Urinary biomarker of kidney function

Abstract

A method for assessing kidney injury in a subject, comprising (a) measuring the concentration of human phospholipase B-like 1 precursor or a fragment thereof (HPLBII-P) in a urine sample from the subject; (b) comparing the measured value to a relevant reference value; and (c) detecting the presence of kidney injury, or determining the severity of kidney injury, based on difference in the measured concentration compared to the reference value. A method of treatment, comprising (a) assessing the severity of kidney injury in a subject with the aforementioned method; and (b) applying or adjusting treatment given to the subject based on the results of the assessment. A use of HPLBII-P in urine as a marker for kidney injury, particularly in diabetes.

Description

[0001] URINARY BIOMARKER OF KIDNEY FUNCTION

[0002] TECHNICAL FIELD

[0003] The present invention relates to biomarkers of kidney function in urine, and their uses for diagnostic purposes, particularly in diabetic subjects.

[0004] BACKGROUND TO THE INVENTION

[0005] Kidney diseases are a heterogeneous group of disorders that impair the renal function, which is essential for maintaining fluid and electrolyte balance, blood pressure regulation, and waste excretion. Kidney diseases can lead to severe complications, such as cardiovascular disease, cerebrovascular disease, and mortality. Some of the common categories of kidney diseases are chronic kidney disease (CKD), acute kidney injury (AKI), diabetic nephropathy, nephrolithiasis (kidney stones), and urinary tract infections (UTIs).

[0006] Kidney diseases have a high global burden, affecting people of all ages and backgrounds, especially those with comorbidities, such as diabetes, hypertension, obesity, and genetic predisposition. The prevalence of kidney diseases varies depending on the definition, measurement, and population studied. According to certain estimates, CKD affects approximately 10% of the global population, and millions of people die annually due to lack of access to affordable renal replacement therapy. In the US, about 37 million adults (14% of the adult population) are thought to have CKD, but only 10% of them are aware of their condition. The prevalence of CKD increases with age, reaching 20% in men and 25% in women aged 65-74 years worldwide.

[0007] Kidney injury can be diagnosed early by performing urine and blood tests to detect markers of renal damage, such as protein in urine or elevated serum creatinine. Early detection and intervention can help prevent or delay the deterioration of renal function and reduce the risk of adverse outcomes. Some of the interventions for kidney diseases include pharmacotherapy, lifestyle modification, dialysis, and kidney transplantation. People with kidney diseases can also take measures to preserve their renal function, such as controlling their glycemia and blood pressure, following a renal-friendly diet, engaging in physical activity, quitting smoking, and avoiding nephrotoxic agents.

[0008] Thus, an object of the present invention is the provision of alternative and / or improved methods for assessing kidney injury. DEFINITIONS

[0009] The term "comprising" is to be interpreted as including, but not being limited to.

[0010] Numerical values in the specification and claims of this application should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

[0011] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of "from 2 to 10" is inclusive of the endpoints, 2 and 10, and all the intermediate values).

[0012] The term "about" can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range, "about" also discloses the range defined by the absolute values of the two endpoints, e.g. "about 2 to about 4" also discloses the range "from 2 to 4." The term "about" may refer to plus or minus 10% of the indicated number.

[0013] The term kidney injury is to be interpreted broadly in the present context, encompassing all forms of pathological alterations of the kidney, be they structural, functional, or both. The term encompasses thus both acute and chronic kidney diseases and all forms of renal damage.

[0014] The term HPLBII-P refers to the protein with the official name human phospholipase B-like 1 precursor having GenPept accession number NP_079105 (version 4 as of 13 November 2023), as well as its relevant fragments. In the context of the present invention when the concentration of "HPLBII-P" is measured, this may mean measurement of a fragment of the polypeptide of NP_079105.4, or the entire protein. The fragment in this context may mean fragments of at least 500 amino acids of the total 553 on the full-length precursor, at least 400 aa, at least 300 aa, at least 200 aa, at least 100 aa, at least 80 aa, at least 60 aa, at least 50 aa, at least 40 aa, at least 30 aa or at least 20 aa fragments of the full-length precursor. The fragment preferably contains an epitope for an antibody raised against human phospholipase B-like 1 precursor. BRIEF DESCRIPTION OF THE FIGURES

[0015] Figure 1. Immunohistochemistry staining of kidney tissue from healthy subjects. Polyclonal antibodies against HPLBII-P were used. The figure shows distinct staining of glomerular cells presumably podocytes and also faint staining of some tubular cells.

