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

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 herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically and is incorporated herein by reference in its entirety. The Sequence Listing is provided as 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. Therefore, loss of kidney function due to injury and / or disease results in substantial morbidity and mortality. A detailed discussion of renal failure can be found 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. Kidney 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 kidney 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 kidney function over months to years."

[0004] Acute kidney injury (AKI, also known as acute renal failure or ARF) is a sudden (typically detected within approximately 48 hours to 1 week) decrease in glomerular filtration. AKI is a leading cause of both morbidity and mortality worldwide. It is estimated that at least half of AKI cases recover within 72 hours. AKI cases that recover within 72 hours tend to have significantly better outcomes compared with cases 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 experience a dramatically reduced survival rate 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 (incorporated herein by reference in its entirety). Persistence of AKI for more than one week, termed acute kidney disease (AKD), is significant in that it increases an individual's risk of developing chronic kidney disease and its consequences. 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 attempt 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(1):58-66 (each of which is incorporated by reference in its entirety).Therefore, early identification of individuals at risk for AKD will allow for appropriate delivery of these proposed interventions, but will also identify individuals who can be targeted with newer therapies to attenuate AKI.

[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 relies almost entirely on clinical expectations regarding the likelihood of recovery in the absence of commercially available diagnostics to aid this decision-making process. Thus, considerable debate exists around the timing of RRT, with studies indicating that some patients can benefit from early RRT initiation, while other studies indicate that some individuals undergoing RRT may not require such treatment because they quickly recover renal function. (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 could allow for treatment stratification and avoid the current risks of extracorporeal therapy. Summary of the Invention

[0006] These challenges highlight the need for better methods to detect and assess 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 for assessing renal function in a subject are provided. As described herein, measurements 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 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) determining that the subject is at elevated risk of developing sustained AKI based on the level of CCL14 detected in the urine sample being greater than a threshold concentration of CCL14 of about 1.3 ng / ml.

[0009] In another aspect, a method for assessing a subject's high risk of developing sustained acute kidney injury (AKI) is provided, 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 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 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.

[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 the subject at high or elevated risk of having persistent AKI with one or more renal replacement therapies (RRT), withdrawing compounds known to be kidney damaging, performing procedures known to be kidney damaging with a delay of 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.

[0013] In one embodiment, the 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 persistent KDIGO stage 2 or 3 AKI.

[0017] In another embodiment, the subject is determined to be at elevated or high risk for persistent 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, wherein 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, hi 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 the 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 the CCL14 threshold concentration of about 1.3 ng / ml.

[0023] In another aspect, a method of assessing a subject's high risk of developing persistent acute kidney injury (AKI) 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 a high risk for the subject developing persistent 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 high risk of developing persistent 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 high risk of developing persistent 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 persistent AKI with one or more renal replacement therapies (RRT), withdrawing compounds known to be kidney damaging, performing procedures known to be kidney damaging with a delay of at least about 48 hours after obtaining the sample, altering and / or optimizing diuretic administration, altering and / or optimizing 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.

[0027] In one embodiment, the 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 persistent KDIGO stage 2 or 3 AKI.

[0031] In one embodiment, the subject is determined to be at elevated or high risk for persistent 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, wherein 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, hi 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 less than a CCL14 threshold concentration of about 1.3 ng / ml.

[0037] In one embodiment, a method for assessing whether a subject has an increased risk of developing sustained acute kidney injury (AKI) 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, wherein 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 has an increased risk of sustained AKI based on an upward trend in the levels of CCL14 detected in the urine samples.

[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, wherein at least a second urine sample is obtained from the subject within about 72 hours after the first urine sample is obtained; (b) determining whether the level of CCL14 detected in the urine samples is low (low), moderate, or high (high) based on a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14, wherein: (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) (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, wherein 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 about 24 hours after the first urine sample, the second urine sample is obtained about 24 hours after the first urine sample, the second urine sample is obtained about 36 hours after the first urine sample, the second urine sample is obtained about 36 hours after 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 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 method further comprises obtaining the second urine sample about 12 hours after the first urine sample is obtained, and obtaining the third urine sample about 24 hours after the first urine sample is obtained.

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

[0046] In one embodiment, the first and second urine samples 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 and third urine samples 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 low levels of CCL14, a second urine sample has intermediate levels of CCL14, and a third sample has high levels 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 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 to 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 persistent AKI with one or more renal replacement therapies (RRT), withdrawing compounds known to be kidney damaging, performing procedures known to be kidney damaging at least about 48 hours after obtaining the first 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.

