPC) ratio. Regardless of the potential cause, the active damage degree of the dog suffering from CKD can serve as a surrogate marker of progression rate.

[0044] Stage 1 chronic kidney disease (CKD) based on the IRIS guidelines is generally more stable than advanced CKD. However, some IRIS stage 1 CKD dogs, especially those with progressive CKD, may have active persistent damage throughout the follow-up period, as reflected by elevated uCysB values. In addition, uCysB can provide an earlier indicator of such damage compared to traditional biomarkers.

[0045] CKD in dogs and cats is a multifactorial disease with multiple related causes. In most animals with CKD, it is not possible to identify or eliminate the underlying cause. In the absence of an identifiable cause that can be eliminated, treatment focuses primarily on dietary intervention (which has been shown to slow the rate of progression) and control of risk factors for rapid progression (e.g., hypertension, proteinuria). Once the disease has progressed to a more advanced stage, treatment turns to a more symptomatic treatment. Therefore, the main therapeutic goal is to identify the disease early in the disease process and slow the rate of progression before substantial irreversible damage occurs.

[0046] The diagnosis of CKD stage 1 is challenging and requires additional methods for detecting kidney damage. Currently, the diagnosis of CKD cannot usually rely on functional markers. Therefore, early diagnosis is based on the presence of persistent non-concentrated urine (after excluding all other potential causes), the presence of renal proteinuria, and ultrasound changes (which are usually negligible and uncertain at this stage of the disease). Many of these techniques require multiple visits to the veterinarian. Further diagnosis includes measuring GFR and renal biopsy, both of which are not routinely obtained in clinical settings. As described in this article, 75% of progressive CKD dogs had elevated uCysB at the first visit, compared to only eight percent of stable dogs. In addition, 67% of progressive CKD dogs with elevated uCysB progressed to after the IRIS1 stage. As demonstrated here, uCysB can be used as an early biomarker to distinguish between stable and progressive IRIS1 stage CKD.

[0047] In this study, dogs with rapid progression had higher concentrations of uCysB during follow-up compared with stable dogs. This means that the extent of active persistent damage, regardless of cause and underlying etiology, can be used as a surrogate marker of active damage within the kidney that leads to loss of renal function and may be irreversible (i.e., fibrosis). It is uncertain whether therapeutic interventions can slow its progression, however, the fact that real-time markers of renal damage are available will facilitate real-time evaluation of various therapeutic interventions in animals with CKD.

[0048] To date, therapeutic interventions have typically been monitored using functional markers, which change very slowly. Therefore, evaluating these interventions requires very long follow-up periods, during which other factors (some of which can be identified and controlled, while others cannot) may also affect the rate of progression. Due to the very long time required to evaluate the effects of therapeutic interventions in dogs with early kidney disease, such studies are difficult and costly to conduct. However, these interventions may be more realistic in the early stages of the disease, before substantial irreversible changes have occurred in most kidneys. In this context, real-time markers such as uCysB are valuable and will allow for a better understanding of the intrarenal processes that control CKD and potential interventions to improve these processes.

[0049] The slope of SDMA was used in this article to assess the rate of disease progression. Despite the high prevalence of CKD in dogs and cats, no definition or guidelines have been established to distinguish animals with slow progression rates from those with intermediate or high progression rates. The percentage change or absolute change in creatinine or SDMA concentration can be used to define progression, however, the time at which these changes occur is critical to determining the rate of progression (i.e., if a creatinine increase of 0.5 mg / dL occurs within 2 months, the dog can be defined as rapidly progressive, while if it occurs within 3 years, the dog can be defined as slowly progressive). In this study, the slope of at least 3 time points over at least 3 months was used to account for the time effect and assess the rate of progression.

[0050] uCysB and clusterin (described below) are biomarkers for detecting intrarenal damage in animals with CKD and are potential surrogate markers for progression rate. In stage I CKD, uCysB and clusterin may differentiate stable CKD from progressive CKD, creating an opportunity to halt progression and improve patient management.

[0051] Stable chronic kidney disease and progressive kidney disease

[0052] The compositions and methods described herein can be used to distinguish between progressive CKD and stable CKD in mammals such as canines, felines, and humans who have been diagnosed with CKD. The progress of progressive CKD and stable CKD can also be monitored. Progressive CKD and stable CKD can lead to (1) decreased renal function compared to healthy subjects; or (2) physical damage to the kidneys; or (3) both. In some embodiments, CKD (both progressive and stable) does not include cancer or does not include renal cancer. The markers of cancer including renal cancer may be different from the markers of stable or progressive CKD. In some embodiments, stable or progressive CKD does not cover cancer, such as renal cancer or bladder cancer.

[0053] CKD is a disease characterized by a gradual loss of kidney function over time. CKD can exist in a stable or progressive form. Progressive or stable CKD can be glomerular CKD or tubular CKD. As CKD worsens, waste products build up to high levels in the blood, and high blood pressure, anemia, weak bones, poor nutritional health, and nerve damage may develop. CKD increases the risk of developing cardiovascular disease and may eventually lead to kidney failure. CKD can be caused by, for example, diabetes, high blood pressure, and other conditions. Early detection and treatment can often prevent the disease from getting worse.

[0054] The canine CKD staging established by the International Renal Rights Society ("IRIS") is shown in Table 1.

[0055] Table 1.

[0056]

[0057] The feline CKD staging system established by the International Kidney Rights Association is shown in Table 2.

[0058] Table 2.

[0059]

[0060] Functional renal markers (e.g., creatinine or SDMA) changes over time can be used to detect CKD. However, there are several problems with using these markers to determine whether a subject suffers from stable CKD or progressive CKD. In particular, these markers must be observed over time to determine whether CKD is stable or progressive. Irreversible kidney damage may occur in the time required to observe any trend in marker levels. In addition, the use of blood creatinine as an early indicator of CKD is relatively insensitive. Creatinine levels in healthy dogs and cats may overlap with creatinine values ​​in dogs and cats with mild to moderate kidney disease. In addition, other problems with using creatinine as a marker include: creatinine varies with muscle mass, blood creatinine measurements are not standardized between veterinary diagnostic laboratories, eating a meat diet results in postprandial creatinine absorption, so fasting samples need to be used (ideally fasting for 12 hours), and an increase in blood creatinine concentration must be detected after most of the kidney tissue has been damaged. The use of SDMA as a marker for CKD also has disadvantages, including: an increase in SDMA in dogs and cats with diseases other than CKD (e.g., hyperthyroidism or adrenocortical insufficiency), significant differences in SDMA levels between different breeds of dogs and cats, and hemolysis of samples may cause interference in measuring SDMA. Therefore, a reliable marker that can determine whether a CKD patient has stable or progressive CKD without measuring over time may be helpful in preventing kidney disease.

[0061] Surprisingly, it has been discovered that the use of cystatin B ("CysB") and / or clusterin markers can determine whether a CKD patient has stable or progressive CKD without the need to use two or more tests over time. Prior to the present disclosure, it was unknown whether there were markers that could be used to distinguish stable CKD from progressive CKD at a single time point.

[0062] Although referred to as "stable CKD," stable CKD can progress past IRIS CKD stages I-IV. However, stable CKD that progresses past stage IV is in contrast to progressive CKD, where symptoms can progress or worsen over time at a faster rate than the symptoms of stable CKD.

[0063] Functional renal markers such as creatinine or SDMA in patients with stable CKD do not substantially change over time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36 months or longer (and any range between about 1 to about 36 months, e.g., about 1 to about 3 months, about 2 to about 4 months, about 3 to about 6 months, about 6 to about 12 months, or about 1-36 months). No substantial change in a functional renal marker means that the amount of a functional renal marker in a CKD patient increases by less than about 2%. , 5%, 10%, 20% or 25%. The increase of the amount of the functional kidney marker of progressive CKD can be over time (for example, in 5 days, 1 week, 2 weeks, 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36 months or longer (and in any range between about 5 days to about 36 months, for example, about 1 to about 3 months, about 2 to about 4 months, about 3 to about 6 months, about 6 to about 12 months, or about 1-36 months) increase by about 21%, 25%, 30%, 35%, 40% or more.

[0064] Provided herein is a method for determining whether a patient diagnosed with CKD suffers from stable CKD or progressive CKD by measuring, for example, the level of cystatin B, clusterin or both at a single time point. In some embodiments, a mammalian CKD patient is in an IRIS I phase or has an SDMA value of up to 20. When a mammalian CKD patient has a urine cystatin B level higher than about 50, 60, 70, 80, 90, 100 ng / mL or more cystatin B (or at any range between about 50 to 100 ng / mL or more (e.g., about 50-100 ng / mL or more, about 50-80 ng / mL or more, about 70-100 ng / mL or more)), the patient is diagnosed as suffering from progressive CKD. These are considered to be "the amount of increase" of cystatin B. When a mammalian CKD patient has a urine level of greater than about 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 ng / mL or more of clusterin (or any range between about 250 and 350 ng / mL or more (e.g., about 250-300 ng / mL or more, about 275-325 ng / mL or more, about 325-350 ng / mL or more)), the patient is diagnosed as having progressive CKD. These are considered "elevated amounts" of clusterin.

[0065] Also provided herein is a method for determining whether a patient diagnosed with CKD suffers from stable CKD by measuring, for example, the level of cystatin B, clusterin or both at a single time point. In some embodiments, the mammal CKD patient is in IRIS phase I or has an SDMA value of up to 20. When the mammal CKD patient has a urine cystatin B level of about 20, 30, 40, 50, 60, 70, 80, 90, 100 ng / mL or more cystatin B (e.g., any range between about 20 to 100 ng / mL), the patient is diagnosed as suffering from stable CKD. When the mammal CKD patient has a urine cystatin B level of about 100, 90, 80, 70, 60, 50, 40, 30, 20 ng / mL or less cystatin B (e.g., any range between about 100 to 20 ng / mL), the patient is diagnosed as suffering from stable CKD.

[0066] Using a single time point of SDMA or creatinine did not provide the same results as using the cystatin B or clusterin markers as described herein.

[0067] Peptides

[0068] IDEXX Laboratories, Inc.'s previous discoveries and work related to cystatin B and clusterin have been described in U.S. Patent No. 10,725,052 (Methods and compositions for the detection and diagnosis of renal disease and periodontal disease); U.S. Patent No. 10,436,797 (Markers for renal disease); and U.S. Patent Publication 2018 / 0142009 (Specific Detection of Clusterin isoforms). IDEXX's previous work validating cystatin B and clusterin in canine renal injury also included the development of an in vitro cell model of cultured canine kidney cells treated with nephrotoxic gentamicin. The clusterin isoforms present in the cultured cell supernatant have a unique glycosylation pattern compared to clusterin isoforms isolated from plasma, enabling the generation of antibodies that specifically target the kidney-specific isoforms. Purification of canine cystatin B from cultured canine kidney cells was determined by trypsin digestion followed by LC-MS, allowing the creation of a recombinant protein that matched the native protein extracted from canine kidney cells using LC-MS.Clinical validation of cystatin B has previously been demonstrated in dogs with renal damage.

[0069] Cystatin B (CysB) is an intracellular protein belonging to the cysteine ​​protease inhibitor family. CysB is an intracellular protein and therefore does not circulate at high serum concentrations. Cystatin A and B are members of family 1 of the cystatin superfamily and are relatively small proteins with a size of about 11 kDa. In humans, these proteins are monomeric with a size of about 11 kDa. They are not glycosylated and do not have the disulfide bridges seen in other cystatin superfamilies. They also lack signal sequences and are generally intracellular proteins confined to cells. See Ochieng & Chaudhuri, J Health Care Poor Underserved 2010, 21 (1 Suppl): 51. A certain amount of cystatin B is present in extracellular fluid (including human urine). Cystatin B has been shown to inhibit members of the cathepsin family lysosomal cysteine ​​protease, particularly cathepsin B, H and L. See Green et al., Biochem J 1984 218:939; D'Amico et al., J Transl Med 2014, 12:350; Jarvinen & Rinne, Biochim Biophys Acta 1982, 708:210-217.

[0070] Canine cystatin B (SEQ ID NO: 1) is:

[0071] MMCGAPSASQPATADTQAIADQVKAQLEERENKKYTTFKAVTFRSQVVAGTXYFIKVQVDDDEFVHLRVFQSLPHENKPLALSSYQTNKAKHDELAYF

[0072] (wherein X may be any amino acid, or where X may be P or N).

