Methods and compositions for distinguishing progressive from stable chronic kidney disease - Patents.com

Abstract

The present disclosure provides methods and compositions for the detection and differentiation of stable and progressive CKD in a mammal.

Description

[Technical field]

[0001] Priority This application claims the benefit of U.S. Patent Application No. 63 / 267,181, filed January 26, 2022, and U.S. Patent Application No. 63 / 354,878, filed June 23, 2022, which are incorporated by reference in their entireties. [Background technology]

[0002] Chronic kidney disease (CKD) may be associated with increased water consumption, frequent urination, loss of appetite, weight loss, and muscle atrophy. Generally, by the time clinical symptoms of CKD appear, irreversible kidney damage has occurred. Early detection allows for early treatment, thus slowing disease progression. Current treatments include, for example, dialysis, resolution of any precipitating factors, and diets low in phosphorus and protein. Early detection is important for improving longevity and quality of life.

[0003] In humans, the progression of CKD is divided into five stages. In companion animals, such as dogs and cats, the progression of CKD is divided into four stages as described by the International Renal Interest Society. Current methods for detecting CKD in mammals, such as dogs, cats, and humans, include renal ultrasound, biopsy, blood creatinine and symmetric dimethylarginine (SDMA) measurements, and urinary protein and creatinine measurements. Biopsy is invasive, limiting its ability as an early screening or diagnostic test. Blood creatinine levels increase only after substantial decline in glomerular function and are poorly sensitive for early, mild, or moderate CKD. Urine and blood creatinine are also affected by muscle mass, diet, and tubular secretion. Currently available methods for detecting and diagnosing CKD are unable to distinguish stable CKD from progressive CKD in a timely manner. Summary of the Invention

[0004] One embodiment provides a method for distinguishing stable from progressive CKD in a subject diagnosed with chronic kidney disease ("CKD") by (a) determining the amount of cystatin B polypeptide, clusterin polypeptide, or both in a sample from the subject at a single time point, and (b) comparing the amount of cystatin B polypeptide, clusterin polypeptide, or both in the sample with a control sample or control standard, wherein an increased level of cystatin B polypeptide or clusterin polypeptide, or both in the sample compared to the control sample or control standard is indicative of progressive CKD.

[0005] In some embodiments, the subject may be diagnosed with CKD stage I of the International Renal Interest Society or CKD with a symmetric dimethylarginine (SDMA) value of up to 20 μg / dl prior to step (a). In some embodiments, the method further comprises administering to the subject one or more treatments, including surgery for obstructed ureter, obstructed kidney stones, or urinary tract stones, dietary management, administration of intestinal phosphate binders, administration of 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 combinations thereof. In some embodiments, the method may further comprise determining the amount of cystatin B polypeptide, clusterin polypeptide, or both in a sample from the subject at two, three, four, five, or more additional time points. In some embodiments, the amount of cystatin B polypeptide may be determined using one or more antibodies or specific binding fragments that specifically bind to a cystatin B polypeptide, or one or more polypeptides set forth in SEQ ID NOs: 1-2, 5-32, fragments thereof, or combinations thereof. In some embodiments, clusterin polypeptide may be determined using one or more antibodies or specific binding fragments that specifically bind to a clusterin polypeptide, or one or more polypeptides set forth in SEQ ID NOs: 3-4, fragments thereof, or combinations thereof. In some embodiments, determining 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 a cystatin B polypeptide, a clusterin polypeptide, or one or more polypeptides set forth in SEQ ID NOs: 1-32 under conditions suitable for the formation of a complex between the cystatin B polypeptide or clusterin polypeptide and the one or more antibodies or specific binding fragments thereof, and detecting the complex between the cystatin B polypeptide or clusterin polypeptide and the one or more antibodies or specific binding fragments thereof. In some embodiments, the antibodies or specific binding fragments thereof may be immobilized to a solid support.In some embodiments, the antibody or specific binding fragment thereof may be conjugated to one or more labels. In some embodiments, the method may further comprise contacting the complex of the cystatin B or clusterin polypeptide and one or more antibodies or specific binding fragments thereof with an indicator. In some embodiments, the subject may be a non-human mammal. In some embodiments, the non-human mammal may be a dog or a cat. In some embodiments, the sample may be blood, serum, plasma, or urine. In some embodiments, the amount of the cystatin B or clusterin polypeptide may be determined by immunoassay, competitive immunoassay, sandwich immunoassay, enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunoassay (RIA), or Western blot assay. In some embodiments, determining the amount of the cystatin B or clusterin polypeptide may comprise subjecting the sample to mass spectrometry, LC-MS, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatographic (HPLC) mass balance assay, or a combination thereof. In some embodiments, the control sample or control standard may be derived from a normal healthy subject. In some embodiments, the control sample may be from a subject with stable CKD.

[0006] Specific embodiments will become apparent from the following more detailed description of certain preferred embodiments and the claims. [Brief description of the drawings]

[0007] [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 larger uCysB values ​​compared to control and stable CKD dogs. Sequential measurements for a given dog do not appear to be independent. [Diagram 2]Y-intercepts calculated from linear regression per dog are shown, with urinary cystatin B as the dependent variable and follow-up time as the independent variable. Y-intercepts for control and stable CKD dogs were relatively similar, whereas y-intercepts for progressive CKD dogs were consistently higher. Differences in y-intercepts between groups were detected using Kruskal-Wallis ANOVA (p<10-4). [Diagram 3] Slopes calculated from linear regression per dog are shown, with log(uCysB) as the dependent variable and follow-up time as the independent variable. Differences between slopes in control, stable, and progressive CKD dogs were detected by Kruskal-Wallis ANOVA (p=0.006). [Figure 4] First uCysB measurements by CKD group are shown. Only eight dogs had advanced IRIS stage 1 CKD at first visit, but six (75%) had a first uCysB value above the reference range (RR) of 50, of which four (67%) progressed beyond IRIS stage 1 CKD. [Diagram 5] FIG. 1 shows differentiation of stable from progressive CKD using cystatin B levels in canine International Renal Interest Society stage I CKD patients. [Figure 6] FIG. 1 shows differentiation of stable from progressive CKD using cystatin B levels in canine CKD patients with SDMA values ​​up to 20. [Figure 7] FIG. 1 shows differentiation of stable from progressive CKD using clusterin levels in canine International Renal Interest Society stage I CKD patients. [Figure 8] 1 shows differentiation of stable from progressive CKD using clusterin levels in canine CKD patients with SDMA values ​​up to 20. [Figure 9] FIG. 1 shows the reverse creatinine slope cutoff for distinguishing between stable renal function in healthy dogs (HD) and impaired renal function in progressive CKD resulting from X-linked hereditary nephropathy (XLHN). The slope cutoff is indicated by the grey dashed line. [Figure 10]FIG. 1 shows the inverse SDMA slope cutoff for distinguishing between stable renal function in healthy dogs (HD) and renal dysfunction in progressive CKD resulting from XLHN. The slope cutoff is indicated by the grey dashed line. [Figure 11] 1 illustrates a method for determining normal biomarker ranges for reverse creatinine. [Figure 12] 1 shows a method for determining normal biomarker ranges for reverse SDMA. [Figure 13] A method for determining the minimum change in reverse creatinine slope from normal to abnormal is presented. [Figure 14] A method for determining the minimum change in inverse SDMA slope from normal to abnormal is presented. [Figure 15] Representative time series s-creatinine findings in 30 randomly selected dogs from a cohort of 113 healthy dogs are shown. [Figure 16] Representative time series of reverse creatinine in 30 randomly selected dogs from a cohort of 113 healthy dogs are shown. [Figure 17] 1 shows the reverse creatinine slope plot for the HD cohort. [Figure 18] Representative time series of sSDMA in 30 randomly selected dogs from a cohort of 113 healthy dogs are shown. [Figure 19] Time series of inverse SDMA in 30 randomly selected dogs and slopes from a cohort of 113 healthy dogs are shown. [Figure 20] 1 shows the inverse SDMA slope plot for the HD cohort. [Figure 21] Figure 1 shows the time course of s-creatinine in the XLHN cohort. [Figure 22] Serial changes in s-creatinine in the XLHN cohort are shown. [Diagram 23] 1 shows the time course of reverse creatinine in the XLHN cohort. [Figure 24] 1 shows the reverse creatinine slope plot for the XLHN cohort. [Diagram 25]The longitudinal findings of sSDMA in the XLHN cohort are shown. [Figure 26] Serial changes in sSDMA in the XLHN cohort are shown. [Figure 27] Shows time course of reverse SDMA in the XLHN cohort. [Figure 28] 1 shows the inverse SDMA slope plot for the XLHN cohort. [Figure 29] FIG. 1 shows the reverse creatinine slope cutoffs for distinguishing between stable renal function in healthy dogs (HD) and renal dysfunction in progressive CKD resulting from X-linked hereditary nephropathy (XLHN). [Diagram 30] 1 shows the inverse SDMA slope cutoff for distinguishing between stable renal function in healthy dogs (HD) and impaired renal function in progressive CKD resulting from X-linked hereditary nephropathy (XLHN). [Diagram 31] Box blots showing slope cutoffs (dotted lines) for reversal creatinine (left panel) and reversal SDMA (right panel) to distinguish between stable renal function in healthy dogs (HD) and impaired renal function in progressive CKD resulting from X-linked hereditary nephropathy (XLHN).

[0008] These and other objects and features will be better understood from the following detailed description taken in conjunction with the drawings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The terms used herein generally have their ordinary meanings in the art, within the context of the compositions and methods described herein, and in the specific context in which each term is used. Some terms are more specifically defined herein to provide additional guidance to the practitioner regarding the description of the compositions and methods.

[0010] 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 predominate. CKD tends to be irreversible and progressive in nature. CKD is typically diagnosed based on changes in functional markers such as blood creatinine and symmetric dimethylarginine (SDMA) concentrations, which are also used to monitor disease progression. However, these functional markers may be less sensitive in early CKD due to their nonlinear relationship with glomerular filtration rate (GFR) and due to relatively wide reference ranges for both creatinine and SDMA. Markers of active and ongoing injury (acute kidney injury, AKI) may also be increased in some dogs with CKD, indicating that active and ongoing injury is also present in dogs with CKD, even if the disease appears stable based on functional markers. Thus, CKD and AKI are not completely separate processes and may share some common features.

[0011] CKD tends to be progressive in all dogs, but the degree of progression varies considerably between dogs. These differences in progression can be observed in the rate of change or slope of functional renal biomarkers (e.g., creatinine, SDMA). When assessing CKD progression, functional renal biomarker values ​​can be converted to their reciprocals ("inverse biomarkers"), from which slopes ("inverse slopes") can be calculated. Several risk factors for CKD progression have been identified, including the degree of renal proteinuria, as reflected by urinary protein:creatinine (UPC) ratio, hypertension, and hyperphosphatemia. The degree of activity injury in dogs with CKD can serve as a surrogate marker for the rate of progression, regardless of the underlying cause.

[0012] Stage 1 chronic kidney disease (CKD) based on the IRIS guidelines is typically associated with greater stability than more advanced stages of CKD. However, some IRIS stage 1 CKD dogs, particularly those with advanced CKD, may have active, ongoing injury as reflected by elevated uCysB levels throughout the follow-up period. Furthermore, uCysB may provide an earlier indicator of such injury compared to conventional biomarkers.

[0013] CKD in dogs and cats is a multifactorial disorder with multiple contributing etiologies. In the majority of animals with CKD, the underlying cause cannot be identified or excluded. In the absence of an identifiable cause that can be excluded, therapy focuses on dietary interventions that have been shown to slow the rate of progression and control risk factors for rapid progression (e.g., hypertension, proteinuria). As the disease progresses to more advanced stages, treatment becomes more symptomatic in focus. Thus, the primary therapeutic goal is to identify the disease early in its course and slow the rate of progression before significant irreversible damage occurs.