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

[0060] In another aspect, 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, wherein 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 has a 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, wherein 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 whether the level of CCL14 detected in the urine samples is low (low), moderate, or high (high) based on a first predetermined threshold concentration of CCL14 and a second predetermined threshold concentration of CCL14, wherein (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. (iii) 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) the level of CCL14 in the first urine sample is moderate and the level of CCL14 in the second urine sample is low; (v) 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) 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.

[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 about 24 hours after the first urine sample, the second urine sample is obtained about 24 hours after the first urine sample, the second urine sample is obtained about 36 hours after the first urine sample, the second urine sample is obtained about 36 hours after 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 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 method further comprises obtaining the second urine sample about 12 hours after the first urine sample is obtained, and 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 intermediate levels of CCL14. In one embodiment, the first and second urine samples have high levels of CCL14, and the third urine sample has low levels of CCL14. In one embodiment, the first and second urine samples have intermediate levels of CCL14, and the third urine sample has low levels of CCL14. In one embodiment, the first urine sample has intermediate levels of CCL14, and the second and third urine samples have low levels of CCL14. In one embodiment, the first urine sample has high levels of CCL14, and the second and third urine samples have low levels of CCL14. In one embodiment, the first urine sample has high levels of CCL14, and the second and third urine samples have intermediate levels 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 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 to 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 the 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 administration, altering and / or optimizing 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.

[0074] In one embodiment, a method includes assessing a subject's risk of developing persistent acute kidney injury (AKI) by performing an assay to detect levels of CC 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 the first urine sample is obtained, and determining that the subject has an increased risk of persistent AKI if the levels of CCL14 detected in the urine samples trend upward, and determining that the subject has a decreased risk of persistent AKI if the levels of CCL14 detected in the urine samples trend downward. [Brief explanation of the drawings]

[0075] [Figure 1] FIG. 1 shows a flow diagram of the pooled analysis of patients from the Ruby and Sapphire studies. [Figure 2] A comparison of CCL14 concentrations in the four populations is shown. [Figure 3] Figure 1 shows the risk of the primary endpoint of persistent severe AKI stratified by CCL14 levels below, between, and above 1.3 and 13 ng / mL. [Figure 4] Figure 1 shows the risk of persistent severe AKI stratified by CCL14 levels <1.3 ng / mL and >1.3 ng / mL. [Figures 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. [Figures 6A-6C] Figure 1 shows the cumulative incidence of RRT, death, and RRT or death within 90 days for 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-to-severe AKI in the primary analysis cohort are shown. [Figure 9] Change in CCL14 category stratified by initial CCL14 category after diagnosis of moderate-to-severe AKI. Sensitivity analysis including patients with CCL14 values ​​< -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 INVENTION

[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 (documentation of 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 at least 48-72 hours before sustained reversal. Reversal of AKI generally must last a minimum of 48 hours for subsequent episodes of AKI to be considered separate episodes rather than a continuation of the initial episode. Definitions of various stages of kidney 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 specific stages of AKI (e.g., KDIGO stage 3 AKI) can also be 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 "increased risk" of persistent 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 persistent AKI means that the level of CCL14 in the sample is higher than the CCL14 threshold concentration of 13.0 ng / ml.

[0080] "Low risk" of persistent 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 "confirmatory 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 an elevated 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 rule out" 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 persistent AKI means that the level of CCL14 is trending downward 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 to be elevated" 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" 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, 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 additional samples obtained from the same subject.

[0090] One or more serial 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, about 6-12 hours, about 12-18 hours, about 18-24 hours, about 24-36 hours, about 36-48 hours, 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 approximately one week. Contrast-induced nephropathy (CIN, or AKI caused by radiocontrast agents) is thought to be caused by intrarenal vasoconstriction (leading to ischemic injury) and 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 (peak within 3–5 days, resolve within one week) elevations in blood urea nitrogen and serum creatinine.

[0092] The commonly reported criterion 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 elevated serum creatinine to define and detect AKI is well established, the magnitude of the serum creatinine rise and the time period measured to define AKI vary considerably among publications. Traditionally, relatively large increases in serum creatinine, such as 100%, 200%, an increase of at least 100% 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 increases in serum creatinine to define AKI. The relationship between elevated serum creatinine, 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, along with the references cited therein, are incorporated herein by reference in their entirety. As described in these publications, acutely worsening kidney function (AKI) and increased risk of mortality and other adverse outcomes are now known to be associated with very small increases in serum creatinine. These increases can be determined as relative (percent) values ​​or nominal values. While relative increases in serum creatinine as small as 20% from pre-injury values ​​have been reported to indicate acutely worsening kidney function (AKI) and increased health risks, a more commonly reported value defining 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 durations for serum creatinine to rise to these thresholds have been used to define AKI, ranging from 2 days, 3 days, 7 days, or a variable duration defined as the time the patient spends in the hospital or intensive care unit. These studies demonstrate that there is no specific threshold of serum creatinine elevation (or duration of elevation) that worsens renal function or AKI, but rather there is a continuous increase in risk with increasing magnitude of serum creatinine elevation.