[0073] SEQ ID NO: 1 can be used to detect feline or canine cystatin B.

[0074] Fragments of canine cystatin B may include: MMCGAPSASQPATADTQAIAD (SEQ ID NO:5) QTNKAKHDELAYF (SEQ ID NO:6) Cystatin BC terminal "peptide 9" CGAPSASQPATADTQAIA (SEQ ID NO:7) Cystatin B N-terminal "peptide 3-20" CGAPSASQ (SEQ ID NO:8) Cystatin B N-terminal "peptide 3-10" CAIADQVKA (SEQ ID NO:9) Cystatin B N-terminal "peptide 18-25" FQSLPHENKPLALSS (SEQ ID NO:10) Cystatin B "peptide 2" SQVVAGTPYFIKVQVDDD (SEQ ID NO:11) Cystatin B "peptide 1" KHDELAYF (SEQ ID NO:12) MMCGAPSASQPATADTQAIADQVKAQLEE (SEQ ID NO:13) AIADQVKA (SEQ ID NO:14) NO:14)SQVVAGTNYFIKVQVDDD(SEQ ID NO:15)

[0075] Feline cystatin B (NCBI reference sequence: XP_023094432.1) is shown below

[0076] MMCGAPSATQ PATAETQAIA DQVKPQLEEQ ENKKYTTFKA VEFRSQVVAG RNYFIKVQVDDDEFVHIRVF QSLPHENKPL ALSSYQTHKA RHDELAYF(SEQ ID NO:2)

[0077] Fragments of canine cystatin B may include:

[0078] SQVVAGRNYFIKVQVDDD(SEQ ID NO:16)

[0079] AIADQVKP (SEQ ID NO: 17)

[0080] MMCGAPSATQPATAETQAIADQVKPQLEE(SEQ ID NO:18)

[0081] RHDELAYFSEQ ID NO:19

[0082] SQVVAGRNYFIKVQVDDDSEQ ID NO:20

[0083] FQSLPHENKPLALSS SEQ ID NO:10

[0084] QAIADQVKP SEQ ID NO:21

[0085] CGAPSATQ SEQ ID NO:22

[0086] CGAPSATQPATAETQAIA SEQ ID NO:23

[0087] QTHKARHDELAYF SEQ ID NO:24

[0088] MMCGAPSATQPATAETQAIAD SEQ ID NO:25

[0089] Clusterin or apolipoprotein J is a 75-80 kDa disulfide-linked heterodimeric protein. Clusterin is part of many physiological processes, including sperm maturation, lipid transport, complement inhibition, tissue remodeling, membrane recycling, stabilization of stress proteins, and promotion of apoptosis inhibition. Clusterin polypeptides can be detected using any suitable method, including, for example, immunoassays. In some embodiments, clusterin can be detected using a combination of lectins and anti-clusterin antibodies and lectins. See, for example, U.S. Patent Publication 20180142009. Lectins that specifically bind to carbohydrates on human, canine, feline, equine, bovine, ovine, or ape clusterin isoforms can be used. Lectins that specifically bind to one or more plasma, serum, or kidney clusterin isoforms without binding to other clusterin isoforms can also be used.

[0090] Lectins are proteins that recognize and bind to specific monosaccharide or oligosaccharide structures (carbohydrates). Lectins usually contain two or more binding sites for carbohydrate units. The carbohydrate binding specificity of a particular lectin is determined by the amino acid residue that binds the carbohydrate. The strength of lectin binding to carbohydrates can increase with the number of molecular interactions. The dissociation constant for lectin binding to carbohydrates is about 10 -5 Up to 10 -7 K d .

[0091] Lectins can be labeled with any type of label known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal, radioisotope, and bioluminescent labels.

[0092] In some embodiments, a lectin that specifically binds to the kidney's unique clusterin and does not specifically bind to plasma or serum clusterin can be used. In some embodiments, a lectin that specifically binds to N-acetylglucosamine can be used in the method. Such lectins include, for example, WGA (wheat germ agglutinin), WGA1, WGA2, WGA3, sWGA, DSL lectin (Datura stramonium agglutinin), mannose binding lectin, PHA-L (Phaseolus vulgaris leukocyte agglutinin), PHA-E (Phaseolus vulgaris red agglutinin (erythoagglutanin)) and LEL (tomato (tomato) lectin). Other lectins that can be used include, for example, jacalin, STR lectin (potato), LCA lectin (lentil), PSA lectin (pea lectin), ECL lectin (erythrina), RCA lectin (castor), DBA lectin (Double Flower Lentil), SBA lectin (soybean) and CONA lectin (concanavalin). Lectins are commercially available from, for example, Vector Laboratories.

[0093] In some embodiments, clusterin can be detected using an anti-clusterin antibody or a specific binding fragment thereof and a combination of one or more lectins that specifically bind to clusterin (e.g., a kidney-specific clusterin isoform). See, e.g., U.S. Patent Publication 20180142009. Some non-limiting examples of anti-clusterin antibodies are clusterin canine, sheep polyclonal antibodies from BioVendorLaboratory Medicine, Inc. Commercially available feline antibodies to clusterin can be, for example, but not limited to, clusterin / APOJ mouse anti-human, bovine, canine, feline, porcine, DyLight TM 650. Clone: ​​Hs-3, Novus Biologicals TMIn some embodiments, clusterins can be detected by contacting a sample with one or more antibodies or antigen binding fragments thereof that specifically bind to clusterins and one or more lectins that specifically bind to carbohydrate portions of kidney-specific clusterins and do not specifically bind to carbohydrate portions of other clusterin isoforms (e.g., plasma clusterins, serum clusterins, or hematogenous non-kidney-specific clusterins). A complex of kidney-specific clusterins, the one or more antibodies or antigen binding fragments thereof that specifically bind to clusterins, and the one or more lectins can be detected. The lectin can specifically bind to N-acetylglucosamine. The lectin can be, for example, Phaseolus vulgaris leukocyte agglutinin (PHA-L), wheat germ agglutinin (WGA), WGA1, WGA2, WGA3, sWGA, Phaseolus vulgaris lectin-E (PHA-E), tomato agglutinin (LEL), Datura stramonium agglutinin (DSL), pea agglutinin (PSA), or Dolichos biflorus lectin (DBA). One or more antibodies or antigen binding fragments thereof can be fixed to a support. The sample and one or more detectably labeled lectins can be added to the support. The lectin can be fixed to the support. The sample and one or more detectably labeled antibodies or antigen-binding fragments thereof can be added to the support. The one or more antibodies or antigen-binding fragments thereof, the one or more lectins (e.g., plasma clusterin, serum clusterin or blood-derived non-kidney-specific clusterin) or both can be labeled with a detectable label.

[0094] The canine clusterin precursor polypeptide UniProtKB-P25473 is shown below:

[0095] MMKTLLLLVG LLLTWDNGRV LGDQAVSDTE LQEMSTEGSK YINKEIKNAL

[0096] KGVKQIKTLI EQTNEERKSL LSNLEEAKKK KEDALNDTKD SETKLKASQG

[0097] VCNDTMMALWEECKPCLKQTCMKFYARVCRSGSGLVGHQLEEFLNQSSPF

[0098] YFWMNGDRID SLLENDRQQT HALDVMQDSF NRASSIMDEL FQDRFFTREP

[0099] QDTYHYSPFS LFQRRPFFNP KFRIARNIIP FPRFQPLNFH DMFQPFFDMI HQAQQAMDVNLHRIPYHFPI EFPEEDNRTV CKEIRHNSTG CLKMKDQCEK

[0100] CQEILSVDCS SNNPAQVQLR QELSNSLQIA EKFTKLYDEL LQSYQEKMFN

[0101] TSSLLKQLNE QFSWVSQLAN LTQSEDPFYL QVTTVGSQTS

[0102] DSNVPVGFTK

[0103] VVVKLFDSDP ITVMIPEAVS RNNPKFMETV AEKALQEYRQ KHREE(SEQ ID NO:3)

[0104] The precursor polypeptide chain is proteolytically cleaved to remove the 22-mer secretory signal peptide and subsequently cleaved between residues 227 / 228 to generate the α and β chains. These chains assemble in antiparallel to give a heterodimeric molecule with a cysteine-rich core connected by five disulfide bridges and flanked by two predicted coiled-coil α-helices and three predicted amphipathic α-helices.

[0105] The feline clusterin precursor UniProtKB-M3WKP2 is shown below:

[0106] MTVSCSLQTEACWDSRIGEPGTMKTLLLLVGLLLTCENGRVLGDKAVSDAELQEMSTEGS

[0107] KYINKEIKNALKGVKQIKTLIEQTNEERKSLLSNLEEAKKKKEDALSDTKDSEMKLKASE

[0108] GVCNDTMMALWEECKPCLKQTCMKFYARVCRSGSGLVGQQLEEFLNQSSPFYFWINGDRI

[0109] DSLLENDRQQTHALDVMQDSFNRASRIMDELFQDRFFTREPQDTYHYSPFSSLQRRPFFF

[0110] NPKSRFARNVMPFPAFQPLNFHDMFQPFFDMIHQAQQAMDINLQRIPYHFPMEFTEEEDNQ

[0111] DRMVCKEIRHNSTGCLRMKDQCDKCQEILSVDCSASNPSQVLLRQELNNSLQMAEKFTKL

[0112] YDELLRSYQEKMFNTSSSLKQLNEQFSWVSQLANLTQSEDPFYLQVTTVSSQTSDSNVPS

[0113] GFTKVVVKLFDSDPISVMVPEEVSRNNPKFMETVAEKALQEYRQKNGEK(SEQ ID NO:4)

[0114] The precursor polypeptide chain is proteolytically cleaved to remove the 43-mer secretory signal peptide.

[0115] Antibodies or specific binding fragments thereof that specifically bind to one or more polypeptides of SEQ ID NOs: 1-32 can be used to detect these polypeptides in a sample.

[0116] Provided herein are methods for detecting and quantifying cystatin B and clusterin polypeptides (e.g., SEQ ID NOs: 1-4) and fragments thereof (e.g., SEQ ID NOs: 5-32). One embodiment provides a purified polypeptide comprising SEQ ID NOs: 1-32 or a fragment thereof. The polypeptide fragment of SEQ ID NOs: 1-32 can be composed of less than about 95, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10 (or any range between about 10 to about 95) consecutive amino acids. In one embodiment, the polypeptide fragment consists of more than about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or 95 consecutive amino acids of SEQ ID NOs: 1-32. In one embodiment, the polypeptide or fragment thereof is non-naturally occurring.

[0117] A polypeptide is a polymer of three or more amino acids covalently linked by amide bonds. The polypeptide may be modified post-translationally. A purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used in polypeptide synthesis, or a combination thereof. A polypeptide preparation that is substantially free of cellular material, culture medium, chemical precursors, chemicals used in polypeptide synthesis has less than about 30%, 20%, 10%, 5%, 1% or more of other polypeptides, culture medium, chemical precursors, and / or other chemicals used in synthesis. Thus, a purified polypeptide is about 70%, 80%, 90%, 95%, 99% or more pure.

[0118] The term "polypeptide" may refer to one or more of a type of polypeptide (a group of polypeptides). "Polypeptide" may also refer to a mixture of two or more different types of polypeptides (i.e., a mixture of polypeptides including but not limited to full-length proteins, truncated polypeptides, or polypeptide fragments). The term "polypeptides" or "polypeptide" may also refer to "one or more polypeptides".

[0119] Polypeptide variants differ from the polypeptides set forth in SEQ ID NOs: 1-32 or fragments thereof by about, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues (e.g., amino acid additions, substitutions or deletions). Where such comparisons require alignment, the sequences are aligned for maximum homology. The variant sites may occur anywhere in the polypeptide.

[0120] Variant polypeptides can generally be identified by modifying one of the polypeptide sequences described herein and evaluating the properties of the modified polypeptide to determine whether it is a bioequivalent. If the variant reacts substantially the same as the polypeptide described herein in an assay such as an immunohistochemical assay, an enzyme-linked immunosorbent assay (ELISA), a turbidimetric immunoassay, a particle-enhanced turbidimetric immunoassay, a radioimmunoassay (RIA), an immunoenzymatic assay, or a Western blot assay, for example, 90-110% of the activity of the original polypeptide, the variant is a bioequivalent. In one embodiment, the assay is a competitive assay, in which the bioequivalent polypeptide is able to reduce the binding of the polypeptide described herein to the corresponding reactive antigen or antibody by about 80%, 95%, 99%, or 100%. Antibodies that specifically bind to the corresponding polypeptide also specifically bind to the variant polypeptide.