[0014] Diagnosis of CKD stage 1 is difficult, and additional methods of kidney injury detection are needed. Currently, diagnosis of CKD often cannot rely on functional markers. Therefore, early diagnosis is based on the presence of persistently unconcentrated urine (after excluding all other possible causes), the presence of renal proteinuria, and ultrasound changes (which are often subtle and inconclusive at this stage of the disease). Many of these techniques require multiple visits to the veterinarian. Further diagnosis includes measurement of GFR and renal biopsy, neither of which are routinely obtained in clinical practice. As described herein, 75% of progressive CKD dogs had elevated uCysB at first visit, compared to only 8 percent of stable dogs. Furthermore, 67% of progressive CKD dogs with elevated uCysB progressed beyond IRIS stage 1. As shown here, uCysB can be applied as an early biomarker to distinguish stable from progressive CKD at IRIS stage 1.

[0015] In this study, concentrations of uCysB during the follow-up period were higher in dogs with rapid progression compared to stable dogs. This means that regardless of the cause and underlying etiology, the degree of active, ongoing injury can be used as a surrogate marker of active injury within the kidney that leads to loss of kidney function (i.e., fibrosis) that is likely to be irreversible. Whether this progression can be delayed by therapeutic intervention remains to be determined, but the fact that a real-time marker of kidney injury is available will facilitate real-time evaluation of various therapeutic interventions in animals with CKD.

[0016] To date, therapeutic interventions are typically monitored using functional markers that change very slowly. This requires very long follow-up periods to evaluate these interventions, during which other factors (some of which can be identified and controlled, others not) may also affect the rate of progression. The very long time it takes to evaluate the effects of therapeutic interventions in dogs with early renal disease makes such studies difficult and expensive to perform. However, these interventions are likely to be more meaningful in the early stages of the disease before the bulk of the kidney has undergone substantial irreversible changes. In this context, real-time markers such as uCysB are beneficial and allow a better understanding of the intrarenal processes that manage CKD and possible interventions to improve these processes.

[0017] Herein, the slope of SDMA is used to evaluate disease progression rate.Despite the high prevalence of CKD in dogs and cats, no definition or guideline has been established to distinguish slow-progressing animals from moderate or fast-progressing animals.Although the rate or absolute change of creatinine or SDMA concentration can be used to define progression, the time at which these changes occur is important to determine progression rate (i.e., a creatinine increase of 0.5mg / dL can be defined as a fast-progressing dog if it occurs over 2 months, and as a slow-progressing dog if it occurs over 3 years).In this study, the slope of at least 3 time points over at least 3 months is used to evaluate progression rate, taking into account time effect.

[0018] uCysB and clusterin (described below) are biomarkers for the detection of intrarenal injury in animals with CKD and possible surrogate markers for the rate of progression. In stage I CKD, uCysB and clusterin allow for the differentiation of stable from progressive CKD, creating opportunities for halting progression and improving patient management.

[0019] Stable Chronic Kidney Disease and Progressive Kidney Disease The compositions and methods described herein can be used to distinguish progressive CKD from stable CKD in mammals, such as dogs, cats, and humans, diagnosed with CKD. Progressive and stable CKD can also be monitored for progression. Progressive and stable CKD can result in (1) decreased renal function compared to healthy subjects, or (2) physical damage to the kidney, or (3) both. In some embodiments, CKD (both progressive and stable) does not include cancer or does not include renal cancer. Markers of cancer, including renal cancer, can be different from markers of stable CKD or progressive CKD. In some embodiments, stable CKD or progressive CKD does not include cancer, such as renal cancer or bladder cancer.

[0020] CKD is a condition characterized by the gradual loss of kidney function over time. CKD may exist in a stable or progressive form. Progressive or stable CKD may be glomerular or tubular. As CKD worsens, waste products accumulate to high levels in the blood, which may result in high blood pressure, anemia, weakened bones, poor nutritional health, and nerve damage. CKD increases the risk of heart and vascular diseases, and may eventually result in kidney failure. CKD may be caused by, for example, diabetes, high blood pressure, and other disorders. Early detection and treatment can often prevent the disease from worsening.

[0021] The stages of CKD in dogs, as established by the International Renal Interest Society ("IRIS"), are shown in Table 1. [Table 1]

[0022] The stages of CKD in cats, as established by the International Renal Interest Society, are shown in Table 2. [Table 2]

[0023] Changes in functional renal markers (e.g., creatinine or SDMA) over time can be used to detect CKD. However, there are several problems with using these markers to determine whether a subject has stable or progressive CKD. Most notably, these markers must be monitored over time to determine whether CKD is stable or progressive. During the time required to observe any trends in the levels of the markers, irreversible kidney damage may occur. In addition, the use of blood creatinine is relatively insensitive as an early indicator of CKD. Creatinine levels in healthy dogs and cats may overlap with creatinine values ​​in dogs and cats with mild to moderate renal disease. Further, other problems with the use of creatinine as a marker include that creatinine can vary based on muscle mass, there is no standardization of blood creatinine measurements across veterinary diagnostic laboratories, the consumption of a meat-based diet can result in postprandial absorption of creatinine, necessitating the use of fasting samples (ideally 12 hours fasting), and elevated blood creatinine concentrations are detectable only after a significant amount of kidney tissue has been damaged. The use of SDMA as a marker of CKD has drawbacks, including increased SDMA in dogs and cats with diseases other than CKD (e.g., hyperthyroidism or hypoadrenocorticism), significant differences in SDMA levels in different dog and cat breeds, and hemolysis of samples can cause interference with the measurement of SDMA. Therefore, a reliable marker that does not need to be measured over time to determine whether a CKD patient has stable or progressive CKD can help prevent kidney disease.

[0024] Surprisingly, it has been discovered that by using the cystatin B ("CysB") and / or clusterin markers, it is possible to determine whether a CKD patient has stable or progressive CKD without the use of more than one test over time. Prior to the present disclosure, it was unclear whether markers existed that could be used to distinguish stable from progressive CKD at a single time point.

[0025] Although referred to as "stable CKD," stable CKD may progress through IRIS CKD stages I-IV. However, stable CKD progressing through the four stages may be contrasted with progressive CKD, whose symptoms may progress or worsen over time at a faster rate than symptoms of stable CKD.

[0026] Patients with stable CKD have no substantial change in functional renal markers, such as creatinine or SDMA, over time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36 months or more (and any range from about 1 to about 36 months, e.g., about 1 to about 3, about 2 to about 4, about 3 to about 6, about 6 to about 12, or about 1 to 36 months). No substantial change in functional renal markers indicates that an increase in the amount of functional renal markers in CKD patients is greater than or equal to about 2, By "advanced CKD" we mean less than 5, 10, 20, or 25 percent. The increase in the amount of functional renal marker in progressive CKD can be an increase of about 21, 25, 30, 35, 40% or more over time (e.g., 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 more (and any range from about 5 days to about 36 months, e.g., about 1 to about 3, about 2 to about 4, about 3 to about 6, about 6 to about 12, or about 1 to 36 months).

[0027] Provided herein are methods for determining whether a patient diagnosed with CKD has stable or progressive CKD by determining the levels of cystatin B, clusterin, or both, for example, at a single time point. In some embodiments, the mammalian CKD patient is in IRIS stage I or has an SDMA value of up to 20. The patient is diagnosed with progressive CKD when the mammalian CKD patient has a cystatin B level of about 50, 60, 70, 80, 90, 100 ng / mL or more (or any range of about 50-100 ng / mL or more, such as about 50-100 ng / mL or more, about 50-80 ng / mL or more, about 70-100 ng / mL or more)). These are considered "elevated amounts" of cystatin B. A mammalian CKD patient is diagnosed with progressive CKD when they have urinary levels of about 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 ng / mL or greater of clusterin (or any range of about 250-350 ng / mL or greater, e.g., about 250-300 ng / mL or greater, about 275-325 ng / mL or greater, about 325-350 ng / mL or greater). These are considered "elevated amounts" of clusterin.

[0028] Also provided herein are methods for determining whether a patient diagnosed with CKD has stable CKD by determining the levels of cystatin B, clusterin, or both, for example, at a single time point. In some embodiments, the mammalian CKD patient is in IRIS stage I or has an SDMA value of up to 20. The mammalian CKD patient is diagnosed with stable CKD if he has a urinary cystatin B level of about 20, 30, 40, 50, 60, 70, 80, 90, 100 ng / mL or more of cystatin B (or any range between about 20-100 ng / mL). The mammalian CKD patient is diagnosed with stable CKD if he has a urinary cystatin B level of about 100, 90, 80, 70, 60, 50, 40, 30, 20 ng / mL or less of cystatin B (or any range between about 100-20 ng / mL).

[0029] The use of SDMA or creatinine at a single time point does not provide the same results as the use of the cystatin B or clusterin markers described herein.

[0030] Polypeptides Previous discoveries and research by IDEXX Laboratories, Inc. on cystatin B and clusterin are 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 No. 2018 / 0142009 (Specific Detection of Clusterin isoforms). Previous IDEXX research examining cystatin B and clusterin in canine renal injury included the development of an in vitro cell model of cultured canine renal cells treated with the nephrotoxic gentamicin. Clusterin isoforms present in cultured cell supernatants have unique glycosylation patterns compared to clusterin isoforms isolated from plasma, allowing for the generation of antibodies specifically targeting the renal-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 identical to the native protein extracted from canine kidney cells using LC-MS. Clinical validation of cystatin B was previously confirmed in dogs exhibiting renal injury.

[0031] Cystatin B (CysB) is an intracellular protein that belongs to the family of cysteine ​​protease inhibitors. CysB does not circulate at high serum concentrations because it is an intracellular protein. 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 and about 11 kDa in size. They are not glycosylated and do not have the disulfide bridges found in other members of the cystatin superfamily. They also lack signal sequences and are generally intracellular proteins that are restricted to the cell. See Ochieng & Chaudhuri, J Health Care Poor Underserved 2010,21(1 Suppl):51. Some amount of cystatin B is present in extracellular fluids, including human urine. Cystatin B has been shown to inhibit members of the lysosomal cysteine ​​proteinase, cathepsin family, specifically 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.

[0032] Canine cystatin B (SEQ ID NO: 1) is MMCGAPSASQPATADTQAIADQVKAQLEERENKKYTTFKAVTFRSQVVAGTXYFIKVQVDDDEFVHLRVFQSLPHENKPLALSSYQTNNKAKHDELAYF (X can be any amino acid, alternatively, X can be P or N).

[0033] SEQ ID NO:1 can be used to detect feline or canine cystatin B.

[0034] Fragments of canine Cystatin B may include: MMCGAPSASQPATADTQAIAD (SEQ ID NO: 5) QTNKAKHDELAYF (SEQ ID NO: 6) Cystatin BC terminus "peptide 9" CGAPSASQPATADTQAIA (SEQ ID NO: 7) Cystatin B N-terminus "peptides 3-20" CGAPSASQ (SEQ ID NO: 8) Cystatin B N-terminus "peptides 3-10" CAIADQVKA (SEQ ID NO: 9) Cystatin B N-terminus "peptides 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) SQVVAGTNYFIKVQVDDD (SEQ ID NO: 15)

[0035] Feline cystatin B is shown below (NCBI reference sequence: XP_023094432.1). MMCGAPSATQ PATAETQAIA DQVKPQLEEQ ENKKYTTFKA VEFRSQVVAG RNYFIKVQVD DDEFVHIRVF QSLPHENKPL ALSSYQTHKA RHDELAYF (SEQ ID NO:2)

[0036] Fragments of canine Cystatin B may include: SQVVAGRNYFIKVQVDDD (SEQ ID NO: 16) AIADQVKP (SEQ ID NO: 17) MMCGAPSATQPATAETQAIADQVKPQLEE (SEQ ID NO: 18) RHDELAYF SEQ ID NO:19 SQVVAGRNYFIKVQVDDD SEQ ID NO:20 FQSLPHENKPLALSS SEQ ID NO:10 QAIADQVKP SEQ ID NO:21 CGAPSATQ SEQ ID NO:22 CGAPSATQPATAETQAIA SEQ ID NO:23 QTHKARHDELAYF SEQ ID NO:24 MMCGAPSATQPATAETQAIAD SEQ ID NO:25

[0037] Clusterin or apolipoprotein J is a 75-80 kDa disulfide-linked heterodimeric protein. Clusterin is part of many physiological processes including promoting sperm maturation, lipid transport, complement inhibition, tissue remodeling, membrane recycling, stabilization of stressed proteins, and inhibition of apoptosis. Clusterin polypeptides can be detected using any suitable method, including, for example, immunoassays. In some embodiments, a combination of lectins and anti-clusterin antibodies and lectins can be used to detect clusterin. See, for example, U.S. Patent Publication No. 2018 / 0142009. Lectins that specifically bind carbohydrates on human, canine, feline, equine, bovine, ovine, or monkey clusterin isoforms can be used. Lectins that specifically bind one or more plasma, serum, or kidney clusterin isoforms and do not bind other clusterin isoforms can also be used.