[0016] Figure 2. The staining of a nephron with the polyclonal anti-HPLBII-P antibodies. Most staining is seen in the glomeruli and probably associated with the podocytes, but also some in other structures such as the collecting ducts and tubuli.

[0017] Figure 3. The concentrations of HPLBII-P in urine of COVID-19 patients and healthy controls. The statistical difference between the groups was evaluated by Mann-Whitney U- test and the significance is shown in the figure. The insert (duplicated as Fig 3-2) shows the results of COVID-19 patients without signs of AKI in comparison to healthy controls.

[0018] Figure 4. The HPLBII-P concentrations in urine of COVID-19 patients with and without AKI.

[0019] The difference of the groups was evaluated by the Mann-Whitney U-test and the significance shown in the figure.

[0020] Figure 5. Regression analysis of the correlations between urine concentrations of HPLBII-P and urine Albumin (panel A) and urine HNL (763 / 8F) (panel B). The regression equations and r-values are shown in the figures.

[0021] Figure 6. The urine concentrations of HPLBII-P in COVID-19 patients with or without diabetes mellitus. The difference was evaluated by the Mann-Whitney U-test and the significance given in the figure. The insert (duplicated as Fig 6-2) shows the results in the same cohorts but without AKI.

[0022] Figure 7. Regression analysis of the correlations between urine concentrations of HPLBII-P and Plasma Glucose concentrations. The regression equation and r-value are shown in the figure.

[0023] Figure 8. Panel A shows the urine concentrations of HPLBII-P in the male populations of the ULSAM and PIVUS cohorts. The urine concentrations were identical. Panel B shows the urine concentrations of HPLBII-P in males and females of the PIVUS cohort. The concentrations in the female population were significantly higher than in the male population (p<0.0001, Mann-Whitney U-test). Figure 9. Panel A shows the comparisons of urine concentrations of the female population of the PIVUS cohort as compared to a healthy younger female population. The concentrations were significantly higher in the PIVUS cohort (p<0.0001, Mann-Whitney u- test). Panel B shows the comparisons of urine concentrations of the male population of the PIVUS cohort as compared to a healthy younger female population. The concentrations were significantly higher in the PIVUS cohort (p=0.005, Mann-Whitney u-test).

[0024] Figure 10. The urine concentrations of HPLBII-P in subjects with diabetes (BM) and no diabetes (No DM) in the ULSAM cohort. The statistical difference is indicated on the figure. Also shown are the differences between the healthy younger male population and the two ULSAM populations.

[0025] Figure 11. The correlation between plasma-glucose concentrations and urine HPLBII-P concentrations in the ULSAM cohort (r=0.20, p<0.001). The results of the diabetic subjects are shown by the open squares (n=43, r=0.58, p<0.001).

[0026] Figure 12. The correlation between serum concentrations of Cathepsin S and urine HPLBII- P concentrations in the ULSAM cohort. The insert (duplicated as Fig 12-2) shows the results with the diabetic population of the ULSAM cohort. The statistics are given on the figures

[0027] Figure 13. The correlation between blood-HbAlc percentage and urine concentrations of HPLBII-P in the diabetic subpopulation of the ULSAM cohort. The statistics are given on the figure (n=44, r=0.42, p=0.004).

[0028] Figure 14. ROC analysis of whole COVID cohort vs. healthy controls (left) and diabetic COVID cohort vs. healthy controls.

[0029] Figure 15. ROC analysis of whole elderly ULSAM cohort vs. young healthy controls (left) and diabetic elderly ULSAM cohort vs. young healthy controls.

[0030] SUMMARY OF THE INVENTION

[0031] The inventors have shown that HPLBII-P is expressed in kidney glomeruli (Example 1), and that the urinary concentration of HPLBII-P correlates with kidney injury in COVID-19 patients with or without diabetes (Examples 2 and 3), as well as in elderly community cohorts (Example 4). The urinary concentration of HPLBII-P correlates highly with serum level of Cathepsin-S, which is a known marker of kidney function. Significant correlations were also found to the kidney tubular biomarkers NGAL and KIM-1 in urine as well as to the general glomerular biomarker albumin in urine (Example 4, Table 3).

[0032] The present invention therefore provides novel methods for diagnosing, assessing and / or detecting kidney injury, including qualitative assessments on the degree of severity.

[0033] The present invention relates to the following items. The subject matter disclosed in the items below should be regarded disclosed in the same manner as if the subject matter were disclosed in patent claims.