[0093] One study (Lassnigg et al., J Am Soc Nephrol 15:1597-1605, 2004) examined both increases and decreases in serum creatinine. Patients with a mild decrease in serum creatinine of -0.1 to -0.3 mg / dL after cardiac surgery had the lowest mortality rate. Patients with a larger decrease 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 (detectable by small creatinine changes within 48 hours of surgery) can have a profound impact on patient outcomes. In an effort to reach 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: "Risk": serum creatinine increased 1.5 times above baseline or urine production <0.5 ml / kg body weight / hour for 6 hours; "Disorder": serum creatinine increased 2.0 times above baseline or urine production <0.5 ml / kg / h for 12 hours; "Failure": Serum creatinine increased 3.0 times 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 the 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 numerous 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 more severe RIFLE stage of AKI (i.e., subjects who do not have renal impairment, or subjects who have renal impairment but have not progressed to meet the threshold criteria for RIFLE stage R or any more severe RIFLE stage of AKI).

[0096] More recently, Mehta et al., Crit Care 11:R31 (doi:10.1186.cc5713), 2007 (incorporated herein by reference in its entirety) proposed the following similar classification (AKIN) for stratifying AKI patients, modified from RIFLE: "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": a serum creatinine increase of more than 200% (>2-fold) from baseline or a urine output of less than 0.5 mL / kg per hour for more than 12 hours; "Stage III": A serum creatinine increase of more than 300% (>3-fold) from baseline or a serum creatinine ≥ 4.0 mg / dL (≥ 354 μmol / L) with an acute increase of at least 0.5 mg / dL (44 μmol / L) or a urine output of less than 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 (hereby incorporated 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% increase in serum creatinine to define contrast-induced nephropathy (a type of AKI). Although various groups have proposed slightly different criteria for using serum creatinine to detect AKI, the consensus is that a small change in serum creatinine, such as 0.3 mg / dL or 25%, is sufficient to detect AKI (worsening renal function), and the magnitude of the serum creatinine change is an indicator of the severity of AKI and the risk of death.

[0102] These classification systems for AKI generally include serum creatinine and urine output criteria for each stage. Wherever specified herein, any stage of AKI may be considered equivalent (i.e., substituted for) any of the individual criteria that qualify 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., likelihood of reaching a particular AKI stage or likelihood of persistent AKI at a particular stage), where a particular AKI stage may be defined by satisfying both a serum creatinine criterion that qualifies the subject as being at that particular stage and a urine output criterion that qualifies the subject as being at that particular stage. In some embodiments, a particular AKI stage may be defined by satisfying all criteria (i.e., both all serum creatinine criteria and all urine output criteria). All methods disclosed herein may define AKI stages 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 interchangeable with one another with respect to the use of biomarkers disclosed herein, unless the context dictates 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] Serial measurement of serum creatinine over several days is a common method for detecting and diagnosing AKI and is considered one of the most important tools for evaluating patients with AKI. However, serum creatinine is generally considered to have several limitations in diagnosing, evaluating, and monitoring patients with AKI. The time it takes for serum creatinine to rise to a diagnostic value for AKI (e.g., a 0.3 mg / dL or 25% increase) can be 48 hours or longer, depending on the definition used. Because cellular injury in AKI can occur over several hours, elevated serum creatinine detected at or beyond 48 hours may be a late indicator of injury, and therefore relying on serum creatinine may delay the diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of accurate renal status and the 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- or long-term), and some experience other adverse outcomes, including death, major adverse cardiac events, and chronic kidney disease. Because serum creatinine is a marker of filtration rate, 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., tubular, glomerular, or interstitial origin). Urine output is similarly limited, knowledge of which can be crucial in the management and treatment of patients with AKI.

[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. Furthermore, while the subject is preferably a living 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 receiving 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 from a biological material intended to be provided to a subject. For example, a sample can be obtained from a kidney being evaluated for possible transplantation into a subject, and an analyte measurement can be obtained that is used to evaluate the kidney for existing damage. A preferred sample is a body fluid sample.

[0109] As used herein, the term "body fluid sample" 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, e.g., RRT. Preferred body fluid samples include blood (including whole blood, serum, and plasma), cerebrospinal fluid, urine, saliva, sputum, pleural effusion, hemofiltrate, and ultrafiltrate. Furthermore, one skilled in the art will understand that certain body fluid samples are more easily analyzed after a fractionation or purification procedure, e.g., separation of whole blood into serum or plasma components.