[0038] Lectins are proteins that recognize and bind to specific mono- 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 residues that bind to the carbohydrate. The binding strength of lectins to carbohydrates can increase with the number of molecular interactions. The dissociation constant for lectin binding to carbohydrates is approximately 10 -5 ~10 -7 K d It is.

[0039] The lectins can be labeled with any type of label known in the art, including, for example, fluorescent labels, chemiluminescent labels, radioactive labels, enzyme labels, colloidal metal labels, radioisotope labels, and bioluminescent labels.

[0040] In some embodiments, lectins that specifically bind to renal specific clusterin and do not specifically bind to plasma or serum clusterin can be used. In some embodiments, lectins that specifically bind to N-acetylglucosamine are useful in the present methods. Such lectins include, for example, WGA (wheat germ layer agglutinin), WGA1, WGA2, WGA3, sWGA, DSL lectin (Datura stramonium lectin), mannose-binding lectin, PHA-L (Phaseolus vulgaris leucoagglutanin), PHA-E (Phaseolus vulgaris erythoagglutanin), and LEL (Lycopersicon esculentum (tomato) lectin). Other lectins that can be used include, for example, jacalin, STL lectin (Solanum tuberosum), LCA lectin (Lens culinaris), PSA lectin (Pisum sativum agglutinin), ECL lectin (Erythina cristagalli), RCA lectin (Ricin communis), DBA lectin (Dolichos biflorus), SBA lectin (soybean), and CONA lectin (concanavlin). Lectins are commercially available, for example, from Vector Laboratories.

[0041] In some embodiments, clusterin can be detected using a combination of an anti-clusterin antibody or specific binding fragment thereof and one or more lectins that specifically bind clusterin, e.g., kidney-specific clusterin isoforms. See, e.g., U.S. Patent Publication No. 2018 / 0142009. Some non-limiting examples of anti-clusterin antibodies are Clusterin Canine, Sheep Polyclonal Antibody from BioVendor Laboratory Medicine, Inc. Commercially available feline antibodies for clusterin can be, for example, but are not limited to, Clusterin / APOJ Mouse anti-Human, Bovine, Canine, Feline, Porcine, DyLight™ 650, Clone:Hs-3, Novus Biologicals™. In some embodiments, clusterin can be detected by contacting a sample with one or more antibodies or antigen-binding fragments thereof that specifically bind to clusterin, and one or more lectins that specifically bind to the carbohydrate moiety of renal-specific clusterin and do not specifically bind to the carbohydrate moiety of other clusterin isoforms (e.g., plasma clusterin, serum clusterin, or blood-derived non-renal-specific clusterin). A complex of renal-specific clusterin, one or more antibodies or antigen-binding fragments thereof that specifically bind to clusterin, and one or more lectins can be detected. The lectins can specifically bind to N-acetylglucosamine. The lectin can be, for example, Phaseolus vulgaris leucoaglutanin (PHA-L), wheat germ agglutinin (WGA), WGA1, WGA2, WGA3, sWGA, Phaseolus vulgaris agglutinin-E (PHA-E), Lycopersicon esculentum lectin (LEL), Datura stramonium lectin (DSL), Pisum sativum agglutinin (PSA), or Dolichos biflorus lectin (DBA). One or more antibodies or antigen-binding fragments thereof can be immobilized to the support. The sample and one or more detectably labeled lectins can be added to the support.The polypeptide may be immobilized on a support. The sample and one or more detectably labeled antibodies or antigen-binding fragments thereof may be added to the support. One or more antibodies or antigen-binding fragments thereof, one or more lectins (e.g., plasma clusterin, serum clusterin, or blood-derived non-renal specific clusterin), or both may be labeled with a detectable label.

[0042] The canine clusterin precursor polypeptide UniProtKB-P25473 is shown below. MMKTLLLLVG LLLTWDNGRV LGDQAVSDTE LQEMSTEGSK YINKEIKNAL KGVKQIKTLI EQTNEERKSL LSNLEEAKKK KEDALNDTKD SETKLKASQG VCNDTMMALW EECKPCLKQT CMKFYARVCR SGSGLVGHQL EEFLNQSSPF YFWMNGDRID SLLENDRQQT HALDVMQDSF NRASSIMDEL FQDRFFTREP QDTYHYSPFS LFQRRPFFNP KFRIARNIIP FPRFQPLNFH DMFQPFFDMI HQAQQAMDVN LHRIPYHFPI EFPEEDNRTV CKEIRHNSTG CLKMKDQCEK CQEILSVDCS SNNPAQVQLR QELSNSLQIA EKFTKLYDEL LQSYQEKMFN TSSLLKQLNE QFSWVSQLAN LTQSEDPFYL QVTTVGSQTS DSNVPVGFTK VVVKLFDSDP ITVMIPEAVS RNNPKFMETV AEKALQEYRQ KHREE (SEQ ID NO: 3)

[0043] The precursor polypeptide chain is proteolytically cleaved to remove the 22-mer secretory signal peptide and then cleaved between residues 227 / 228 to generate the α- and β-chains, which assemble in an antiparallel manner to yield a heterodimeric molecule with a cysteine-rich core linked by five disulfide bridges and flanked by two predicted coiled-coil a-helices and three predicted amphipathic a-helices.

[0044] The feline clusterin precursor is shown below: UniProtKB-M3WKP2.: MTVSCSLQTEACWDSRIGEPGTMKTLLLLVGLLLTCENGRVLGDKAVSDAELQEMSTEGS KYINKEIKNALKGVKQIKTLIEQTNEERKSLLSNLEEAKKKKEDALSDTKDSEMKLKASE GBCNDTMMALWEECKPCLKQTCMKFYARVCRSGSGLVGQQLEEFLNQSSPFYFWINGDRI DSLLENDRQQTHALDVMQDSFNRASRIMDELFQDRFFTREPQDTYHYSPFSSLQRRPFFF NPKSRFARNVMPFPAFQPLNFHDMFQPFFDMIHQAQQAMDINLQRIPYHFPMEFTEEDNQ DRMVCKEIRHNSTGCLRMKDQCDKCQEILSVDCSASNPSQVLLRQELNNSLQMAEKFTKL YDELLRSYQEKMFNTSSLLKQLNEQFSWVSQLANLTQSEDPFYLQVTTVSSQTSDSNVPS GFTKVVVKLFDSDPISVMVPEEVSRNNPKFMETVAEKALQEYRQKNGEK (SEQ ID NO: 4)

[0045] The precursor polypeptide chain is proteolytically cleaved to remove the 43-mer secretory signal peptide.

[0046] Antibodies or specific binding fragments thereof that specifically bind to one or more of the polypeptides of SEQ ID NOs: 1-32 can be used to detect these polypeptides in a sample.

[0047] Provided herein are methods for the detection and quantification of 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 or fragment thereof comprising SEQ ID NOs: 1-32. A polypeptide fragment of SEQ ID NOs: 1-32 can consist of less than about 95, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10 (or any range of about 10 to about 95) consecutive amino acids. In one embodiment, a 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 of non-natural origin.

[0048] A polypeptide is a polymer of three or more amino acids covalently linked by amide bonds. Polypeptides may be post-translationally modified. A purified polypeptide is a polypeptide preparation that is substantially free of cellular material, other types of polypeptides, chemical precursors, chemicals used to synthesize the polypeptide, or combinations thereof. A polypeptide preparation that is substantially free of cellular material, culture medium, chemical precursors, chemicals used to synthesize the polypeptide has less than about 30%, 20%, 10%, 5%, 1% 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.

[0049] The term "polypeptide" can refer to one or more of a type of polypeptide (a set of polypeptides). "Polypeptide" can 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 terms "polypeptide" (singular or plural) can each mean "one or more polypeptides."

[0050] Polypeptide variants differ by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid residues (e.g., amino acid additions, substitutions, or deletions) from the peptides set forth in SEQ ID NOs: 1-32 or fragments thereof. Where this comparison requires alignment, the sequences are aligned for maximum homology. Mutation sites can occur anywhere in the polypeptide.

[0051] 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 biological equivalent. A variant is biologically equivalent if it reacts substantially the same as a polypeptide described herein in an assay such as an immunohistochemistry assay, enzyme-linked immunosorbent assay (ELISA), immunoturbidimetry, particle-enhanced immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunoassay (RIA), enzyme antibody assay, or Western blot assay, e.g., has 90-110% of the activity of the original polypeptide. In one embodiment, the assay is a competitive assay, in which a biologically equivalent polypeptide has the ability to reduce binding of a polypeptide described herein to a corresponding reactive antigen or antibody by about 80, 95, 99, or 100%. An antibody that specifically binds to the corresponding polypeptide also specifically binds to the variant polypeptide.

[0052] 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 set forth in SEQ ID NOs: 1-32. For example, variant polypeptides of SEQ ID NOs: 1-32 can be at least about 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 90%, 87%, 84%, or 81% identical to SEQ ID NOs: 1-32. Variant polypeptides have one or more conservative amino acid mutations, or other minor modifications, and retain biological activity, i.e., are biologically functional equivalents of SEQ ID NOs: 1-32. Biologically active equivalents have substantially equivalent function when compared to the corresponding polypeptides.

[0053] Methods for introducing mutations into amino acid sequences are well known to those skilled in the art. For example, see 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™ Site-Directed Mutagenesis Kit (Stratagene). Generating functionally active variant polypeptides by replacing amino acids that do not affect the function of the polypeptide can be achieved by those skilled in the art.

[0054] A variant polypeptide may have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. A conservative substitution is one in which an amino acid is replaced with another amino acid having similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic index properties of the polypeptide to remain substantially unchanged. In general, the following groups of amino acids represent conservative changes: (1) alanine, proline, glycine, glutamic acid, aspartic acid, glutamine, asparagine, serine, threonine, (2) cysteine, serine, tyrosine, threonine, (3) valine, isoleucine, leucine, methionine, alanine, phenylalanine, (4) lysine, arginine, histidine, and (5) phenylalanine, tyrosine, tryptophan, histidine. In one embodiment, the polypeptide has no more than about 1, 2, 3, 4, 5, 10, 20 conservative amino acid substitutions.

[0055] The terms "sequence identity" or "percent identity" are used interchangeably herein. To determine the percent identity of two polypeptide molecules or two polynucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first polypeptide or polynucleotide sequence for optimal alignment with the second polypeptide or polynucleotide sequence). The amino acids or nucleotides at corresponding amino acid or nucleotide positions are then compared. If a 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, and in some embodiments, at least 90% or 100%. In some embodiments, the two sequences are the same length.

[0056] Desirable ranges of sequence identity are approximately 80% to 100% and integer values ​​therebetween. The percent identity between the disclosed and claimed sequences 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%. In general, a perfect match indicates 100% identity over the length of the reference sequence (e.g., SEQ ID NO:1).

[0057] Polypeptides and polynucleotides that are sufficiently similar to the polypeptides and polynucleotides described herein (e.g., cystatin B or clusterin polypeptides) can be used herein. Polypeptides and polynucleotides that are about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 99.5% or more identical to the polypeptides and polynucleotides described herein can also be used herein.

[0058] For example, a polynucleotide may have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NOs:1-32.