[0034] 1. A method for assessing kidney injury in a subject, comprising: a. Measuring the concentration of human phospholipase B-like 1 precursor or an at least 20 amino-acid fragment thereof (HPLBII-P) in a urine sample from the subject; b. Comparing the measured value to a relevant reference value; and c. Detecting the presence of kidney injury, or determining the severity of kidney injury, based on difference in the measured concentration compared to the reference value.

[0035] 2. The method according to item 1, wherein the reference value is based on urinary HPLBII-P concentration in a healthy reference subject.

[0036] 3. The method according to item 1, wherein the reference value is comparably measured urinary HPLBII-P concentration in a sample from the same subject taken at a different point in time.

[0037] 4. The method according to item 3, wherein the method is further for following kidney function over time in a subject, wherein elevation in the measured concentration indicates debut or worsening of kidney injury, and / or wherein reduction in the measure concentration indicates recovery of kidney injury.

[0038] 5. The method according to item 1, wherein the reference value is comparably measured urinary HPLBII-P concentrations in a reference sample from a reference subject free of kidney injury.

[0039] 6. The method according to item 1, wherein the reference value is a mean value of comparably measured urinary HPLBII-P concentrations in a set of reference samples from a set of reference subjects free of kidney injury. 7. The method according to item 6, wherein the set comprises at least 10 reference subjects.

[0040] 8. The method according to any one of items 2 or 5-7, wherein the reference subject(s) is / are sex and / or age matched with the subject.

[0041] 9. The method according to any one of the preceding items, wherein the method is for detecting kidney injury.

[0042] 10. The method according to any one of the preceding items, wherein kidney injury is detected, if the measured concentration is at least 2 times, preferably 3 times, more preferably 5 times, most preferably 10 times higher compared to the reference value.

[0043] 11. The method according to any one of the preceding items, wherein kidney injury is detected, if the measured concentration is higher than the mean of a set of comparably measured reference samples from a set of reference subjects free of kidney injury by at least 2 times the standard deviation of the set of reference samples.

[0044] 12. The method according to any one of the preceding items, wherein kidney injury is detected, if the measured concentration is statistically significantly higher than the reference value with a p value of less than 0.05 calculated with Student's T-test.

[0045] 13. The method according to any one of the preceding items, wherein kidney injury is detected, if the measured concentration is statistically significantly higher than the reference value with a p value of less than 0.05 calculated with Mann-Whitney U- test.

[0046] 14. The method according to any one of the preceding items, wherein the subject is a diabetic subject.

[0047] 15. The method according to any one of the preceding items, wherein the subject has an ongoing SARS-COV-2 infection.

[0048] 16. The method according to any one of the preceding items, wherein the subject has, or is being tested for potentially having, one or more of: diabetic nephropathy, chronic kidney disease, acute kidney injury, Alport syndrome, Fabry disease, Focal Segmental Glomerulosclerosis, glomerulonephritis, IgA nephropathy, interstitial cystitis, kidney stones, kidney infection, polycystic kidney disease, pre-eclampsia. 17. A method of treatment, comprising: a. assessing the severity of kidney injury in a subject with method according to any one of the preceding items; b. applying or adjusting a treatment given to the subject based on the results of the assessment.

[0049] 18. The method according to item 17, wherein the subject is diabetic, and upon detecting the presence of kidney injury, the patient is administered a further line of diabetes treatment compared to the currently administered line of treatment.

[0050] 19. The method according to item 18, wherein the further line of diabetes treatment entails adding to the patient's current treatment one or more of: metformin, insulin, a DPP-4 inhibitor, a GLP-1 receptor agonist, a SGLT2-inhibitor, sulfonylureas, a meglitinide.

[0051] 20. The method according to item 17, wherein the subject has one or more of the following conditions: diabetic nephropathy, chronic kidney disease, acute kidney injury, Alport syndrome, Fabry disease, Focal Segmental Glomerulosclerosis, glomerulonephritis, IgA nephropathy, interstitial cystitis, kidney stones, kidney infection, polycystic kidney disease, pre-eclampsia; and the subject is administered intensified therapy appropriate for the condition(s) the patient is suffering from, if progression of kidney injury is detected by the assessment.

[0052] 21. A use of HPLBII-P in urine as a marker for kidney injury.