[0121] Variant polypeptides are at least about 80%, or about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the polypeptide sequences shown in SEQ ID NO: 1-32. For example, the variant polypeptides of SEQ ID NO: 1-32 may be about at least 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 90%, 87%, 84% or 81% identical to SEQ ID NO: 1-32. Variant polypeptides have one or more conservative amino acid changes or other minor modifications and retain biological activity, i.e., are biological functional equivalents of SEQ ID NO 1-32. When compared to the corresponding polypeptide, biologically active equivalents have substantially equivalent functions.

[0122] Methods for introducing mutations into amino acid sequences are well known to those skilled in the art. See, for example, Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, NY (1989)). Mutations can also be introduced using commercially available kits such as the QuikChange TM The "Stratagene Site-Directed Mutagenesis Kit" was introduced. Those skilled in the art can generate functionally active variant polypeptides by replacing amino acids that do not affect the function of the polypeptide.

[0123] Variant polypeptides may have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. Conservative substitutions are substitutions in which one amino acid is replaced by another amino acid of similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic / hydrophobic nature of the polypeptide to be substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. In one embodiment, the polypeptide has about 1, 2, 3, 4, 5, 10, 20 or fewer conservative amino acid substitutions.

[0124] The terms "sequence identity" or "percent identity" are used interchangeably herein. In order to determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for the best comparison purpose (e.g., a gap can be introduced in the sequence of the first polypeptide or polynucleotide to be optimally aligned with the second polypeptide or polynucleotide sequence). The amino acids or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e., overlapping positions) x 100). In some embodiments, the length of the reference sequence (e.g., SEQ ID NO: 1) aligned for comparison purposes is at least 80% of the length of the comparison sequence, in some embodiments at least 90% or 100%. In one embodiment, the two sequences have the same length.

[0125] The range of the desired sequence identity degree is about 80% to 100% and integer values ​​therebetween. The percentage identity between the disclosed sequence and the claimed sequence can be at least 80%, at least 83%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5% or at least 99.9%. Typically, an exact match indicates 100% identity over the length of the reference sequence (e.g., SEQ ID NO: 1).

[0126] Polypeptides and polynucleotides sufficiently similar to the polypeptides and polynucleotides described herein (e.g., cystatin B or clusterin polypeptides) can be used herein. Polypeptides and polynucleotides with about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or higher identity to the polypeptides and polynucleotides described herein can also be used herein.

[0127] For example, the polynucleotide can be 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 1-32.

[0128] A polypeptide or antibody can be covalently or non-covalently linked to an amino acid sequence that the polypeptide or antibody is not usually associated with in nature. In addition, a polypeptide or antibody can be covalently or non-covalently linked to a compound or molecule other than an amino acid. For example, a polypeptide or antibody can be linked to an indicator reagent, an amino acid spacer, an amino acid linker, a signal sequence, a termination transfer sequence, a transmembrane domain, a protein purification ligand, or a combination thereof. In one embodiment, a protein purification ligand can be one or more C amino acid residues at, for example, the amino terminal or carboxyl terminal of a polypeptide. An amino acid spacer is an amino acid sequence that is not usually associated with a polypeptide or antibody in nature. An amino acid spacer can include about 1, 5, 10, 20, 100, or 1,000 amino acids.

[0129] The polypeptide can be produced recombinantly. The polynucleotide encoding the polypeptide can be introduced into a recombinant expression vector, which can be expressed in a suitable expression host cell system using techniques well known in the art. There are a variety of bacterial, yeast, plant, mammalian and insect expression systems in the art, and any such expression system can be used. Optionally, the polynucleotide encoding the polypeptide can be translated in a cell-free translation system. The polypeptide can be lyophilized, dehydrated or dried, such as freeze-dried.

[0130] Polynucleotide

[0131] One embodiment includes a polynucleotide encoding one or more of the polypeptides disclosed herein. The polynucleotide contains less than the entire genome and can be a single-stranded or double-stranded nucleic acid. The polynucleotide can be RNA, DNA, cDNA, genomic DNA, chemically synthesized RNA or DNA or a combination thereof. The polynucleotide can be purified, free of other components such as proteins, lipids and other polynucleotides. For example, the polynucleotide can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99% or 100% purified. In one embodiment, the polynucleotide encodes a polypeptide or a fragment thereof shown in SEQ ID NO: 1-32.

[0132] The polynucleotide may contain other nucleotide sequences, such as sequences encoding linkers, signal sequences, TMR terminator transfer sequences, transmembrane domains, or ligands useful for protein purification such as glutathione-S-transferase, histidine tags, and Staphylococcal protein A.

[0133] Polynucleotides can be isolated. An isolated polynucleotide is a naturally occurring polynucleotide that is not adjacent to one or both of the 5' and 3' flanking genomic sequences with which it is naturally associated. An isolated polynucleotide can be, for example, a recombinant DNA molecule of any length, provided that the nucleic acid sequences that are usually found adjacent to the flanking recombinant DNA molecule in the naturally occurring genome are removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules.

[0134] Degenerate nucleotide sequences encoding polypeptides are also contemplated herein. Degenerate nucleotide sequences are polynucleotides encoding polypeptides as described herein or fragments thereof, but due to the degeneracy of the genetic code, their nucleic acid sequences are different from wild-type polynucleotide sequences. Complementary DNA (cDNA) molecules, species homologs, and polynucleotide variants encoding biologically functional polypeptides are also contemplated herein.

[0135] The polynucleotide may comprise a coding sequence for a naturally occurring polypeptide, or may encode a sequence of an alteration not present in nature. If desired, the polynucleotide may be cloned into an expression vector comprising an expression control element, including, for example, a replication origin, a promoter, an enhancer, or other regulatory elements that drive the expression of the polynucleotide in a host cell. The expression vector may be, for example, a plasmid such as pBR322, pUC, or ColE1, or an adenovirus vector such as an adenovirus type 2 vector or a type 5 vector. Optionally, other vectors may be used, including but not limited to Sindbis virus, Simian virus 40, alpha virus vectors, poxvirus vectors, cytomegalovirus, and retrovirus vectors, such as murine sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and Rous sarcoma virus. Minichromosomes such as MC and MC1, bacteriophages, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes, viral particles, virus-like particles, cosmids (plasmids into which the phage lambda cos site has been inserted) and replicons (genetic elements that can replicate under their own control in cells) may also be used.

[0136] Methods for preparing polynucleotides operably linked to expression control sequences and expressing them in host cells are well known in the art. See, for example, U.S. Patent No. 4,366,246. A polynucleotide is operably linked when it is positioned adjacent to or near one or more expression control elements that direct the transcription and / or translation of the polynucleotide.

[0137] Polynucleotides can be used, for example, as probes or primers, such as PCR primers, to detect the presence of polynucleotides in a test sample such as a biological sample. Probes are molecules that can interact with a target nucleic acid, usually in a sequence-specific manner, such as by hybridization. Primers are subsets of probes that can support enzymatic manipulations and can hybridize with target nucleic acids so that enzymatic manipulations occur. Primers can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art that do not interfere with enzymatic manipulations.

[0138] A probe or primer can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or more contiguous nucleotides encoding a polypeptide described herein.

[0139] Antibody

[0140] Antibodies and their specific binding fragments include antibody molecules that specifically bind to cystatin B and clusterin polypeptides described herein, variant cystatin B polypeptides and variant clusterin polypeptides described herein, or fragments thereof. Antibodies can specifically bind to a variety of polypeptides. The term "antibody" also includes any type of antibody molecule or specific binding fragment or molecule that specifically binds to one or more cystatin B polypeptides or fragments thereof or one or more clusterin polypeptides or fragments thereof (e.g., SEQ ID NO: 1-32). The term "antibody" also includes any type of antibody molecule or specific binding fragment that specifically binds to one or more cystatin B polypeptides or clusterin polypeptides (e.g., SEQ ID NO: 1-32). Antibodies can be naturally occurring, non-naturally occurring, synthetic or genetically engineered. As used herein, the term "antigen binding portion" of an antibody, "antigen binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptides, glycoproteins or immunoglobulins that specifically bind to cystatin B polypeptides (e.g., SEQ ID NO: 1-32) to form a complex.

[0141] The antibody or its specific binding fragment binds to the epitope of the polypeptide described herein. Antibodies can be prepared in vivo in suitable experimental animals or in vitro using recombinant DNA technology. Means for preparing and characterizing antibodies are well known in the art. See, for example, Dean, Methods Mol. Biol. 80:23-37 (1998); Dean, Methods Mol. Biol. 32:361-79 (1994); Baileg, Methods Mol. Biol. 32:381-88 (1994); Gullick, Methods Mol. Biol. 32:389-99 (1994); Drenckhahn et al., Methods Cell. Biol. 37:7-56 (1993); Morrison, Ann. Rev. Immunol. 10:239-65 (1992); Wright et al., Crit. Rev. Immunol. 12:125-68 (1992). For example, polyclonal antibodies can be produced by administering the polypeptides described herein to animals such as humans or other primates, mice, rats, rabbits, guinea pigs, goats, pigs, dogs, cattle, sheep, donkeys or horses. Serum from the immunized animals is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate followed by chromatography such as affinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art.

[0142] The antibodies can be of any isotype, including IgG (IgGl, IgG2, IgG2a, Ig2b, IgG3, IgG4), IgM, IgA (IgAl and IgA2), IgD, and IgE.

[0143] Antibodies can be monoclonal antibodies, polyclonal antibodies, chimeric antibodies or their specific binding fragments. Monoclonal antibodies are antibodies obtained from a group of substantially homogeneous antibodies. A group of substantially homogeneous antibodies may contain a small amount of mutants or variants. Monoclonal antibodies are highly specific and interact with a single antigenic site. Each monoclonal antibody typically targets a single epitope, while a polyclonal antibody population typically includes various antibodies targeting a group of different epitopes. Monoclonal antibodies can be produced by many methods, including, for example, hybridoma methods (Kohler and Milstein, Nature 256: 495, 1975), recombinant methods (U.S. Patent No. 4,816,567) and separation from phage antibody libraries (Clackson et al., Nature 352: 624-628, 1991; Marks et al., J. Mol. Biol. 222: 581-597, 1991).

[0144] Antibodies that specifically bind to mammalian (e.g., human, canine, and feline) cystatin B and clusterin are known in the art. In some embodiments, commercially available antibodies that bind to canine or feline clusterin or cystatin B can be used. Commercially available canine antibodies to clusterin can be, for example, but not limited to, anti-clusterin / APOJ antibodies (STJ70776) (EKALQEYRKKHREE SEQ ID NO: 34) from St John's Laboratory or clusterin canine, sheep polyclonal antibodies from BioVendorLaboratory Medicine, Inc. Commercially available feline antibodies to clusterin can be, for example, but not limited to, clusterin / APOJ mouse anti-human, bovine, canine, feline, porcine, DyLight TM 650. Clone: ​​Hs-3, Novus Biologicals TM Commercially available antibodies to canine cystatin B can be, for example, but not limited to, goat anti-cystatin B / Stefin B antibody (QTNKAKHDELTYF SEQ ID NO: 35) from MyBioSource.com or goat anti-human cystatin B / Stefin B (C-terminal) from RayBiotech.

[0145] Chimeric antibodies, or antigen-binding portions thereof, have a portion of a heavy chain and / or light chain derived from a particular species or a particular antibody class or subclass, and the remainder of the chain is derived from another species or another antibody class or subclass. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715, 4,816,567, and 4,816,397.

[0146] Chimeric antibodies can be produced using a variety of techniques, including, for example, CDR grafting (EP 239,400; PCT Publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28:489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., PNAS 96:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

[0147] In one embodiment, a chimeric antibody may comprise variable regions and constant regions of species different from each other, for example, an antibody may comprise a heavy chain and light chain variable region of a mammal and heavy chain and light chain constant regions from different animals (such as mice, rabbits, canines, felines or humans). A chimeric antibody may comprise additional amino acids that are not included in the CDR introduced into the receptor antibody, nor in the framework sequence. These amino acids may be introduced to more accurately optimize the ability of the antibody to recognize and bind antigens. For example, as needed, the amino acids in the framework region of the variable region of the antibody may be replaced so that the CDR of the remodeled antibody forms an appropriate antigen binding site. See Sato et al., Cancer Res. (1993) 53: 851-856.