[0059] A polypeptide or antibody can be covalently or non-covalently linked to an amino acid sequence with which the polypeptide or antibody is not normally associated in nature. In addition, a polypeptide or antibody can be covalently or non-covalently linked to compounds or molecules other than amino acids. 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 stop transport sequence, a transmembrane domain, a protein purification ligand, or a combination thereof. In one embodiment, a protein purification ligand can be, for example, one or more C amino acid residues at the amino or carboxy terminus of a polypeptide. An amino acid spacer is a sequence of amino acids that is not normally 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.

[0060] 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. A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available 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, e.g., freeze-dried.

[0061] Polynucleotides An embodiment includes an isolated polynucleotide encoding one or more of the polypeptides disclosed herein. The polynucleotide comprises less than an 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 to be 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 set forth in SEQ ID NOs: 1-32 or a fragment thereof.

[0062] The polynucleotide may contain other nucleotide sequences, for example, sequences encoding linkers, signal sequences, TMR stop transport sequences, transmembrane domains, or ligands useful for protein purification, such as glutathione-S-transferase, histidine tags, and Staphylococcal protein A.

[0063] A polynucleotide can be isolated. An isolated polynucleotide is a naturally occurring polynucleotide that is not immediately contiguous with 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 naturally occurring nucleic acid sequences immediately adjacent to the recombinant DNA molecule in the natural genome are removed or absent. Isolated polynucleotides also include non-naturally occurring nucleic acid molecules.

[0064] Degenerate nucleotide sequences encoding the polypeptides are also contemplated herein. Degenerate nucleotide sequences are polynucleotides that encode the polypeptides described herein or fragments thereof, but that differ in nucleic acid sequence from the wild-type polynucleotide sequence due to the degeneracy of the genetic code. Complementary DNA (cDNA) molecules, species homologs, and variants of the polynucleotides that encode biologically functional polypeptides are also contemplated herein.

[0065] The polynucleotide may comprise a coding sequence of a natural polypeptide or may encode a modified sequence that does not occur in nature. If desired, the polynucleotide may be cloned into an expression vector that comprises expression control elements, including, for example, an origin of replication, a promoter, an enhancer, or other regulatory elements that drive 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 type 5 vector. Optionally, other vectors may be used, including, but not limited to, Sindbis virus, simian virus 40, alphavirus vectors, poxvirus vectors, and cytomegalovirus and retrovirus vectors, such as mouse 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, virus particles, virus-like particles, cosmids (plasmids into which a phage lambda cos site has been inserted), and replicons (genetic elements capable of replicating in a cell under its own control) can also be used.

[0066] Methods for preparing polynucleotides operably linked to expression control sequences and expressing them in host cells are well known in the art. See, e.g., U.S. Patent No. 4,366,246. A polynucleotide is operably linked when it is positioned adjacent to or in close proximity to one or more expression control elements that direct the transcription and / or translation of the polynucleotide.

[0067] Polynucleotides can be used, for example, as probes or primers, for example, PCR primers, to detect the presence of polynucleotides in test samples, such as biological samples. Probes are typically molecules that can interact with target nucleic acids in a sequence-specific manner, for example, through hybridization. Primers are a subset of probes that can assist enzymatic manipulation and can hybridize with target nucleic acids to allow enzymatic manipulation to 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 manipulation.

[0068] Probes or primers 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 that encode a polypeptide described herein.

[0069] antibody Antibodies and specific binding fragments thereof include antibody molecules that specifically bind to cystatin B and clusterin polypeptides described herein, variant cystatin B and variant clusterin polypeptides described herein, or fragments thereof. An antibody can specifically bind to multiple polypeptides. The term "antibody" also includes any kind 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, such as SEQ ID NOs: 1-32. The term "antibody" also includes any kind of antibody molecule or specific binding fragment that specifically binds to one or more cystatin B or clusterin polypeptides (e.g., SEQ ID NOs: 1-32). Antibodies can be natural, non-natural, synthetic, or genetically engineered. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any natural, enzymatically obtainable, synthetic, or genetically engineered polypeptide, glycoprotein, or immunoglobulin that specifically binds to a cystatin B polypeptide (e.g., SEQ ID NOs: 1-32) to form a complex.

[0070] The antibody or its specific binding fragment binds to the epitope of the polypeptide described herein.The antibody can be produced in vivo in a suitable experimental animal or in vitro using recombinant DNA technology.The means for preparing and characterizing the antibody are well known in the art. 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 a polypeptide described herein to an animal such as a human or other primate, mouse, rat, rabbit, guinea pig, goat, pig, dog, cow, sheep, donkey, or horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma, for example, by precipitation with ammonium sulfate followed by chromatography, such as affinity chromatography. Techniques for producing and processing polyclonal antibodies are known in the art.

[0071] The antibody can be of any isotype, including IgG (IgG1, IgG2, IgG2a, Ig2b, IgG3, IgG4), IgM, IgA (IgA1 and IgA2), IgD, and IgE.

[0072] The antibody may be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, or a specific binding fragment thereof. A monoclonal antibody is an antibody obtained from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies may contain small amounts of mutants or variants. Monoclonal antibodies are highly specific and interact with a single antigenic site. Each monoclonal antibody typically targets a single epitope, whereas a polyclonal antibody population typically contains different antibodies that target a diverse group of epitopes. Monoclonal antibodies can be produced by a number of methods, including, for example, the hybridoma method (Kohler and Milstein, Nature 256:495, 1975), recombinant methods (U.S. Pat. No. 4,816,567), and isolation from phage antibody libraries (Clackson et al., Nature 352:624-628, 1991; Marks et al., J. Mol. Biol. 222:581-597, 1991).

[0073] 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. A commercially available canine antibody against clusterin can be, for example, but not limited to, Anti-Clusterin / APOJ Antibody (STJ70776) (EKALQEYRKKHREE, SEQ ID NO: 34) from St John's Laboratory, or Clusterin Canine, Sheep Polyclonal Antibody from BioVendor Laboratory Medicine, Inc. A commercially available feline antibody against clusterin can be, for example, but not limited to, Clusterin / APOJ Mouse anti-Human, Bovine, Canine, Feline, Porcine, DyLight™ 650, Clone:Hs-3, Novus Biologicals™. Commercially available antibodies to canine Cystatin B can be, for example, but not limited to, Goat anti-Cystatin B / Stefin B Antibody from MyBioSource.com (QTNKAKHDELTYF, SEQ ID NO: 35) or Goat Anti-Human Cystatin B / Stefin B, (C Terminus) from RayBiotech.

[0074] A chimeric antibody, or antigen-binding portion thereof, has a portion of the heavy and / or light chain derived from a particular species or antibody class or subclass, with the remainder of the chain derived from another species or 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. Patent Nos. 5,807,715, 4,816,567, and 4,816,397).

[0075] Chimeric antibodies can be produced using a variety of techniques including, for example, CDR grafting (EP 239,400, PCT Publication No. 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).

[0076] In one embodiment, a chimeric antibody can contain variable and constant regions from different species, for example, an antibody can contain heavy and light chain variable regions from one mammal and heavy and light chain constant regions from a different animal (such as mouse, rabbit, dog, cat, or human). A chimeric antibody can contain additional amino acids that are not included in the CDRs or framework sequences introduced into the recipient antibody. These amino acids can be introduced to more precisely optimize the ability of the antibody to recognize and bind to an antigen. For example, amino acids in the framework regions of the antibody variable regions can be replaced as necessary so that the CDRs of the reshaped antibody form the appropriate antigen binding site. See Sato et al., Cancer Res. (1993) 53:851-856.

[0077] 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 (nanobodies); Fab-like antibodies (antigen-binding fragments comprising the variable regions of the heavy and light chains corresponding to Fab fragments obtained by papain digestion); F(ab')2-like antibodies (antigen-binding fragments comprising two antigen-binding domains corresponding to F(ab')2 fragments obtained by pepsin digestion), multispecific antibodies prepared from antibody fragments, diabodies, bispecific antibodies, multifunctional antibodies, humanized antibodies, caninized antibodies, human antibodies, dog antibodies, feline antibodies, mouse antibodies, rabbit antibodies, synthetic antibodies, CDR-grafted antibodies, and minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity determining regions (CDRs), such as CDR3 peptides), or constrained FR3-CDR3-FR4 peptides. 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, bivalent nanobodies), single chain (Fv)2 (sc(Fv)2), bivalent (sc(Fv)2), tetravalent ([sc(Fv)2]2) scFV antibodies, and small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also considered "antigen-binding fragments or portions" as that term is used herein.

[0078] By "specifically binds", "specifically binds" or "specific to" it is meant that a first antigen, e.g., a polypeptide of SEQ ID NO: 1-32, recognizes and binds to an antibody described herein with higher affinity than it does to other non-specific molecules. By "specifically binds", "specifically binds" or "specific to" it is meant that a first antibody, e.g., an antibody raised against SEQ ID NO: 1-32, recognizes and binds to SEQ ID NO: 1-32 with higher affinity than it does 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, for example, using an enzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), immunoturbidimetry, particle-enhanced immunoturbidimetry, or Western blot assay, or other suitable assay.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to an epitope on a clusterin polypeptide set forth as 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 some embodiments, 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).

[0080] 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 for this purpose. Means for assaying cross-reactivity are well known to those of skill in the art (see, for example, Dowbenko et al. (1988) J. Virol. 62:4703-4711).

[0081] A first antibody is considered to competitively inhibit binding of a second antibody if binding of the second antibody to the antigen is reduced by at least about 30%, 40%, 50%, 60%, 75%, 90% or more in the presence of the first antibody using any of the assays used to assess competitive binding.

[0082] Competitive binding can be determined by providing one or more of the isolated polypeptides set forth in SEQ ID NOs: 1-32 bound to a solid support and assaying the ability of an antibody to bind to the polypeptide or to compete with an antibody described herein for binding to the polypeptide.

[0083] The antibody has a binding affinity to SEQ ID NO: 1-32 or an antigen-binding fragment thereof that (a) competes with a reference antibody for binding to SEQ ID NO: 1-32 or an antigen-binding fragment thereof, (b) binds to the same epitope of SEQ ID NO: 1-32 or an antigen-binding fragment thereof as the reference antibody, and (c) has substantially the same K d and / or (d) antibodies and antigen-binding fragments thereof that bind to SEQ ID NOs: 1-32 or antigen-binding fragments thereof with substantially the same dissociation rate as a reference antibody, wherein the reference antibody is at least 10 7 Binding affinity K of ≥ 1 / mol a and is an antibody or an antigen-binding fragment thereof that specifically binds to the polypeptides of SEQ ID NOs: 1 to 32 or antigen-binding fragments thereof.

[0084] The affinity of an antibody or antigen-binding fragment thereof for its polypeptide partner can be expressed by the dissociation constant (Kd). The equilibrium dissociation constant (Kd) is k off / k on Affinity constants are calculated by assigning a specific K to the reference antibody. See Chen, Y. et al., 1999, J. Mol. Biol. 293:865-881. Various methods are known in the art for measuring affinity constants. In certain embodiments, the reference antibody has a specific K on Rate / Association Rate or K off In one embodiment, the antibody or antigen-binding fragment thereof has binding affinity to the polypeptides of SEQ ID NOs: 1 to 32 at a rate of 6×10 5 M -1 s -1 More than K on Antibodies that bind specifically at 5 x 10 -6 s -1 More than K off The antibody specifically binds with a rate or with a binding affinity of 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 40 pM, 30 pM, 20 pM or greater.

[0085] Antibodies that specifically bind to SEQ ID NOs: 1-32 or SEQ ID NOs: 26-32 are particularly useful for detecting the presence of cystatin B and clusterin polypeptides and fragments thereof present in samples such as serum, blood, plasma, cells, tissues, saliva, plaque, gingival crevicular fluid, gingival biopsy, tongue swab, 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 and polyclonal antibodies. Immunoassay protocols can be based, for example, on competitive, direct reaction, or sandwich assays using, for example, labeled antibodies. The antibody can be labeled with any type of label known in the art, including, for example, fluorescent labels, chemiluminescent labels, radioactive labels, enzyme labels, colloidal metal labels, radioisotope labels, and bioluminescent labels.