[0053] DETAILED DESCRIPTION

[0054] Diagnostic methods and markers for kidney injury

[0055] In a first aspect of the present invention, there is provided a method for assessing kidney injury in a subject, comprising: a. Measuring the concentration of human phospholipase B-like 1 precursor or a fragment thereof in a urine sample from the subject; b. Comparing the measured value to a relevant reference value; and c. Detecting the presence of kidney injury, or determining the severity of kidney injury, based on difference in the measured concentration compared to the reference value.

[0056] The fragment may be at least 500 amino acids of the total 553 on the full-length precursor, at least 400 aa, at least 300 aa, at least 200 aa, at least 100 aa, at least 80 aa, at least 60 aa, at least 50 aa, at least 40 aa, at least 30 aa or at least 20 aa. The fragment may contain an epitope for an antibody raised against human phospholipase B-like 1 precursor.

[0057] In certain embodiments, the method is for detecting kidney injury. The urine sample may be morning urine.

[0058] The method may be further for following kidney function over time in a subject, wherein elevation in the measured concentration indicates debut or worsening of kidney injury, and / or wherein reduction in the measure concentration indicates recovery of kidney injury.

[0059] In a second aspect, the present invention provided the use of HPLBII-P (or a fragment thereof as defined above for the first aspect) in urine as a marker for kidney injury.

[0060] The measurement of HPLBII-P can be carried out with any means known in the art, including ELISA, RIA, corresponding fluorescence-based assays and the like. For the immunoassays, polyclonal and / or monoclonal antibodies raised against HPLBII-P may be used. Suitable antibodies can be generated following the procedure in Xu S, Cai L, Zhao L, Douhan- Hakansson L, Kristjansson G, Pauksen K, Venge P. Tissue localization and the establishment of a sensitive immunoassay of the newly discovered human phospholipase B-precursor (PLB- P). J Immunol Methods 2010 Feb 28;353(l-2):71-7. The measurement can also be carried out with non-immunological methods including mass spectrometry-based methods such as LC-MS, LC-MS / MS or SELDI, although these methods are currently less cost-effective for routine clinical use.

[0061] Reference value

[0062] The reference value is relevant when it represents a reference point relevant for the status and / or change in the subject's health. The reference value is preferably based on urinary HPLBII-P concentration in a healthy reference subject. Alternatively, the reference value may be comparably measured urinary HPLBII-P concentration in a sample from the same subject taken at a different point in time. The reference value may be comparably measured urinary HPLBII-P concentration in a reference sample from a reference subject free of kidney injury. The reference value may also be a mean value of comparably measured urinary HPLBII-P concentrations in a set of reference samples from a set of reference subjects free of kidney injury. Preferably, the set comprises at least 10 reference subjects. Preferably, reference subject(s) is / are suitably matched with the subject, more preferably sex and / or age matched with the subject.

[0063] Diagnostic applications

[0064] The diagnostic utility of the method of the first aspect was shown in the Examples 2-4. As is well-known from the commonly used method called the receiver-operator-characteristic (ROC)-analysis, there is always a trade-off between sensitivity and selectivity. If the threshold for concluding the presence of a pathology is set lower, the sensitivity is increased (i.e. the number of false negatives is reduced), but at the cost of lowering selectivity (i.e. the number of false positives is increased). If the threshold is raised instead, the sensitivity is decreased, and selectivity increased. Depending on the setting, the tolerances for false negatives and false positives will differ. Examples of ROC curves generated from Examples 2- 4 are shown in Figs 13 and 14. For instance, in a population-wide screening where millions of people are tested, the number of false positives must be very low, otherwise the number of patients needing a follow-up will be overwhelming. On the other hand, if a diagnostic test is used as part of a specialist evaluation of a single patient, where several other factors will be weighed in before reaching a diagnosis, a much larger proportion of false positives may be regarded tolerable. Thus, the threshold for making a conclusion will is most cases need to be set considering the setting in which the analysis is made, and determining a generally applicable threshold is neither appropriate nor necessary.

[0065] Kidney injury may be detected in the method of the first aspect, if the measured concentration is at least 2 times, preferably 3 times, more preferably 5 times, most preferably 10 times higher compared to the reference value.

[0066] Kidney injury may be detected in the method of the first aspect, if the measured concentration is higher than the mean of a set of comparably measured reference samples from a set of reference subjects free of kidney injury by at least 2 times the standard deviation of the set of reference samples. Kidney injury may be detected in the method of the first aspect, if the measured concentration is statistically significantly higher than the reference value with a p value of less than 0.05 calculated with Student's T-test.

