Methods and compositions for assessing and treating persistent acute kidney injury based on CC motif chemokine ligand 14 measurement - Patents.com

Abstract

The present invention relates to methods and compositions for assessing the risk of developing persistent acute kidney injury, and methods of treating a subject based on the assessment. In particular, disclosed herein are methods and compositions for detecting CC motif chemokine ligand 14 (CCL14) for predicting the risk of persistent acute kidney injury based on changes in the levels of CCL14 in two or more measurements of CCL14 in a subject, and / or based on two cut-off levels of CCL14.

Description

[Technical field]

[0001] Cross-Referenced Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 316,377, filed March 3, 2022, U.S. Provisional Patent Application No. 63 / 318,338, filed March 9, 2022, and U.S. Provisional Patent Application No. 63 / 319,206, filed March 11, 2022, each of which is incorporated by reference in its entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted in electronic format and is incorporated herein by reference in its entirety. The Sequence Listing is provided in a file named DN02084SeqListPCT.xml, created on March 1, 2023, which is 2,924 bytes in size. [Background technology]

[0003] The kidneys are responsible for the excretion of water and solutes from the body. Their functions include maintaining acid-base balance, regulating electrolyte concentrations, regulating blood volume, and regulating blood pressure. Thus, loss of renal function due to injury and / or disease results in substantial morbidity and mortality. A detailed discussion of renal impairment is provided in Harrison's Principles of Internal Medicine, 17 th Ed., McGraw Hill, New York, pages 1741-1830, which is incorporated herein by reference in its entirety. Renal diseases and / or disorders can be acute or chronic. Acute and chronic kidney diseases are described as follows (Current Medical Diagnosis & Treatment 2008, 47 thEd, McGraw Hill, New York, pages 785-815, incorporated herein by reference in its entirety): "Acute renal failure is the deterioration of renal function over hours to days, resulting in the retention of nitrogenous waste products (e.g., urea nitrogen) and creatinine in the blood. The retention of these substances is called azotemia. Chronic renal failure (chronic kidney disease) results from an abnormal loss of renal function over months to years."

[0004] Acute kidney injury (AKI, also known as acute renal failure, or ARF) is a sudden (typically detected within about 48 hours to 1 week) decrease in glomerular filtration. AKI is a leading global cause of both morbidity and mortality. It is estimated that at least half of AKI cases recover within 72 hours. Cases of AKI that recover within 72 hours tend to have significantly better outcomes compared to those that persist for at least 72 hours, especially for severe AKI cases. Oliguria lasting at least 72 hours has been identified as a criterion for initiating renal replacement therapy (RRT). See Gaudry S, Hajage D, Schortgen F, Martin-Lefevre L, Pons B, Boulet E, et al. The New England Journal of Medicine. 2016;375(2):122-33, which is incorporated herein by reference in its entirety. Recent evidence suggests that two-thirds of patients with AKI recover from renal dysfunction within 3-7 days, but those who persist have dramatically reduced survival over the next year. See Kellum JA, Sileanu FE, Bihorac A, Hoste EA, Chawla LS. Am J Respir Crit Care Med. 2017;195(6):784-91, which is incorporated herein by reference in its entirety. Persistence of AKI for more than one week, referred to as acute kidney disease (AKD), is of major importance in that it increases the risk and consequences of an individual developing chronic kidney disease. This association with chronic kidney disease (CKD) has been established over the past decade, and specific recommendations for the management of patients with AKD have been proposed to try to influence this transition. See Chawla LS, Bellomo R, Bihorac A, Goldstein SL, Siew ED, Bagshaw SM, et al., Nat Rev Nephrol. 2017;13(4):241-57; Chawla LS, Eggers PW, Star RA, Kimmel PL, The New England Journal of Medicine 2014;371(l):58-66, each of which is incorporated by reference in its entirety.Thus, early identification of individuals at risk for AKD will allow for appropriate delivery of these proposed interventions, but will also identify individuals to whom newer therapies to attenuate AKI can be targeted.

[0005] Not only is the persistence of AKI related to long-term outcomes, but clinical decision-making is also critically influenced by physician expectations surrounding renal recovery and the decision of when to initiate renal replacement therapy (RRT). Currently, this is almost entirely dependent on clinical expectations regarding the likelihood of recovery in the absence of commercially available diagnostic agents to aid this decision-making process. Thus, a great deal of debate exists around the timing of RRT, with studies indicating that some patients may benefit from early RRT initiation, while other studies indicate that some individuals undergo RRT who will recover renal function quickly and may not require such treatment. (Bagshaw SM, Lamontagne F, Joannidis M, Wald R. Critical care 2016;20(l):245; Forni LG, Joannidis M. Nat Rev Nephrol 2019;15(1):5-6.) Early and reliable identification of those who will recover renal function would allow for stratifying treatment and avoiding the current risks of extracorporeal therapy. Summary of the Invention

[0006] These challenges highlight the need for better methods to detect and evaluate AKI, especially in the early and asymptomatic stages, but also in later stages when renal recovery and repair can occur. Additionally, there is a need to better identify patients at risk for having persistent AKI.

[0007] Methods and compositions are provided for assessing renal function of a subject.As described herein, the measurement of CC motif chemokine ligand 14 (CCL14) can be used to assess the risk of sustained acute kidney injury (AKI) in subjects diagnosed with AKI.

[0008] In one embodiment, a method for assessing an increased risk of developing sustained acute kidney injury (AKI) in a subject is provided. The method includes: (a) performing an assay to detect a level of CC motif chemokine ligand 14 (CCL14) in a urine sample obtained from the subject; and (b) determining that the subject is at increased risk of developing sustained AKI based on the level of CCL14 detected in the urine sample being higher than a CCL14 threshold concentration of about 1.3ng / ml.

[0009] In another embodiment, a method for assessing a high risk of developing sustained acute kidney injury (AKI) in a subject is provided. The method includes: (a) performing an assay to detect the level of CC motif chemokine ligand 14 (CCL14) in a urine sample obtained from the subject; and (b) determining that the subject is at high risk of developing sustained AKI based on the level of CCL14 detected in the urine sample exceeding a CCL14 threshold concentration of about 13.0 ng / ml.

[0010] In some embodiments, the level of CCL14 detected in a urine sample is above a threshold concentration of CCL14 of about 1.3 ng / ml and below a threshold concentration of CCL14 of about 13.0 ng / ml.

[0011] In one embodiment, the method further comprises seeking further analysis and / or treatment from a nephrologist or specialist.

[0012] In some embodiments, the methods further include treating a subject at high or elevated risk of having sustained AKI with one or more renal replacement therapies (RRT), removing compounds known to be kidney damaging, performing procedures known to be kidney damaging at least about 48 hours after obtaining the sample, altering diuretic dosing, altering dosing of renally cleared compounds, and / or administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels or hemodynamics.

[0013] In one embodiment, RRT includes one or more of continuous RRT, intermittent RRT, hemodialysis, peritoneal dialysis, hemofiltration, and renal transplantation.

[0014] In one embodiment, the subject is diagnosed with AKI.

[0015] In one embodiment, the subject has KDIGO stage 1 AKI. In one embodiment, the subject has KDIGO stage 2 AKI. In another embodiment, the subject has KDIGO stage 3 AKI.

[0016] In one embodiment, the subject is determined to be at elevated or high risk for sustained KDIGO stage 2 or 3 AKI.

[0017] In another embodiment, the subject is determined to be at elevated or high risk for sustained KDIGO stage 3 AKI.

[0018] In one embodiment, the method includes determining that the subject has an elevated or high risk of developing persistent KDIGO stage 3 AKI within 48 hours of the time the urine sample was obtained, where the duration of persistent KDIGO stage 3 AKI comprises a 72 hour period in which the minimum KDIGO stage is KDIGO 3.

[0019] In one embodiment, the subject is in an intensive care unit.

[0020] In one embodiment, the method further comprises contacting the urine sample with a binding reagent that binds to CCL14. In one embodiment, the binding reagent is an antibody.

[0021] In one embodiment, the assay is an immunoassay.

[0022] In one aspect, a method of assessing an elevated risk of developing sustained acute kidney injury (AKI) in a subject is provided, the method comprising: (a) performing an assay to detect a level of CC motif chemokine ligand 14 (CCL14) in a urine sample obtained from the subject; and (b) correlating the assay result with an elevated risk for the subject developing sustained AKI by comparing the assay result to a CCL14 threshold concentration of about 1.3 ng / ml, wherein the correlation is used as a rule-in test for an elevated risk for the subject developing sustained AKI if the assay result is above a CCL14 threshold concentration of about 1.3 ng / ml, or the correlation is used as a rule-out test for a subject at elevated risk of developing sustained AKI if the assay result is below a CCL14 threshold concentration of about 1.3 ng / ml.

[0023] In another aspect, a method of assessing an elevated risk of developing sustained acute kidney injury (AKI) in a subject is provided, the method comprising: (a) performing an assay to detect a level of CC motif chemokine ligand 14 (CCL14) in a urine sample obtained from the subject; and (b) correlating the assay result with an elevated risk for the subject developing sustained AKI by comparing the assay result with a CCL14 threshold concentration of about 13.0 ng / ml, wherein the correlation is used as a confirmatory diagnostic test for a subject at elevated risk of developing sustained AKI if the assay result is above the CCL14 threshold concentration of about 13.0 ng / ml, or the correlation is used as a rule-out diagnostic test for a subject at elevated risk of developing sustained AKI if the assay result is below the CCL14 threshold concentration of about 13.0 ng / ml.

[0024] In one embodiment, the level of CCL14 detected in the urine sample is above a threshold concentration of CCL14 of about 1.3 ng / ml and below a threshold concentration of CCL14 of about 13.0 ng / ml.

[0025] In one embodiment, the method further comprises seeking further analysis and / or treatment from a nephrologist or specialist.

[0026] In some embodiments, the methods further include treating the subject at high or elevated risk of having sustained AKI with one or more renal replacement therapies (RRT), removing compounds known to be kidney damaging, performing procedures known to be kidney damaging delayed at least about 48 hours after obtaining the sample, altering and / or optimizing diuretic dosing, altering and / or optimizing dosing of renally cleared compounds, and / or administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels or hemodynamics.

[0027] In one embodiment, RRT includes one or more of continuous RRT, intermittent RRT, hemodialysis, peritoneal dialysis, hemofiltration, and renal transplantation.

[0028] In one embodiment, the subject is diagnosed with AKI.

[0029] In one embodiment, the subject has KDIGO stage 1 AKI. In another embodiment, the subject has KDIGO stage 2 AKI. In another embodiment, the subject has KDIGO stage 3 AKI.

[0030] In one embodiment, the subject is determined to be at elevated or high risk for sustained KDIGO stage 2 or 3 AKI.

[0031] In one embodiment, the subject is determined to be at elevated or high risk for sustained KDIGO stage 3 AKI.

[0032] In one embodiment, the method includes determining that the subject has an elevated or high risk of developing persistent KDIGO stage 3 AKI within 48 hours of the time the urine sample was obtained, where the duration of persistent KDIGO stage 3 AKI comprises a 72 hour period in which the minimum KDIGO stage is KDIGO 3.

[0033] In one embodiment, the subject is in an intensive care unit.

[0034] In one embodiment, the method further comprises contacting the urine sample with a binding reagent that binds to CCL14. In one embodiment, the binding reagent is an antibody.

[0035] In one embodiment, the assay is an immunoassay.

[0036] In another aspect, a method for assessing a low risk of developing sustained acute kidney injury (AKI) in a subject is provided, the method comprising: (a) performing an assay to detect a level of CC motif chemokine ligand 14 (CCL14) in a urine sample obtained from the subject; and (b) determining that the subject is at low risk of developing sustained AKI based on the level of CCL14 detected in the urine sample being below a CCL14 threshold concentration of about 1.3 ng / ml.

[0037] In one embodiment, a method for assessing an increased risk of developing sustained acute kidney injury (AKI) in a subject is provided, the method comprising: (a) performing an assay to detect levels of CC motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, where at least a second urine sample is obtained from the subject within about 72 hours after the first urine sample is obtained; and (b) determining that the subject is at increased risk of sustained AKI based on the level of CCL14 detected in the urine samples trending upward.

[0038] In one embodiment, the method includes: (a) performing an assay to detect levels of CC motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, where at least a second urine sample is obtained from the subject within about 72 hours after a first urine sample is obtained; (b) determining that the level of CCL14 detected in the urine samples is low, moderate, or high based on a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14, where (i) if the level of CC motif chemokine 14 in the urine sample is below the first predetermined threshold concentration of CCL14, then the level of CCL14 in the urine sample is low; (ii) if the level of CCL14 in the urine sample is above a second predetermined threshold concentration of CCL14, the level of CCL14 in the urine sample is high; or (iii) if the level of CCL14 in the urine sample is between the first predetermined threshold concentration of CCL14 and the second predetermined threshold concentration of CCL14, the level of CCL14 in the urine sample is moderate; (c)(iv) if the level of CCL14 in the first urine sample is low and the level of CCL14 in the second urine sample is moderate or high; or (v) if the level of CCL14 in the first urine sample is moderate and the level of CCL14 in the second urine sample is high, correlating the level of CCL14 with an increased risk that the subject will develop persistent KDIGO stage 1, 2 or 3 AKI.

[0039] In some embodiments, the subject has AKI that meets the definition of KDIGO stage 1, the subject has AKI that meets the definition of KDIGO stage 2, or the subject has AKI that meets the definition of KDIGO stage 3.