[0086] The antibody or antigen-binding fragment thereof can be bound to a support and used to detect the presence or amount of polypeptide present in a sample in certain diseases and conditions. 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, e.g., freeze-dried.

[0087] The antibody can further be used to isolate the polypeptide by immunoaffinity column. The antibody can be attached to a solid support, for example, by absorption or by covalent bonding, so that the antibody retains its immunoselective activity. Optionally, a spacer group can be included so that the antigen-binding site of the antibody remains accessible. The immobilized antibody can then be used to specifically bind to SEQ ID NO: 1-32 or a fragment thereof from biological samples, including, but not limited to, saliva, plaque, gingival crevicular fluid, gingival biopsy, tongue swab, serum, blood, and urine.

[0088] Antibodies can also be used in immunolocalization studies to analyze the presence and distribution of the polypeptides described herein during various cellular events or physiological conditions. Antibodies can also be used to identify molecules involved in passive immunization and to identify molecules involved in the biosynthesis of non-protein antigens. Identification of such molecules can be useful in vaccine development. Antibodies, including, for example, monoclonal and single chain antibodies, can be used to monitor the progress of amelioration of a particular disease or condition. By measuring the increase or decrease in the amount of SEQ ID NO: 1-32 or SEQ ID NO: 26-32 or fragments thereof in a test sample from an animal, it can be determined whether a particular therapeutic regimen aimed at ameliorating a disorder is effective. Antibodies can be detected and / or quantified using direct binding assays, such as, for example, RIA, ELISA, or Western blot assays.

[0089] Methods for detecting and quantifying polypeptides One embodiment provides a method for detecting and quantifying cystatin B, clusterin, SDMA, or creatinine polypeptides in a sample, e.g., a mammalian urine sample. Methods for detecting and quantifying polypeptides include, for example, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatographic (HPLC) mass balance assays, liquid chromatography mass spectrometry (LC-MS), and mass spectrometry. qNMR provides a method for detecting and quantifying polypeptides, regardless of chemical structure. 1It takes advantage of its inherent ability to achieve equal degrees of response from magnetic nuclei such as H. AAA is based on the quantification of stable amino acids after peptide hydrolysis. HPLC selectively quantifies analytes of interest against reference standards of the same analyte. In LC-MS, sample components are separated by liquid chromatography (LC) and then introduced into a mass spectrometer (MS). The MS creates and detects charged ions. LC / MS data may be used to provide information on the molecular weight, structure, identity, and quantity of specific sample components. Mass spectra obtained by mass spectrometry are plots of ion signals as a function of mass-to-charge ratio. These spectra are used to determine the elemental or isotopic characteristics of a sample, the mass of a polypeptide, and to elucidate the chemical identity or structure of the polypeptide. Immunoassays (IA) and competitive IA can also be used to quantify polypeptides.

[0090] Polypeptides can also be detected and quantified in immunoassays, for example, using antibodies or specific binding fragments thereof (e.g., polyclonal antibodies, monoclonal antibodies, and specific binding fragments thereof, or combinations). Such methods can include contacting a sample with, for example, one or more antibodies or specific binding fragments thereof specific for SEQ ID NOs: 1-32 or fragments thereof, under conditions suitable for the formation of a complex between a cystatin B polypeptide or clusterin polypeptide and one or more antibodies or specific binding fragments thereof. Complexes between a cystatin B polypeptide or clusterin polypeptide and one or more antibodies or specific binding fragments thereof specific for SEQ ID NOs: 1-32 are detected and quantified.

[0091] SDMA and creatinine can be detected, for example, using antibodies and / or ELISA kits. Examples of ELISA kits include CREA ELISA kit product number MBS748495 MyBioSource (San Diego CA) and Canine Creatinine ELISA kit product number CNEB0472 AssayGenie (Dublin IE). Creatinine can also be detected, for example, using electrochemical methods (US Patent Publication No. 2017 / 0284954, US Patent Publication No. 2018 / 0149612), polymer matrix (US Patent Publication No. 2015 / 0160242), and fluorescence methods (US Patent Publication No. 2004 / 0132200). SDMA can be detected, for example, using antibodies (U.S. Patent Publication No. 2016 / 0187348), product number AB1N7845543 (Antibodies Online, Pottstown, PA), product number ABIN870728 (Antibodies Online, Pottstown, PA). Also see U.S. Patent Publication No. 2021 / 0403603 for detection of SDMA.

[0092] In one embodiment, methods are provided for detecting and quantifying polypeptides including cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32, and fragments thereof. The relative amounts of the polypeptides can be used to distinguish mammals with stable CKD from mammals with progressive CKD. Any method known in the art for detecting a polypeptide can be used in the methods described herein.

[0093] Assay methods used in conjunction with the antibodies described herein may include direct and indirect labeling techniques, immunoaffinity columns, immunomagnetic beads, fluorescence activated cell sorting (FACS), enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), agglutination assays, turbidimetric assays, quantitative turbidimetric assays, Western blots, IFA, hemagglutination (HA), immunoturbidimetry, particle-enhanced immunoturbidimetry, fluorescence polarization immunoassays (FPIA), and microtiter plate assays (any assay carried out in one or more wells of a microtiter plate). Some assays include reversible flow chromatography binding assays, such as the SNAP® assay. See, for example, U.S. Patent No. 5,726,010.

[0094] The antibody or its specific binding fragment can be detectably labeled with a fluorescent label having excitation and emission wavelengths adapted for detection using commercially available instruments such as, for example, a fluorescence activated cell sorter. Examples of fluorescent labels include phycoerythrin (PE), fluorescein isothiocyanate (FITC), rhodamine (RH), Texas Red (TX), Cy3, Hoechst 33258, and 4',6-diamidino-2-phenylindole (DAPI). Such labels can be conjugated to the antibody using standard techniques (Maino et al., 1995, Cytometry 20:127-133).

[0095] The methods described herein can be used to detect cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32, or fragments thereof, and the antibody or antigen-binding antibody fragment specifically binds to cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32.

[0096] As used herein, a "patient" or "subject" can mean a human or a non-human animal, including a cat, cow, pig, horse, or dog.

[0097] The terms "sample," "test sample," "patient sample," or "subject sample," as used herein, include, but are not limited to, a blood, serum, plasma, saliva, plaque, gingival crevicular fluid, gingival biopsy, tongue swab, or urine sample obtained from a subject. A test sample can be unprocessed, precipitated, fractionated, separated, diluted, concentrated, or purified.

[0098] The assay may include a solid phase or substrate, or may be performed by immunoprecipitation or any other method that does not utilize a solid phase. When a solid phase or substrate is used, one or more polypeptides or antibodies (or specific binding fragments thereof) are directly or indirectly bound to a solid support or substrate, such as a microtiter well, a magnetic bead, a non-magnetic bead, a column, a matrix, a membrane, a fibrous mat composed of synthetic or natural fibers (e.g., glass or cellulosic materials, or polyethylene, polypropylene, or polyester thermoplastic polymers), a sintered structure composed of particulate materials (e.g., glass or various thermoplastic polymers), or a cast membrane (generally of a synthetic nature) composed of nitrocellulose, nylon, polysulfone, and the like. In one embodiment, the substrate is a sintered microparticle of polyethylene, commonly known as porous polyethylene, e.g., 10-15 micron porous polyethylene manufactured by Chromex Corporation (Albuquerque, NM). All of these substrate materials may be used in any suitable shape, such as a film, sheet, or plate, or may be coated or bonded or laminated onto a suitable inert carrier, such as paper, glass, plastic film, or fabric. Suitable methods for immobilizing antibodies on a solid phase include ionic, hydrophobic, covalent interactions, and the like.

[0099] In one embodiment, the method includes contacting the test sample with one or more of the following antibodies that specifically bind to cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or fragments thereof under conditions that allow a polypeptide / antibody complex, i.e., an immune complex, to form. That is, the antibody specifically binds to one or more of the following polypeptides: cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or fragments thereof located in the sample. Those skilled in the art are familiar with assays and conditions used to detect antibody / polypeptide complex binding. The formation of a complex between a polypeptide and an antibody in the sample is detected. The formation of an antibody / polypeptide complex is an indication that the polypeptide is present in a certain amount in the CKD patient sample.

[0100] In one embodiment, the polypeptide / antibody complex is detected when an indicator reagent, such as an enzyme conjugate bound to the antibody, catalyzes a detectable reaction. Optionally, an indicator reagent, including a signal-generating compound, can be applied to the polypeptide / antibody complex under conditions that allow for the formation of a polypeptide / antibody / indicator complex. The polypeptide / antibody / indicator complex is detected. Optionally, the polypeptide or antibody can be labeled with an indicator reagent prior to the formation of the polypeptide / antibody complex. The method can optionally include a positive or negative control. A positive control can include one or more polypeptides that specifically bind to a cystatin B, clusterin, SDMA, or creatinine specific antibody, resulting in a positive result. A negative control does not include any polypeptide or other moiety that specifically binds to or cross-reacts with a cystatin B, clusterin, SDMA, or creatinine polypeptide, or a cystatin B, clusterin, SDMA, or creatinine specific antibody.

[0101] In one embodiment, one or more antibodies are covalently or non-covalently immobilized to a solid phase or substrate. A sample that may contain 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 in the solid phase or from a different source or species, and can be linked 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 to develop color. The color reaction is stopped and the color can be quantified, for example, using a spectrophotometer.

[0102] In another embodiment, one or more antibodies are immobilized on a solid phase or substrate. A test sample that may contain cystatin B, clusterin, SDMA, or creatinine polypeptides is added to the substrate. Second anti-species antibodies that specifically bind to cystatin B, clusterin, SDMA, or creatinine polypeptides are added. These second anti-species antibodies are from a different species than the antibody immobilized on the solid phase. A third anti-species antibody is added that specifically binds to the second anti-species antibody and does not specifically bind to the antibody immobilized on the solid phase. The third anti-species antibody may include an indicator reagent, such as an enzyme conjugate. A wash step may be performed before each addition. A chromophore or enzyme substrate is added to develop color. The color reaction is stopped and the color can be quantified, for example, using a spectrophotometer.

[0103] In one embodiment, the one or more capture antibodies may specifically bind to one or more epitopes of the polypeptide described herein. The capture antibody or antibodies may be used to immobilize one or more of the polypeptides of Cystatin B, Clusterin, SDMA, Creatinine, SEQ ID NO: 1-32 or fragments thereof, for example, to a solid support. The one or more detection antibodies may specifically bind to the same one or more epitopes or different one or more epitopes of the polypeptide. The detection antibodies may be used to detect or visualize the immobilization of the polypeptide to the solid support. This embodiment is advantageous because it is more specific and sensitive than assays that use only one antibody for both capture and detection functions.

[0104] 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 with an indicator reagent comprising a signal generating compound conjugated to an antibody or antibody fragment specific for the polypeptide for a time and under conditions sufficient to form an antigen / antibody complex of either the test sample polypeptide or the antibody of the solid phase to the indicator reagent compound conjugated to an antibody specific for the polypeptide. Binding of the indicator reagent conjugated to an anti-polypeptide antibody to the solid phase can be quantitatively measured. A measurable change in signal compared to the signal generated from a control sample or control standard indicates the presence of a polypeptide comprising cystatin B, clusterin, SDMA, creatinine, SEQ ID NO: 1-32 or a fragment thereof. This type of assay allows the amount of polypeptide in the test sample to be quantified.

[0105] In another type of assay format, one or more antibodies are coated onto a support or substrate. The antibodies are conjugated to an indicator reagent and added to a test sample. This 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 the indicator reagent and one or more antibodies conjugated to the one or more antibodies immobilized on the support. The polypeptide / antibody / indicator complex is then detected. This type of assay allows the amount of polypeptide in a test sample to be quantified.

[0106] In another type of assay format, one or more antibodies are coated onto a support or substrate. The test sample is applied to the support or substrate and incubated. Unbound components from the sample are washed away by washing the solid support with a wash solution. If polypeptides including cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32 or fragments thereof are present in the test sample, they will bind to the antibodies coated onto 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 reagent complex is then detected. This type of assay allows the amount of polypeptide in the test sample to be quantified.