[0067] Kidney injury may be detected in the method of the first aspect, if the measured concentration is statistically significantly higher than the reference value with a p value of less than 0.05 calculated with Mann-Whitney U-test.

[0068] Test subjects

[0069] The invention according to the first or second aspects is in principle applicable to assessing any subject. The invention may be used for screening apparently healthy subjects without particular suspicion of any pathology, screening of subjects having elevated risk for kidney disease (such as diabetics), or assessing kidney function (or changes in kidney function over time) in patients already known to have a form of kidney injury.

[0070] Preferably, the subject is a diabetic. In other preferred embodiments, subject has an ongoing SARS-COV-2 infection.

[0071] The subject may have, suspected to have, or may be tested for potentially having, one or more of: diabetic nephropathy, chronic kidney disease, acute kidney injury, Alport syndrome, Fabry disease, Focal Segmental Glomerulosclerosis, glomerulonephritis, IgA nephropathy, interstitial cystitis, kidney stones, kidney infection, polycystic kidney disease, pre-eclampsia, or any other form of kidney injury.

[0072] Methods of treatment

[0073] The results obtained from the assessment of kidney injury using the invention according to the first or second aspects may be used to direct a treatment administered to the subject.

[0074] Thus, in a third aspect, the present invention provides a method of treatment, comprising: a. Assessing and / or quantifying the severity of kidney injury in a subject with method according to the first aspect; b. applying or adjusting treatment given to the subject based on the results of the assessment / quantification.

[0075] The subject may be a diabetic, and upon detecting kidney injury, the patient is administered a further line of diabetes treatment compared to the currently administered line of treatment, and / or a treatment involving an increased intensity of the current treatment.

[0076] The further line of diabetes treatment may entail adding to the patient's current treatment one or more of: metformin, insulin, a DPP-4 inhibitor, a GLP-1 receptor agonist, a SGLT2- inhibitor, sulfonylureas, a meglitinide. An increased intensity may involve a higher dose of the patient's current treatment.

[0077] In other embodiments, the subject has one or more of the following conditions: diabetic nephropathy, chronic kidney disease, acute kidney injury, Alport syndrome, Fabry disease, Focal Segmental Glomerulosclerosis, glomerulonephritis, IgA nephropathy, interstitial cystitis, kidney stones, kidney infection, polycystic kidney disease, pre-eclampsia; and the subject is administered intensified therapy appropriate for the condition(s) the patient is suffering from, if progression of kidney injury is detected.

[0078] General aspects relating to the present disclosure

[0079] The arrangement of the present disclosure into sections with headings and subheadings is merely to improve legibility and is not to be interpreted limiting in any way, in particular, the division does not in any way preclude or limit combining features under different headings and subheadings with each other. All references are hereby incorporated by reference.

[0080] EXAMPLES

[0081] The following examples are not to be regarded as limiting. For further information on the experimental details, the skilled reader is directed to a separate section titled Materials and Methods.

[0082] Example 1: HPLBII-P is expressed in the kidney glomeruli

[0083] Staining of kidney tissue with the polyclonal antibodies raised against HPLBII-P showed distinct staining of cells in the glomeruli (Figure 1). Staining was also seen in some tubular cells.

[0084] Staining of kidney tissue with the polyclonal antibodies raised against HPLBII-P showed distinct staining of podocytes in the glomeruli (Figure 2). Weak staining was also seen in some collecting ducts, but in few tubular cells. Example 2: Urinary HPLBII-P in COVID patients with acute kidney injury (AKI)

[0085] By means of the HPLBII-P ELISA the concentrations of HPLBII-P were measured in urine of 60 healthy persons and of patients with COVID-19. The COVID-19 patients were all admitted to the ICU because of respiratory failure. It is shown in Figure 3 that the urine concentrations in samples from COVID-19 patients were highly and significantly raised as compared to the results of healthy persons (p<0.0001). This was also the case when we only looked at COVID- 19 patients without any sign of AKI (Figure 3 insert). It is further indicated in Figure 4 and Table 1 that the concentrations were higher in the COVID-19 patients with AKI (p<0.02) and with a relation to AKI stages (p=0.04, Kruskal-Wallis ANOVA) (Not shown). In Table 1 we show the concentrations of several other biomarkers in urine in COVID-19 patients with or without AKI. None of the biomarkers were elevated in patients with AKI, whereas the serum concentrations of Cystatin C were significantly higher in those patients with AKI (p=0.0002). The relationships of these biomarkers to HPLBII-P were evaluated by Spearman rank correlation analysis. Significant correlations were found with albumin and HNL (763 / 8F) in urine as illustrated in Figures 5 A and B. A correlation was also found to KIM-1 (rs=0.28, p=0.01).