[0110] The term "about" when referring to a number or numerical range means that the referenced number or numerical range is approximate within experimental variation (or within statistical experimental error), and thus the number or numerical range may vary, for example, 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 in 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 indicate 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 disease probability, 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, clinicians can accept substantial diagnostic uncertainty, so few tests are required. On the other hand, in situations where treatment options are less effective and have higher risks, clinicians often require greater diagnostic certainty. Therefore, a cost / benefit analysis is involved in selecting a diagnostic threshold.

[0113] Appropriate threshold can be determined in various ways.For example, one recommended diagnostic threshold for diagnosing acute myocardial infarction using cardiac troponin is the 97.5th percentile of the concentration found in normal population.Another method can be to examine serial samples from the same patient, where previous "baseline" results are used to monitor the time-varying changes in biomarker levels.

[0114] Population studies can also be used to select a decision threshold. The receiver operating characteristic ("ROC") arose from the field of signal detection theory, developed during World War II for the analysis of radar images. ROC analysis is often used to select a threshold that can best distinguish a "diseased" subpopulation from a "non-diseased" subpopulation. A false positive, in this case, occurs when a person tests positive but does not actually have the disease. A false negative, on the other hand, occurs when a person tests negative, suggesting they are healthy, but actually has the disease. To plot an ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is continuously varied. Because the TPR is equivalent to sensitivity and the FPR is equal to 1 minus specificity, the ROC graph is sometimes called a sensitivity versus (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 threshold is selected to provide acceptable levels of specificity and sensitivity.

[0115] In this context, "diseased" refers to a population having one characteristic (the presence of a disease or symptom, or the occurrence of some consequence), and "non-diseased" refers to a population lacking that characteristic. While 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 can be assigned with a relatively high degree of confidence, and above a second threshold, the presence of disease can also be assigned with a relatively high degree of confidence. Between two thresholds, the range can 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 non-occurrence, likelihood of outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods, which can generate a probability value representing the degree to which a subject belongs to one of multiple classes.

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

[0118] As mentioned above, a suitable test may exhibit 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 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 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 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, even more 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, 0.5 or less, more preferably 0.3 or less, and most preferably 0.1 or less.

[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 for persistent AKI or at low risk for persistent AKI. Treatment options may vary for each subject, and treatments may be prioritized or deprioritized based on the individual subject's clinical assessment and other underlying symptoms. Non-limiting examples of treatment options for subjects at high risk for persistent AKI or at low risk for 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 various stages of AKI. The 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 radiographic procedures.

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

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

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

[0123] Renal replacement therapy (RRT) is an option for the management of patients with renal dysfunction, including AKI, persistent AKI, AKD, or CKD. As used herein, RRT is interchangeable with renal replacement therapy (KRT) and includes kidney transplantation and 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 used. The various types of dialysis are generally classified as 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 RT Ther Clin Risk Manag. 2005;1(2):141-50 (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 extended-duration dialysis (EDD). Some types can be performed in the subject's home or while traveling, while some require a clinical setting with the assistance of a medical professional.

[0124] In some embodiments, treatment options further include accelerating the initiation of RRT or other treatment or procedures, referring the patient for further analysis to determine an appropriate therapeutic regimen, withdrawing compounds known to be kidney damaging, performing procedures known to be kidney damaging with a delay of at least about 48 hours after obtaining the sample, altering and / or optimizing diuretic administration, altering and / or optimizing 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.

[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 urinary sediment, proteinuria, and renal ultrasound; renal biopsy; therapeutic drug monitoring or optimization; hemodynamic monitoring and / or optimization; evaluation of AKI etiology for persistent AKI; and further analysis to diagnose and / or treat rarer causes of AKI (interstitial nephritis, tumor lysis syndrome, thrombotic thrombocytopenic purpura, and cholesterol embolism syndrome). [Example]

[0126] Example 1: Introduction In the Ruby study, we enrolled critically ill patients within 36 hours of a 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 predicting persistent (≥72 hours) KDIGO (Kidney Disease Improving Global Outcomes) stage 3 (severe) AKI. In this preplanned 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 cutoff values ​​on the CCL14 receiver operating characteristic (ROC) curve for predicting persistent severe AKI. We selected one cutoff for high sensitivity and another 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 along with clinical variables from the Ruby study to assess whether the biomarker added value 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 study and the reference population study have been previously described (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 urgent need of RRT, were receiving palliative care only, or had 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-specified 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 representatives) 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 of stage 3 AKI before reaching 72 hours were considered a positive endpoint. Patients with stage 2 AKI at enrollment who received RRT or initiated 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 Astute 140® Meter (Astute Medical, San Diego, CA) by an operator blinded to clinical data. NEPHROCLEAR™ CCL14 Test concentration results are traceable to a reference material containing a defined mass of CCL14 protein according to EN ISO 17511.