[0107] The formation of a polypeptide / antibody complex or a polypeptide / antibody / indicator complex can be detected, for example, by radiometric, colorimetric, fluorometric, size separation, or precipitation methods. Optionally, detection of the polypeptide / antibody complex is by adding a secondary antibody that binds to an indicator reagent that includes a signal-generating compound. The indicator reagent that includes a signal-generating compound (label) associated with the polypeptide / antibody complex can be detected using the methods described above and includes color developers, catalysts such as enzyme conjugates, fluorescent compounds such as fluorescein and rhodamine, chemiluminescent compounds such as dioxetanes, acridinium, phenanthridinium, ruthenium, and luminol, radioactive elements, direct visual labels, as well as cofactors, inhibitors, magnetic particles, and the like. Examples of enzyme conjugates include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. The choice of a particular label is not critical, but it can generate a signal alone or in conjunction with one or more additional substances.

[0108] The phrase "determining the amount" as used herein refers to measuring or identifying the level of one or more polypeptides in a sample. This can be accomplished by methods well known in the art for detecting polypeptides, including the use of antibodies, including, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoturbidimetry, particle-enhanced immunoturbidimetry, or Western blot assay, or immunohistochemistry assay. Alternatively, the polypeptides of cystatin B, clusterin, SDMA, creatinine, SEQ ID NOs: 1-32 or fragments thereof can be determined by mass spectrometry, LC-MS, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatographic (HPLC) mass balance assay, or similar methods known to those skilled in the art. Determining the amount of polypeptide present in a sample is accomplished by such in vitro analyses and experimental procedures.

[0109] How is it diagnosed? One embodiment provides a method for diagnosing stable CKD or progressive CKD renal disease in a subject. The subject is first diagnosed with CKD, and then the amount of cystatin B or clusterin polypeptide in a sample from the 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 the subject, where the amount of cystatin B or clusterin polypeptide is determined, for example, by one or more antibodies specifically binding to cystatin B, clusterin, SEQ ID NOs: 1-32, or fragments thereof, or using other methods described herein. The amount of cystatin B or clusterin polypeptide in the sample is compared to a control sample or a control standard, and a certain level of cystatin B or clusterin polypeptide in the sample compared to the control sample or the control standard is indicative of either stable CKD or progressive CKD renal disease in the subject.

[0110] Some embodiments provide a method for diagnosing or detecting stable CKD or progressive CKD. The method includes determining the amount of cystatin B or clusterin polypeptide in a sample from a subject, the amount of cystatin B polypeptide or clusterin being determined using one or more antibodies (or specific binding fragments) that specifically bind to one or more polypeptides or fragments thereof as set forth in SEQ ID NOs: 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 to a control sample or a control standard, and an elevated level of cystatin B or clusterin polypeptide in the sample compared to the control sample or the control standard is indicative of stable CKD or progressive CKD.

[0111] In some embodiments, a method that does not rely on an antibody can be used to diagnose or detect stable or progressive CKD. The method can include determining the amount of cystatin B or clusterin polypeptide in a sample from a subject, where 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 to a control sample or a control standard, and an elevated level of cystatin B or clusterin polypeptide in the sample compared to the control sample or the control standard is indicative of stable or progressive CKD.

[0112] The elevated level of cystatin B polypeptide may be about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500% or more higher than a control sample or a control standard. The elevated level of cystatin B polypeptide may 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, or about 100 to 500% or more than a control sample or a control standard.

[0113] An elevated level of a cystatin B or clusterin polypeptide can also be a level that is a statistically significantly increased amount when compared to a control sample or control standard.

[0114] As noted above, an elevated level of cystatin B polypeptide can also be about 50, 60, 70, 80, 90, 100 ng / mL or more of cystatin B (or any range of about 50-100 ng / mL or more, such as about 50-100 ng / mL or more, about 50-80 ng / mL or more, about 70-100 ng / mL or more). A control level or standard of cystatin B polypeptide can be about 49, 40, 30, 25, 20, 15, 10, 5, 1 ng / mL or less.

[0115] As noted above, an elevated level of clusterin polypeptide can also be about 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350 ng / mL or more of clusterin (or any range of about 250-350 ng / mL or more, such as about 250-300 ng / mL or more, about 275-325 ng / mL or more, about 325-350 ng / mL or more). A control level or standard of cystatin B polypeptide can be about 249, 240, 230, 225, 200, 150, 100, 50 ng / mL or less.

[0116] Elevated levels of cystatin B or clusterin polypeptides can be compared to a control sample or standard determined using normal control subjects who do not have any type of renal disease or condition or bacterial infection.

[0117] Surprisingly, the use of reverse SDMA (1 / SDMA) slope or reverse creatinine (1 / creatinine) slope can also be used to distinguish whether a CKD patient has stable or progressive CKD. No substantial change in functional renal markers means that the change from the reverse SDMA or reverse creatinine slope, respectively, of a healthy population over a specified period of time is less than 200%. The period of time can be, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36 months or more (or any range from about 1 week to about 36 months, for example, about 1 to about 3, about 2 to about 4, about 3 to about 6, about 6 to about 12, or about 1 to 36 months). In these cases, the subject has stable chronic kidney disease.

[0118] In contrast, CKD patients with advanced CKD had a mean blood glucose level of approximately 0.0002222411 (μg / dL) -1 / week or more (e.g., about 0.0001, 0.00015, 0.0002, 0.00025 or more) decrease in the inverse SDMA (1 / SDMA) slope (Figure 14) or about 0.01860675 (mg / dL) -1 / week or more (e.g., about 0.010, 0.015, 0.018, 0.020, 0.025 or more) decrease in the reverse creatinine (1 / creatinine) slope ( FIG. 13 ) over time (e.g., 1 week, 2 weeks, 3 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36 months or more, and any range from about 5 days to about 36 months, e.g., over about 1 to about 3, about 2 to about 4, about 3 to about 6, about 6 to about 12, or about 1 to 36 months).

[0119] The stable vs. progressive slope cutoff is approximately -0.0117 for 1 / creatinine (mg / dL) -1 / week (e.g., about -0.010, -0.012, -0.015, or -0.02 (mg / dL) -1 / week), and 1 / SDMA is approximately -0.0007 (μg / dL) -1 / week (e.g., about -0.0005, -0.0006, -0.0007, or -0.0008 (μg / dL) -1 / week).

[0120] Accordingly, there is provided a method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD, comprising: (a) determining a reverse SDMA (1 / SDMA) slope in a sample from the subject at two or more time points (e.g., a first time point, then 1 week, 2 weeks, 3 weeks, 4 weeks or more later); (b) Comparing the reverse SDMA (1 / SDMA) slopes in the samples to each other, which were approximately 0.0002222411 (μg / dL) over time. -1 / week or more (e.g., approximately 0.0001, 0.00015, 0.0002, 0.00025 (μg / dL) -1 / week or more) is indicative of progressive CKD.

[0121] The subject may be diagnosed with CKD stage I according to the International Renal Interest Society or CKD with a symmetric dimethylarginine (SDMA) level of up to 20 μg / dl prior to step (a). The subject may be administered one or more treatments for CKD disease.

[0122] A method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD includes: (a) measuring the concentration of SDMA in a sample obtained from the subject at three or more time points (e.g., a first time point, then 1 week, 2 weeks, 3 weeks, 4 weeks or more); (b) determining the inverse SDMA (1 / SDMA) slope in samples from the subject at three or more time points; (c) comparing the reverse SDMA (1 / SDMA) slope with a minimum slope of a cohort of healthy control animals, wherein a reverse SDMA (1 / SDMA) slope less than the minimum slope of the cohort of healthy control animals indicates that the subject has progressive CKD, and a reverse SDMA (1 / SDMA) slope greater than the minimum slope of the cohort of healthy control animals indicates that the subject has stable CKD.

[0123] The minimum slope for the cohort of healthy control animals was approximately -0.0007 (μg / dL) for 1 / SDMA. -1 / week (e.g., about -0.0005, -0.0006, -0.0007, or -0.0008 (μg / dL) -1 / week).

[0124] Also provided is a method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD, comprising: (a) determining a reverse creatinine (1 / creatinine) slope in a sample from the subject at two or more time points (e.g., a first time point, then 1 week, 2 weeks, 3 weeks, 4 weeks or more); (b) comparing the reverse creatinine (1 / creatinine) slopes in the samples to each other, and determining whether the reverse creatinine (1 / creatinine) slope over time is greater than or equal to about 0.01860675 (mg / dL); -1 / week or more (e.g., about 0.010, 0.015, 0.018, 0.020, 0.025 (mg / dL) -1 / week or more) is indicative of progressive CKD.

[0125] The subject may be diagnosed with CKD stage I according to the International Renal Interest Society or CKD with a symmetric dimethylarginine (SDMA) level of up to 20 μg / dl prior to step (a). The subject may be administered one or more treatments for CKD disease.

[0126] A method for distinguishing stable chronic kidney disease ("CKD") from progressive CKD in a subject diagnosed with CKD includes: a) measuring the concentration of creatinine in a sample obtained from the subject at three or more time points (e.g., a first time point, then 1 week, 2 weeks, 3 weeks, 4 weeks or more); b) determining the reverse creatinine (1 / creatinine) slope in samples from the subject at three or more time points; and c) comparing the reverse creatinine (1 / creatinine) slope with a minimum slope of a cohort of healthy control animals, wherein a reverse creatinine (1 / creatinine) slope less than the minimum slope of the cohort of healthy control animals indicates that the subject has progressive CKD, and a reverse creatinine (1 / creatinine) slope greater than the minimum slope of the cohort of healthy control animals indicates that the subject has stable CKD.

[0127] The minimum slope for the cohort of healthy control animals was approximately -0.0117 for 1 / creatinine (mg / dL). -1 / week (e.g., about -0.010, -0.012, -0.015, or -0.02 (mg / dL) -1 / week).

[0128] In some embodiments, the level of cystatin B, clusterin, SDMA, or creatinine in the test sample is compared to the level of cystatin B or clusterin (or both) in a control sample from one or more normal control subjects. Typically, the control level measured in the control sample is then compared to 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 to a previously determined or predetermined control level ("control standard"). For example, the control standard of cystatin B, clusterin, SDMA, or creatinine polypeptide can be calculated from data, for example, data including the levels of cystatin B, clusterin, SDMA, or creatinine in control samples from multiple normal or healthy control subjects. The normal or healthy control subjects and the test subjects under evaluation can be of the same species.

[0129] In some embodiments, the level of cystatin B, clusterin, SDMA, or creatinine polypeptide in the test sample is compared to the level of cystatin B, clusterin, SDMA, or creatinine polypeptide in a control sample from one or more control subjects, where the control subjects have stable CKD. Typically, the control level measured in the control sample is then compared to 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 to a previously determined or predetermined control level ("control standard"). For example, the control standard of cystatin B, clusterin, SDMA, or creatinine polypeptide can be calculated from data, for example, data including the level of cystatin B, clusterin, SDMA, or creatinine polypeptide in a control sample from a plurality of control subjects having stable CKD. The stable CKD control subject and the test subject being evaluated can be of the same species.

[0130] Certain embodiments provide reagents and methods for identifying certain diseases or conditions in mammals, for example, dogs, cats, and humans. Certain embodiments provide methods for providing diagnosis and prognosis to CKD patients. Identification and quantification of Cystatin B polypeptide in a subject sample can be an independent predictor of progressive CKD. The method advantageously allows for the diagnosis and identification of progressive CKD and is not influenced or confounded by the age or weight of the CKD patient. Thus, additional embodiments are directed to using the progressive CKD diagnosis determined by the methods described herein to select appropriate therapy.

[0131] The embodiments further include methods for predicting CKD patient health, monitoring disease progression, and / or assessing / monitoring treatment effectiveness by identifying levels of certain polypeptides in CKD patient samples. In some aspects, the methods can be performed at a single time point. In other aspects, the methods can be performed at multiple time points (e.g., about 2, 3, 4, 5, or more time points), e.g., to assess disease progression or treatment effectiveness. In certain embodiments, the methods can be performed at the time of diagnosis and then at a particular time point after treatment, when a particular therapy should result in a reduction or amelioration of disease progression.