[0086] Table 1. The table shows the results of all investigated biomarkers in urine and Cystatin C in serum of patients with SARS-Cov-2 infections and the comparisons of the concentrations in patients with or without AKI (left) and in patients with or without diabetes mellitus (right). Differences were evaluated by the Mann-Whitney U-test and the results given in the table.

[0087] Biomarker No AKI AKI P-value Non-Diabetes Diabetes P-value

[0088] Urine Median (95% Cl) Median (95% Cl) Mann-Whitney Median (95% Cl) Median (95% Cl) Mann-Whitney

[0089] HPLBII-P, mg / L 0.19 (0.15-0.24) n=62 0.25 (0.23-0.35) n=67 P=0.019 0.19 (0.15-0.23) n=88 0.28 (0.24-0.44) n=41 P=0.0008

[0090] HNL (763 / 8F), mg / L 25 (20-39) n=61 29 (20-54) n=67 Ns 23 (16-35) n=87 41 (24-99) n=41 P=0.0056

[0091] NGAL, mg / L 45 (21-69) n=19 42 (28-85) n=29 Ns 35 (24-53) n=36 112 (38-213) n=12 P=0.007

[0092] TIM P-2, ng / L 3.7 (2.5-4-8) n=19 4.4 (2.4-5.9) n=30 Ns 4.2 (2.7-4.9) n=36 4.1 (1.7-7.2) n=13 Ns

[0093] KIM-1, ng / L 2.3 (1.6-5.2) n=47 2.6 (1.6-3.1) n=60 Ns 2.5 (1.9-3.2) n=77 2.4 (1.3-4.7) n=30 Ns

[0094] Cystatin C, mg / L 0.56 (0.42-0.83) n=47 0.87 (0.58-1.01) n=60 Ns 0.68 (0.53-0.91) n=77 0.76 (0.42-1.46) n=30 Ns

[0095] Albumin, mg / L 68 (50-112) n=47 94 (58-130) n=60 Ns 85 (59-130) n=77 71 (42-111) n=30 Ns

[0096] Biomarker No AKI AKI P-value Non-Diabetes Diabetes P-value

[0097] Serum Median (95% Cl) Median (95% Cl) Mann-Whitney Median (95% Cl) Median (95% Cl) Mann-Whitney

[0098] Cystatin C, mg / L 0.98 (0.80-1.05) n=33 1.19 (1.11-1.35) n=47 P=0.0002 1.07 (0.99-1.12) n=58 1.22 (1.06-1.80) n=23 P=0.055

[0099] Example 3: Urinary HPLBII-P in COVID patients with Diabetes Mellitus

[0100] In Figure 6 we show the highly and significantly raised concentrations of HPLBII-P in patients with diabetes mellitus (p=0.0008). In the insert in figure 6 we show the increased concentrations in diabetes mellitus in COVID-19 patients without signs of AKI (p=0.007). Higher concentrations in urine in patients with diabetes mellitus were also seen for HNL (763 / 8F) (p=0.0056) and NGAL (p=0.007), but not for the other investigated biomarkers shown in Table 1. Raised concentrations of HNL (763 / 8F) and NGAL in diabetes mellitus were only seen in those patients with AKI, p=0.002 and p=0.01, respectively. A significant correlation (rs=0.29, p=0.001) was seen between the plasma glucose concentrations at admission day and the HPLBII-P concentrations (Figure 7).

[0101] Example 4: Urinary HPLBII-P in elderly community cohorts

[0102] Figure 8 shows the distribution of HPLBII-P in urine of the ULSAM and PIVUS cohorts. The concentrations of HPLBII-P were similar in the male ULSAM cohort and in the male sub cohort of PIVUS. A highly significant difference (p<0.0001), however, was observed between males and females in the PIVUS cohort. It is shown in Figure 9 that the concentrations were significantly elevated in the PIVUS cohorts, both in males an in females, as compared to a group of younger healthy controls [females; age 35 years (95% Cl 27-45) and males; age 27 years (95% Cl 26-34)]. No difference between genders was seen in the control group. Also, the concentrations in the ULSAM cohort were significantly elevated as compared to the male subgroup of the healthy controls (p<0.0001) (Figure 10).