[0040] In some embodiments, the subject is determined to be at increased risk for persistent KDIGO stage 2 or 3 AKI. In one embodiment, the subject is determined to be at increased risk for persistent KDIGO stage 3 AKI.

[0041] In one embodiment, the method includes determining that the subject is at increased risk of developing persistent KDIGO stage 3 AKI within 48 hours of the first urine sample being obtained, where the duration of persistent KDIGO stage 3 AKI comprises a 72 hour period in which the minimum KDIGO stage is KDIGO 3.

[0042] In one embodiment, the first threshold concentration of CCL14 is less than or equal to 1.3 ng / mL of CCL14, hi another embodiment, the second threshold concentration of CCL14 is greater than 13 ng / mL.

[0043] In some embodiments, the second urine sample is obtained within about 12 hours of the first urine sample, the second urine sample is obtained about 12 hours after the first urine sample, the second urine sample is obtained within about 24 hours of the first urine sample, the second urine sample is obtained about 24 hours after the first urine sample, the second urine sample is obtained within about 36 hours of the first urine sample, the second urine sample is obtained about 36 hours after the first urine sample, the second urine sample is obtained within about 48 hours of the first urine sample, the second urine sample is obtained about 48 hours after the first urine sample, the second urine sample is obtained about 72 hours after the first urine sample, or the second urine sample is obtained about 72 hours after the first urine sample.

[0044] In one embodiment, the method further comprises obtaining a third urine sample. In one embodiment, the second and third urine samples are obtained within about 12 hours of the first urine sample. In another embodiment, the second and third urine samples are obtained within about 24 hours of the first urine sample. In another embodiment, the second urine sample is obtained about 12 hours after the first urine sample is obtained, and the method further comprises obtaining the third urine sample about 24 hours after the first urine sample is obtained.

[0045] In one embodiment, the first urine sample and the second urine sample have low levels of CCL14 and the third urine sample has intermediate levels of CCL14.

[0046] In one embodiment, the first urine sample and the second urine sample have low levels of CCL14 and the third urine sample has high levels of CCL14.

[0047] In one embodiment, the first and second urine samples have intermediate levels of CCL14 and the third urine sample has high levels of CCL14.

[0048] In one embodiment, the first urine sample has low levels of CCL14 and the second urine sample and the third urine sample have intermediate levels of CCL14.

[0049] In one embodiment, the first urine sample has a moderate level of CCL14 and the second urine sample and the third urine sample have high levels of CCL14.

[0050] In one embodiment, a first urine sample has a low level of CCL14, a second urine sample has a moderate level of CCL14, and a third sample has a high level of CCL14.

[0051] In one embodiment, the first urine sample has low levels of CCL14 and the second urine sample and the third urine sample have high levels of CCL14.

[0052] In one embodiment, the subject is in an intensive care unit.

[0053] In one embodiment, the subject is diagnosed with AKI.

[0054] In one embodiment, the subject has had AKI for less than 36 hours before the first urine sample was obtained.

[0055] In one embodiment, the method further comprises contacting the urine sample with a binding reagent that binds CCL14.

[0056] In one embodiment, the binding reagent is an antibody.

[0057] In one embodiment, the assay is an immunoassay.

[0058] In one embodiment, the method further comprises treating the subject at increased risk of having sustained AKI with one or more renal replacement therapies (RRT), withdrawing compounds known to be renal damaging, performing procedures known to be renal damaging at least about 48 hours after obtaining the first sample, altering diuretic dosing, altering dosing of renally cleared compounds, and / or administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels or hemodynamics.

[0059] In one embodiment, RRT includes one or more of continuous RRT, intermittent RRT, hemodialysis, peritoneal dialysis, hemofiltration, and renal transplantation.

[0060] In another embodiment, a method for assessing a reduced risk of developing sustained acute kidney injury (AKI) in a subject is provided, the method comprising: (a) performing an assay to detect levels of CC motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, where at least a second urine sample is obtained from the subject within about 72 hours after the first urine sample is obtained; and (b) determining that the subject is at reduced risk of sustained AKI based on a downward trend in the levels of CCL14 detected in the urine samples.

[0061] In one embodiment, the method includes (a) performing an assay to detect levels of CC motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, where at least a second urine sample is obtained from the subject within about 72 hours after a first urine sample is obtained, and (b) determining whether the level of CCL14 detected in the urine samples is low, moderate, or high based on a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14, where (i) if the level of CC motif chemokine 14 in the urine sample is below the first predetermined threshold concentration of CCL14, the level of CCL14 in the urine sample is low; (ii) if the level of CCL14 in the urine sample is below the second predetermined threshold concentration of CCL14, the level of CCL14 in the urine sample is high. if the level of CCL14 in the urine sample is above a predetermined threshold concentration, the level of CCL14 in the urine sample is high; or (iii) if the level of CCL14 in the urine sample is between a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14, the level of CCL14 in the urine sample is moderate; (c)(iv) if the level of CCL14 in the first urine sample is moderate and the level of CCL14 in the second urine sample is low; (v) if the level of CCL14 in the first urine sample is high and the level of CCL14 in the second urine sample is moderate; or (vi) if the level of CCL14 in the first urine sample is high and the level of CCL14 in the second urine sample is low, correlating the level of CCL14 with a decreased risk that the subject will develop sustained KDIGO stage 1, 2 or 3 AKI.

[0062] In one embodiment, the subject has AKI that meets the definition of KDIGO Stage 1. In one embodiment, the subject has AKI that meets the definition of KDIGO Stage 2. In one embodiment, the subject has AKI that meets the definition of KDIGO Stage 3.

[0063] In one embodiment, the first threshold concentration of CCL14 is less than or equal to 1.3 ng / mL of CCL14, hi another embodiment, the second threshold concentration of CCL14 is greater than 13 ng / mL.

[0064] In some embodiments, the second urine sample is obtained within about 12 hours of the first urine sample, the second urine sample is obtained about 12 hours after the first urine sample, the second urine sample is obtained within about 24 hours of the first urine sample, the second urine sample is obtained about 24 hours after the first urine sample, the second urine sample is obtained within about 36 hours of the first urine sample, the second urine sample is obtained about 36 hours after the first urine sample, the second urine sample is obtained within about 48 hours of the first urine sample, the second urine sample is obtained about 48 hours after the first urine sample, the second urine sample is obtained about 72 hours after the first urine sample, or the second urine sample is obtained about 72 hours after the first urine sample.

[0065] In one embodiment, the method further comprises obtaining a third urine sample. In one embodiment, the second and third urine samples are obtained within about 12 hours of the first urine sample. In one embodiment, the second and third urine samples are obtained within about 24 hours of the first urine sample. In one embodiment, the second urine sample is obtained about 12 hours after the first urine sample is obtained, and the method further comprises obtaining the third urine sample about 24 hours after the first urine sample is obtained.

[0066] In one embodiment, the first and second urine samples have high levels of CCL14 and the third urine sample has a medium level of CCL14. In one embodiment, the first and second urine samples have high levels of CCL14 and the third urine sample has a low level of CCL14. In one embodiment, the first and second urine samples have a medium level of CCL14 and the third urine sample has a low level of CCL14. In one embodiment, the first urine sample has a medium level of CCL14 and the second and third urine sample has a low level of CCL14. In one embodiment, the first urine sample has a high level of CCL14 and the second and third urine sample has a low level of CCL14. In one embodiment, the first urine sample has a high level of CCL14 and the second and third urine sample has a medium level of CCL14. In one embodiment, the first urine sample has a high level of CCL14 and the second and third urine sample has a medium level of CCL14. In one embodiment, a first urine sample has a high level of CCL14, a second urine sample has a moderate level of CCL14, and a third urine sample has a low level of CCL14.

[0067] In one embodiment, the subject is in an intensive care unit.

[0068] In one embodiment, the subject is diagnosed with AKI.

[0069] In one embodiment, the subject has had AKI for less than 36 hours before the first urine sample was obtained.

[0070] In one embodiment, the method further comprises contacting the urine sample with a binding reagent that binds CCL14.

[0071] In one embodiment, the binding reagent is an antibody.

[0072] In one embodiment, the assay is an immunoassay.

[0073] In one embodiment, the method further includes treating a subject at reduced risk of sustained AKI by one or more of administering a compound known to be kidney damaging, performing a procedure known to be kidney damaging, altering and / or optimizing diuretic dosing, altering and / or optimizing dosing of a renally cleared compound, and administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels, or hemodynamics.

[0074] In one embodiment, the method includes assessing a risk of developing sustained acute kidney injury (AKI) in a subject by performing an assay to detect levels of CC motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, where at least a second urine sample is obtained from the subject within about 72 hours after the first urine sample is obtained, determining that the subject is at increased risk of sustained AKI if the levels of CCL14 detected in the urine samples are trending upward, and determining that the subject is at decreased risk of sustained AKI if the levels of CCL14 detected in the urine samples are trending downward. [Brief description of the drawings]

[0075] [Figure 1] FIG. 1 shows a flow diagram of the pooled analysis of patients from the Ruby and Sapphire studies. [Diagram 2] A comparison of CCL14 concentrations in the four populations is shown. [Diagram 3] The risk of the primary endpoint of persistent severe AKI stratified by CCL14 levels below, between, and above 1.3 and 13 ng / mL is shown. [Figure 4] Figure 1 shows the risk of sustained severe AKI stratified by CCL14 levels <1.3 ng / mL and >1.3 ng / mL. [Figure 5A-5C]Figure 1 shows the cumulative incidence of RRT, death, and RRT or death within 90 days of Ruby Study Enrollment stratified by CCL14 concentrations below, between, and above 1.3 and 13 ng / mL. [Figure 6A-6C] Figure 1 shows the cumulative incidence of RRT, death, and RRT or death within 90 days of the Ruby Study Enrollment stratified by CCL14 concentrations <1.3 ng / mL and >1.3 ng / mL. [Figure 7] A comparison of CCL14 concentrations in the four populations is shown. [Figure 8] CCL14 trajectories stratified by initial CCL14 category after diagnosis of moderate-severe AKI in the primary analysis cohort are shown. [Figure 9] Change in CCL14 category stratified by initial CCL14 category after diagnosis of moderate-severe AKI. Sensitivity analysis including patients with CCL14 values ​​below -3. [Figure 10] 1 shows an extended measurement of CCL14 over 6 days with the first change from the initial category. [Figure 11] The ability of absolute urinary CCL14 levels to predict the rolling study endpoints (sustained severe AKI, RRT or death beginning within the next 48 hours) is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] As used herein, "acute kidney injury" or "AKI" is a rapid (e.g., within about 14 days, e.g., within about 7 days, within about 72 hours, or within about 48 hours) decrease in renal function identified by an absolute increase in serum creatinine of 0.3 mg / dL or more (>= 26.4 μmol / l), a percentage increase in serum creatinine of 50% or more (1.5 times baseline), or a decrease in urine output (documented oliguria of less than 0.5 ml / kg for at least 6 hours).

[0077] As used herein, "persistent AKI" refers to an episode of AKI that lasts for at least 48-72 hours before sustained reversal. Reversal of AKI must generally last a minimum of 48 hours in order for subsequent episodes of AKI to be considered separate episodes rather than a continuation of the first episode. Definitions of various stages of renal injury, including persistent AKI, as well as methods of assessment and treatment, can be found in Nat Rev Nephrol. 2017 Apr;13(4):241-257, which is incorporated herein by reference in its entirety. Persistence of a particular stage of AKI (e.g., KDIGO stage 3 AKI) can be similarly defined, where a minimum stage of AKI must be maintained for 48-72 hours before sustained recovery from that stage. "Sustained severe AKI" refers to a subject whose minimum AKI stage during a 72-hour period is KDIGO stage 3.

[0078] An "elevated risk" of sustained AKI means that the level of CCL14 in the sample exceeds the CCL14 threshold concentration of 1.3 ng / ml.

[0079] A "high risk" of sustained AKI means that the level of CCL14 in the sample is greater than the CCL14 threshold concentration of 13.0 ng / ml.

[0080] "Low risk" of sustained AKI means that the level of CCL14 in the sample is below the CCL14 threshold concentration of 1.3 ng / ml.

[0081] In certain embodiments, the level of CCL14 is used as a "definitive diagnosis" for an elevated risk of developing persistent AKI. In these embodiments, the measured level of CCL14 exceeds a threshold concentration of CCL14 of about 1.3 ng / ml.

[0082] In certain embodiments, the level of CCL14 is used as a "definitive diagnosis" for high risk of developing persistent AKI. In these embodiments, the measured level of CCL14 exceeds a threshold concentration of CCL14 of about 13.0 ng / ml.

[0083] In certain embodiments, the level of CCL14 is used as a "diagnosis of exclusion" for an elevated risk of developing persistent AKI. In these embodiments, the measured level of CCL14 is below a threshold concentration of CCL14 of about 1.3 ng / ml.

[0084] In certain embodiments, the level of CCL14 is used as a "diagnosis of rule out" for an increased risk of developing persistent AKI. In these embodiments, the measured level of CCL14 is below a threshold concentration of CCL14 of about 13.0 ng / ml.

[0085] An "elevated risk" of persistent AKI means that the level of CCL14 tends to be elevated in one or more consecutive samples obtained from the subject compared to a reference level (e.g., a baseline level or threshold level) of CCL14 obtained from the subject (e.g., a first CCL14 measurement).