[0148] Non-limiting examples of specific binding portions or fragments of antibodies include: Fab fragments; Fab' fragments, Fab'-SH fragments, F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; sdAb fragments (nanoantibodies); Fab-like antibodies (an antigen-binding fragment containing the variable regions of the heavy and light chains, which is equivalent to the Fab fragment obtained by papain digestion); F(ab')2-like antibodies (an antigen-binding fragment containing two antigen-binding domains, which is equivalent to the F(ab')2 fragment obtained by pepsin digestion), multispecific antibodies prepared from antibody fragments, diabodies, bispecific antibodies, multifunctional antibodies, humanized antibodies, caninized antibodies, human antibodies, canine antibodies, feline antibodies, mouse antibodies, rabbit antibodies, synthetic antibodies, CDR-grafted antibodies, and a minimum recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementary determining region (CDR), such as a CDR3 peptide) or a restricted FR3-CDR3-FR4 peptide. Other engineered molecules, such as domain-specific antibodies, single domain antibodies, domain-deleted antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., monovalent nanobodies, divalent nanobodies), single chain (Fv)2 (sc(Fv)2); divalent (sc(Fv)2); tetravalent ([sc(Fv)2]2) scFV antibodies and small modular immunopharmaceuticals (SMIPs) as well as shark variable IgNAR domains are also considered as "antigen-binding fragments or portions" as used herein.

[0149] "Specifically binds," "specifically bind," or "specific for" means that a first antigen, such as a polypeptide of SEQ ID NO: 1-32, recognizes and binds to an antibody described herein with a greater affinity than to other non-specific molecules. "Specifically binds," "specifically bind," or "specific for" also means that a first antibody, such as an antibody raised against SEQ ID NO: 1-32, recognizes and binds to SEQ ID NO: 1-32 with a greater affinity than to other non-specific molecules. A non-specific molecule is an antigen that does not share a common epitope with the first antigen. Specific binding can be tested using, for example, an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), a turbidimetric immunoassay, a particle-enhanced turbidimetric immunoassay, or a Western blot assay, or other suitable assay.

[0150] In one embodiment, the antibody or antigen-binding fragment thereof specifically binds to an epitope on a clusterin polypeptide as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In one embodiment, the epitope is AYF, LAYF (SEQ ID NO: 26), ELAYF (SEQ ID NO: 27), DELAYF (SEQ ID NO: 28), HDELAYF (SEQ ID NO: 29), KHDELAYF (SEQ ID NO: 12), AKHDELAYF (SEQ ID NO: 30), KAKHDELAYF (SEQ ID NO: 31), NKAKHDELAYF (SEQ ID NO: 32), TNKAKHDELAYF (SEQ ID NO: 33), or QTNKAKHDELAYF (SEQ ID NO: 6).

[0151] In one embodiment, the antibody or antigen-binding fragment thereof competes with a second or reference antibody for binding to SEQ ID NO: 1-32 or a fragment thereof. Any competitive binding assay can be used to measure competition between two antibodies for the same antigen. For example, a sandwich ELISA assay can be used to achieve this purpose. Means for determining cross-reactivity are well known to those skilled in the art (see, e.g., Dowbenko et al., (1988) J. Virol. 62: 4703-4711).

[0152] A first antibody is considered to competitively inhibit binding of a second antibody if, in the presence of the first antibody, binding of the second antibody to the antigen is reduced by at least about 30%, 40%, 50%, 60%, 75%, 90% or more using any assay for assessing competitive binding.

[0153] Competitive binding can be determined by providing one or more of the isolated polypeptides set forth in SEQ ID NOs: 1-32 attached to a solid support and determining the ability of an antibody to bind to the polypeptide or to compete with an antibody described herein for binding to the polypeptide.

[0154] Antibodies include antibodies and antigen-binding fragments thereof that (a) compete with a reference antibody for binding to SEQ ID NO: 1-32 or an antigen-binding fragment thereof; (b) bind to the same epitope of SEQ ID NO: 1-32 or an antigen-binding fragment thereof as the reference antibody; (c) bind to the same epitope of SEQ ID NO: 1-32 or an antigen-binding fragment thereof with substantially the same K as the reference antibody dand / or (d) binds to SEQ ID NO: 1-32 or an antigen-binding fragment thereof with substantially the same off-rate as a reference antibody, wherein the reference antibody is an antibody having an off-rate of 10 7 l / mol or higher binding affinity K a An antibody or antigen-binding fragment thereof that specifically binds to a polypeptide of SEQ ID NO: 1-32 or an antigen-binding fragment thereof.

[0155] The affinity of an antibody or its antigen-binding fragment for its polypeptide partner can be expressed as the dissociation constant (Kd). off / k on See Chen, Y. et al., 1999, J. Mol. Biol. 293:865-881. A variety of methods are known in the art for measuring affinity constants. In a specific embodiment, the reference antibody is a on Rate / Association Rate or K off The rate of the antibody or antigen-binding fragment thereof having binding affinity for the polypeptide of SEQ ID NO: 1-32. In one embodiment, the antibody is at 6 x 10 5 M -1 s -1 or better K on Specific binding; Antibodies at 5x 10 - 6 s -1 or better K off or the antibody specifically binds with a binding affinity of 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM or better.

[0156] Antibodies that specifically bind to SEQ ID NO: 1-32 or SEQ ID NO: 26-32 are particularly useful for detecting the presence of cystatin B and clusterin polypeptides and fragments thereof in samples such as serum, blood, plasma, cells, tissues, saliva, plaque, sulcus fluid, gum biopsy, tongue swabs or urine samples from animals. Immunoassays can utilize one antibody or several antibodies. Immunoassays can use, for example, monoclonal antibodies specific for one epitope, a combination of monoclonal antibodies specific for epitopes of one polypeptide, monoclonal antibodies specific for epitopes of different polypeptides, polyclonal antibodies specific for the same antigen, polyclonal antibodies specific for different antigens, or a combination of monoclonal antibodies and polyclonal antibodies. Immunoassay protocols can be based on, for example, competition, direct reaction or sandwich assays, using, for example, labeled antibodies. Antibodies can be labeled with any type of marker known in the art, including, for example, fluorescent, chemiluminescent, radioactive, enzyme, colloidal metal, radioisotope and bioluminescent markers.

[0157] The antibody or antigen-binding fragment thereof can be bound to a support and used to detect the presence or amount of a polypeptide present in a sample in certain diseases and patients. Supports include, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose, and magletite. The antibody or antigen-binding fragment thereof can be lyophilized, dehydrated, or dried, such as freeze-dried.

[0158] Antibodies can also be used to separate polypeptides by immunoaffinity columns. Antibodies can be fixed to a solid support, for example, by absorption or covalent attachment, so that the antibody retains its immunoselective activity. Optionally, a spacer group can be included to keep the antigen binding site of the antibody accessible. The fixed antibody can then be used to specifically bind to SEQ ID NO: 1-32 or a fragment thereof from a biological sample, including, but not limited to, saliva, plaque, sulcus fluid, gingival biopsy, tongue swab, serum, blood, and urine.

[0159] Antibodies can also be used in immunolocalization studies to analyze the presence and distribution of polypeptides described herein during various cell events or physiological conditions. Antibodies can also be used to identify molecules involved in passive immunity and to identify molecules involved in the biosynthesis of non-protein antigens. The identification of such molecules can be used in vaccine development. Antibodies, including, for example, monoclonal antibodies and single-chain antibodies, can be used to monitor the improvement process of certain diseases or patients. By measuring the increase or decrease of the amount of SEQ ID NO:1-32 or SEQ ID NO:26-32 or its fragment in the test sample from the animal, it can be determined whether the specific treatment regimen intended to improve the disease is effective. For example, direct binding assays such as RIA, ELISA or Western blot assays can be used to detect and / or quantify antibodies.

[0160] Peptide detection and quantification methods

[0161] One embodiment provides a method for detecting and quantifying cystatin B, clusterin, SDMA or creatinine polypeptides in a sample, such as a mammalian urine sample. Methods for detecting and quantifying polypeptides include, for example, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatography (HPLC) mass balance assays, liquid chromatography-mass spectrometry (LC-MS) and mass spectrometry. qNMR utilizes its unique ability to obtain a polypeptide from a magnetic nucleus such as 1 H obtains a response of equal magnitude, regardless of chemical structure. AAA is based on quantifying stable amino acids after peptide hydrolysis. HPLC selectively quantifies the analyte of interest based on a reference standard of the same analyte. In LC-MS, liquid chromatography (LC) separates the sample components and then introduces them into a mass spectrometer (MS). MS produces and detects charged ions. LC / MS data can be used to provide information about the molecular weight, structure, identity and amount of specific sample components. The mass spectrum obtained by mass spectrometry is a graph of ion signals as a function of mass-to-charge ratio. These spectra are used to determine the elemental or isotopic characteristics of the sample, the mass of the polypeptide, and to elucidate the chemical identity or structure of the polypeptide. Immunoassays (IA) and competitive IA can also be used to quantify polypeptides.

[0162] The polypeptides can also be detected and quantified using, for example, antibodies or specific binding fragments thereof (e.g., polyclonal antibodies, monoclonal antibodies, specific binding fragments thereof, or combinations thereof) in immunoassays. Such methods may include contacting a sample with one or more antibodies or specific binding fragments thereof that are specific for, for example, SEQ ID NOs: 1-32 or fragments thereof under conditions suitable for the formation of a complex of the cystatin B polypeptide or clusterin polypeptide with one or more antibodies or specific binding fragments thereof. Detect and quantify a complex of the cystatin B polypeptide or clusterin polypeptide with one or more antibodies or specific binding fragments thereof that are specific for SEQ ID NOs: 1-32.

[0163] SDMA and creatinine can be detected using, for example, antibodies and / or ELISA kits. Examples of ELISA kits include CREA ELISA kit product number MBS748495 MyBioSource (San Diego, California) and canine creatinine ELISA kit product number CNEB0472 AssayGenie (Dublin, Ireland). Creatinine can also be detected using, for example, electrochemical methods (U.S. Patent Publication No. 20170284954; U.S. Patent Publication No. 20180149612), polymer matrices (U.S. Patent Publication No. 20150160242) and fluorescent methods (U.S. Patent Publication No. 20040132200). SDMA can be detected using, for example, antibodies (U.S. Patent Publication No. 20160187348), Product No. AB1N7845543 (Antibodies Online, Pottstown, Pennsylvania), Product No. ABIN870728 (Antibodies Online, Pottstown, Pennsylvania); for detection of SDMA, see also U.S. Patent Publication No. 20210403603.

[0164] In one embodiment, methods for detecting and quantifying polypeptides comprising cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32 and fragments thereof are provided. The relative amount of the polypeptide can be used to distinguish mammals with stable CKD from mammals with progressive CKD. Any method known in the art for detecting polypeptides can be used in the methods described herein.

[0165] Assay methods used in conjunction with the antibodies described herein can include direct and indirect labeling techniques, immunoaffinity columns, immunomagnetic beads, fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), agglutination assay, scatter photometry, quantitative scatter photometry, Western blot, IFA, hemagglutination (HA), turbidimetric immunoassay, particle enhanced turbidimetric immunoassay, fluorescence polarization immunoassay (FPIA), and microtiter plate assays (any assay performed in one or more wells of a microtiter plate). One assay includes a reversing flow chromatography binding assay, e.g. See, e.g., U.S. Pat. No. 5,726,010.

[0166] Antibodies or their specific binding fragments can be detectably labeled with, for example, fluorescent markers having excitation and emission wavelengths suitable for detection using commercially available instruments such as fluorescence activated cell sorters. Examples of fluorescent markers include phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine (RH), Texas Red (TX), Cy3, Hoechst 33258, and 4', 6-diamidino-2-phenylindole (DAPI). Such markers can be conjugated to antibodies using standard techniques (Maino et al., 1995, Cytometry 20: 127-133).

[0167] The methods described herein can be used to detect cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32 or fragments thereof, wherein the antibody or antigen-binding antibody fragment specifically binds to cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32.