[0103] In Figure 10 the HPLBII-P concentrations in urine are shown in two populations of the ULSAM cohort i.e. in those with no diagnosis of diabetes mellitus and in those with the diagnosis of diabetes mellitus. The concentrations were significantly higher in diabetes mellitus (p<0.0001). In the PIVUS cohort a significant elevation of the concentrations of HPLBII-P was only seen among males (p=0.018), but not in females (Table 2). In the group of females significantly higher concentrations were observed in those subjects treated with insulin as compared to those with no insulin treatment, either in comparison with the whole cohort without insulin (p=0.026) or in comparison with the subjects who were treated with oral antidiabetics only (p=0.028). A similar trend was seen in the ULSAM population although the number of insulin-treated subjects was low i.e. n=3. Plasma glucose and HbAlc were recorded in the ULSAM study. Figure 11 shows a significant positive correlation in the ULSAM cohort between HPLBII-P and plasma glucose (r=0.20, p<0.001). However, as suggested on the figure this correlation was mainly due to findings in those subjects with diabetes mellitus. By the calculation of correlations on the subgroup of diabetics separately a strong correlation (r=0.58, p<0.001) was found to plasma glucose, but also to the proportion of HbAlc in blood (r=0.42, p=0.004) (Figure 11)

[0104] In Table 3 we show results of selected biomarkers and their correlations to HPLBII-P.

[0105] Significant correlations were found to the kidney tubular biomarkers NGAL and KIM-1 in urine as well as to the glomerular biomarker albumin in urine. However, the strongest correlations were seen between Cathepsin S in serum and the urine concentrations of HPLBII-P (Figure 12).

[0106] able 2. Urine output of HPLBII-P in the PIVUS and ULSAM cohorts in relation to diabetes mellitus

[0107] Table 3. Biomarkers in urine and serum in the subpopulations of diabetes and non-diabetes in the ULSAM cohort

[0108] MATERIALS AND METHODS

[0109] Diabetes study

[0110] The urine samples of the ULSAM cohort of men were collected at 77 years of age (n=839). The collection was finalized 2001 (sampling period 1998-2001). The urine samples of the PIVUS cohort were collected when 75 years of age and contained 401 samples from women and 387 samples from men. All samples were stored in aliquots at -80°C.

[0111] The studies were approved by the ethics committee of Uppsala University and the Declaration of Helsinki and its subsequent revisions were followed.

[0112] Immunohistochemistry of kidney tissue was performed as previously described (Xu S, Cai L, Zhao L, Douhan-Hakansson L, Kristjansson G, Pauksen K, Venge P. Tissue localization and the establishment of a sensitive immunoassay of the newly discovered human phospholipase B- precursor (PLB-P). J Immunol Methods 2010 Feb 28;353(l-2):71-7) using a rabbit polyclonal antibody raised against HPLBII-P.

[0113] HPLBII-P was measured by ELISA. The assay was based on antibodies developed for an earlier radioimmunoassay (Xu S, Cai L, Zhao L, Douhan-Hakansson L, Kristjansson G, Pauksen K, Venge P. Tissue localization and the establishment of a sensitive immunoassay of the newly discovered human phospholipase B-precursor (PLB-P). J Immunol Methods 2010 Feb 28;353(1-2) :71-7). The assay was a double polyclonal antibody-based assay with the same polyclonal antibody raised against HPLBII-P both as capture antibody bound to a microplate surface and as the detecting antibody. The detecting antibody was biotinylated and reacted with HRP-conjugated streptavidin. The colour reaction was developed by addition of H2O2 and the colour was read by a spectrophotometer and directly proportional to the concentration of HPLBII-P in the sample. The sensitivity of the assay was 0.075 ug / L. The calibration curve extended from 0.070 to 5 ug / L.

[0114] The measurement of Cathepsin S (DY1183), NGAL (DY1757) and KIM-1 (DY1750) were performed by ELISA kits purchased from R&D systems (Minneapolis, MN, USA). All clinical data were blinded to the analysing personal. The analytical performances of the ELISA:s were acceptable with CVs (Coefficient of Variation) in the range of 4-10% of duplicate samples. Albumin, Creatinine, Cystatin C were all measured in urine as part of the clinical routine and performed by the clinical chemistry laboratory at the University Hospital, Uppsala, Sweden.

[0115] COVID study

[0116] Data found in this research are part of the PronMed study approved by the National Ethical Review Agency (Dnr 2017 / 043, with amendments 2020-01623, 2020-02719, 2020-05730, 2021-01469, and 2022-00526-01) and listed at ClinicalTrials.gov (NCT03720860). Informed consent was obtained from the patient or next of kin. The Declaration of Helsinki and its subsequent revisions were followed.