[0086] A "reduced risk" of sustained AKI means a trend toward a downward trend in the level of CCL14 in one or more consecutive samples obtained from the subject compared to a reference level (e.g., a baseline level or threshold level) of CCL14 obtained from the subject (e.g., a first CCL14 measurement).

[0087] By "tends toward increased" it is meant that the second measurement or level of CCL14 is higher than the first measurement or level of CCL14, e.g., at least about 10% higher, or 20% higher, or 30% higher, or 40% higher, or 50% higher, or 60% higher, or 70% higher, or 80% higher, or 90% higher, or more; or 2-fold or more, or 5-fold or more, or 10-fold or more, or 20-fold or more, or 100-fold or more, or more.

[0088] By "downward trend" it is meant that a first measurement or level of CCL14 is higher than a second measurement or level of CCL14, e.g., at least about 10% higher, or 20% higher, or 30% higher, or 40% higher, or 50% higher, or 60% higher, or 70% higher, or 80% higher, or 90% higher, or more; or 2-fold or more, or 5-fold or more, or 10-fold or more, or 20-fold or more, or 100-fold or more.

[0089] CCL14 levels can be measured in one or more consecutive samples obtained from the same subject to monitor changes in CCL14 levels over time. For example, CCL14 levels can be measured in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples obtained from the same subject. In one embodiment, CCL14 levels are measured in two samples obtained from the same subject. In another embodiment, CCL14 levels are measured in three samples obtained from the same subject. The CCL14 level in the first sample can be used as a baseline level to monitor changes in CCL14 levels in further samples obtained from the same subject.

[0090] One or more consecutive samples can be obtained from the subject at any time after the first sample is obtained. Samples can be obtained about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, 7 days, 30 days or more after the first sample is obtained. The sample can be obtained about 1-3 hours, about 2-4 hours, about 3-5 hours, about 4-6 hours, about 5-7 hours, about 6-8 hours, about 7-9 hours, about 8-10 hours, about 9-11 hours, about 10-12 hours, about 11-13 hours, about 12-14 hours, about 13-15 hours, about 14-16 hours, about 15-17 hours, about 16-18 hours, about 17-19 hours, about 18-20 hours, about 19-21 hours, about 20-22 hours, about 21-23 hours, about 22-24 hours or more after the first sample is obtained. In some embodiments, the sample is obtained within about 1-6 hours, within about 6-12 hours, within about 12-18 hours, within about 18-24 hours, within about 24-36 hours, within about 36-48 hours, within about 48-72 hours, or more, after the first sample is obtained.

[0091] AKI can be caused by radiocontrast agents (also called contrast media) and other nephrotoxins such as cyclosporine, antibiotics including aminoglycosides, and anticancer drugs such as cisplatin, and typically manifests over a period of several days to about a week. Contrast-induced nephropathy (CIN, which is AKI caused by radiocontrast agents) is thought to be caused by intrarenal vasoconstriction (leading to ischemic injury) and from the generation of reactive oxygen species that are directly toxic to renal tubular epithelial cells. CIN classically presents with acute (onset within 24-48 hours) but reversible (peaks 3-5 days, resolves within 1 week) elevations in blood urea nitrogen and serum creatinine.

[0092] The commonly reported criteria for defining and detecting AKI is a sudden (typically within about 2-7 days or within the hospital stay) rise in serum creatinine. Although the use of serum creatinine rise to define and detect AKI is well established, the magnitude of serum creatinine rise and the time measured to define AKI vary considerably between publications. Traditionally, relatively large increases in serum creatinine such as 100%, 200%, at least a 100% increase to a value greater than 2 mg / dL, and other definitions have been used to define AKI. However, the recent trend has been to use smaller serum creatinine rises to define AKI. The relationship between serum creatinine elevation, AKI and associated health risks is reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270, 2005 and Chertow et al, J Am Soc Nephrol 16:3365-3370, 2005, which are incorporated herein by reference in their entirety, along with the references cited therein. As described in these publications, acutely worsening renal function (AKI) and increased risk of death and other adverse outcomes are now known to be associated with very small increases in serum creatinine. These increases can be determined as relative (percentage) values ​​or nominal values. Relative increases in serum creatinine as small as 20% from pre-injury values ​​have been reported to indicate acutely worsening renal function (AKI) and increased health risks, although a more commonly reported value that defines AKI and increased health risks is a relative increase of at least 25%. Nominal increases of 0.3 mg / dL, 0.2 mg / dL, or even 0.1 mg / dL have been reported to indicate worsening renal function and increased risk of death. Various periods during which serum creatinine rises to these thresholds have been used to define AKI, such as 2 days, 3 days, 7 days, or a range of variable periods defined as the time the patient is in the hospital or intensive care unit. These studies indicate that there is no specific threshold of serum creatinine rise (or period of rise) that worsens renal function or AKI, but rather a continuous increase in risk with increasing magnitude of serum creatinine rise.

[0093] One study (Lassnigg et al, J Am Soc Nephrol 15:1597-1605, 2004) looked at both increases and decreases in serum creatinine. Patients with mild decreases in serum creatinine of -0.1 to -0.3 mg / dL after cardiac surgery had the lowest mortality rates. Patients with larger decreases in serum creatinine (-0.4 mg / dL or greater) or any increase in serum creatinine had a greater mortality rate. These findings led the authors to conclude that even very subtle changes in renal function (detected by small creatinine changes within 48 hours of surgery) have a profound effect on patient outcomes. In an effort to reach a consensus on a unified classification system for using serum creatinine to define AKI in clinical trials and clinical practice, Bellomo et al., Crit Care. 8(4):R204-12, 2004, which is incorporated herein by reference in its entirety for the RIFLE criteria, propose the following classification for stratifying AKI patients: "At risk": serum creatinine increased 1.5 times baseline or urine production <0.5 ml / kg body weight / hour for 6 hours; "Disorder": serum creatinine increased 2.0-fold to baseline or urine production <0.5 ml / kg / hour for 12 hours; "Failure": Serum creatinine increased 3.0-fold above baseline, or creatinine ≥ 4.0 mg / dL (355 μmol / l) with an acute rise ≥ 0.5 mg / dL (44 μmol / l), or urine output < 0.3 ml / kg / h for 24 hours, or anuria for at least 12 hours.

[0094] In addition, two clinical outcomes were included: "Loss": The need for sustained renal replacement therapy for more than 4 weeks. "ESRD": End Stage Renal Disease - the need for dialysis for more than three months.

[0095] These criteria are called RIFLE criteria, which provide a useful clinical tool for classifying renal status. As discussed in Kellum, Crit Care Med.36:S 141-45,2008 and Ricci et al., Kidney Int.73,538-546,2008 (each of which is incorporated by reference in its entirety), the RIFLE criteria provide a uniform definition of AKI that has been validated in multiple studies.As understood in the art, RIFLE stage 0 can be used to classify subjects who do not meet the criteria for RIFLE stage R or any of the more severe RIFLE stages of AKI (i.e., subjects who do not have renal impairment, or subjects who have renal impairment but have not progressed to meet any of the threshold criteria for RIFLE stage R or more severe RIFLE stages of AKI).

[0096] More recently, Mehta et al., Crit Care 11:R31 (doi:10.1186.cc5713), 2007, which is incorporated herein by reference in its entirety, have proposed the following similar classification (AKIN), modified from RIFLE, for stratifying AKI patients: "Stage I": an increase in serum creatinine of 0.3 mg / dL or more (≥ 26.4 μmol / L) or an increase of 150% or more (1.5-fold) from baseline or a urine output of less than 0.5 mL / kg per hour for more than 6 hours; "Stage II": greater than 200% (>2-fold) increase in serum creatinine from baseline or urine output less than 0.5 mL / kg per hour for greater than 12 hours; "Stage III": An increase in serum creatinine of more than 300% (>3-fold) from baseline OR serum creatinine ≥ 4.0 mg / dL (≥ 354 μmol / L) with an acute increase of at least 0.5 mg / dL (44 μmol / L) OR urine output < 0.3 mL / kg / hour for 24 hours or anuria for 12 hours.

[0097] As is understood in the art, AKIN stage 0 can be used to classify subjects who do not meet the criteria for AKIN stage I or any of the more severe AKIN stages of AKI (i.e., subjects who do not have renal impairment, or subjects who have renal impairment but have not progressed to meeting any of the threshold criteria for AKIN stage I or more severe AKIN stages of AKI).

[0098] Similarly, the Kidney Disease: Improving Global Outcomes (KDIGO) Acute Kidney Injury Work Group. KDIGO Clinical Practice Guideline for Acute Kidney Injury, Kidney Inter., Suppl. 2012;2:1-138, incorporated herein by reference in its entirety, refers to both RIFLE and AKIN and provides the following AKI staging guidelines:

[0099] [Table 1]

[0100] As is understood in the art, KDIGO stage 0 can be used to classify subjects who do not meet the criteria for KDIGO stage 1 or any more severe KDIGO stage of AKI (i.e., subjects who do not have renal impairment, or subjects who have renal impairment but have not progressed to meeting any of the threshold criteria for KDIGO stage 1 or more severe KDIGO stages of AKI).

[0101] The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med. 2006;7(4):177-197, incorporated herein by reference in its entirety) uses a 25% serum creatinine rise to define contrast-induced nephropathy, which is a type of AKI. Although various groups have proposed slightly different criteria for using serum creatinine to detect AKI, the consensus is that small changes in serum creatinine, such as 0.3 mg / dL or 25%, are sufficient to detect AKI (worsening renal function) and the magnitude of serum creatinine change is an indicator of the severity of AKI and risk of mortality.

[0102] These classification systems of AKI generally include serum creatinine and urine output criteria for each stage. Whenever specified herein, any stage of AKI may be considered equivalent (i.e., substituted) for any of the individual criteria that identify a subject as being at that particular stage of AKI. In some embodiments, the methods disclosed herein may also be used to correlate renal status defined by a particular AKI stage (e.g., the likelihood of reaching a particular AKI stage or the likelihood of sustained AKI at a particular stage), where a particular AKI stage may be defined by meeting both a serum creatinine criterion that identifies a subject as being at that particular stage and a urine output criterion that identifies a subject as being at that particular stage. In some embodiments, a particular AKI stage may be defined by meeting all criteria (i.e., both all serum creatinine criteria and all urine output criteria). All methods disclosed herein may define a stage of AKI according to any of these embodiments, unless otherwise specified. It will be understood in the art that similarly defined stages of AKI may generally be interchanged with one another with respect to the use of biomarkers disclosed herein, unless the context indicates otherwise. That is, RIFLE stage R, AKIN stage I, and KDIGO stage 1 may generally be interchangeable; RIFLE stage I, AKIN stage II, and KDIGO stage 2 may generally be interchangeable. RIFLE stage F, AKIN stage III, and KDIGO stage 3 may generally be interchangeable.

[0103] Although serial measurement of serum creatinine over several days is a common method of detecting and diagnosing AKI and is considered one of the most important tools for evaluating AKI patients, serum creatinine is generally considered to have some limitations in diagnosing, evaluating and monitoring AKI patients. The time for serum creatinine to rise to a value at which AKI is diagnosed (e.g., 0.3 mg / dL or 25% increase) can be 48 hours or more depending on the definition used. Because cellular injury in AKI can occur over several hours, serum creatinine elevation detected at or beyond 48 hours may be a late indicator of injury, and thus relying on serum creatinine may delay the diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of the exact renal status and need for treatment during the most acute phase of AKI when renal function is rapidly changing. Some patients with AKI recover completely, some require dialysis (either short-term or long-term), and some have other adverse outcomes including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration volume, it does not distinguish between causes of AKI (prerenal, intrinsic renal, postrenal obstructive, atheroembolic, etc.) or the category or location of injury in intrinsic renal disease (e.g., of tubular, glomerular, or interstitial origin). Urine output is similarly limited. Knowing these things can be crucial in the management and treatment of AKI patients.

[0104] As used herein, the term "CC motif chemokine 14" refers to the CC motif chemokine 14 precursor (human sequence: Swiss-Prot Q16627 (SEQ ID NO: 1)): MKISVAAIPF FLLITIALGT KTESSSRGPY HPSECCFTYT TYKIPRQRIM 50 DYYETNSQCS KPGIVFITKR GHSVCTNPSD KWVQDYIKDM KEN 93 refers to one or more polypeptides present in a biological sample derived from

[0105] The following domains have been identified in the CC motif chemokine 14:

[0106] [Table 2]

[0107] The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary diseases. Additionally, while the subject is preferably an organism, the invention described herein can also be used for post-mortem analysis. A preferred subject is a human, and most preferably a "patient," as used herein, refers to a living human undergoing medical care for a disease or condition. This includes individuals who are not clearly ill but are being investigated for signs of a medical condition.

[0108] Preferably, the analyte (e.g., CCL14) is measured in a sample. Such a sample may be obtained from a subject or may be obtained from a biological material intended to be provided to a subject. For example, a sample may be obtained from a kidney that is being evaluated for possible transplantation into a subject, and an analyte measurement may be obtained that is used to evaluate the kidney for existing damage. A preferred sample is a body fluid sample.

[0109] The term "body fluid sample" as used herein refers to a sample of body fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample can be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition, such as RRT. Preferred body fluid samples include blood (including whole blood, serum, and plasma), cerebrospinal fluid, urine, saliva, sputum, pleural effusion, hemofiltrate, and ultrafiltrate. Furthermore, those skilled in the art will understand that certain body fluid samples are more easily analyzed after a fractionation or purification procedure, such as separation of whole blood into serum or plasma components.