[0130] Cutoff Selection Two cutoff points on the urinary CCL14 receiver operating characteristic 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 were candidates for measures recommended as a high priority in the KDIGO Clinical Practice Guideline due to their increased risk of adverse AKI outcomes. The types of actions considered when determining the appropriate balance between sensitivity and specificity included consultation with specialists (e.g., nephrology, pharmacy, intensive care), additional diagnostic workup, and prioritized assessment of the need for more intensive monitoring (including hemodynamic / fluid monitoring and possible ICU admission) or renal replacement therapy. In selecting the cutoff points, we assumed that the test would not be used in isolation 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 would 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 for treatment as 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 the 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 subjects with and without the primary endpoint were presented in box and whisker plots. The Kruskal-Wallis test was used to detect concentration differences 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 levels stratified by two cutoffs improved risk prediction beyond clinical variables alone, a reference logistic regression model was constructed as previously described. When CCL14 was analyzed as a categorical variable in the regression analysis, a CCL14 level of ≤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 using the Kruskal-Wallis, Cochran-Amitage, and Fisher's exact tests, respectively. Confidence intervals for sensitivity, specificity, positive predictive value, and negative predictive value were calculated using the Clopper-Pearson exact method, and confidence intervals for positive likelihood ratios, risks, and relative risks were calculated using the asymptotic method (normal approximation). A two-sided p value of <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 urinary CCL14 cutoff value of 1.3 ng / ml 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 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 patients (62.9%) had CCL14 levels >1.3 ng / ml, and 54 patients (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 entire cohort) did not meet the 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 the 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 pre-enrollment 24-hour urine output with CCL14 for predicting the primary outcome. Tables 3A and 3B demonstrate the poor performance of urine output for predicting the primary outcome (AUC = 0.63) and 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 selected to achieve high sensitivity (91%), i.e., to identify most subjects who would progress to persistent severe AKI. Similarly, a high cutoff of 13 ng / mL was chosen to achieve high specificity (93%), ie, identify most subjects who would not progress to persistent severe AKI.

[0134] Figure 2 shows the operating characteristics from the Ruby study for the two cutoffs and cutoff locations for the distribution of urinary CCL14 levels in the Ruby study and reference population cohorts. These cohorts consisted of healthy (378), chronically ill without acute illness (366), Ruby without persistent severe AKI (225), and Ruby with persistent severe AKI (110). The lower and upper whiskers in the figure represent the 10th and 90th percentiles of CCL14 concentrations for that group, respectively. The lower and upper boxes represent the first and third quartiles, respectively. The middle bar represents the median. The horizontal dashed lines correspond to cutoffs of 1.3 ng / mL and 13 ng / mL. The P value was calculated using the Kruskal-Wallis test and was <0.0001. The cutoff at 13 ng / mL is higher 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% (with a negative predictive value of 75%). Both cutoffs substantially exceed the urinary CCL14 range for an 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 0.2–30 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 within 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 significantly increased with increasing CCL14 levels, and the individual components, RRT and persistently elevated serum creatinine or oliguria, were similarly stratified (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 levels ≤ 1.3, whereas patients with CCL14 levels > 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. 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 to 13 ng / mL is 124, 157, and 54, respectively. A log-rank test for trend was used to calculate p-values ​​for differences between strata. Higher CCL14 levels were associated with increased risk of both death and RRT, as well as the combined risk of the two (log-rank p<0.001). These findings persisted when the cohort was dichotomized at a CCL14 level of 1.3 ng / mL (Figures 6A-6C). Referring to the data shown in Figures 6A, 6B, and 6C, the number of patients with CCL14 concentrations below 1.3 ng / mL and above 1.3 ng / mL is 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 biomarker cutoffs in comparison with clinical variables, we performed multivariate logistic regression analysis using persistent AKI as the endpoint with and without CCL14 as a covariate (Table 6). 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 analyses, we found that the use of 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 described 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 or 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 AKI at enrollment for the two cutoffs and cutoff locations for the distribution of urinary CCL14 levels in this cohort and the reference population cohort. The operating characteristics remained 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 illness (366), the full ITD cohort (335 patients, of which 225 did not develop persistent severe AKI), and those adjudicated as having stage 2 or 3 disease 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 first and third quartiles, respectively. The middle bar represents the median. The horizontal dashed lines correspond to cutoffs of 1.3 ng / mL and 13 ng / mL. P values ​​<0.0001 were 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 before 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 highly sensitive cutoff of 1.3 ng / ml of CCL14, we were able to identify the majority of patients who will develop persistent severe AKI and are therefore candidates for treatment recommended as a high priority in the KDIGO Clinical Practice Guideline for those at increased risk for adverse outcomes of AKI. Using a highly specific cutoff of 13 ng / ml, we were able to identify the highest-risk patients who most urgently require evaluation for further intervention. The clinical relevance of these two cutoffs is demonstrated by their ability to stratify the risk of adverse events (RRT or death) over a 90-day period and add significant predictive information beyond that available clinically. Importantly, although CCL14 has already been validated as a biomarker for persistent AKI, this is the first report to validate a specific cutoff using a standardized CCL14 assay developed for routine clinical use. This study represents an important step in framing the clinical utility of CCL14 for identifying those at risk for persistent severe AKI.