[0168] As used herein, "patient" or "subject" may refer to a human or a non-human animal, including a feline, bovine, porcine, equine, or canine.

[0169] As used herein, the term "sample", "test sample", "patient sample" or "subject sample" includes, but is not limited to, blood, serum, plasma, saliva, plaque, sulcus fluid, gum biopsy, tongue swab or urine sample obtained from a subject. The test sample can be untreated, precipitated, fractionated, separated, diluted, concentrated or purified.

[0170] The assay may include a solid phase or substrate, or may be performed by immunoprecipitation or any other method that does not employ a solid phase. Where a solid phase or substrate is used, one or more polypeptides or antibodies (or their specific binding fragments) are attached directly or indirectly to a solid support or substrate such as a microtiter well, magnetic beads, non-magnetic beads, columns, matrix, membrane, a fiber mat consisting of synthetic or natural fibers (e.g., glass or cellulose-based materials or thermoplastic polymers such as polyethylene, polypropylene or polyester), a sintered structure consisting of a granular material (e.g., glass or various thermoplastic polymers), or a cast film consisting of nitrocellulose, nylon, polysulfone, etc. (usually synthetic in nature). In one embodiment, the substrate is sintered polyethylene fine particles, commonly referred to as porous polyethylene, such as 10-15 micron porous polyethylene from Chromex Corporation (Albuquerque, New Mexico). All of these substrate materials can be used in a suitable shape, such as a film, sheet or plate, or they can be coated on a suitable inert carrier such as paper, glass, plastic film or fabric or bonded or laminated to a suitable inert carrier such as paper, glass, plastic film or fabric. Suitable methods for fixing antibodies on a solid phase include ionic, hydrophobic, covalent interactions, etc.

[0171] In one embodiment, the method includes contacting the test sample with one or more antibodies that specifically bind to cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or a fragment thereof under conditions that allow the formation of a polypeptide / antibody complex, i.e., an immune complex. That is, the antibody specifically binds to one or more polypeptides of cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or a fragment thereof located in the sample. Those skilled in the art are familiar with assays and conditions for detecting antibody / polypeptide complex binding. The formation of a complex between the polypeptide and the antibody in the test sample. The formation of an antibody / polypeptide complex indicates that the polypeptide is present in a certain amount in the CKD patient sample.

[0172] In one embodiment, the polypeptide / antibody complex is detected when an indicator reagent such as an enzyme conjugate that is bound to the antibody catalyzes a detectable reaction. Optionally, an indicator reagent comprising a signal generating compound can be applied to the polypeptide / antibody complex under conditions that allow the formation of a polypeptide / antibody / indicator complex. Detect the polypeptide / antibody / indicator complex. Optionally, the polypeptide or antibody can be labeled with an indicator reagent before the polypeptide / antibody complex is formed. The method can optionally include a positive control or a negative control. The positive control can contain one or more polypeptides that will specifically bind to an antibody specific for cystatin B, clusterin, SDMA or creatinine and provide a positive result. The negative control does not contain any cystatin B, clusterin, SDMA or creatinine polypeptides or any polypeptides or other components that will specifically bind to or cross-react with an antibody specific for cystatin B, clusterin, SDMA or creatinine.

[0173] In one embodiment, one or more antibodies are covalently or non-covalently fixed to a solid phase or substrate. A sample potentially containing cystatin B, clusterin, SDMA or creatinine polypeptides is added to the substrate. One or more antibodies (or specific binding fragments) specific for cystatin B, clusterin, SDMA or creatinine are added to the substrate. The antibodies can be the same antibodies used on the solid phase, or can be from different sources or species and can be connected to an indicator reagent, such as an enzyme conjugate. A washing step can be performed before each addition. A chromophore or enzyme substrate is added and allowed to develop color. The color reaction is stopped and the color can then be quantified using, for example, a spectrophotometer.

[0174] In another embodiment, one or more antibodies are fixed to a solid phase or substrate. A test sample potentially containing cystatin B, clusterin, SDMA or creatinine polypeptide is added to the substrate. A second anti-species antibody that specifically binds to cystatin B, clusterin, SDMA or creatinine polypeptide is added. These second anti-species antibodies are from species different from the antibodies fixed to the solid phase. A third anti-species antibody that specifically binds to the second anti-species antibody but does not specifically bind to the antibody fixed to the solid phase is added. The third anti-species antibody may contain an indicator reagent such as an enzyme conjugate. A washing step may be performed before each addition. A chromophore or enzyme substrate is added and allowed to develop color. The color reaction is stopped and the color can then be quantified using, for example, a spectrophotometer.

[0175] In one embodiment, one or more capture antibodies can specifically bind to one or more epitopes of polypeptides described herein. The one or more capture antibodies can be used to fix one or more polypeptides of cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or its fragments to, for example, a solid support. One or more detection antibodies can specifically bind to the same one or more epitopes or different one or more epitopes of the polypeptide. The detection antibody can be used to detect or visualize the situation that the polypeptide is fixed to a solid support. This embodiment is advantageous because it is more specific and more sensitive than the assay method that only uses a kind of antibody to realize capture and detection of two functions.

[0176] In one type of assay format, one or more antibodies can be coated onto a solid phase or substrate. A test sample suspected of containing a polypeptide comprising cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or a fragment thereof is incubated for a certain time under conditions sufficient to form an antigen / antibody complex of any antibody on a solid phase and a test sample polypeptide or an indicator reagent compound conjugated to an antibody specific for the polypeptide. The binding of the indicator reagent conjugated to the anti-polypeptide antibody to the solid phase can be quantitatively measured. A measurable change in the signal compared to the signal generated from a control sample or a control standard indicates the presence of a polypeptide comprising cystatin B, clusterin, SDMA or creatinine, SEQ ID NO: 1-32 or a fragment thereof. This type of assay can quantify the amount of polypeptide in a test sample.

[0177] In another type of assay format, one or more antibodies are coated onto a support or substrate. The antibody is conjugated to an indicator reagent and added to a test sample. The mixture is applied to a support or substrate. If cystatin B, clusterin, SDMA or creatinine polypeptides are present in the test sample, they will bind to one or more antibodies conjugated to the indicator reagent and one or more antibodies fixed to the support. The polypeptide / antibody / indicator complex can then be detected. This type of assay can quantify the amount of polypeptide in a test sample.

[0178] In another type of assay format, one or more antibodies are coated onto a support or substrate. The test sample is applied to a support or substrate and incubated. Unbound components are washed off from the sample by washing the solid support with a washing solution. If there are polypeptides comprising cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or fragments thereof in the test sample, they will bind to the antibodies coated on the solid phase. This polypeptide / antibody complex can be detected using a second anti-species specific antibody conjugated to an indicator reagent. The polypeptide / antibody / anti-species antibody indicator complex can then be detected. This type of assay can quantify the amount of polypeptide in the test sample.

[0179] The formation of polypeptide / antibody complex or polypeptide / antibody / indicator complex can be detected by, for example, radiometric method, colorimetry, fluorescence method, size separation method or precipitation method. Optionally, the detection of polypeptide / antibody complex is carried out by adding a second antibody coupled to an indicator reagent comprising a signal generating compound. The indicator reagent comprising a signal generating compound (label) associated with the polypeptide / antibody complex can be detected using the method described above and includes a developer, a catalyst such as an enzyme conjugate, a fluorescent compound such as fluorescein and rhodamine, a chemiluminescent compound such as dioxetane, acridinium, phenanthridinium, ruthenium and luminol, a radioactive element, a direct visual marker and a cofactor, an inhibitor, a magnetic particle, etc. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, etc. The selection of a specific marker is not critical, but it can generate a signal alone or in combination with one or more additional substances.

[0180] As used herein, the expression "determine the amount of ..." refers to measuring or identifying the level of one or more polypeptides in a sample. This can be achieved by polypeptide detection methods including the use of antibodies well known in the art, including, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), turbidimetric immunoassay, particle-enhanced turbidimetric immunoassay, protein blot assay or immunohistochemical assay. Alternatively, the polypeptide of cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or its fragment can be determined by mass spectrometry, LC-MS, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatography (HPLC) mass balance assay or similar methods known to those skilled in the art. The determination of the amount of polypeptide present in a sample can be completed by such in vitro analysis and experimental operation.

[0181] Diagnostic Methods

[0182] One embodiment provides a method for diagnosing stable CKD or progressive CKD nephropathy in a subject. First, the subject is diagnosed with CKD, and then the amount of cystatin B or clusterin polypeptide in a sample from a subject diagnosed with CKD is determined. The method may include determining the amount of cystatin B polypeptide, clusterin polypeptide, or both in a sample from a subject, wherein one or more antibodies that specifically bind to cystatin B, clusterin, SEQ ID NO: 1-32 or a fragment thereof, or other methods as described herein, are used to determine the amount of cystatin B or clusterin polypeptide. The amount of cystatin B or clusterin polypeptide in the sample is compared with a control sample or a control standard, wherein certain levels of cystatin B or clusterin polypeptide in the sample compared with the control sample or the control standard will indicate whether the subject suffers from stable CKD or progressive CKD nephropathy.

[0183] Some embodiments provide a method for diagnosing or detecting stable or progressive CKD. The method includes determining the amount of cystatin B or clusterin polypeptide in a sample from a subject, wherein the amount of cystatin B polypeptide or clusterin is determined using one or more antibodies (or specific binding fragments), and the antibody (or specific binding fragment) specifically binds to one or more polypeptides or fragments thereof as described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32. The amount of cystatin B or clusterin polypeptide in the sample is compared with a control sample or a control standard, wherein the level of cystatin B or clusterin polypeptide in the sample increased compared with the control sample or the control standard will indicate whether it is stable CKD or progressive CKD.

[0184] In some embodiments, antibody-independent methods can be used to diagnose or detect stable or progressive CKD. The method may include determining the amount of cystatin B or clusterin polypeptide in a sample from a subject, wherein the amount of cystatin B or clusterin is determined using an LC-MS assay or other assays described herein. The amount of cystatin B or clusterin polypeptide in the sample is compared with a control sample or a control standard, wherein the level of cystatin B or clusterin polypeptide increased in the sample compared with the control sample or the control standard will indicate whether it is stable CKD or progressive CKD.

[0185] The elevated level of cystatin B polypeptide can be about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more than the control sample or control standard. The elevated level of cystatin B polypeptide can be about 10% to 500% or more; about 20% to 500% or more; about 30% to 500% or more; about 40% to 500% or more; about 50% to 500% or more; about 60% to 500% or more; about 100% to 500% or more than the control sample or control standard.

[0186] An elevated level of a cystatin B or clusterin polypeptide may also be a level that is a statistically significant increased amount when compared to a control sample or control standard.

[0187] As described above, the elevated level of cystatin B polypeptide can also be above about 50, 60, 70, 80, 90, 100 ng / mL or more cystatin B (or any range between about 50 and 100 ng / mL or more (e.g., about 50-100 ng / mL or more, about 50-80 ng / mL or more, about 70-100 ng / mL or more)), and the control level or control standard of cystatin B polypeptide can be about 49, 40, 30, 25, 20, 15, 10, 5, 1 or less ng / mL.

[0188] As described above, the elevated level of clusterin polypeptide can also be above about 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 ng / mL or more clusterin (or any range between about 250 and 350 ng / mL or more (e.g., about 250-300 ng / mL or more, about 275-325 ng / mL or more, about 325-350 ng / mL or more)). The control level or control standard of cystatin B polypeptide can be about 249, 240, 230, 225, 200, 150, 100, 50 or less ng / mL.

[0189] The elevated levels of cystatin B or clusterin polypeptide can be compared to a control sample or control standard measured using a normal control subject not suffering from any type of kidney disease or disorder or bacterial infection.

[0190] Surprisingly, it is also possible to use SDMA reciprocal (1 / SDMA) slope or creatinine reciprocal (1 / creatinine) slope to distinguish whether CKD patients suffer from stability CKD or progressive CKD. Functional kidney markers mean that in a given time period, respectively, the SDMA reciprocal or creatinine reciprocal slopes are compared with the SDMA reciprocal of healthy populations without substantial changes and change less than 200%. This time period can be, for example, in 1 week, 2 weeks, 3 weeks, surrounding, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months or longer (or any scope between approximately 1 week to approximately 36 months, for example, approximately 1 month to approximately 3 months, approximately 2 months to approximately 4 months, approximately 3 months to approximately 6 months, approximately 6 months to approximately 12 months or approximately 1-36 months). In these cases, the experimenter suffers from stability chronic kidney disease.