[0110] The term "about" when referring to a number or numerical range means that the number or numerical range referred to is approximate within experimental variation (or within statistical experimental error), such that the number or numerical range may vary, for example, by between 1% and 15% of the stated number or numerical range.

[0111] The term "correlating" as used herein with respect to the use of biomarkers refers to comparing the presence or amount of a biomarker in a patient with its presence or amount in individuals known to have or be at risk for a given condition; or individuals known to be free of a given condition. Often this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold value selected to be indicative of the occurrence or non-occurrence of a disease or the likelihood of some future outcome.

[0112] Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, the distribution of true and false diagnoses at different testing thresholds, and an estimate of the outcome of treatment (or treatment failure) based on the diagnosis. For example, when considering administering a particular treatment that is highly effective and has a low level of risk, the clinician can accept substantial diagnostic uncertainty, so little testing is required. On the other hand, in situations where treatment options are less effective and risky, the clinician often requires higher diagnostic certainty. Thus, a cost / benefit analysis is involved in the selection of a diagnostic threshold.

[0113] The appropriate threshold can be determined in various ways.For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5th percentile of the concentration found in normal population.Another method can be to examine consecutive samples from the same patient, where the previous "baseline" results are used to monitor the change in biomarker level over time.

[0114] Population studies can also be used to select decision thresholds. Receiver operating characteristics ("ROC") arise from the field of signal detection theory, developed during World War II for the analysis of radar images, and ROC analysis is often used to select thresholds that can best distinguish "diseased" from "non-diseased" subpopulations. A false positive in this case occurs when a person tests positive but does not actually have the disease. On the other hand, a false negative occurs when a person tests negative, suggesting they are healthy, but actually has the disease. To plot the ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is continuously varied. Since the TPR is equivalent to the sensitivity and the FPR is equal to 1-specificity, the ROC graph is sometimes called a sensitivity vs. (1-specificity) plot. A perfect test has an area under the ROC curve of 1.0; a random test has an area of ​​0.5. The thresholds are selected to provide acceptable levels of specificity and sensitivity.

[0115] In this context, "diseased" means a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome), and "non-diseased" means a population lacking that characteristic. Although a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with a relatively high degree of confidence, and above a second threshold, the presence of disease may also be assigned with a relatively high degree of confidence. Between the two thresholds may be considered indeterminate. This is meant to be merely exemplary in nature.

[0116] In addition to threshold comparisons, other methods for correlating assay results with patient classification (such as disease occurrence or nonoccurrence, likelihood of outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods, which can generate a probability value that represents the degree to which a subject belongs to one of multiple classifications.

[0117] Test accuracy measures are described in Fischer et al., Intensive Care Med. 29:1043-51, 2003, and are used to determine the validity of a given biomarker. These measures include sensitivity and specificity, predictive value, likelihood ratio, diagnostic odds ratio, and ROC curve area. The area under the curve ("AUC") of the ROC plot is equal to the probability that the classifier will rank a randomly selected positive instance higher than a randomly selected negative instance. The area under the ROC curve can be considered to be equivalent to the Mann-Whitney U test (which tests the median difference between the scores obtained in the two groups considered when the groups are continuous data), or the Wilcoxon rank test.

[0118] As mentioned above, a suitable test may show one or more of the following results for these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, even more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95, with a corresponding sensitivity of greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, even more preferably at least 0.5, even more preferably at least 0.6, even more preferably greater than 0.7, even more preferably greater than 0.8, more preferably greater than 0.9, and most preferably at least 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, even more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95, with a corresponding specificity of greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, even more preferably at least 0.5, even more preferably at least 0.6, even more preferably greater than 0.7, even more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of at least 75% combined with a specificity of at least 75%; an ROC curve area of ​​greater than 0.5, preferably at least 0.6, more preferably 0.7, even more preferably at least 0.75, even more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, even more preferably at least about 4 or more or about 0.25 or less, and most preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity / (1-specificity)) of greater than 1, at least 2, more preferably at least 3, even more preferably at least 5, and most preferably at least 10; and / or a negative likelihood ratio (calculated as (1-sensitivity) / specificity) of less than 1, less than 0.5, more preferably less than 0.3, and most preferably less than 0.1.

[0119] The compositions and methods of the present invention can be used by clinicians to determine treatment options for subjects determined to be at high risk of persistent AKI or at low risk of persistent AKI. Treatment options may vary from subject to subject, and treatments may be prioritized or deprioritized based on the clinical evaluation and other underlying symptoms of the individual subject. Non-limiting examples of treatment options for subjects at high risk of persistent AKI or at low risk of persistent AKI are disclosed in Chawla LS, et al., Nat Rev Nephrol. 2017; 13(4): 241-57; Kellum JA, Critical Care Med., 2015; 43(8): 1785-86; Kashani, K B., et al., Intensive Care Med. 2020; 46: 1036-38, and Kashani K, et al., Critical Care, 2013; 17(R25): 1-12, each of which is incorporated herein by reference in its entirety. The KDIGO Clinical Practice Guideline for Acute Kidney Injury. Kidney Inter., Suppl. 2012;2:8-12, incorporated herein by reference in its entirety, discloses treatments for the various stages of AKI. Treatments suggested therein for AKI and high risk of AKI stages 1, 2, and 3 include: 1. Discontinue all nephrotoxic medications if possible; 2. Ensure volume status and perfusion pressure; 3. Consider functional hemodynamic monitoring; 4. Monitor serum creatinine and urine output; 5. Avoid hyperglycemia; and 6. Consider alternatives to radiological contrast procedures.

[0120] Treatments proposed therein for AKI stages 1, 2 and 3 include: 1. Non-invasive diagnostic work-up; and 2. Consider an invasive diagnostic workup

[0121] Treatments proposed therein for AKI stages 2 and 3 include: 1. Check for changes in medication; 2. Consider renal replacement therapy (RRT); and 3. Consider ICU admission.

[0122] For AKI stage 3, it is also recommended to avoid subclavian catheters if possible.

[0123] Renal replacement therapy (RRT) is an option for the management of patients suffering from renal dysfunction, including AKI, persistent AKI, AKD, or CKD. RRT, as used herein, is interchangeable with renal replacement therapy (KRT) and includes kidney transplants as well as various types of dialysis. Dialysis filters and removes waste products, electrolytes, and water from the body, similar to the function of the kidneys. Multiple dialysis protocols are in use. The various types of dialysis are generally included in the categories of hemodialysis and peritoneal dialysis. Hemodialysis removes solutes from the blood by diffusion across an artificial membrane using a concentration gradient. Peritoneal dialysis, which uses the peritoneal membrane as a semipermeable membrane to remove solvents, is also used clinically. Unlike hemodialysis, which directly filters blood, peritoneal dialysis involves infusing fluid into the peritoneal cavity. The peritoneal membrane acts as a filter, and the fluid is then removed along with the accompanying waste products, electrolytes, and excess water. The timing of dialysis has been shown to be associated with patient outcomes. Reviewed by Pannu N. and Noel Gibney RTTher Clin Risk Manag. 2005;1(2):141-50, which is incorporated herein by reference in its entirety. More specific dialysis procedures include intermittent renal replacement therapy (IRRT) and continuous renal replacement therapy (CRRT). IRRT includes intermittent hemodialysis, intermittent hemofiltration, and intermittent hemodiafiltration. CRRT includes continuous hemofiltration and continuous hemodiafiltration. There is also a hybrid dialysis protocol called prolonged intermittent renal replacement therapy (PIRRT). These include continuous low-efficiency dialysis (SLED) and chronic dialysis (EDD). Some types can be performed at the subject's home or while traveling, but some require a clinical setting with the assistance of a medical professional.

[0124] In some embodiments, treatment options further include accelerating initiation of RRT or other treatment or procedures, referring the patient for further analysis to determine an appropriate therapeutic regimen, removing compounds known to be renal damaging, performing procedures known to be renal damaging with a delay of at least about 48 hours after obtaining the sample, altering and / or optimizing diuretic dosing, altering and / or optimizing dosing of renally cleared compounds, and / or administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels or hemodynamics.

[0125] In some cases, the subject may seek further analysis and / or treatment from a nephrologist or specialist. In one embodiment, further analysis includes performing additional tests. Non-limiting examples of analyses that may be performed include evaluation of urine sediment, proteinuria, and renal ultrasound; renal biopsy; therapeutic drug monitoring or optimization; hemodynamic monitoring and / or optimization; performing evaluation of AKI etiology for persistent AKI; further analysis to diagnose and / or treat rarer causes of AKI (interstitial nephritis, tumor lysis syndrome, thrombotic thrombocytopenic purpura, and cholesterol embolism syndrome). EXAMPLES

[0126] Example 1: Introduction In the Ruby study, we enrolled critically ill patients within 36 hours of clinical diagnosis of KDIGO (Kidney Disease Improving Global Outcomes) stage 2 (moderate) or 3 (severe) AKI. We reported on the performance of urinary CC motif chemokine ligand 14 (CCL14) for prediction of persistent (>=72 hours) KDIGO (Kidney Disease Improving Global Outcomes) stage 3 (severe) AKI. In this pre-planned secondary analysis of the Ruby study, we report the performance of a standardized clinical assay, the NEPHROCLEAR™ CCL14 test, along with the derivation and operating characteristics of two different cut-off values ​​on the CCL14 receiver operating characteristic (ROC) curve for prediction of persistent severe AKI. We selected one cut-off for high sensitivity and another cut-off for high specificity. We compared renal replacement therapy (RRT) or mortality across CCL14 strata defined by cutoffs over the first 90 days after enrollment. We also evaluated the CCL14 cutoffs together with clinical variables from the Ruby study to assess whether there was added value of the biomarker in clinical models. Finally, we examined the distribution of urinary CCL14 levels relative to the cutoffs in a reference population that included apparently healthy subjects and subjects with stable chronic comorbidities.

[0127] method subject Both the Ruby and reference population studies have been described previously (Hoste E, et al. Intensive Care Med, 2020; 46: 943-953 and Chindarkar NS, et al. Clinica Chimica Acta, 2016; 452: 32-37, both of which are incorporated herein by reference in their entirety). Ruby enrolled adult ICU patients with established KDIGO stage 2-3 AKI at 21 sites in Europe and the United States from June 2013 to May 2014. Patients were excluded if they had previously undergone a kidney transplant, were receiving or were in imminent need of RRT, were receiving palliative care only, or had a known infection with human immunodeficiency virus or active hepatitis. The reference population study enrolled two cohorts of adult subjects at six sites between April 2012 and November 2012: (A) apparently healthy subjects, and (B) subjects with pre-identified stable chronic conditions without acute illness. Protocols for both studies were approved by the Institutional Review Board or Ethics Committee as required by each participating site, and all subjects (or their surrogates) provided written informed consent.

[0128] Study Endpoints The primary endpoint of the Ruby study was persistent severe AKI, defined as KDIGO stage 3 AKI for at least 72 consecutive hours. Patients who received RRT or died before reaching 72 hours with stage 3 AKI were considered a positive endpoint. Patients with stage 2 AKI at enrollment who received RRT or started RRT within 48 hours and progressed to persistent severe AKI were also considered a positive endpoint. Reference serum creatinine was determined by expert adjudication blinded to biomarker results, as previously described. Secondary endpoints included RRT initiation, death, and a combination of RRT initiation or death within 90 days.

[0129] Sample collection and testing In both the Ruby and Reference cohorts, urine samples were collected at enrollment and centrifuged. Supernatants were flash frozen within 2 hours of collection and stored at ≦−70° C. Thawed samples were analyzed for CCL14 using the NEPHROCLEAR™ CCL14 Test on an Astute140® Meter (Astute Medical, San Diego, Calif.) by an operator blinded to clinical data. Concentration results of the NEPHROCLEAR™ CCL14 Test are traceable to a reference material containing a defined mass of CCL14 protein according to EN ISO 17511.

[0130] Cutoff Selection Two cutoffs on the urinary CCL14 ROC curve were selected for risk assessment of the development of persistent severe AKI. A low CCL14 concentration cutoff was selected to achieve high sensitivity with reasonable specificity, allowing early recognition of the majority of patients who subsequently developed persistent severe AKI. This cutoff was selected to identify patients who would be candidates for measures recommended as high priority in the KDIGO Clinical Practice Guideline as an increased risk of adverse outcomes of AKI. The types of actions considered in determining the appropriate balance between sensitivity and specificity included consultation from specialists (nephrology, pharmacy, intensive care, etc.), additional diagnostic workup, and prioritized assessment of the need for higher intensity monitoring (including hemodynamic / fluid monitoring and possible ICU admission) or renal replacement therapy. In selecting the cutoffs, we assumed that the test would not be used in an independent manner as the sole basis for initiating invasive procedures, which should only be ordered after careful consideration of all clinical and laboratory information to ensure that the potential benefits outweigh the risks. A second, higher concentration cutoff was selected to allow for the identification of patients who will develop persistent severe AKI with high specificity. This high specificity cutoff was selected to identify the subgroup of patients at highest risk for persistent severe AKI and therefore most urgently in need of evaluation to undergo measures recommended in the KDIGO Clinical Practice Guideline. Importantly, values ​​between these cutoffs are not "indeterminate," nor do values ​​in this range represent a "gray zone." Instead, this portion of the ROC curve reflects a transition from high sensitivity to high specificity.