[0140] We demonstrated that urinary CCL14 provides significant information regarding the likelihood of persistent 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 a 10.4-fold increased odds of persistent 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 assessments to provide the most comprehensive view of a patient's acute renal status and potential course without further intervention. The high negative predictive value (92%) at a cutoff of 1.3 ng / mL ensures that nearly all patients with a negative test will not develop persistent AKI. Furthermore, values ​​above 1.3 ng / mL are associated with a nearly 1 in 2 risk of developing persistent 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 significant clinical impact on the care of critically ill patients. A growing body of literature demonstrates 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 NSAIDS 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 initial clinical management, making it very difficult to know which patients require discontinuation of nephrotoxic medications or more intensive evaluation of hemodynamic / volume status. We suggest that tools such as CCL14, which can better predict persistent severe AKI, could serve as a tool to determine which patients require additional renal intensive care beyond early management. It is reasonable to assume that patients with persistent severe AKI are likely to benefit from strict adherence to the renal care bundle. For example, consider an ICU patient who develops stage 1 AKI. Clinicians review their medication list and assess their volume status. They determine that the patient requires additional boluses of balanced fluids, adjustments to vancomycin administration based on therapeutic monitoring, and a change from piperacillin-tazobactam to cefepime. Now, when considering whether this patient will progress to stage 2 AKI, we realize that changes in serum creatinine often reflect damage already occurring. Thus, the clinician is uncertain as to whether additional management changes are necessary. If CCL14 is measured and is >1.3 ng / ml, it may be appropriate to consider more aggressive clinical interventions (more intensive hemodynamic monitoring, discontinuation / change of other medications, or nephrology consultation). However, if CCL14 is ≤1.3 ng / ml, the clinician may be inclined to continue the current management and forgo further interventions.

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

[0143] In summary, we have validated a standardized clinical assay for CCL14, the NEPHROCLEAR™ CCL14 test, and used this test to derive and characterize a CCL14 cutoff, enabling the identification of elevated patients at high risk for developing severe, persistent AKI. These results have immediate utility 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] 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 levels 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 the subjects described in Example 1 herein. Sapphire enrolled critically ill adult patients with cardiac or respiratory dysfunction who were not known to have stage 2-3 AKI at the time of enrollment from 35 centers in 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 one week of enrollment were included in this analysis. For the purposes of this analysis, the Sapphire cohort included serial biomarker measurements from the time patients met stage 2-3 AKI criteria, mirroring the time course of enrollment and biomarker measurements in the Ruby study. For both studies, patients without 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 enrolled patients were included on an intention-to-diagnose 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 before sample testing. The primary analysis utilized CCL14 concentrations from the first three scheduled urine collections in Ruby and the first three urine collections after the onset of KDIGO stage 2 or 3 AKI in Sapphire for each patient. Technicians blinded to clinical data measured CCL14 concentrations in 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 persistent severe AKI, as previously described. Briefly, patients who developed stage 3 AKI for 72 consecutive hours starting within 48 hours of the first sample collection, or who died after stage 3 AKI or received RRT 48 hours before the 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, each patient's baseline serum creatinine 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 regarding 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, Kidney 360. 2022 Mar 24;3(7):1158-1168) to stratify CCL14 concentrations in the first three urine collections into three levels: low (≤1.3 ng / mL), intermediate (>1.3 to ≤13 ng / mL), and high (>13 ng / mL). A highly sensitive cutoff of 1.3 ng / mL CCL14 was derived to identify the majority of patients at risk for persistent severe AKI, and a highly specific cutoff of 13 ng / mL was derived to identify the highest-risk patients most likely to develop PS-AKI. To evaluate the effectiveness of using the pooled dataset, we used the Cochran Q test to assess the heterogeneity of relative risk (RR) 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 persistent 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 of <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 a complete set of three CCL14 measurements over a 24-hour period to allow for assessment of true trajectories rather than point measurements, because 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 used alternative pairs of sensitivity and specificity cutoffs of 1.3 and 13 ng / mL to explore potential differences in our primary conclusions.