[0132] For example, a method for monitoring disease progression includes: (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; (b) treating the subject for CKD with one or more of the CKD treatments described herein; and (c) 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 second time point; (d) comparing the amount of cystatin B, clusterin, SDMA, or creatinine polypeptide, or a combination thereof, at the first and second time points.

[0133] A lower level of cystatin B or clusterin polypeptide, or both, at the second time point indicates that the treatment is slowing the progression or stable CKD. The slope will be less steep in this situation. Additional treatments and time points (e.g., 3, 4, 5, 6 or more time points) may be provided.

[0134] The methods described herein can also indicate the amount or quantity of a polypeptide comprising cystatin B, clusterin, SDMA, or creatinine, SEQ ID NOs: 1-32 or fragments thereof. For many indicator reagents, such as enzyme conjugates, the amount of polypeptide present is proportional to the signal generated. Depending on the type of test sample, it can be diluted with a suitable buffer reagent, concentrated, or contacted with a solid phase without any manipulation. For example, previously diluted urine, serum, or plasma samples, or concentrated specimens such as urine, can be used to determine the presence of a polypeptide and / or the amount of polypeptide present.

[0135] The polypeptides and assays described herein can be combined with other polypeptides or assays for detecting the presence of renal disease. For example, the polypeptides and assays can be combined with reagents suitable for detecting or measuring creatinine, SDMA, or general protein levels.

[0136] Treatment methods Certain embodiments provide a method for treating a condition in a subject. The method includes calling for a test to determine the amount of a cystatin B polypeptide in a sample from the subject, providing an analytical result. If the sample contains an elevated amount of the cystatin B or clusterin polypeptide (or both) described herein compared to a control sample or control standard for the condition, treatment for the condition is administered to the subject.

[0137] The condition includes stable CKD and progressive CKD. In one embodiment, the condition is not cancer or renal cancer.

[0138] Treatments for advanced CKD include, for example, surgery for obstructed ureters, obstructing kidney stones, or urinary stones, dietary management (dietary reduction in protein, phosphorus, salt, or a combination thereof), administration of intestinal phosphate binders, antiproteinuric drugs (e.g., administration of angiotensin-converting enzyme inhibitors (ACEIs), omega-3 fatty acids), administration of antihypertensive agents (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.

[0139] Treatment using dietary management can be through a number of diets, including reduced protein, reduced phosphorus, and reduced salt diets. Specialty pet food formulations are routinely recommended by veterinarians. Specialty pet food formulations include Hill's® Prescription Diet® k / d®, Royal Canin® Veterinary Diet Canine Renal Support, Purina® ProPlan® Veterinary Diet NF Kidney Function®, and Rayne Clinical Nutrition™ Adult Health-RSS™. Reduction of dietary protein appears to slow the progression of CKD by reducing the load on the kidneys to excrete protein waste. Less protein means less need for excretion of the protein itself through the kidney's filtering mechanism, which helps preserve kidney function. The recommended range of protein on a dry matter basis for dogs with CKD is, for example, about 14-20% or less. The recommended range of protein on a dry matter basis for cats with CKD is, for example, 28-35% or less. Restricting dietary phosphorus in dogs and cats with CKD appears to help slow the progression of CKD, although the exact mechanism is unclear. The recommended phosphorus range on a dry matter basis for dogs with CKD is, for example, about 0.2-0.5%. The recommended phosphorus range on a dry matter basis for cats with CKD is, for example, 0.3-0.6%. Since phosphorus content is related to protein content, low levels of phosphorus usually require restriction of protein content. To reduce the load on the kidneys, dietary sodium levels are mildly restricted (for example, on a dry matter basis, about 0.3% or less in dogs and 0.4% or less in cats). This, in turn, helps to maintain reasonable blood pressure. Dietary omega-3 fatty acids contribute to slowing the progression of CKD by helping to reduce the production of inflammatory compounds that cause oxidative stress in affected kidney tissues. It works by reducing protein "leakage" through the kidneys. Recommended omega-3 fatty acids on a dry matter basis for dogs and cats with CKD, for example, are 0.4-2.5%.

[0140] Management of acidosis may involve monitoring CKD patients for metabolic acidosis by measuring blood bicarbonate or total CO2. If necessary, renal nutrition therapy may be supplemented with oral sodium or potassium bicarbonate to maintain blood bicarbonate concentrations in the range of approximately 18-24 mmol / L.

[0141] Correction of electrolyte abnormalities may involve correction of blood sodium concentration, which may be normal, increased, or decreased in renal failure. Hypernatremia before fluid therapy indicates excessive free water loss. Administration of sodium bicarbonate or hypertonic saline may contribute to hypernatremia. Hyponatremia may indicate excessive sodium loss associated with vomiting or may represent transient dilutional hyponatremia after administration of mannitol, hypertonic dextrose, or colloid solutions. Sodium-deficient solutions (e.g., dextrose about 5%, total parenteral nutrition, enteral formulas) may contribute to hyponatremia. In many situations, dehydration is initially caused by isobaric fluid loss, and CKD patients have normal blood sodium concentrations. Initial fluid loss can be replaced by isobaric solutions such as lactated Ringer's solution, 0.9% saline, or Plasmalyte-148. Continuous administration of these solutions over several days may lead to hypernatremia. Sodium-depleting fluids such as half-diluted lactated Ringer's solution or 0.45% saline with 2.5% dextrose may be a more appropriate fluid choice after the initial rehydration phase. Serum sodium concentrations can be monitored periodically and fluid selection adjusted as needed. Hypokalemia may also occur, but is more common in cats. 20%-30% of cats with CKD have hypokalemia. Potassium excretion may be impaired in renal failure, so treatment in this situation requires judicious replacement with careful monitoring. In hospitalized CKD patients who cannot tolerate orally administered medications, potassium chloride may be added to the IV fluids. The rate of potassium replacement may be approximately 0.5 mEq / kg / h or less. Blood potassium concentrations may decrease during initial fluid therapy despite replacement due to extracellular fluid volume expansion, increased distal tubular flow, and cellular uptake, especially if potassium is administered with dextrose-containing fluids. Additionally, metabolic acidosis increases the ionized calcium fraction, but more than 50% of dogs with CKD and metabolic acidosis are hypocalcemia. Hypercalcemia may respond to fluid therapy. Normal saline (0.9% NaCl) is a good fluid choice because its high sodium content facilitates increased calcium excretion in the urine.Furosemide also promotes urinary calcium loss. Sodium bicarbonate therapy reduces the concentration of ionized calcium in the blood because more calcium ions are bound to blood proteins.

[0142] Holistic treatments may include, for example, administration of fish oil, B vitamins, iron, pet multivitamins, fresh parsley, kali chloricum, arsenicum album, silicea, mastica, aluminum hydroxide, or glandular therapy.

[0143] In alternative embodiments, the methods described herein can be used to assess the effectiveness of a composition or treatment regimen (either a composition or a diet) for ameliorating disease progression. Similarly, the methods described herein can be used to assess the activity of a composition or treatment regimen on CKD patient levels of clusterin or cystatin B polypeptide.

[0144] The compositions and methods are described in more detail below, and the examples described herein are intended to be illustrative only, since numerous modifications and variations will be apparent to those skilled in the art.Terms used herein generally have their ordinary meanings in the art within the context of the compositions and methods described herein, and in the specific context in which each term is used.Some terms are more specifically defined herein to provide additional guidance to practitioners regarding the description of the compositions and methods.

[0145] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. As used in the description of this specification and throughout the claims that follow, the meaning of "a," "an," and "the" includes singular as well as plural references, unless otherwise clear by context. The term "about" in connection with a numerical value means that the value varies above and below by 5%. For example, a value of about 100 means 95 to 105 (or any value between 95 and 105).

[0146] All patents, patent applications, and other scientific or technical literature mentioned anywhere in this specification are incorporated herein by reference in their entirety. The embodiments illustratively described herein can be suitably practiced in the absence of any element(s), limitation(s) specifically or non-specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms while retaining their ordinary meaning. The terms and expressions used are used as terms of description and not of limitation, and in the use of such terms and expressions, there is no intention to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the claims. Thus, although the present methods and compositions are specifically disclosed by embodiments and optional features, it will be understood that modifications and variations of the concepts disclosed herein may be made by those skilled in the art, and that such modifications and variations are considered to be within the scope of the compositions and methods as defined by the description and the appended claims.

[0147] Any single term, element, phrase, group of terms, phrases, or elements described herein may each be specifically excluded from the claims.

[0148] Whenever a range is given herein, for example a temperature range, time range, composition range, or concentration range, all intermediate ranges and subranges, as well as all individual values ​​included in the given range, are intended to be included in the disclosure. It will be understood that any subrange or individual value within a range or subrange included in the description herein may be excluded from the aspects of the description herein. It will be understood that any element or step included in the description herein may be excluded from the claimed composition or method.

[0149] In addition, where features or aspects of the present compositions and methods are described in terms of a Markush group or other grouping of alternatives, one of skill in the art will recognize that the compositions and methods also are described in terms of any individual member or subgroup of members of the Markush group or other group.

[0150] The following are provided for illustrative purposes only and are not intended to limit the scope of the embodiments broadly described above. EXAMPLES

[0151] The following examples are illustrative of certain embodiments of the present disclosure and various uses thereof, which are provided for purposes of illustration only and should not be construed as limiting.

[0152] Example 1: Methods Study population Data for control patients were drawn from an archived longitudinal study of common vector-borne diseases in pets of students, faculty, and staff at the University Health Center at the University of Missouri. All pets were enrolled with owner consent, and Institutional Animal Care and Use Committee approval for the study was obtained through the University of Missouri. Dogs were aged 1-10 years and reported by their owners to be healthy at the time of enrollment. Follow-up was for 3 years. Evaluation frequency ranged from every 4-6 months. Patients with data available for at least three visits were included in the study.

[0153] Because CysB is not exclusively present in the urinary system and is a small 11 kD solute that can be freely filtered in the glomerulus when detectable in the circulation, we cannot exclude that in some dogs the origin of uCysB may not be from renal tubular epithelial cell injury but from active cell injury elsewhere in the body. To reduce this possibility, dogs with concurrent inflammatory conditions or concurrent systemic diseases that may have contributed to active cell injury in other organs were not included in this study, further reducing the possibility that the origin of uCysB in this study was from extrarenal cell injury.

[0154] Data on stage 1 CKD dogs were collected prospectively from four additional academic institutions: University of Minnesota, North Carolina State University, Hebrew University, and University of California Veterinary Medical Center, San Diego. Dogs older than 6 months of age for evaluation of IRIS stage 1 CKD were eligible for inclusion. All patient owners were provided with information regarding the study goals, protocol, and responsibilities. Owner consent was required for enrollment, and Animal Care and Use Committee approval for the study was obtained through each academic institution, respectively. Dogs with primary adrenal or exocrine pancreatic disease, concurrent liver or cardiac disease, or other systemic disease expected to promote CKD progression were excluded. Evaluation of enrolled dogs included physical examination, CBC, comprehensive biochemistry profile, urinalysis, urine culture, and UPC ratios. Each dog was reevaluated and IRIS CKD stage determinations were performed at monthly intervals for 6 months.

[0155] Classification of Stage 1 Stable CKD vs. Progressive CKD Classification of stage 1 IRIS CKD patients into stable or progressive CKD was based on the slope calculated from linear regression with inverse SDMA as the dependent variable and follow-up time as the independent variable for each patient. Slope cutoff of -0.0007

number

[0156] uCysB technique uCysB was measured using a research sandwich format ELISA at IDEXX Headquarters (Westbrook, ME) as previously described. Urine sample aliquots for uCysB measurements were frozen at -80°C, shipped in frozen batches to IDEXX, and stored in batches at -80°C until testing while the study progressed.