[0131] statistical analysis To evaluate clinical performance at different cutoffs, operating characteristics were calculated, including sensitivity, specificity, negative and positive predictive values, and negative and positive likelihood ratios for the primary endpoint across the range of CCL14 concentrations. Differences in CCL14 concentrations between healthy subjects, subjects with chronic conditions, and Ruby subjects who did and did not develop the primary endpoint were presented in box and whisker plots. The Kruskal-Wallis test was used to detect differences in concentrations between the four groups. The risk and relative risk of developing sustained severe AKI were calculated by dividing the cohort by the cutoff. The reference level for relative risk consisted of patients with CCL14 concentrations below the lower (high sensitivity) cutoff. The Cochran-Amitage test was used to determine trends across risk strata defined by the cutoffs. Cumulative incidence curves for RRT initiation within 90 days, death, and the combination of RRT initiation or death were estimated using the Kaplan-Meier method, and the log-rank test was used to compare groups defined by the two cutoffs. To examine whether urinary CCL14 stratified by the two cutoffs improved risk prediction beyond clinical variables alone, a reference logistic regression model was constructed as described previously. When CCL14 was analyzed as a categorical variable in the regression analysis, CCL14 ≤ 1.3 ng / mL (lower limit) was used as the reference level. The integrated discrimination improvement (IDI) and category-free net reclassification (cfNRT) were used to evaluate the enhancement of risk prediction by CCL14. Continuous, dichotomous, and polytomous baseline variables were compared by CCL14 stratification by Kruskal-Wallis, Cochran-Amitage, and Fisher's exact tests, respectively. Confidence intervals for sensitivity, specificity, positive predictive value, and negative predictive value were calculated by the Clopper-Pearson exact method, and confidence intervals for positive likelihood ratio, risk, and relative risk were calculated by the asymptotic method (normal approximation). A two-sided p value of less than 0.05 was considered statistically significant. Statistical analyses were performed using R 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria), and IDI and cfNRI were calculated using the “Hmisc” package.

[0132] result A cutoff value of 1.3 ng / ml urinary CCL14 was determined to achieve high sensitivity (91% (95% Cl: 84%-96%)), and a cutoff of 13 ng / ml was found to achieve high specificity (93% (89%-96%)), with the range in between reflecting the transition from high sensitivity to high specificity. Baseline characteristics of all Ruby study patients at enrollment are shown in Table 1, stratified by the two cutoffs (≦1.3, 1.3-13, and >13 ng / mL). Patients with higher CCL14 levels (>13 ng / ml) were less likely to have a history of coronary artery disease but were more likely to be admitted to the ICU for respiratory failure or sepsis. 211 (62.9%) patients had CCL14 >1.3 ng / ml and 54 (16.1%) had values ​​>13 ng / ml. Although there were no differences across CCL14 strata in retrospectively adjudicated prehospital baseline serum creatinine, patients with CCL14 concentrations >13 ng / mL had significantly higher serum creatinine at study enrollment compared with patients with CCL14 ≤1.3 ng / mL (p<0.001 between CCL14 ≤1.3 and CCL14>13). Using the aforementioned retrospective adjudication of baseline serum creatinine, 55 patients (16.4% of the total cohort) did not meet criteria for stage 2-3 AKI at enrollment. Patients with higher CCL14 levels had more severe AKI at study enrollment (p<0.001). Table 2 provides baseline characteristics of the cohort dichotomized by CCL14 concentrations higher or lower than 1.3 ng / mL.

[0133] Enrollment serum creatinine (SCr) was higher in those with elevated CCL14 concentrations, providing an AUC of 0.81 for the primary endpoint of persistent severe AKI. Adding urinary CCL14 to enrollment SCr as a categorical variable stratified by the two cutoffs significantly increased the AUC to 0.85 (p=0.02). We also compared the performance of 24-hour urine output before enrollment with CCL14 for predicting the primary outcome. Tables 3A and 3B demonstrate the poor performance of urine output (AUC=0.63) to predict the primary outcome as well as its non-significant odds ratio (95% CI) in the multivariate model 0.76 (0.57-1.00): P=0.053. A low cutoff of 1.3 ng / mL was chosen to achieve a high sensitivity (91%), i.e., to identify most of the subjects who would progress to persistent severe AKI. Similarly, a high cutoff of 13 ng / mL was selected to achieve high specificity (93%), ie, to identify most subjects who would not progress to sustained severe AKI.

[0134] Operating characteristics from the Ruby study for the two cutoffs and the location of the cutoffs for the distribution of urinary CCL14 levels in the Ruby study and reference population cohorts are shown in Figure 2. These cohorts were healthy (378), chronically ill without acute disease (366), Ruby did not develop persistent severe AKI (225), and Ruby developed persistent severe AKI (110). The lower and upper whiskers in the figure represent the 10th and 90th percentiles of CCL14 concentrations in that group, respectively. The lower and upper boxes represent the 1st and 3rd quartiles, respectively. The middle bar is the median. The horizontal dashed lines correspond to cutoffs of 1.3 ng / mL and 13 ng / mL. P value < 0.0001 calculated using the Kruskal-Wallis test. The cutoff at 13 ng / mL is higher (with a negative predictive value of 75%) than the majority (93%) of CCL14 values ​​from the Ruby study patients who did not develop sustained severe AKI and has a high positive predictive value of 72%. Both cutoffs substantially exceed the urinary CCL14 range for the apparently healthy, stable chronic comorbidity reference cohort. Depending on clinical need, different cutoffs can be selected to achieve different balances between sensitivity and specificity. For example, Table 4 provides operating characteristics for urinary CCL14 values ​​of 0.2 to 30 ng / mL in the Ruby study population.

[0135] Figure 3 shows the risk of developing persistent severe AKI across three strata of CCL14 levels defined by two cutoffs. Within each CCL14 stratum, the individual components of the composite endpoint are displayed. Endpoint components are shown based on the first criterion met (e.g., serum creatinine or urine output). Endpoints were confirmed within 5 days of enrollment. Shading in each stratum indicates the relative contribution of patients who met the persistent severe AKI composite endpoint by initiating renal replacement therapy (RRT), death, or persistently (>72 hours) elevated serum creatinine or oliguria. The risk of the composite endpoint increased significantly with increasing CCL14 levels, stratifying the individual components, RRT and persistently elevated serum creatinine or oliguria, similarly (p<0.0001 for both). The increased risk of death as a component of the primary endpoint was not statistically significant (p=0.13). Patients with CCL14 concentrations between 1.3 ng / mL and 13 ng / mL were 4.8 (2.6-9.0) times more likely to develop persistent severe AKI compared with patients with CCL14 ≤ 1.3, whereas patients with CCL14 > 13 ng / mL were 9.0 (4.8-17) times more likely to develop persistent severe AKI. The risk of developing persistent severe AKI across two strata of CCL14 levels defined by a 1.3 ng / mL cutoff is shown in Figure 4. With reference to Figure 4, within each CCL14 stratum, the individual components of the composite endpoint are displayed. Endpoint components are shown based on the first criterion met (e.g., serum creatinine or urine output). Endpoints were confirmed within 5 days of enrollment.

[0136] Figures 5A-5C show the individual and combined rates of RRT and death in the cohort across CCL14 strata over the first 90 days after enrollment. In the graphs shown in Figures 5A, 5B, and 5C, the number of patients with CCL14 concentrations below, between, and above 1.3 ng / mL-13 ng / mL are 124, 157, and 54, respectively. A log-rank test for trend was used to calculate p-values ​​for differences between strata. Higher CCL14 values ​​were associated with increased risk of both death and RRT as well as the combined of the two (log-rank p<0.001). These findings persisted when the cohort was dichotomized at 1.3 ng / mL CCL14 (Figures 6A-6C). Referring to the data shown in Figures 6A, 6B, and 6C, the number of patients with CCL14 concentrations below and above 1.3 ng / mL are 124 and 211, respectively. The log-rank test was used to calculate p-values ​​for differences between strata. Table 5 shows the median time (days from enrollment) until subjects received RRT or died.

[0137] To evaluate the biomarker cutoffs compared to clinical variables, we performed a multivariate logistic regression analysis with persistent AKI as the endpoint with and without CCL14 as a covariate (Table 6). The results demonstrate that CCL14 concentrations were significant even after accounting for clinical variables previously shown to be associated with persistent AKI. Using IDI and cfNRI analysis, we found that the use of the two cutoffs added value to the clinical model (Table 7). Adding these CCL14 concentrations to the model significantly increased the AUC (95% CI) from 0.86 (0.82-0.90) to 0.88 (0.85-0.92) (p=0.02).

[0138] Finally, as mentioned above, we retrospectively adjudicated baseline creatinine for all patients. Using this retrospective baseline creatinine to determine AKI stage, 55 patients were determined not to have stage 2 or 3 AKI at enrollment. Thirty-seven of these 55 patients (67%) had stage 2-3 AKI within 24 hours prior to enrollment, but their AKI stage improved. Sensitivity analyses were performed excluding these 55 patients. Table 8 demonstrates that excluding these patients did not significantly change the odds ratios for any strata of CCL14 concentration (compared to Table 6). Furthermore, Figure 7 shows the operating characteristics of subjects adjudicated to have stage 2 or 3 at enrollment for the two cutoffs and the location of the cutoffs for the distribution of urinary CCL14 levels in this cohort and the reference population cohort. The operating characteristics remain similar to those shown in the intention-to-diagnose (ITD) cohort (Figure 2). The cohorts shown in Figure 7 are healthy (378), chronically symptomatic without acute disease (366), the Ruby vs. Diagnosis (ITD) complete cohort (335 patients, of which 225 did not develop persistent severe AKI), and those adjudicated to have stage 2 or 3 at enrollment (280 patients, of which 170 did not develop persistent severe AKI). The lower and upper whiskers represent the 10th and 90th percentiles of CCL14 concentrations for that group, respectively. The lower and upper boxes represent the 1st and 3rd quartiles, respectively. The middle bar is the median. The horizontal dashed lines correspond to cutoffs of 1.3 ng / mL and 13 ng / mL. P-values ​​<0.0001 calculated using the Kruskal-Wallis test.

[0139] Consideration We derived and characterized two cutoffs for urinary CCL14 using a standardized clinical assay to aid in clinical risk assessment of the development of persistent severe AKI. Assay standardization is essential for clinical implementation and is required prior to specifying cutoffs. These cutoffs were determined by optimizing the operating characteristics (sensitivity, specificity, negative and positive predictive values) based on the intended clinical use of the NEPHROCLEAR™ CCL14 test. Using a high sensitivity cutoff at 1.3 ng / ml CCL14, the majority of patients who will develop persistent severe AKI can be identified, and therefore, patients are candidates for treatments recommended as high priority in the KDIGO Clinical Practice Guideline as at increased risk for adverse outcomes of AKI. Using a high specificity cutoff of 13 ng / ml, the highest risk patients who are most urgently in need of evaluation for further intervention can be identified. The clinical relevance of these two cutoffs is demonstrated by their ability to stratify risk of adverse events (RRT or death) over 90 days and add significant predictive information beyond that clinically available. Importantly, although CCL14 has already been validated as a biomarker for persistent AKI, this is the first report to validate specific cutoffs using a standardized CCL14 assay developed for routine clinical use. This study represents an important step in the framework of the clinical utility of CCL14 to identify those at risk for persistent severe AKI.

[0140] We have demonstrated that urinary CCL14 provides significant information regarding the likelihood of sustained AKI that cannot be inferred from existing laboratory tests such as serum creatinine or clinical variables alone. In our multivariate model (Table 6), CCL14 remained statistically significant, with elevated values ​​associated with 10.4-fold increased odds of sustained AKI. Similarly, IDI and cfNRI were both statistically significant (p<0.001 for both) (Table 7). Thus, the test provides important new information that can be used in conjunction with existing laboratory tests and clinical evaluations to provide the most comprehensive view of a patient's acute renal status and likely course without further intervention. The high negative predictive value (92%) at the cutoff of 1.3 ng / mL ensures that nearly all patients with a negative test will not develop sustained AKI. Furthermore, values ​​above 1.3 ng / mL are associated with a risk of nearly 1 in 2 of developing sustained AKI (positive predictive value (PPV) 48%). At a higher cutoff (>13 ng / mL), the PPV further increased to 72%, which was consistent with the PPV performance of other biomarkers such as B-type native uric acid peptide (100 pg / mL had a PPV of 79% for acute heart failure) and high-sensitivity troponin I (>30 pg / mL had a PPV of 75% for acute coronary syndrome).