[0169] CCL-14's extended trajectory Measurement of CCL14 in urine samples collected at least daily allowed for evaluation of changes in CCL14 trajectories over a longer time period and their relationship with day 7 outcomes. We performed a rolling assessment 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 beginning within 48 hours or death or RRT at 48 hours of that measurement. The ability of urinary CCL14 (as a continuous variable) at any time point to discriminate between rolling endpoints was assessed 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 for the "unchanged" value, reflecting the inclusion of patients whose measurements were truncated due to death or need for RRT (Figure 9), with a similar pattern of association in logistic regression (Table 12).

[0170] result patient The Ruby study recruited 364 patients within 36 hours of diagnosis of stage 2-3 AKI, whereas 212 of the 723 critically ill patients without AKI enrolled in Sapphire 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). Furthermore, 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 patients (18%) reached the primary endpoint of PS-AKI (72-hour sustained stage 3 AKI or death before recovery of RRT or renal function); in a sensitivity analysis, 135 patients (26%) reached the endpoint, often reflecting early death or anuria that prevented the 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 the Ruby cohort (Table 13), reflecting a decrease in overall severity of AKI in the Sapphire cohort, with AKI stage 3 at the time of first sample collection in 30% versus 4%. 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 did not differ significantly between patients with and without PS-AKI endpoints, but fluid balance on day 1 after enrollment was significantly more favorable 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 trajectory at day 1 and primary composite endpoint Initial CCL14 levels were low (≤1.3 ng / mL) in 196 patients (47%), moderate (>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 strongly correlated with the primary endpoint. Importantly, the association between CCL14 category and PS-AKI outcome was essentially unchanged at 12 and 24 hours. In line with this observation, in the majority of cases (66%), CCL14 category remained unchanged from 0 to 24 hours, with only one patient experiencing a two-level change (Figure 8, Table 10). The graph in Figure 8 shows the change from initial CCL14 category and the risk of sustained severe AKI. When changes occurred, they were generally consistent in direction, with only 24 of 417 patients (6%) exhibiting a fluctuating CCL14 category over time (Table 10). In patients experiencing a change in CCL14 category, we classified patients as either decreasing or increasing in the CCL14 category 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 the risk of PS-AKI (Figure 8). We evaluated the additional information provided by changes in CCL14 level, accounting for initial values, in multivariate logistic regression. Considering 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). When tested, there was no significant interaction between initial status and change (p=0.36 for the interaction term)—Table 15.

[0172] Sensitivity analysis In sensitivity analyses including patients with missing CCL14 values, the associations between changes in CCL14 stratified by baseline were essentially unchanged apart from the overall worse outcome of "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 a second sensitivity analysis using alternative cutoff values, two additional cutoffs above and below 1.3 and 13 were considered, yielding a total of six cutoffs and nine different combinations of low and high cutoffs (Table 16). Additional cutoff values ​​of 0.8, 1.8, 9, and 17 were selected based on their sensitivity and specificity relative to the original predetermined cutoffs of 1.3 and 13 ng / mL (Table 4 in Example 1). Different combinations of cutoffs resulted in CCL14 categories that were strongly correlated with the persistent severe AKI endpoint, similar to that observed with the original cutoffs of 1.3 and 13 ng / mL (compare Tables 14 and 17). Furthermore, odds ratios in multivariate logistic regression of PS-AKI endpoints showed a similar pattern, in which both initial and change in CCL14 categories were statistically significant independent predictors of the endpoint across all combinations of cutoffs (compare Tables 15 and 18). Finally, a trend of 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 for 7 days after enrollment in surviving patients who remained in the ICU with and without urinary catheterization (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, CCL14 values ​​tended to decrease rather than increase over time (Figure 10), with 67% of patients having low levels of CCL14 as their last measurement. Referring to the graph in Figure 11, the receiver operating characteristic curve (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 maximum of 7 days, with right-censored data imputed using a carry-forward of the last observation. Rolling assessment of the ability to predict the PS-AKI composite endpoint starting within 48 hours demonstrated a sustained ability to discriminate between endpoints well, with a ROCAUC of >0.8 up to 144 hours (Figure 11).