[0157] Descriptive Analysis Descriptive analysis was performed to understand the relationship between serial uCysB values ​​within dogs and their CKD status (control, stable, progressive). This also provided insight into the dependence of serial samples for each dog, as well as the influence of CKD group on uCysB values. Kruskal-Wallis ANOVA was used to gain insight into whether differences existed in dog uCysB expression within and between CKD groups. To understand the influence of CKD group on the y-intercept and slope, least squares regression between uCysB (dependence) and follow-up date (independence) was performed for each dog. Post-hoc descriptive analysis was performed to understand the potential use of uCysB as an early biomarker for progressive CKD.

[0158] statistical analysis A linear mixed-effects model with random slopes and coefficients was used to assess the relationship between follow-up duration and uCysB. Random intercepts were used to account for non-independence of serial uCysB measurements within each individual. Random coefficients were used to account for non-independence within each CKD group. An unstructured covariance structure was used to allow for unique patterns of variation for each individual. Plots of regression lines and standard error bars for each CKD group were generated based on predicted uCysB values ​​for each follow-up day (days 0 to 851). All statistical analyses were performed in R (version 4.1.0).

[0159] Example 2. Demographics Demographics of the enrolled dogs are provided in Table 3. Fifty-five (73%) were controls, 12 (16%) were stable CKD dogs, and 8 (11%) were progressive CKD dogs. Over half (56%, 42 / 75) of the enrolled dogs were 5-10 years old. The majority of dogs were spayed females or neutered males. The top five breeds are listed in Table 3. [Table 3]

[0160] Example 3. Descriptive analysis There were differences in log(uCysB) expression between dogs in the same study group (p 対照 <0.02, p 安定 <10 -7 , p 進行性 <10 -6 ) (Figure 1). The log(uCysB) values ​​in dogs with progressive CKD appeared to be higher than those in controls and stable CKD dogs. The Y-intercepts calculated based on linear regression per dog also differed between CKD groups (p<10 -4 ) (Figure 2). The y-intercepts for progressive CKD dogs tended to be higher than those for control and stable CKD dogs. A difference in slope between control, stable, and progressive CKD dogs (p=0.006) (Figure 3) was detected. Post-hoc analysis (Figure 4) showed that 75% (6 / 8) of dogs with progressive CKD had elevated uCysB values ​​at first visit. In contrast, only 6 percent (3 / 52) and 8 percent (1 / 12) of control and stable dogs had elevated uCysB values ​​at first visit.

[0161] The results of the linear mixed-effects model are shown in Table 4. [Table 4]

[0162] Advanced CKD dogs had significantly higher uCysB values ​​than stable CKD and control dogs. A plot of the linear mixed effects model is shown in FIG.

[0163] Example 4: Distinguishing between Cystatin B and Clusterin Urinary cystatin B (uCysB), a novel renal injury biomarker, has the potential to predict early CKD progression.

[0164] Early identification of dogs with progressive and stable chronic kidney disease (CKD) may provide an opportunity for intervention to slow progression. However, currently there are no surrogate biomarkers to reliably predict CKD progression. High urinary cystatin B ("uCystatin B" or "uCysB") levels in canine patients without diagnosed CKD may indicate acute kidney injury (AKI). Therefore, the differentiation of cystatin B and clusterin in stable versus progressive CKD was examined in patients with diagnosed CKD.

[0165] Animals: 20 dogs for evaluation of International Renal Interest Society Stage (IRIS) I CKD (Stage I) with IDEXX SDMA® up to 17 μg / dL and no systemic comorbidities, and 107 clinically healthy dogs (controls).

[0166] Methods: A prospective longitudinal study was conducted from 2016 to 2021 to evaluate the pattern of uCysB expression in stage I CKD and control dogs. Dogs were followed for up to 3 years. Stage I was subgrouped as stable or progressive CKD by the 1 / IDEXX SDMA® slope calculated from a minimum of three monthly time points during the first 120 days. Slope of -0.0007 (μg / dL) -1 Dogs with > or < 1 week progression were classified as stable or progressive, respectively. Mixed-effects modeling was used to assess the association between uCysB and rate of progression.

[0167] RESULTS: Estimated uCysB was 16 ng / mL in controls, 23 ng / mL in stable CKD, and 204 ng / mL in progressive CKD, predicting active and ongoing injury (Figure 5, p<0.05, dashed lines represent 95% confidence intervals). Thus, uCysB differentiated stable from progressive stage I CKD. Early identification of dogs with progressive CKD provides an opportunity for intervention to halt progression and improve patient management.

[0168] Cystatin B and clusterin levels were determined by ELISA in the urine of canine patients. The minimum inverse SDMA slope observed in a population of healthy dogs over a period of up to 3 years was -0.0007 (μg / dL). -1 / week, which was assigned 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 numerical value. As mentioned above, in the group of patients diagnosed with CKD, -0.0007 (μg / dL) -1 Patients with a slope of more or less than 1 / week were classified as stable or progressive, respectively.

[0169] Mixed-effects modeling was used to evaluate the association between clusterin and progression rate. The IRIS stage I subgroup analysis included CKD patients with initial SDMA up to 17. The study findings were then found to hold true up to initial SDMA of up to 20, spanning the early stages of IRIS stage II. In other words, cystatin B can distinguish between stable and progressive disease in CKD patients with SDMA values ​​up to 20, encompassing stage I (<18 μg / dl) and part of stage II (18-35 μg / dl).

[0170] Cystatin B distinguishes stable from progressive CKD in Renal Interest Society stage I CKD patients, as shown in Figure 5. Cystatin B distinguishes stable from progressive CKD in patients with SDMA values ​​up to 20, as shown in Figure 6.

[0171] Clusterin distinguishes stable from progressive CKD in Renal Interest Society stage I CKD patients, as shown in FIG.

[0172] Clusterin distinguishes stable from progressive CKD in patients with SDMA values ​​up to 20, as shown in Figure 8).

[0173] Example 5: Differentiating between stable renal function and progressive dysfunction in dogs Markers of renal function have been variably used to define whether chronic kidney disease (CKD) is stable or progressive, but quantitative criteria to distinguish these populations are lacking. Here, we evaluated the slope cutoffs of 1 / creatinine and 1 / IDEXX SDMA® to distinguish stable from progressive CKD.

[0174] Animals: 113 clinically healthy dogs and 29 male colony 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 status of clinically healthy dogs for Lyme, Ehrlichia, and Anaplasma. The HD cohort consisted of dogs aged 1-10 years owned by students or staff at the University of Missouri. Health status was assessed at each sample collection by medical history, CBC, serum chemistry profile, urinalysis, and serology. 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 profile of XLHN. XLHN is a rapidly progressive glomerulopathy caused by genetic mutations in the α5 chain of type IV collagen. Methods: Intact male pups (homozygous affected) from a single family of purpose-bred colony dogs housed at Texas A&M University. Monthly blood samples were obtained until death from CKD.

[0175] Analysis: Archived serum samples were stored at -80°C for the HD (clinically healthy dog) cohort from 2012 to 2014 and for the XLHN cohort from 2002 to 2012. The functional marker s-creatinine was analyzed by a modified Jaffe method from IDEXX Columbus, OH (HD) and an enzymatic method from Texas A&M (XLHN). The functional marker sSDMA was analyzed by LC-MS (IDEXX Westbrook, ME). [Table 5] [Table 6]

[0176] METHODS:A retrospective analysis combining 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 colony dogs progressing from HD, assigned a slope cutoff for stable renal function. Continuous 1 / creatinine and 1 / SDMA conversions for each dog were plotted against follow-up period.

[0177] The slopes of the 1 / creatinine and 1 / SDMA transformations (dependent variables) versus follow-up period (independent variable) for each dog were calculated using simple linear regression. The minimum slope value for HD was set as the cut-off value for stable versus progressive renal dysfunction. Relative 1 / creatinine and 1 / SDMA slopes were plotted with assigned cohort-specific intercepts for visual comparison.

[0178] Results: The slope cutoff for stable vs. progressive was 1 / creatinine = -0.0117 (mg / dL). -1 / week, 1 / day for IDEXX SDMA® -0.0007 (μg / dL) -1 / week (Figures 9 and 10, gray dashed lines).

[0179] In the CKD population studied, progressive dysfunction can be differentiated from stable renal function by using the slope of 1 / creatinine or 1 / IDEXX SDMA®. These criteria may be useful to characterize CKD in other cohorts of dogs and to establish guidelines for the rate of progression in dogs with naturally acquired CKD.

[0180] The functional marker sCreatinine remained stable in healthy dogs over the 2+ year observation period (Table 7 and Figure 15).Similarly, the functional marker sSDMA remained stable in healthy dogs over the 1,096 day observation period (Table 8 and Figure 18). [Table 7] [Table 8]

[0181] 1 / creatinine remained stable over time in healthy dogs. The median slope (days) was 1 / creatinine = 0.00001436 * [(mg / dl) -1 / day] * (number of days) + 0.9324022. The median slope (weeks) was 1 / creatinine = 0.00010052 * [(mg / dl) -1 / week] * (number of weeks) + 0.9324022 (Figures 16 and 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 (Figures 19 and 20).

[0182] In contrast to healthy dogs, the functional marker sCreatinine showed a progressive and exponential increase in the XLHN cohort (Figure 21, Figure 22, and Table 9). Similarly, the functional marker sSDMA showed a progressive and exponential increase in the XLHN cohort (Figure 25, Figure 26, and Table 10). [Table 9] [Table 10]

[0183] 1 / creatinine showed a systemic and linear decline in all XLHN dogs over a median observation period of 181 days. 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 (Figures 23 and 24). Similarly, 1 / SDMA showed a systemic and 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] * (number of weeks) + 0.07788298 (Figures 27 and 28).

[0184] The slope cutoff for stable versus progressive disease is 1 / creatinine, -0.0117 (mg / dL) -1 / week, -0.0007 (μg / dL) for SDMA® -1 / week (Figures 29, 30, and 31).

[0185] In the cohort studied, stable renal function in healthy dogs could be differentiated from progressive renal dysfunction in XLHN by the slope of the 1 / creatinine or 1 / SDMA curves.

[0186] When extended to other populations with CKD, short-term (2-3 months) assessment of 1 / creatinine or 1 / SDMA slopes may allow identification of dogs with stable versus progressive CKD.

[0187] Discrete values ​​of 1 / creatinine or 1 / SDMA slopes can define renal function as "stable" or "progressing" and serve as guidelines (cut-offs) to guide appropriate therapy for each condition.

Claims

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

2. 2. The method of claim 1, wherein the subject is diagnosed with International Renal Interest Society CKD stage I or CKD with a symmetric dimethylarginine (SDMA) level of up to 20 μg / dl prior to step (a).

3. 3. The method of claim 1 or 2, further comprising administering to the subject one or more treatments including surgery for obstructed ureter, obstructing kidney stones, or urinary tract stones, dietary management, administration of intestinal 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 the cystatin B polypeptide, clusterin polypeptide, or both in a sample from the subject at two, three, four, five, or more additional time points.

5. 3. The method of claim 1 or 2, wherein the amount of cystatin B polypeptide is 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 NOs: 1-2, 5-32, fragments thereof, or combinations thereof.

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

7. The method of claim 1 or 2, wherein determining the amount of the cystatin B polypeptide or clusterin polypeptide in a sample from the subject comprises contacting the sample with one or more antibodies, or specific binding fragments thereof, that specifically bind to a cystatin B polypeptide, a clusterin polypeptide, or one or more polypeptides set forth in SEQ ID NOs: 1-32, under conditions suitable for the formation of a complex between the cystatin B polypeptide or clusterin polypeptide and the one or more antibodies, or specific binding fragments thereof, and detecting the complex between the cystatin B polypeptide or clusterin polypeptide and the one or more antibodies, or specific binding fragments thereof.

8. The method of claim 7, wherein the antibody or specific binding fragment thereof is immobilized on 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. 8. 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 dog or a cat.

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

14. The method of 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), immunoturbidimetry, particle-enhanced immunoturbidimetry, radioimmunoassay (RIA), or Western blot assay.

15. The method of claim 1 or 2, wherein determining the amount of the cystatin B or clusterin polypeptide comprises subjecting the sample to mass spectrometry, LC-MS, quantitative nuclear magnetic resonance (qNMR), amino acid analysis (AAA), chromatographic (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 from a subject with stable CKD.

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