[0141] The ability to detect persistence early in the course of severe AKI could have a major clinical impact on the care of critically ill patients. A growing body of literature shows that patients with established AKI do not receive guideline-based care. In multiple large retrospective single-center cohorts and one multicenter international cohort, patients with known KDIGO AKI continued to receive nephrotoxins (including NS AIDS and aminoglycosides) and experienced persistent hypotension (e.g., mean arterial pressure <55 mmHg) even in the setting of established stage 2 AKI. One of the most significant barriers to delivering these recommended clinical actions is that most AKI resolves spontaneously or with early clinical management, and it is very difficult to know which patients will require discontinuation of nephrotoxic drugs or more intensive evaluation of hemodynamic / volume status. We state that a tool such as CCL14, which can better predict persistent severe AKI, could serve as a tool to determine which patients will require additional renal intensive care beyond early management. It is reasonable to assume that patients destined for persistent severe AKI will likely benefit from strict adherence to the renal care bundle. For example, consider an ICU patient who develops stage 1 AKI. The clinician reviews their medication list and assesses their volume status. They determine that the patient needs additional boluses of balanced fluids, adjustments to vancomycin dosing based on therapeutic monitoring, and changing piperacillin-tazobactam to cefepime. Now, when considering whether this patient will progress to stage 2 AKI, we see that the change in serum creatinine often reflects damage that has already occurred. Thus, the clinician is uncertain as to whether additional management changes are necessary. If CCL14 is measured and is >1.3ng / ml, it may be appropriate to consider more aggressive clinical maneuvers (more intensive hemodynamic monitoring, stopping / changing other medications, or consulting a nephrologist). However, if CCL14 is ≤1.3ng / ml, the clinician may be inclined to continue the current management and not make further interventions.

[0142] Another clinical use of CCL14 could be around the initiation of RRT in the ICU. Ruby cohort CCL14 results stratify patients regarding initiation of short-term (Figure 3) or longer-term (Figures 5A-5C) RRT. Clinical testing alone is not expected to indicate which patients should or should not receive RRT, but CCL14 can identify patients for whom additional clinical evaluation is appropriate in conjunction with medical history, physical exam, fluid balance, blood chemistry, etc.

[0143] In summary, we have validated a standardized clinical assay for CCL14, the NEPHROCLEAR™ CCL14 test, and used this test to derive and characterize cutoffs for CCL14, allowing for the identification of elevated patients at high risk for the development of severe, persistent AKI. These results are of immediate value in helping to guide patient care and can facilitate future clinical trials.

[0144] [Table 3]

[0145] [Table 4]

[0146] [Table 5]

[0147] [Table 6]

[0148] [Table 7]

[0149] [Table 8]

[0150] [Table 9]

[0151] [Table 10]

[0152] [Table 11]

[0153] [Table 12]

[0154] [Table 13]

[0155] For categorical CCL14 variables, CCL14 ≤ 1.3 ng / mL was the reference level

[0156] All numerical variables were standardized by subtracting the mean and dividing by the standard deviation; N = 312 (34% persistent). Clinical variables in the reference model were selected based on their association with persistent severe AKI as previously described. 9

[0157] [Table 14]

[0158] The clinical variables in the reference model were body mass index, extrarenal APACHE III score, serum creatinine trajectory, KDIGO stage at enrollment, and diabetes.

[0159] [Table 15]

[0160] For categorical CCL14 variables, CCL14 ≤ 1.3 ng / mL was the reference level

[0161] All numerical variables were standardized by subtracting the mean and dividing by the standard deviation; N = 260 (40% persistent). Clinical variables in the reference model were selected based on their association with persistent severe AKI as previously described. 9

[0162] Example 2: method Research Background We performed a preplanned analysis of serial measurements of urinary CCL14 early after the onset of moderate to severe acute kidney injury in a mixed population of critically ill adults. The analytical cohort consisted of a pooled cohort of patients from the Ruby and Sapphire studies.

[0163] subject Ruby enrolled subjects as described in Example 1 herein. Sapphire enrolled critically ill adult patients with cardiac or respiratory dysfunction without known stage 2-3 AKI at the time of enrollment from 35 centers across Europe and North America (summarized in Figure 1). (Kashani K, et al., Critical Care, 2013;17(R25):1-12). Samples from the subset of Sapphire patients who developed stage 2-3 AKI within 1 week of enrollment were included in the present analysis. For the purposes of this analysis, in the Sapphire cohort, serial biomarker measurements were included from the time the patient met stage 2-3 AKI criteria, mirroring the time course of enrollment and biomarker measurements in the Ruby study. For both studies, patients who did not have CCL14 concentrations for all three samples or who underwent RRT or died before the third sample were excluded from the primary analysis. Patients were recruited based on AKI assessment by local investigators using creatinine and urine output criteria, and all patients enrolled were included on an intention-to-diagnoses basis.

[0164] Sample collection and testing In the Ruby study, urine samples were collected twice daily for 3 days from enrollment, then once daily for 4 days. In the Sapphire study, urine samples were collected twice daily for 4 days from enrollment, then once daily for 3 days. In both studies, urine samples were centrifuged, flash frozen, stored at -70°C or below, and thawed prior to sample testing. The primary analysis utilized CCL14 concentrations from the first 3 scheduled urine collections in Ruby and the first 3 urine collections after the onset of KDIGO stage 2 or 3 AKI in Sapphire for each patient. Technicians blinded to the clinical data measured CCL14 concentrations in the samples using the NEPHROCLEAR™ CCL14 Test on an Astute 140® Meter (Astute Medical Inc., San Diego, CA).

[0165] Clinical Endpoints The primary endpoint of this analysis was the development of sustained severe AKI, as previously described. Briefly, patients who developed stage 3 AKI for 72 consecutive hours starting within 48 hours of first sample collection, or who died after stage 3 AKI or received RRT before 48 hours of first sample collection, or who received RRT within 72 hours of stage 3 AKI, were considered to have a positive endpoint. For the purpose of determining the primary endpoint, the baseline serum creatinine of each patient was determined as described in the relevant study.

[0166] Research Question We hypothesized that urinary CCL14, as a biomarker of underlying renal inflammation during tissue injury and repair, would be informative about the clinical course of AKI. Therefore, we sought to characterize the distribution of CCL14 levels over time and the clinical correlates of various patterns.

[0167] statistical analysis We used previously determined clinical risk cutoffs (Koyner, JL, Kidney360.2022 Mar 24;3(7):1158-1168) to stratify CCL14 concentrations in the first three urine collections into three levels: low (≦1.3ng / mL), intermediate (>1.3 to ≦13ng / mL), and high (>13ng / mL). A high sensitivity cutoff at 1.3ng / mL CCL14 was derived to identify the majority of patients at risk for sustained severe AKI, and a high specificity cutoff at 13ng / mL was derived to identify the highest risk patients most likely to have PS-AKI. To evaluate the validity of using the pooled dataset, we used the Cochran Q test to assess the heterogeneity of relative (RR) risk between individual time points across Ruby and Sapphire. Patients in the pooled analysis cohort were then grouped by the pattern of CCL14 levels across the three samples. Trajectories of CCL14 levels when compared based on the proportion of patients positive for sustained severe AKI within each group. Continuous and categorical variables were analyzed using Wilcoxon rank sum and Fisher's exact tests, respectively. A two-sided p-value <0.05 was considered statistically significant. All analyses were performed using R 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria).

[0168] Sensitivity analysis Our primary analysis was based on having a complete set of three CCL14 measurements over a 24-hour period to allow for assessment of true trajectories rather than point measurements, as missing CCL14 values ​​may not have been missing at random. However, we also performed a sensitivity analysis including all individuals with one or more CCL14 measurements after enrollment to assess the consistency of our findings in this population. In a second sensitivity analysis, we explored potential differences in our main conclusions using alternative pairs of cutoff values ​​that differed from the sensitivity and specificity of 1.3 and 13 ng / mL.

[0169] CCL-14 extension trajectory CCL14 measurements in urine samples collected at least daily allowed for evaluation of changes in CCL14 trajectories over longer periods of time and their relationship to outcomes at day 7. We performed rolling assessments of the PS-AKI composite endpoint over time - i.e., at each measurement time point, we assessed the occurrence of 72-hour persistent stage 3 AKI starting within 48 hours or death or RRT at 48 hours of the measurement. The ability of urinary CCL14 (as a continuous variable) at any time point to discriminate the rolling endpoint was evaluated as the area under the receiver operating curve (ROC-AUC) at 95% confidence determined by the DeLong method. In sensitivity analyses including patients with missing CCL14 values, the association between changes in CCL14 stratified by baseline was essentially unchanged apart from a worsening overall outcome with "unchanged" values ​​reflecting the inclusion of patients whose measurements were truncated by death or need for RRT (Figure 9), with a similar pattern of associations in logistic regression (Table 12).

[0170] result patient The Ruby study recruited 364 patients within 36 hours of stage 2-3 diagnosis, whereas of the 723 critically ill patients without AKI enrolled in Sapphire, 212 developed stage 2-3 AKI and were eligible for inclusion in this pooled analysis. After further excluding patients without three consecutive measurements of urinary CCL14 after fulfilling the enrollment criteria, a total of 417 patients, including 268 patients from the Ruby study and 149 patients from Sapphire, were included in our primary analysis (Figure 1). In addition, an additional 111 patients had one or two valid urine measurements within 36 hours of enrollment and were included in the sensitivity analysis of 528 patients. Of the pooled population, 75 (18%) reached the primary endpoint of PS-AKI (sustained stage 3 AKI at 72 hours or death before recovery of RRT or renal function), whereas in sensitivity analysis, 135 (26%) reached the endpoint, often reflecting the effect of early death or anuria that prevented collection of a complete set of study samples. In the Sapphire cohort, only 7.4% of patients reached the primary endpoint compared with 24% in Ruby (Table 13), reflecting a reduction in the overall severity of AKI in the Sapphire cohort, with 30% vs. 4% having AKI stage 3 at the time of first sample collection. Conversely, the Sapphire cohort had higher extrarenal APACHE-2 scores (62 vs. 52) and use of mechanical ventilation (85 vs. 54%). In the pooled primary data set, the median age was 65 years and 59% were male (Table 13). Baseline renal function, demographics, disease severity, and organ support were not significantly different between patients with and without PS-AKI endpoints, but fluid balance on day 1 after enrollment was significantly more positive in patients who developed PS-AKI (2.3 L vs. 3.4 L, p=0.04). Overall, despite differences between studies, relative risk analysis showed similar behavior across CCL14 strata at each time point justifying pooled cohort analysis (Table 9).

[0171] CCL14 trajectories at day 1 and primary composite endpoint Initial CCL14 levels were low (≦1.3 ng / ml) in 196 patients (47%), intermediate (>1.3 to ≦13 ng / ml) in 180 patients (43%), and high (>13 ng / ml) in 41 patients (9.8%) (Table 14). Initial CCL14 category correlated strongly with the primary endpoint. Importantly, the association of CCL14 category with PS-AKI outcome was essentially unchanged at 12 and 24 hours. In line with this observation, in the majority of cases (66%), the CCL14 category remained unchanged at 0 to 24 hours, with a change of 2 levels occurring in only one patient (Figure 8, Table 10). The graph in Figure 8 shows the change from the initial CCL14 category and the risk of sustained severe AKI. When changes occurred, they were generally consistent in direction, with only 24 / 417 patients (6%) showing a fluctuating course in the CCL14 category (Table 10). In patients experiencing a change in CCL14 category, we classified patients as CCL14 category decreasing or increasing between 0 and 24 hours and stratified these by initial CCL14 level. Across all initial categories, changes in CCL14 category were associated with a corresponding change in risk of PS-AKI (Figure 8). We evaluated the additional information provided by changes in CCL14 levels, accounting for initial values ​​in multivariate logistic regression. Considering the initial CCL14 category, a decrease in CCL14 category in the first 24 hours was associated with a decreased risk of sustained severe AKI, odds ratio (OR) 0.2 (95% CI 0.08-0.45, p<0.001). An increase in category was associated with an increased risk, OR 4.04 (1.75-9.46, p=0.001). There was no significant interaction between initial status and change when tested (p=0.36 for the interaction term) - Table 15.

[0172] Sensitivity analysis In sensitivity analyses including patients with missing CCL14 values, associations between changes in CCL14 stratified by baseline were essentially unchanged apart from the overall worse outcome for "no change" values ​​reflecting the inclusion of patients whose measurements were truncated by death or need for RRT (Figure 9), with a similar pattern of associations in logistic regression (Table 12).

[0173] In the second sensitivity analysis using alternative cut-off values, two additional cut-offs above and below 1.3 and 13 were considered, resulting in a total of six cut-offs and nine different combinations of low and high cut-offs (Table 16). Additional cut-off values ​​of 0.8, 1.8, 9 and 17 were selected based on their sensitivity and specificity relative to the original predefined cut-offs of 1.3 and 13 ng / mL (Table 4 in Example 1). Different combinations of cut-offs resulted in CCL14 categories that were strongly correlated with persistent severe AKI endpoints, similar to those observed with the original cut-offs of 1.3 and 13 ng / mL (compare Tables 14 and 17). Moreover, odds ratios in multivariate logistic regression of PS-AKI endpoints showed a similar pattern, where both initial and change in CCL14 categories were statistically significant independent predictors of the endpoint across all combinations of cut-offs (compare Tables 15 and 18). Finally, a trend for increasing risk of the primary endpoint with increasing change in CCL14 category within the initial CCL14 category can be observed across all combinations of low and high cutoff values ​​(compare Figure 8 and Table 19).

[0174] Extended CCL14 locus Urinary CCL14 measurements were performed at least daily until 7 days after enrollment in surviving patients remaining in the ICU with urinary catheter and without RRT. At 36 hours, 355 of the original 417 patients had available measurements, which gradually declined to 148 by 144 hours. Compared to the original values, over time, CCL14 values ​​tended to decrease rather than increase (Figure 10), with 67% of patients having low levels of CCL14 as their last measurement. Referring to the graph in Figure 11, ROC-AUC is shown for each time point with 95% CI for CCL14 over the study period. Endpoint assessment was based on clinical data collected over a period of up to 7 days, with carry-forward of the last observation used to imput right-censored data. Rolling assessment of the ability to predict the PS-AKI composite endpoint starting within 48 hours demonstrated a sustained ability to discriminate the endpoints well with ROCAUC>0.8 up to the 144 hour time point (Figure 11).