[0191] In contrast, a CKD patient with progressive CKD will exhibit a slope of the inverse SDMA (1 / SDMA) of about 0.0002222411 (μg / dL) over time (e.g., within 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 24 months, 36 months, or longer; and any range between about 5 days to about 36 months, such as within about 1 month to about 3 months, within about 2 months to about 4 months, within about 3 months to about 6 months, within about 6 months to about 12 months, or within about 1-36 months). -1 / week or more (e.g., about 0.0001, 0.00015, 0.0002, 0.00025 or more) decrease ( Fig.14 ) or the inverse of creatinine (1 / creatinine) with a slope of approximately 0.01860675 (mg / dL) -1 / week or more (e.g., about 0.010, 0.015, 0.018, 0.020, 0.025 or more) decrease ( Fig.13 ).

[0192] For 1 / creatinine, the slope cutoff for stability and progression can be about -0.0117 (mg / dL) -1 / week (e.g., about -0.010, -0.012, -0.015, or -0.02 (mg / dL) -1 / week), while for 1 / SDMA, it can be about -0.0007 (μg / dL) -1 / week (e.g., about -0.0005, -0.0006, -0.0007, or -0.0008 (μg / dL) -1 / week).

[0193] Accordingly, provided herein are methods for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD by the following steps:

[0194] (a) determining the slope of the reciprocal SDMA (1 / SDMA) in a sample from a subject at two or more time points (e.g., at a first time point, then 1 week later, 2 weeks later, 3 weeks later, 4 weeks later, or longer);

[0195] (b) Comparison of the slope of the reciprocal SDMA (1 / SDMA) in the samples, where the slope is about 0.0002222411 (μg / dL) over time -1 / week or greater (e.g., about 0.0001, 0.00015, 0.0002, 0.00025 (μg / dL) -1 A decrease in CKD (≥ 1 / week or greater) is indicative of progressive CKD.

[0196] Prior to step (a), the subject may be diagnosed with International Kidney Rights Association CKD stage I or CKD with a symmetric dimethylarginine (SDMA) value of up to 20 μg / dl. The subject may be administered one or more treatments for the CKD disease.

[0197] A method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD may comprise:

[0198] (a) measuring the concentration of SDMA in samples obtained from a subject at three or more time points (e.g., at a first time point, then 1 week later, 2 weeks later, 3 weeks later, 4 weeks later or longer);

[0199] (b) determining the slope of the reciprocal SDMA (1 / SDMA) in samples from the subject at three or more time points; and

[0200] (c) comparing the SDMA reciprocal (1 / SDMA) slope to the minimum slope of a healthy control animal cohort, wherein a SDMA reciprocal (1 / SDMA) slope less than the minimum slope of a healthy control animal cohort indicates that the subject has progressive CKD, and wherein a SDMA reciprocal (1 / SDMA) slope greater than the minimum slope of a healthy control animal cohort indicates that the subject has stable CKD.

[0201] For 1 / SDMA, the minimum slope for the healthy control animal cohort could be approximately -0.0007 (μg / dL) -1 / week (e.g., about -0.0005, -0.0006, -0.0007, or -0.0008 (μg / dL) -1 / week).

[0202] Also provided herein are methods for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD by the following steps:

[0203] (a) determining the reciprocal creatinine (1 / creatinine) slope in samples from a subject at two or more time points (e.g., at a first time point, then 1 week later, 2 weeks later, 3 weeks later, 4 weeks later, or longer);

[0204] (b) The slopes of the reciprocal creatinine (1 / creatinine) in the samples were compared with each other, wherein the slope of the reciprocal creatinine (1 / creatinine) over time was approximately 0.01860675 (mg / dL) -1 / week or greater (e.g., about 0.010, 0.015, 0.018, 0.020, 0.025 (mg / dL) -1 A decrease in CKD (≥ 1 / week or greater) is indicative of progressive CKD.

[0205] Prior to step (a), the subject may be diagnosed with International Kidney Rights Association CKD stage I or CKD with a symmetric dimethylarginine (SDMA) value of up to 20 μg / dl. The subject may be administered one or more treatments for the CKD disease.

[0206] A method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD may comprise:

[0207] a) measuring creatinine concentration in samples obtained from a subject at three or more time points (e.g., at a first time point, then 1 week later, 2 weeks later, 3 weeks later, 4 weeks later, or longer);

[0208] b) determining the reciprocal creatinine (1 / creatinine) slope in samples from the subject at three or more time points; and

[0209] c) comparing the reciprocal creatinine (1 / creatinine) slope to the minimum slope of a cohort of healthy control animals, wherein a reciprocal creatinine (1 / creatinine) slope less than the minimum slope of a cohort of healthy control animals indicates that the subject has progressive CKD, and wherein a reciprocal creatinine (1 / creatinine) slope greater than the minimum slope of a cohort of healthy control animals indicates that the subject has stable CKD.

[0210] For 1 / creatinine, the minimum slope for the healthy control animal cohort can be about -0.0117 (mg / dL) -1 / week (e.g., about -0.010, -0.012, -0.015, or -0.02 (mg / dL) -1 / week).

[0211] In some embodiments, the level of cystatin B, clusterin, SDMA or creatinine in the test sample is compared with the level of cystatin B or clusterin (or both) in the control sample from one or more normal control subjects. Typically, the measured control level in the control sample is then compared with the cystatin B, clusterin, SDMA or creatinine level measured in the test sample. Alternatively, the level of cystatin B, clusterin, SDMA or creatinine polypeptide in the test sample is compared with a previously measured or predefined control level ("control standard"). For example, the control standard of cystatin B, clusterin, SDMA or creatinine polypeptide can be calculated from data, such as data including the level of cystatin B, clusterin, SDMA or creatinine in the control sample from multiple normal or healthy control subjects. The normal or healthy control subject and the test subject being evaluated can be the same species.

[0212] In some embodiments, the level of cystatin B, clusterin, SDMA or creatinine polypeptide in the test sample is compared with the level of cystatin B, clusterin, SDMA or creatinine polypeptide in the control sample from one or more control subjects, wherein the control subject suffers from stable CKD. Typically, the measured control level in the control sample is then compared with the cystatin B, clusterin, SDMA or creatinine polypeptide level measured in the test sample. Alternatively, the level of cystatin B, clusterin, SDMA or creatinine polypeptide in the test sample is compared with a previously measured or predefined control level ("control standard"). For example, the control standard of cystatin B, clusterin, SDMA or creatinine polypeptide can be calculated from data, such as data including the level of cystatin B, clusterin, SDMA or creatinine polypeptide in the control sample from a plurality of control subjects suffering from stable CKD. The stable CKD control subject and the test subject being evaluated can be the same species.

[0213] Specific embodiments provide reagents and methods for identifying certain diseases or patients in mammals, such as dogs, cats and humans. Certain embodiments provide methods for providing diagnosis and prognosis for CKD patients. Identification and quantification of the cystatin B polypeptide in the subject sample can be an independent predictor of progressive CKD. The method advantageously allows diagnosis and identification of progressive CKD without being affected or confused by the age or weight of the CKD patient. Therefore, additional embodiments relate to using the progressive CKD diagnosis determined by the method described herein to select appropriate therapy.

[0214] Embodiment also comprises the method for predicting CKD patient health, monitoring disease progression and / or evaluating / monitoring treatment efficacy by the level of specific polypeptide in the identification CKD patient sample.In some respects, described method can be carried out at single time point.In other respects, described method can be carried out at multiple time points (for example, about 2,3,4,5 or more time points) to for example evaluate disease progression or treatment efficacy.In a specific embodiment, described method can be carried out at the specific time point after diagnosis and then treatment, wherein specific therapy should cause the minimization or improvement of disease progression.

[0215] For example, methods for monitoring disease progression may include:

[0216] (a) determining the amount of cystatin B, clusterin, SDMA or creatinine polypeptide, or a combination thereof, in a sample from a subject diagnosed with CKD at a first time point;

[0217] (b) treating the subject's CKD with one or more of the CKD treatments described herein;

[0218] (c) determining the amount of cystatin B, clusterin, SDMA or creatinine polypeptide, or a combination thereof, in a sample from the subject diagnosed with CKD at a second time point;

[0219] (d) comparing the amount of cystatin B, clusterin, SDMA or creatinine polypeptide or a combination thereof at the first time point and the second time point.

[0220] Lower levels of cystatin B or clusterin polypeptide or both at the second time point indicate that the treatment is slowing the progression of progressive or stable CKD. In this case, the slope will become less steep. Additional treatments and time points (e.g., 3, 4, 5, 6 or more time points) can be provided.

[0221] The methods described herein can also indicate the amount or quantity of a polypeptide comprising cystatin B, clusterin, SDMA or creatinine, SEQ ID NO: 1-32 or a fragment thereof. For many indicator reagents, such as enzyme conjugates, the amount of the polypeptide present is proportional to the signal generated. Depending on the type of the test sample, it can be diluted, concentrated or contacted with a solid phase with a suitable buffer reagent without any manipulation. For example, a previously diluted urine, serum or plasma sample or a concentrated sample such as urine can be used to determine the presence of a polypeptide and / or the amount of the polypeptide present.

[0222] The polypeptides and assays described herein can be combined with other polypeptides or assays to detect the presence of kidney disease. For example, the polypeptides and assays can be combined with reagents suitable for detecting or measuring creatinine, SDMA, or general protein levels.

[0223] Treatment

[0224] Certain embodiments provide methods for treating a disease patient in a subject. The method includes requesting a test to provide analytical results to determine the amount of cystatin B polypeptide in a sample from the subject. If the sample contains an elevated amount of cystatin B or clusterin polypeptide (or both) as described herein compared to a control sample or control standard for the disease patient, a treatment for the disease patient is administered to the subject.

[0225] Disease conditions include stable CKD and progressive CKD. In one embodiment, the disease condition is not cancer or renal caner.

[0226] Treatments for progressive CKD include, for example, surgery for ureteral obstruction, obstructive kidney stones, or urolithiasis, dietary management (reducing the amount of dietary protein, phosphorus, salt, or a combination thereof), administration of enteric-coated phosphate binders, antiproteinuric drugs (e.g., angiotensin-converting enzyme inhibitors (ACEIs), administration of omega-3 fatty acids), administration of antihypertensive drugs (e.g., ACEIs, calcium channel antagonists (CCAs)), fluid therapy to correct dehydration, management of acidosis, administration of diuretics, dialysis, correction of electrolyte abnormalities, administration of antiemetics, administration of antacids, and administration of recombinant erythropoietin.

[0227] Treatment using dietary management can be done with a variety of diets, including reduced protein, reduced phosphorus, and reduced salt diets. Veterinarians often recommend special pet food formulas. Special pet food formulas include Prescription Royal Veterinary Diet Canine Renal Support, Veterinary Diet NF Kidney and Rayne ClinicalNutrition TM Adult Health - RSS TM. Reducing dietary protein appears to slow the progression of CKD by reducing the amount of work the kidneys have to do to excrete protein waste. Less protein also means less need to excrete protein itself through the kidney's filtering mechanism, which helps maintain kidney function. For dogs with CKD, the recommended range of protein based on dry matter is less than or equal to, for example, about 14-20%. For cats with CKD, the recommended range of protein based on dry matter is less than or equal to, for example, 28-35%. Restricting dietary phosphorus in dogs and cats with CKD appears to help slow the progression of CKD, but the exact mechanism is unclear. For dogs with CKD, the recommended range of phosphorus based on dry matter is, for example, about 0.2-0.5%. For cats with CKD, the recommended range of phosphorus based on dry matter is, for example, 0.3-0.6%. Since phosphorus content is related to protein content, lower phosphorus levels usually require limiting protein content. Dietary sodium levels are slightly restricted (e.g., based on dry matter, about ≤0.3% for dogs and ≤0.4% for cats) to reduce the workload of the kidneys. This in turn helps maintain reasonable blood pressure. Dietary omega-3 fatty acids help reduce the production of inflammatory compounds that can cause oxidative stress to the tissues of the diseased kidney, thereby helping to slow the progression of CKD. This works by reducing protein "leakage" through the kidneys. For cats with CKD, the recommended range of omega-3 fatty acids on a dry matter basis is, for example, 0.4-2.5%.