[0175] Consideration We demonstrate key clinical features of the CCL14 assay, guiding clinicians in its interpretation and adding further confidence to its interpretation in real-world clinical situations. Importantly, we demonstrate that in two-thirds of patients with moderate-to-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 CCL14 measurement is delayed after the initial diagnosis of stage 2-3 AKI, the results may provide at least as good a prognosis of the clinical course of AKI as those provided by the original clinical trials 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 rather is an indicator of newly emerging but sustained biological processes within the severely injured kidney. Second, serial CCL14 measurements may provide complementary clinical information to refine risk assessment over time by reflecting potentially modifiable pathologies that lead to changes in prognosis over time. In particular, for patients with intermediate CCL14 measurements, moving into the low category was associated with a similar decrease in risk as an initial low measurement, whereas an increase to high was associated with a significant increase in risk. Notably, while CCL14 levels remained stable overall, 45% of those with initial median levels experienced a category shift within the first 24 hours. Thus, for patients with initial measurements in the central risk category, serial measurements offer 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 was 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-risk outcome.Finally, CCL14 appears to retain its ability to discriminate composite endpoints over a 6-day period following initial moderate-to-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 from initial diagnosis through 1 week. Thus, this study represents an important contribution to defining the clinical utility and implementation of the CCL14 assay for identifying those at risk for persistent severe AKI, providing clinicians with 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 more benign (although not entirely) 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 resolved at discharge is associated with a more than fivefold increased risk of death by 1 year, after adjusting for illness severity and demographic differences. The same association has recently been observed in the setting of COVID-19-associated AKI, where persistent AKI after one week was associated with the highest odds ratio of death (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 outcomes. Importantly, this information is not the progression of renal tubular injury in AKI, and cannot be easily 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 persistent 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 medications and assessment of renally excreted medications. Furthermore, the 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, such as 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 easily identified subpopulations of patients likely to benefit in real-world clinical settings. In this regard, CCL14's ability 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 across the time course of AKI. Such uses may include prognosing 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 demonstrated the consistency of CCL14 for identifying patients at high risk for developing severe, persistent AKI across serial sampling and showed where changes occur and parallel clinical outcomes. Therefore, CCL14 is likely to be a useful clinical tool that can be flexibly implemented in determining the prognosis of patients with moderate to severe AKI in real-world settings, 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] Because there was no statistically significant interaction between initial CCL14 values ​​and the rate of concentration change (slope), 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 the initial CCL14 concentration and the rate of change in concentration in the new model were statistically significant (p<0.001). Furthermore, the discriminatory performance of both models for the endpoint, as measured by receiver operating characteristic curve (ROC) area under each variable, was similar: 0.849 for the model using the categorical CCL14 variable versus 0.852 for the model using the continuous CCL14 variable (p=0.78 for the difference in AUC). These results indicate that both the initial CCL14 level and the change in CCL14 level 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 to enable those skilled in the art to make and use it, various alternatives, modifications, and improvements will become apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments and are illustrative only and do not 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 levels 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 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 either of the other two terms. The terms and expressions used are used as terms of description and not of limitation, and in using such terms and expressions, it is not intended to exclude the features shown and described or equivalents thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications and variations of the concepts disclosed herein may be employed by those skilled in the art, and that such modifications and variations are deemed 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 between 1 hour and 72 hours after a first urine sample is obtained; (b) determining that the subject is at increased risk for persistent AKI if the level of CCL14 detected in the urine sample trends upward; (c) 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 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) 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; (d) (i) 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 (ii) 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. A method comprising:

2. 10. The method of claim 1, wherein the subject has AKI that meets the definition of KDIGO Stage 2.

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

4. 10. The method of claim 1, wherein the subject is determined to be at increased risk of developing persistent KDIGO stage 2 or 3 AKI.

5. 3. The method of claim 2, wherein the subject is determined to be at increased risk of developing persistent KDIGO stage 3 AKI.

6. 2. The method of claim 1, wherein the first predetermined threshold concentration of CCL14 is less than or equal to 1.3 ng / mL.

7. 2. The method of claim 1, wherein the second predetermined threshold concentration of CCL14 is greater than 13 ng / mL.

8. 10. The method of claim 1, wherein the second urine sample is obtained from 1 to 12 hours after the first urine sample is obtained.

9. 10. The method of claim 1, wherein the second urine sample is obtained from 1 hour to 24 hours after the first urine sample is obtained.

10. 10. The method of claim 1, wherein the second urine sample is obtained from 1 hour to 36 hours after the first urine sample is obtained.

11. 10. The method of claim 1, wherein the second urine sample is obtained from 1 hour to 48 hours after the first urine sample is obtained.

12. 10. The method of claim 1, wherein the second urine sample is obtained from 1 hour to 72 hours after the first urine sample is obtained.

13. The method of claim 1 , wherein the subject is in an intensive care unit.

14. 10. The method of claim 1, wherein the subject has been diagnosed with AKI.

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