[0175] Consideration We demonstrate key clinical features of the CCL14 assay, guiding clinicians in the interpretation of this assay and adding further confidence to its interpretation in real-world clinical situations. Importantly, we demonstrate that in 2 / 3 of patients with moderate-severe AKI, CCL14 levels remained stable over 24 hours, and when changes occurred, this indicated a corresponding change in risk level for persistent severe AKI. These findings have two important clinical implications. First, even if measurement of CCL14 is delayed after the initial diagnosis of stage 2-3 AKI, the results may provide a prognosis of the clinical course of AKI at least as good as that provided by the original clinical trial from which the assay was derived and validated. In other words, CCL14 does not represent a transient injury or stress signal that declines rapidly during severe injury, but is an indicator of newly arising but sustained biological processes within the severely injured kidney. Second, serial measurements of CCL14 may provide complementary clinical information to modify the assessment of risk over time by reflecting potentially modifiable pathologies that lead to changes in prognosis over time. In particular, for patients with intermediate CCL14 measurements, entering the low category was associated with a similar decrease in risk as an initial low measurement, and conversely, an increase to high was associated with a larger increase in risk. Of note, while CCL14 levels remained stable overall, 45% of those with initial median values ​​experienced a category shift within the first 24 hours. Thus, for patients with initial measurements in the median risk category, serial measurements provide an opportunity to update risk assessment with relatively frequent and clinically significant changes. Conversely, for patients with initial CCL14 in the high-risk category, a change to the low-risk category was associated with a clear decrease in risk but still associated with a >50% incidence of the primary endpoint, suggesting that "watch and wait" is of little value in the setting of a high-category outcome.Finally, CCL14 appears to retain its ability to discriminate composite endpoints over a 6-day period following initial moderate-severe AKI diagnosis, suggesting that CCL14 is potentially a flexible tool that can be used to assess underlying renal pathophysiology throughout the time course of AKI over a 1-week period from initial diagnosis. Thus, this study represents an important contribution towards defining the clinical utility and implementation of the CCL14 assay to identify those at risk for sustained severe AKI, giving clinicians confidence in interpreting AKI over time.

[0176] Persistent severe AKI is a critical clinical event as these patients are at highest risk for adverse cardiovascular and renal outcomes, whereas transient self-limiting AKI has a much (although not entirely) benign prognosis. Several distinct recovery phenotypes have been identified after an episode of stage 2 or 3 AKI; however, patients with recurrent and / or unrecovered AKI have the highest risk of 1-year mortality (up to 45%). Similarly, among patients with septic shock, AKI that persists for >48 hours and has not recovered at discharge is associated with a more than five-fold risk of death by 1 year after adjusting for disease severity and demographic differences. The same association has recently been observed in the context of COVID-19-associated AKI, where persistent AKI after one week was associated with the highest odds ratio of death of 7.6 compared with no AKI. CCL14 fills a diagnostic gap by providing important information about the clinical course of AKI, which is a key factor in determining overall clinical outcome. Importantly, this information is not the progression of renal tubular injury in AKI, and cannot be readily derived from existing AKI criteria, nor from biomarkers directed at the onset of AKI development.

[0177] The ability to classify patients as high or low risk for sustained severe AKI, RRT, or death has the potential to significantly improve patient care. This includes important aspects of routine care such as avoidance of nephrotoxic drugs and assessment of administration of renally excreted drugs. Furthermore, application of guideline-based AKI care is often hindered by insufficient prognostic information and therefore often not delivered in practice. For example, interventions appropriate for early AKI, e.g., aggressive hemodynamic resuscitation, may be futile or even counterproductive in the setting of established AKI, leading to clinical uncertainty regarding treatment selection. Such uncertainty regarding the risk-benefit of interventions may explain the failure of many intervention trials in AKI to improve outcomes. A key feature of any successful intervention study in the ICU is a pragmatic design that ensures that any intervention can be applied to an easily identified subpopulation of patients likely to benefit in a real-world clinical setting. In this context, the ability of CCL14 to provide consistent clinical information over time greatly facilitates study design and inclusion. Similarly, when used as a clinical tool, CCL14 can be similarly applied to a wide range of patients over the time course of AKI. Such uses may include prognostication of the need for RRT in the ICU, where traditional AKI diagnostic criteria and early AKI biomarkers fail to identify patients who would benefit from preemptive RRT initiation.

[0178] In conclusion, we demonstrate the consistency of CCL14 to identify patients at high risk of developing severe persistent AKI across serial sampling and show where changes occur and where these parallel in clinical outcomes. Thus, CCL14 is likely to be a useful clinical tool that can be flexibly implemented in the prognosis of patients with moderate-to-severe AKI in real-world settings and potentially monitored over time. These results can help guide patient care and facilitate future practical clinical trials.

[0179] [Table 16]

[0180] [Table 17]

[0181] [Table 18]

[0182] There was no statistically significant interaction between initial CCL14 values ​​and the rate of concentration change (slope), so the interaction term was removed from the model. Initial CCL14 values ​​were log-transformed. Rates of concentration change were transformed using the Box-Cox method with l=-2.

[0183] Both initial CCL14 levels and rate of change in levels in the new model are statistically significant (p<0.001). Furthermore, the discriminatory performance of both models for the endpoints measured by ROC AUC was similar, 0.849 for the model with categorical CCL14 variables versus 0.852 for the model with continuous CCL14 variables (p=0.78 for difference in AUC). These results indicate that both initial CCL14 levels and change in CCL14 levels are significant predictors of sustained severe AKI, both when CCL14 is classified using cutoffs and when CCL14 is treated as a continuous variable. Distribution of concentration change rates (linear slope) for the three CCL14 values ​​stratified by initial CCL14 category: +*

[0184] [Table 19]

[0185] [Table 20]

[0186] [Table 21]

[0187]

Table 22

[0188]

Table 23

[0189]

Table 24

[0190]

Table 25

[0191]

Table 26

[0192]

Table 27

[0193]

Table 28

[0194]

Table 29

[0195]

Table 30

[0196] Although the present invention has been described and illustrated in sufficient detail for one skilled in the art to make and use it, various alternatives, modifications, and improvements should become apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are illustrative, and are not intended to limit the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are within the spirit of the invention and are defined by the scope of the claims.

[0197] It will be readily apparent to those skilled in the art that varying substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0198] All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which this invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0199] The present invention illustratively described herein may suitably be practiced in the absence of any one or more elements or one or more limitations not specifically disclosed herein. Thus, for example, in each example herein, any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with any of the other two terms. The terms and expressions used are used as terms of description and not of limitation, and in the use of such terms and expressions, it is not intended to exclude the features shown and described or equivalents of any portion thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, although the present invention has been specifically disclosed by preferred embodiments and optional features, it is to be understood that modifications and variations of the concepts disclosed herein may be used by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0200] Other aspects are within the scope of the following claims.

Claims

1. 1. A method for assessing an increased risk of developing sustained acute kidney injury (AKI) in a subject, the method comprising: (a) performing an assay to detect levels of C-C motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, wherein at least a second urine sample is obtained from the subject within about 72 hours after a first urine sample is obtained; (b) determining that the subject is at increased risk for persistent AKI based on an increasing trend in the level of CCL14 detected in the urine sample; A method comprising:

2. (a) performing an assay to detect levels of C-C motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, wherein at least a second urine sample is obtained from the subject within about 72 hours after a first urine sample is obtained; (b) determining whether the level of CCL14 detected in the urine sample is low, medium, or high based on a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14; Where: (i) the level of CCL14 in the urine sample is low if the level of the C-C motif chemokine 14 in the urine sample is below a first predetermined threshold concentration of CCL14; (ii) the level of CCL14 in the urine sample is high if the level of CCL14 in the urine sample is above a second predetermined threshold concentration of CCL14; or (iii) the level of CCL14 in the urine sample is moderate if the level of CCL14 in the urine sample is between a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14; (c) (iv) the level of CCL14 in the first urine sample is low and the level of CCL14 in the second urine sample is moderate or high; or (v) if the level of CCL14 in the first urine sample is intermediate and the level of CCL14 in the second urine sample is high; Correlating the level of CCL14 with an increased risk that the subject will develop persistent KDIGO stage 1, 2 or 3 AKI. The method of claim 1 further comprising:

3. 3. The method of claim 1 or 2, wherein the subject has AKI that meets the definition of KDIGO stage 1.

4. 3. The method of claim 1 or 2, wherein the subject has AKI that meets the KDIGO stage 2 definition.

5. 3. The method of claim 1 or 2, wherein the subject has AKI that meets the definition of KDIGO stage 3.

6. The method of any one of claims 1 to 5, wherein the subject is determined to be at increased risk for developing persistent KDIGO stage 2 or 3 AKI.

7. The method of any one of claims 1 to 6, wherein the subject is determined to be at increased risk for developing persistent KDIGO stage 3 AKI.

8. 8. The method of any one of claims 1-7, comprising determining that the subject is at increased risk of developing persistent KDIGO stage 3 AKI within 48 hours of the first urine sample being obtained, wherein the duration of persistent KDIGO stage 3 AKI comprises a 72 hour period in which a minimum KDIGO stage is KDIGO 3.

9. The method of any one of claims 2 to 8, wherein the first threshold concentration of CCL14 is 1.3 ng / mL or less of CCL14.

10. The method of any one of claims 2 to 9, wherein the second threshold concentration of CCL14 is greater than 13 ng / mL.

11. The method of any one of claims 1 to 10, wherein the second urine sample is obtained within about 12 hours of the first urine sample.

12. The method of any one of claims 1 to 10, wherein the second urine sample is obtained about 12 hours after the first urine sample.

13. The method of any one of claims 1 to 10, wherein the second urine sample is obtained within about 24 hours of the first urine sample.

14. The method of any one of claims 1 to 10, wherein the second urine sample is obtained about 24 hours after the first urine sample.

15. The method of any one of claims 1 to 10, wherein the second urine sample is obtained within about 36 hours of the first urine sample.

16. The method of any one of claims 1 to 10, wherein the second urine sample is obtained about 36 hours after the first urine sample.

17. The method of any one of claims 1 to 10, wherein the second urine sample is obtained within about 48 hours of the first urine sample.

18. The method of any one of claims 1 to 10, wherein the second urine sample is obtained about 48 hours after the first urine sample.

19. The method of any one of claims 1 to 10, wherein the second urine sample is obtained within about 72 hours of the first urine sample.

20. The method of any one of claims 1 to 10, wherein the second urine sample is obtained about 72 hours after the first urine sample.

21. The method of any one of claims 1 to 20, further comprising obtaining a third urine sample.

22. 13. The method of any one of claims 1-12, further comprising obtaining a third urine sample, wherein the second and third urine samples are obtained within about 12 hours of the first urine sample.

23. 15. The method of any one of claims 1-14, further comprising obtaining a third urine sample, wherein the second and third urine samples are obtained within about 24 hours of the first urine sample.

24. 22. The method of any one of claims 1-21, further comprising obtaining the second urine sample about 12 hours after the first urine sample is obtained and obtaining a third urine sample about 24 hours after the first urine sample is obtained.

25. The method of any one of claims 21 to 24, wherein the first urine sample and the second urine sample have a low level of CCL14 and the third urine sample has an intermediate level of CCL14.

26. The method of any one of claims 21 to 24, wherein the first urine sample and the second urine sample have low levels of CCL14 and the third urine sample has high levels of CCL14.

27. The method of any one of claims 21 to 24, wherein the first urine sample and the second urine sample have an intermediate level of CCL14 and the third urine sample has a high level of CCL14.

28. The method of any one of claims 21 to 24, wherein the first urine sample has a low level of CCL14 and the second urine sample and the third urine sample have intermediate levels of CCL14.

29. The method of any one of claims 21 to 24, wherein the first urine sample has a moderate level of CCL14 and the second urine sample and the third urine sample have high levels of CCL14.

30. 25. The method of any one of claims 21 to 24, wherein the first urine sample has a low level of CCL14, the second urine sample has a moderate level of CCL14, and the third sample has a high level of CCL14.

31. The method of any one of claims 21 to 24, wherein the first urine sample has a low level of CCL14 and the second urine sample and the third urine sample have high levels of CCL14.

32. The method of any one of claims 1 to 31, wherein the subject is in an intensive care unit.

33. The method of any one of claims 1 to 32, wherein the subject is diagnosed with AKI.

34. 34. The method of claim 33, wherein the subject had AKI for less than 36 hours before the first urine sample was obtained.

35. The method of any one of claims 1 to 34, wherein the method further comprises contacting the urine sample with a binding reagent that binds to the CCL14.

36. 36. The method of claim 35, wherein the binding reagent is an antibody.

37. The method of any one of claims 1 to 36, wherein the assay is an immunoassay.

38. 38. The method of any one of claims 1 to 37, further comprising treating the subject at increased risk of having persistent AKI with one or more renal replacement therapies (RRT), removing compounds known to be damaging to the kidney, performing a procedure known to be damaging to the kidney at least about 48 hours after obtaining the first or second sample, altering diuretic administration, altering administration of a renally cleared compound, and / or administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels or hemodynamics.