[0228] Management of acidosis can include monitoring CKD patients for metabolic acidosis by measuring serum bicarbonate or total CO2. If necessary, renal dietary therapy can be supplemented with oral sodium or potassium bicarbonate to maintain serum bicarbonate concentrations in the range of approximately 18 to 24 mmol / L.

[0229] Correction of electrolyte abnormalities can include correction of serum sodium concentration, which may be normal, increased or decreased with renal failure. Hypernatremia before fluid therapy indicates excessive free water loss. The administration of sodium bicarbonate or hypertonic saline may cause hypernatremia. Hyponatremia may indicate excessive sodium loss associated with vomiting, or may represent transient dilutional hyponatremia that occurs after the administration of mannitol, hypertonic dextrose or colloid solutions. Sodium-poor solutions (e.g., about 5% dextrose, total parenteral nutrition, enteral preparations) may cause hyponatremia. In many cases, dehydration is initially caused by isotonic fluid loss, while the serum sodium concentration of CKD patients is normal. The initial fluid loss can be replaced with isotonic solutions such as lactated Ringer's solution, 0.9% saline or Plasmalyte-148. Continuous administration of these solutions for several days may cause hypernatremia. After the initial rehydration phase, sodium-poor fluids such as half-strength lactated Ringer's solution or 0.45% saline with 2.5% dextrose may be a more appropriate fluid choice. Serum sodium concentrations can be monitored regularly and fluid selection adjusted as needed. Hypokalemia may also occur, but is more common in cats. Between 20% and 30% of cats with CKD have hypokalemia. Because potassium excretion may be impaired by renal failure, treatment in this case requires cautious supplementation and careful monitoring. Potassium chloride can be added to IV fluids for hospitalized CKD patients who cannot tolerate orally administered medications. The potassium supplementation rate can be about 0.5mEq / kg / h or less. Despite potassium supplementation, serum potassium concentrations may decrease during initial fluid therapy due to extracellular fluid volume expansion, increased distal tubular flow, and cellular uptake, especially if potassium is administered with dextrose-containing fluids. In addition, metabolic acidosis increases the ionized calcium fraction, but more than 50% of dogs with CKD and metabolic acidosis are hypocalcemic. Hypercalcemia may respond to fluid therapy. Normal saline (0.9% NaCl) is a good fluid choice because its high sodium content helps urinary calcium excretion. Furosemide also promotes urinary calcium loss. Sodium bicarbonate therapy decreases serum ionized calcium concentration because more calcium ions are bound to serum proteins.

[0230] Holistic treatment may include, for example, administration of fish oil, B vitamins, iron, pet-friendly multivitamins, fresh parsley, kali chloricum, white arsenic, silica, frankincense, aluminum hydroxide, or glandular therapy.

[0231] In an alternative embodiment, the methods described herein can be used to evaluate the efficacy of a composition or treatment regimen (whether a composition or a diet) in improving disease progression. Similarly, the methods described herein can be used to evaluate the activity of a composition or treatment regimen on clusterin or cystatin B polypeptide levels in CKD patients.

[0232] The compositions and methods are described in more detail below, and the examples set forth herein are intended to be illustrative only, as many modifications and variations therein will be apparent to those skilled in the art. The terms used in this specification in the context of the compositions and methods described herein, and in the specific context in which each term is used, generally have their ordinary meaning in the art. Some terms are more specifically defined herein to provide additional guidance to the practitioner regarding the descriptions of the compositions and methods.

[0233] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As described herein and subsequently used throughout the claims, the meanings of "a", "an", and "the" include plural references as well as singular references, unless the context clearly dictates otherwise. The term "about" associated with a numerical value indicates that the value varies by 5% above or below. For example, for a value of about 100, 95 to 105 (or any value between 95 and 105) is represented.

[0234] All patents, patent applications and other scientific or technical documents mentioned anywhere herein are incorporated herein by reference in their entirety. The embodiments schematically described herein may be appropriately implemented in the absence of any one or more elements, one or more restrictions specifically or not specifically disclosed herein. Thus, for example, in each example herein, any one of the terms "comprising", "consisting essentially of ... " and "consisting of ... " may be replaced with any of the other two terms while retaining their common meaning. The terms and expressions adopted are used as descriptive rather than restrictive terms, and when using such terms and expressions, it is not intended to exclude any equivalents of the features shown and described or parts thereof, but it should be recognized that various modifications may be made within the scope of the claims. Therefore, it should be understood that although the methods and compositions of the present invention have been specifically disclosed by embodiments and optional features, those skilled in the art may adopt modifications and variations of the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the accompanying claims.

[0235] Any individual term, individual element, individual phrase, group of terms, group of phrases or group of elements described herein may each be specifically excluded from the claims.

[0236] Whenever a range is given in the specification, such as a temperature range, time range, composition or concentration range, all intermediate ranges and subranges and all individual values ​​included in the given range are intended to be included in the present disclosure. It should be understood that any subrange or individual value in a range or subrange included in the description herein can be excluded from aspects of the present invention. It should be understood that any element or step included in the description herein can be excluded from the claimed composition or method.

[0237] In addition, where features or aspects of compositions and methods are described in terms of Markush groups or other groupings of alternatives, those skilled in the art will recognize that the compositions and methods are also thereby described in terms of any individual member or subgroup of members of the Markush or other group.

[0238] The following is provided for the purpose of example only and is not intended to limit the scope of the embodiments broadly described above.

[0239] Example

[0240] The following examples illustrate specific embodiments of the present disclosure and various uses thereof. They are for illustrative purposes only and should not be considered as limiting.

[0241] Example 1: Method

[0242] Study Group

[0243] Data for control patients were obtained from an archived longitudinal study of common vector-borne diseases in pets of students, faculty, and staff at the University of Missouri Health Center. All pets were enrolled with owner consent, and approval for the study was obtained from the Institutional Animal Care and Use Committee through the University of Missouri. Dogs were between one and ten years old and were reported healthy by their owners at the time of enrollment. The follow-up period was three years. The frequency of evaluation was every four to six months. Patients with data from at least three visits were included in the current study.

[0244] Because CysB is not only present in the urinary system, but also because it is a small 11 kD solute that is freely filtered in the glomerulus, it cannot be ruled out that the source of uCysB in some dogs, when detectable in the circulation, may be active cell damage elsewhere in the body other than tubular epithelial cell damage. To reduce this possibility, dogs with concurrent inflammatory disease or concurrent systemic illness that could have led to active cell damage in other organs were not included in this study, further reducing the possibility that the uCysB in this study was derived from extrarenal cell damage.

[0245] Data on dogs with stage 1 CKD were prospectively collected from four additional academic institutions: the University of Minnesota, the University of North Carolina, the Hebrew University, and the University of California, San Diego Veterinary Medical Center. Dogs aged 6 months or older undergoing IRIS stage 1 CKD evaluation were eligible for inclusion. All owners were provided with information about the study objectives, protocol, and responsibilities. Owner consent was required for enrollment, and approval for the study was obtained from the Institutional Animal Care and Use Committee through each academic institution, respectively. Dogs with primary adrenal or exocrine pancreatic disease, concurrent liver or cardiac disease, or other systemic disease that would be expected to promote CKD progression were excluded. Evaluation of enrolled dogs included physical examination, CBC, complete biochemistry panel, urinalysis, urine culture, and UPC ratio. Each dog was re-evaluated and IRIS CKD staged monthly for six months.

[0246] Classification of Stage 1 Stable and Progressive CKD

[0247] Patients with stage 1 IRIS CKD were classified as stable CKD or progressive CKD based on the slope calculated from a linear regression of each patient with the inverse of SDMA as the dependent variable and follow-up time as the independent variable. Patients with a slope greater than or equal to this cutoff were classified as having stable CKD. Patients with a slope less than this cutoff were classified as having progressive CKD.

[0248] uCysB Technology

[0249] uCysB was measured using a research sandwich ELISA at IDEXX headquarters (Westbrook, ME) as previously described. Urine sample aliquots for uCysB measurement were frozen at -80°C, shipped frozen in batches to IDEXX, and stored at -80°C until batch testing as the study progressed.

[0250] Descriptive analysis

[0251] Descriptive analyses were performed to understand the relationship between the dog's serial uCysB values ​​and their CKD status (control, stable, progressive). This also provided insight into the dependency of serial samples for each dog and the effect of CKD group on uCysB values. Kruskal-Wallis ANOVA was used to gain insight into whether there were differences in uCysB expression in dogs within and between CKD groups. Least squares regression between uCysB (dependent variable) and follow-up day (independent variable) was performed for each dog to understand the effect of CKD group on the y-intercept and slope. Post hoc descriptive analyses were performed to understand the potential utility of uCysB as an early biomarker for progressive CKD.

[0252] Statistical analysis

[0253] The relationship between follow-up time and uCysB was assessed using a linear mixed effects model with random slope and coefficient. A random intercept was used to explain the non-independence of continuous uCysB measurements in each individual. Random coefficients were used to explain the non-independence in each CKD group. An unstructured covariance structure was used to allow for the unique variation pattern of each individual. The regression line and standard error bar graphs of each CKD group were generated based on the predicted uCysB values ​​of each day (Day 0 to Day 851) of follow-up. All statistical analyses were performed in R (Version 4.1.0).

[0254] Example 2. Demographic Data

[0255] The demographic data of the enrolled dogs are provided in Table 3. There were 55 (73%) controls, 12 (16%) stable CKD dogs, and 8 (11%) progressive CKD dogs. More than half (56%; 42 / 75) of the enrolled dogs were between five and ten years old. Most dogs were spayed females or castrated males. The top five breeds are listed in Table 3.

[0256] Table 3

[0257]

[0258]

[0259] Example 3. Descriptive analysis

[0260] There were differences in log(uCysB) expression between dogs in the same study group (p 对照 <0.02; p 稳定性 <10 -7 ;p 进展性 <10 -6 )( Figure 1 The Log(uCysB) values ​​of dogs with progressive CKD appeared to be higher than those of control and stable CKD dogs. The Y-intercept calculated based on the linear regression for each dog also differed by CKD group (p<10 -4 )( Figure 2 The y-intercepts of dogs with progressive CKD tended to be higher than those of dogs with control and stable CKD. Differences in slopes were detected between control, stable CKD, and progressive CKD dogs (p=0.006) ( Figure 3 Post hoc analysis ( Figure 4 ) showed that 75% (6 / 8) of dogs with progressive CKD had elevated uCysB values ​​at visit 1. In contrast, only six percent (3 / 52) of control dogs and eight percent (1 / 12) of stable dogs had elevated uCysB values ​​at visit 1.

[0261] The results of the linear mixed effects models are shown in Table 4 .

[0262] Table 4. Results of the linear mixed effects model. A random intercept was used to account for the non-independence of repeated measures within individuals. Urinary cystatin B was the dependent variable. Follow-up day was the independent variable. The effect of CKD group was allowed to vary randomly.

[0263]

[0264] Progressive CKD dogs had significantly higher uCysB values ​​than stable CKD and control dogs. Figure 5 A plot of the linear mixed effects model is shown in .

[0265] Example 4: Differentiation between Cystatin B and Clusterin

[0266] Urinary cystatin B (uCysB) is a novel renal injury biomarker with the potential to predict the progression of early CKD.

[0267] Early identification of whether dogs have progressive or stable chronic kidney disease (CKD) may provide an opportunity for intervention to slow progression. However, there are currently no surrogate biomarkers to reliably predict CKD progression. High urinary cystatin B ("uCysB" or "uCysB") levels in canine patients without a diagnosis of CKD may indicate acute kidney injury (AKI). Therefore, the differentiation of stable from progressive CKD by cystatin B and clusterin was investigated in patients with established CKD.

[0268] Animals: Twenty dogs evaluated for International Renal Rights Society (IRIS) I CKD (stage I) with IDEXX up to 17 μg / dL with no systemic comorbidities, and 107 clinically healthy dogs (controls).

[0269] Methods: A prospective longitudinal study was conducted between 2016 and 2021 to evaluate the expression pattern of uCysB in stage I CKD and control dogs. Dogs were followed for up to 3 years. The slope stratifies stage I into stable or progressive CKD subgroups and is calculated from a minimum of three monthly time points during the first 120 days. Slope above or below -0.0007 (μg / dL) -1 Dogs with ≥ 20% uCysB / week were classified as stable or progressive. Mixed effects modeling was used to assess the association between uCysB and progression rate.

[0270] Results: The estimated uCysB predictive of active persistent damage was 16 ng / mL for controls, 23 ng / mL for stable CKD, and 204 ng / mL for progressive CKD ( Figure 5 , p<0.05, dashed lines indicate 95% confidence intervals). Thus, uCysB distinguishes stable from progressive stage I CKD. Early identification of dogs with progressive CKD creates an opportunity to intervene to halt progression and improve patient management.

[0271] Urinary levels of cystatin B and clusterin were measured by ELISA in canine patients. The minimum SDMA reciprocal slope observed in the healthy canine population over a period of up to three years was -0.0007 (μg / dL) -1 / week, which was designated as the slope cutoff for stable renal function. The minimum slope refers to the slope of the individual dog with the most negative slope. In other words, the minimum slope is the slope with the lowest value. As described above, in the group of patients with confirmed CKD, the slope was above or below -0.0007 (μg / dL) -1 Patients with ≥5 years of disease progression were classified as stable or progressive.

[0272] Mixed effects modeling was also used to assess the association between clusterin and progression rate. The IRIS stage I subgroup analysis included CKD patients with an initial SDMA of up to 17. The findings then held true until the initial SDMA was up to 20, which extends into the early stages of IRIS stage II. In other words, cystatin B can distinguish between stable disease and progressive disease in CKD patients with SDMA values ​​up to 20, which covers stage I (<18 μg / dl) and part of stage II (18-35 μg / dl).

[0273] like Figure 5 As shown in , cystatin B can distinguish stable CKD from progressive CKD in patients with International Kidney Rights Association stage I CKD. Figure 6 As shown in , cystatin B can distinguish stable CKD from progressive CKD in patients with SDMA values ​​up to 20.

[0274] like Figure 7 As shown in the literature, clusterin can differentiate stable CKD from progressive CKD in patients with International Kidney Rights Association stage I CKD.

[0275] like Figure 8 As shown in , clusterin can distinguish stable CKD from progressive CKD in patients with SDMA values ​​up to 20.

[0276] Example 5: Differentiation of Stable Renal Function from Progressive Dysfunction in Dogs

[0277] Background: Renal function markers have been variably used to define stable versus progressive chronic kidney disease (CKD), but quantitative criteria to distinguish these groups are lacking. Here, we evaluated 1 / creatinine and 1 / IDEXX The slope cutoff value was used to distinguish stable CKD from progressive CKD.

[0278] Animals: 113 clinically healthy dogs and 29 male social dogs with progressive X-linked hereditary nephropathy (XLHN). The healthy dog ​​cohort (HD) had archived samples from dogs enrolled in a prospective study evaluating the serological forms of Lyme disease, ehrlichiosis, and anaplasmosis in clinically healthy dogs. The HD cohort consisted of dogs between 1 and 10 years of age raised by students and staff at the University of Missouri. Health status was assessed by clinical history, CBC, serum chemistry profile, urinalysis, and serology at each time a sample was collected. Both seropositive and seronegative healthy dogs were included and sampled every 4-6 months for >2 years. The XLHN cohort had archived samples from dogs enrolled in a prospective study evaluating the progressive nature of XLHN. XLHN is a rapidly progressive glomerulopathy caused by mutations in the gene for the alpha 5 chain of type IV collagen. Intact male puppies (homozygous affected) were from a single family of purpose-bred social dogs raised at Texas A&M University. Blood samples were obtained monthly until death from CKD.

[0279] Analysis: Archived serum samples were stored at -80°C from 2012-2014 for the HD (clinically healthy dogs) cohort and 2002-2012 for the XLHN cohort. The functional marker s-creatinine was analyzed at IDEXX, Columbia, OH by a modified Jaffe method (HD) and at Texas A&M by an enzymatic method (XLHN). The functional marker s-SDMA was analyzed by LC-MS (IDEXX, Westbrook, ME).

[0280] Table 5. Characteristics of the HD cohort

[0281]

[0282] Table 6. Characteristics of the XLHN cohort

[0283]

[0284] Methods: Retrospective analysis was combined from two prospective observational studies, one tracking renal function markers in healthy dogs (HD) for up to 3 years and one tracking renal function markers in male social dogs with progressive disease, with the slope cutoff from HD assigned as stable renal function. The temporal 1 / creatinine and 1 / SDMA changes were plotted versus follow-up time for each dog.

[0285] Simple linear regression was used to calculate the slope of the 1 / creatinine and 1 / SDMA transformations (dependent variables) versus follow-up time (independent variable) for each dog. The minimum slope value for HD was set as the cutoff for stable renal function versus progressive renal dysfunction. Relative 1 / creatinine and 1 / SDMA slopes were plotted and cohort-specific intercepts were assigned for visual comparison.

[0286] Results: For 1 / creatinine, the cutoff value for the stability and progression slope was -0.0117 (mg / dL) -1 / week, and for 1 / IDEXX The cut-off value for the stability and progression slope was -0.0007 (μg / dL) -1 / week( Fig. 9 and Fig.10 , grey dashed line).

[0287] In the CKD population studied, either 1 / creatinine or 1 / IDEXX The slope of the curve distinguishes progressive dysfunction from stable renal function. These criteria can be used to characterize CKD in other dog cohorts and establish guidelines for understanding the rate of progression in dogs with naturally acquired CKD.

[0288] The functional marker s-creatinine remained stable in healthy dogs during the >2-year observation period (Table 7 and Fig.15 Similarly, the functional marker sSDMA remained stable in healthy dogs over the 1,096-day observation period (Table 8 and Fig.18 ).

[0289] Table 7: Median s-creatinine in the HD cohort (n=113).

[0290]

[0291] Table 8: Median sSDMA in the HD cohort (n=113).

[0292]

[0293] 1 / creatinine remains stable over time in healthy dogs. The median slope (days) is 1 / creatinine = 0.00001436*[(mg / dl)-1 / day]*(number of days)+0.9324022. The median slope (weeks) is 1 / creatinine = 0.00010052*[(mg / dl)-1 / week]*(number of weeks)+0.9324022( Fig.16 and Fig.17. Similarly, 1 / SDMA remained stable over time in healthy dogs. The median slope (days) was 1 / SDMA = 0.00000175*[(μg / dl)-1 / day]*(number of days)+0.1155601. The median slope (weeks) was 1 / SDMA = 0.00001225*[(μg / dl)-1 / week]*(number of weeks)+0.1155601( Fig.19 and Fig. 20 ).

[0294] The functional marker s-creatinine showed a progressive exponential increase in the XLHN cohort compared with healthy dogs ( Fig.21 , Fig. 22 and Table 9. Similarly, the functional marker sSDMA also showed a progressive exponential increase in the XLHN cohort ( Fig.25 , Fig.26 and Table 10).

[0295] Table 9: Median s-creatinine in the XLHN cohort (n=29).

[0296]

[0297]

[0298] Table 10: Median sSDMA in the XLHN cohort (n=29).

[0299]

[0300] During the median observation period of 181 days, all XLHN dogs showed a progressive linear decline in 1 / creatinine. The median slope (days) was 1 / creatinine = -0.01120215*[(mg / dl)-1 / day]*(days)+2.435009. The median slope (weeks) was 1 / creatinine = -0.07841505*[(mg / dl)-1 / week]*(weeks)+2.435009( Fig.23 and Fig.24 ). Similarly, 1 / SDMA showed a progressive linear decline in all XLHN dogs over a median observation period of 181 days. The median slope (days) was 1 / SDMA = -0.0004527534*[(μg / dl)-1 / day]*(days)+0.07788298. The median slope (weeks) was 1 / SDMA = -0.0031692738*[(μg / dl)-1 / week]*(weeks)+0.07788298( Fig. 27 and Fig.28 ).

[0301] For 1 / creatinine, the stability and progression slope cutoff value is -0.0117 (mg / dL) -1 / week, and for The cut-off value for the stability and progression slope was -0.0007 (μg / dL) -1 / week( Fig.29 , Fig.30 and Fig.31 ).

[0302] In the studied cohort, stable renal function in healthy dogs could be distinguished from progressive renal dysfunction in XLHN by the slope of the 1 / creatinine or 1 / SDMA curve.

[0303] If extended to other CKD populations, short-term (2-3 months) assessment of the 1 / creatinine or 1 / SDMA slope may allow identification of dogs with stable versus progressive CKD.

[0304] Discrete values ​​of the 1 / creatinine or 1 / SDMA slope can serve as a guide (cutoff) to define renal function as "stable" or "progressive" and direct appropriate therapy for each state.

Claims

1. A method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD by the following steps: (a) determining the amount of a cystatin B polypeptide, a clusterin polypeptide, or both in a sample from the subject at a single time point, (b) comparing the amount of the cystatin B polypeptide, the clusterin polypeptide, or both in the sample with a control sample or a control standard, wherein an increased level of the cystatin B polypeptide, the clusterin polypeptide, or both in the sample compared to the control sample or the control standard is indicative of progressive CKD.

2. The method of claim 1, wherein prior to step (a), the subject is diagnosed with International Kidney Rights Association CKD stage I or CKD with a symmetric dimethylarginine (SDMA) value of at most 20 μg / dl.

3. The method of claim 1 or 2, further comprising administering to the subject one or more treatments comprising: Surgery for ureteral obstruction, obstructive kidney stones, or urolithiasis, dietary management, administration of enteric phosphate binders, antiproteinuric drugs, administration of antihypertensive drugs, fluid therapy to correct dehydration, management of acidosis, administration of diuretics, dialysis, correction of electrolyte abnormalities, administration of antiemetics, administration of antacids, administration of recombinant erythropoietin, holistic treatment, or a combination thereof.

4. The method of claim 1 or 2, further comprising determining the amount of cystatin B polypeptide, clusterin polypeptide, or both in samples from the subject at 2, 3, 4, 5 or more additional time points.

5. The method of claim 1 or 2, wherein the amount of the cystatin B polypeptide is determined using one or more antibodies or specific binding fragments that specifically bind to the cystatin B polypeptide or one or more polypeptides set forth in SEQ ID NOs: 1-2, 5-32, fragments thereof, or a combination thereof.

6. The method of claim 1 or 2, wherein the amount of the clusterin polypeptide is determined using one or more antibodies or specific binding fragments that specifically bind to the clusterin polypeptide or one or more polypeptides set forth in SEQ ID NOs: 3-4, fragments thereof, or a combination thereof.

7. A method according to claim 1 or 2, wherein determining the amount of cystatin B polypeptide or clusterin polypeptide in a sample from the subject comprises contacting the sample with one or more antibodies or their specific binding fragments that specifically bind to cystatin B polypeptide, clusterin polypeptide or one or more polypeptides set forth in SEQ ID NO:1-32 under conditions suitable for forming a complex of the cystatin B polypeptide or clusterin polypeptide and the one or more antibodies or their specific binding fragments, and detecting the complex of the cystatin B polypeptide or clusterin polypeptide and the one or more antibodies or their specific binding fragments.

8. The method of claim 7, wherein the antibody or specific binding fragment thereof is immobilized to a solid support.

9. The method of claim 7, wherein the antibody or specific binding fragment thereof is conjugated to one or more labels.

10. The method of claim 7, further comprising contacting the complex of the cystatin B polypeptide or the clusterin polypeptide and the one or more antibodies or specific binding fragments thereof with an indicator reagent.

11. The method of claim 1 or 2, wherein the subject is a non-human mammal.

12. The method of claim 11, wherein the non-human mammal is a canine or a feline.

13. The method of claim 1 or 2, wherein the sample is blood, serum, plasma or urine.

14. The method according to claim 1 or 2, wherein the amount of cystatin B or clusterin polypeptide is determined by immunoassay, competitive immunoassay, sandwich immunoassay, enzyme-linked immunosorbent assay (ELISA), turbidity immunoassay, particle-enhanced turbidity immunoassay, radioimmunoassay (RIA) or Western blot assay.

15. The method of claim 1 or 2, wherein determining the amount of cystatin B or clusterin polypeptide comprises subjecting the sample to mass spectrometry, LC-MS, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatography (HPLC) mass balance assay, or a combination thereof.

16. The method of claim 1 or 2, wherein the control sample or control standard is derived from a normal, healthy subject.

17. The method of claim 1 or 2, wherein the control sample is derived from a subject with stable CKD.

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