39. 39. The method of claim 38, wherein the RRT comprises one or more of continuous RRT, intermittent RRT, hemodialysis, peritoneal dialysis, hemofiltration, and renal transplantation.

40. 1. A method for assessing a reduced risk of developing sustained acute kidney injury (AKI) in a subject, the method comprising: (a) performing an assay to detect levels of C-C motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, wherein at least a second urine sample is obtained from the subject within about 72 hours after a first urine sample is obtained; (b) determining that the subject is at reduced risk of persistent AKI based on a downward trend in the level of CCL14 detected in the urine sample; and A method comprising:

41. (a) performing an assay to detect levels of C-C motif chemokine 14 (CCL14) in two or more urine samples obtained from the subject, wherein at least a second urine sample is obtained from the subject within about 72 hours after a first urine sample is obtained; (b) determining whether the level of CCL14 detected in the urine sample is low, medium, or high based on a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14; Where: (i) the level of CCL14 in the urine sample is low if the level of the C-C motif chemokine 14 in the urine sample is below a first predetermined threshold concentration of CCL14; (ii) the level of CCL14 in the urine sample is high if the level of CCL14 in the urine sample is above a second predetermined threshold concentration of CCL14; or (iii) the level of CCL14 in the urine sample is moderate if the level of CCL14 in the urine sample is between a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14; (c) (iv) the first urine sample has a moderate level of CCL14 and the second urine sample has a low level of CCL14; (v) the first urine sample has a high level of CCL14 and the second urine sample has a moderate level of CCL14; or (vi) if the level of CCL14 in the first urine sample is high and the level of CCL14 in the second urine sample is low; Correlating the level of CCL14 with a reduced risk that the subject will develop persistent KDIGO stage 1, 2 or 3 AKI.

41. The method of claim 40, further comprising:

42. 42. The method of claim 40 or 41, wherein the subject has AKI that meets the KDIGO stage 1 definition.

43. 42. The method of claim 40 or 41, wherein the subject has AKI that meets the KDIGO stage 2 definition.

44. 42. The method of claim 40 or 41, wherein the subject has AKI that meets the definition of KDIGO stage 3.

45. The method of any one of claims 40 to 44, wherein the subject is determined to be at reduced risk of developing persistent KDIGO stage 2 or 3 AKI.

46. The method of any one of claims 40 to 45, wherein the subject is determined to be at reduced risk of developing sustained KDIGO stage 3 AKI.

47. 47. The method of any one of claims 40-46, comprising determining that the subject is at reduced risk of developing persistent KDIGO stage 3 AKI within 48 hours of the first urine sample being obtained, wherein the duration of persistent KDIGO stage 3 AKI comprises a 72 hour period in which a minimum KDIGO stage is KDIGO 3.

48. 48. The method of any one of claims 41 to 47, wherein the first threshold concentration of CCL14 is 1.3 ng / mL or less of CCL14.

49. The method of any one of claims 41 to 48, wherein the second threshold concentration of CCL14 is greater than 13 ng / mL.

50. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained within about 12 hours of the first urine sample.

51. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained about 12 hours after the first urine sample.

52. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained within about 24 hours of the first urine sample.

53. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained about 24 hours after the first urine sample.

54. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained within about 36 hours of the first urine sample.

55. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained about 36 hours after the first urine sample.

56. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained within about 48 hours of the first urine sample.

57. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained about 48 hours after the first urine sample.

58. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained within about 72 hours of the first urine sample.

59. 50. The method of any one of claims 40-49, wherein the second urine sample is obtained about 72 hours after the first urine sample.

60. 60. The method of any one of claims 40 to 59, further comprising obtaining a third urine sample.

61. 52. The method of any one of claims 40-51, further comprising obtaining a third urine sample, wherein the second and third urine samples are obtained within about 12 hours of the first urine sample.

62. 54. The method of any one of claims 40-53, further comprising obtaining a third urine sample, wherein the second and third urine samples are obtained within about 24 hours of the first urine sample.

63. 61. The method of any one of claims 40-53 or 60, further comprising obtaining the second urine sample about 12 hours after the first urine sample is obtained and obtaining a third urine sample about 24 hours after the first urine sample is obtained.

64. 64. The method of any one of claims 60 to 63, wherein the first urine sample and the second urine sample have high levels of CCL14 and the third urine sample has an intermediate level of CCL14.

65. 64. The method of any one of claims 60 to 63, wherein the first urine sample and the second urine sample have high levels of CCL14 and the third urine sample has a low level of CCL14.

66. 64. The method of any one of claims 60 to 63, wherein the first urine sample and the second urine sample have an intermediate level of CCL14 and the third urine sample has a low level of CCL14.

67. 64. The method of any one of claims 60 to 63, wherein the first urine sample has a moderate level of CCL14 and the second urine sample and the third urine sample have low levels of CCL14.

68. 64. The method of any one of claims 60 to 63, wherein the first urine sample has a high level of CCL14 and the second urine sample and the third urine sample have a low level of CCL14.

69. 64. The method of any one of claims 60 to 63, wherein the first urine sample has a high level of CCL14 and the second urine sample and the third urine sample have intermediate levels of CCL14.

70. 64. The method of any one of claims 60 to 63, wherein the first urine sample has a high level of CCL14, the second urine sample has a moderate level of CCL14, and the third urine sample has a low level of CCL14.

71. The method of any one of claims 40 to 70, wherein the subject is in an intensive care unit.

72. The method of any one of claims 40 to 71, wherein the subject is diagnosed with AKI.

73. 73. The method of claim 72, wherein the subject had AKI for less than 36 hours before the first urine sample was obtained.

74. The method of any one of claims 40 to 73, wherein the method further comprises contacting the urine sample with a binding reagent that binds to the CCL14.

75. 75. The method of claim 74, wherein the binding reagent is an antibody.

76. The method of any one of claims 40 to 75, wherein the assay is an immunoassay.

77. 77. The method of any one of claims 40-76, further comprising treating a subject at reduced risk of sustained AKI by one or more of administering a compound known to be kidney damaging, performing a procedure known to be kidney damaging, altering diuretic administration, altering administration of a renally cleared compound, and administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels, or hemodynamics.

78. 1. A method for assessing a high risk of developing sustained acute kidney injury (AKI) in a subject, the method comprising: (a) performing an assay to detect the level of C-C motif chemokine ligand 14 (CCL14) in a urine sample obtained from said subject; (b) determining that the subject is at high risk for developing persistent AKI based on the level of CCL14 detected in the urine sample being greater than a CCL14 threshold concentration of about 1.3 ng / ml; A method comprising:

79. 1. A method for assessing an elevated risk of developing sustained acute kidney injury (AKI) in a subject, the method comprising: (a) performing an assay to detect the level of C-C motif chemokine ligand 14 (CCL14) in a urine sample obtained from said subject; (b) determining that the subject is at high risk for developing persistent AKI based on the level of CCL14 detected in the urine sample being greater than a CCL14 threshold concentration of about 13.0 ng / ml; A method comprising:

80. 79. The method of claim 78, wherein the level of CCL14 detected in the urine sample is above a CCL14 threshold concentration of about 1.3 ng / ml and below a CCL14 threshold concentration of about 13.0 ng / ml.

81. 81. The method of any one of claims 78 to 80, wherein the method further comprises seeking further analysis from a nephrologist or specialist.

82. 82. The method of any one of claims 78-81, further comprising treating the subject at high or elevated risk of having persistent AKI with one or more renal replacement therapies (RRT), removing compounds known to be damaging to the kidney, performing a procedure known to be damaging to the kidney at least about 48 hours after obtaining the sample, altering diuretic administration, altering administration of renally cleared compounds, and / or administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels or hemodynamics.

83. 80. The method of claim 79, wherein the RRT comprises one or more of continuous RRT, intermittent RRT, hemodialysis, peritoneal dialysis, hemofiltration, and renal transplantation.

84. The method of any one of claims 78 to 83, wherein the subject is diagnosed with AKI.

85. The method of any one of claims 78 to 83, wherein the subject has KDIGO stage 1 AKI.

86. The method of any one of claims 78 to 83, wherein the subject has KDIGO stage 2 AKI.

87. The method of any one of claims 78 to 83, wherein the subject has KDIGO stage 3 AKI.

88. 88. The method of any one of claims 78-87, wherein the subject is determined to be at elevated or high risk for persistent KDIGO stage 2 or 3 AKI.

89. 90. The method of any one of claims 78 to 89, wherein the subject is determined to be at elevated or high risk for persistent KDIGO stage 3 AKI.

90. 90. The method of any one of claims 78-89, comprising determining that the subject is at elevated or high risk of developing persistent KDIGO stage 3 AKI within 48 hours of the time the urine sample was obtained, wherein the duration of persistent KDIGO stage 3 AKI comprises a 72 hour period in which the minimum KDIGO stage is KDIGO 3.

91. The method of any one of claims 78 to 90, wherein the subject is in an intensive care unit.

92. The method of any one of claims 78 to 91, wherein the method further comprises contacting the urine sample with a binding reagent that binds to the CCL14.

93. 93. The method of claim 92, wherein the binding reagent is an antibody.

94. The method of any one of claims 78 to 93, wherein the assay is an immunoassay.

95. 1. A method for assessing an elevated risk of developing sustained acute kidney injury (AKI) in a subject, the method comprising: (a) performing an assay to detect the level of C-C motif chemokine ligand 14 (CCL14) in a urine sample obtained from said subject; (b) correlating the assay result with an elevated risk for the subject of developing persistent AKI by comparing the assay result with a CCL14 threshold concentration of about 1.3 ng / ml; wherein said correlation is used as a definitive diagnostic test for said elevated risk for said subject to develop persistent AKI when said assay result exceeds said CCL14 threshold concentration of about 1.3 ng / ml; or The correlation is used as a diagnostic test of exclusion for the subject at elevated risk of developing persistent AKI when the assay result is below the CCL14 threshold concentration of about 1.3 ng / ml.

96. 1. A method for assessing an elevated risk of developing sustained acute kidney injury (AKI) in a subject, the method comprising: (a) performing an assay to detect the level of C-C motif chemokine ligand 14 (CCL14) in a urine sample obtained from said subject; (b) correlating the assay result with an increased risk for said subject of developing persistent AKI by comparing said assay result with a CCL14 threshold concentration of about 13.0 ng / ml. Including, wherein said correlation is used as a definitive diagnostic test for said subject at high risk of developing persistent AKI when said assay result exceeds said CCL14 threshold concentration of about 13.0 ng / ml; or The correlation is used as a diagnostic test of exclusion for those subjects at high risk of developing persistent AKI when the assay result is below the CCL14 threshold concentration of about 13.0 ng / ml.

97. 96. The method of claim 95, wherein the level of CCL14 detected in the urine sample is above a CCL14 threshold concentration of about 1.3 ng / ml and below a CCL14 threshold concentration of about 13.0 ng / ml.

98. 98. The method of any one of claims 95 to 97, wherein the method further comprises seeking further analysis from a nephrologist or specialist.

99. 98. The method of any one of claims 95-97, further comprising treating the subject at high or elevated risk of having persistent AKI with one or more renal replacement therapies (RRT), removing compounds known to be damaging to the kidney, performing a procedure known to be damaging to the kidney at least about 48 hours after obtaining the sample, altering diuretic administration, altering administration of renally cleared compounds, and / or administering one or more agents or measured fluid volumes to restore normal fluid levels, electrolyte levels or hemodynamics.

100. 100. The method of claim 99, wherein the RRT comprises one or more of continuous RRT, intermittent RRT, hemodialysis, peritoneal dialysis, hemofiltration, and renal transplantation.

101. The method of any one of claims 95 to 100, wherein the subject is diagnosed with AKI.

102. The method of any one of claims 95 to 101, wherein the subject has KDIGO stage 1 AKI.

103. The method of any one of claims 95 to 101, wherein the subject has KDIGO stage 2 AKI.

104. The method of any one of claims 95 to 101, wherein the subject has KDIGO stage 3 AKI.

105. 105. The method of any one of claims 95-104, wherein the subject is determined to be at elevated or high risk for persistent KDIGO stage 2 or 3 AKI.

106. The method of any one of claims 92 to 105, wherein the subject is determined to be at elevated or high risk for persistent KDIGO stage 3 AKI.

107. 107. The method of any one of claims 92-106, comprising determining that the subject is at elevated or high risk of developing persistent KDIGO stage 3 AKI within 48 hours of the time the urine sample was obtained, wherein the duration of persistent KDIGO stage 3 AKI comprises a 72 hour period in which the minimum KDIGO stage is KDIGO 3.

108. The method of any one of claims 95 to 107, wherein the subject is in an intensive care unit.

109. The method of any one of claims 95 to 108, wherein the method further comprises contacting the urine sample with a binding reagent that binds to the CCL14.

101. 110. The method of claim 109, wherein the binding reagent is an antibody.

111. The method of any one of claims 95 to 110, wherein the assay is an immunoassay.

112. 1. A method for assessing a low risk of developing sustained acute kidney injury (AKI) in a subject, the method comprising: (a) performing an assay to detect the level of C-C motif chemokine ligand 14 (CCL14) in a urine sample obtained from said subject; (b) determining that the subject is at low risk for developing persistent AKI based on the level of CCL14 detected in the urine sample being less than a CCL14 threshold concentration of about 1.3 ng / ml; A method comprising:

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