[0117] The study included 132 patients admitted to the ICU (Intensive Care Unit) of Uppsala University Hospital with SARS-CoV-2 infections as diagnosed with PCR and signs of organ failure. Detailed information of the patient demographics was given previously (Bulow AS, , et al. J Clin Med 2021 Sep 14;10(18); Luther T et al.. Acta Anaesthesiol Scand 2021 Mar;65(3):364-72.

[0118] Clinical data was collected from the electronic medical records. AKI severity was staged according to Kidney Disease: Improving Global Outcome (KDIGO) creatinine criteria and renal replacement requirement solely (Khwaja A. Nephron Clin Pract 2012;120(4):cl79-cl84).

[0119] Immunohistochemistry of kidney tissue was performed as in the diabetes study.

[0120] HPLBII-P was measured by ELISA as in diabetes study. HNL was measured by ELISA (Diagnostics Development, Uppsala, Sweden). The HNL Elisa was configured with mabs 763 and 8F for the purpose of catching most HNL molecular variants in urine. The analytical performances of the two ELISA:s were acceptable with CVs (Coefficient of Variation) in the range of 4-10% of duplicate samples. The measurements of KIM-1, TIMP-2, NGAL were performed by the ELISA kits DY1750B, DY971, and DY1757 respectively, all purchased from R&D systems, Minneapolis, MN, USA. All clinical data were blinded to the analyzing personal.

[0121] Albumin and Cystatin C in urine and Cystatin C and creatinine in plasma were all measured by the clinical chemistry laboratory at University Hospital, Uppsala, Sweden.

[0122] All data were from results obtained at admission to the ICU. Statistics

[0123] Non-parametric statistics was applied throughout, unless otherwise indicated. For comparison between independent results Mann-Whitney U test was used and for the comparison of the results of multiple groups Kruskal-Wallis ANOVA was used. Correlations between biomarkers were calculated by Spearman rank correlations. The statistical programme Medcalc was used in all calculations: MedCalc® Statistical Software version 20.106 (MedCalc Software Ltd, Ostend, Belgium; https: / / www.medcalc.org; 2022)

Claims

CLAIMS1. A method for assessing kidney injury in a subject, comprising: a. Measuring the concentration of human phospholipase B-like 1 precursor or an at least 20 amino-acid fragment thereof (HPLBII-P) in a urine sample from the subject; b. Comparing the measured value to a relevant reference value; and c. Detecting the presence of kidney injury, or determining the severity of kidney injury, based on difference in the measured concentration compared to the reference value.

2. The method according to claim 1, wherein the reference value is based on urinary HPLBII-P concentration in a healthy reference subject.

3. The method according to claim 1, wherein the reference value is comparably measured urinary HPLBII-P concentration in a sample from the same subject taken at a different point in time.

4. The method according to claim 3, wherein the method is further for following kidney function over time in a subject, wherein elevation in the measured concentration indicates debut or worsening of kidney injury, and / or wherein reduction in the measure concentration indicates recovery of kidney injury.

5. The method according to claim 1, wherein the reference value is comparably measured urinary HPLBII-P concentrations in a reference sample from a reference subject free of kidney injury.

6. The method according to claim 1, wherein the reference value is a mean value of comparably measured urinary HPLBII-P concentrations in a set of reference samples from a set of reference subjects free of kidney injury.

7. The method according to claim 6, wherein the set comprises at least 10 reference subjects.

8. The method according to any one of claims 2 or 5-7, wherein the reference subject(s) is / are sex and / or age matched with the subject.

9. The method according to any one of the preceding claims, wherein the method is for detecting kidney injury.

10. The method according to any one of the preceding claims, wherein kidney injury is detected, if the measured concentration is at least 2 times, preferably 3 times, more preferably 5 times, most preferably 10 times higher compared to the reference value.

11. The method according to any one of claims 1 or 6-10, wherein kidney injury is detected, if the measured concentration is higher than the mean of a set of comparably measured reference samples from a set of reference subjects free of kidney injury by at least 2 times the standard deviation of the set of reference samples.

12. The method according to any one of the preceding claims, wherein kidney injury is detected, if the measured concentration is statistically significantly higher than the reference value with a p value of less than 0.05 calculated with Student's T-test.

13. The method according to any one of the preceding claims, wherein kidney injury is detected, if the measured concentration is statistically significantly higher than the reference value with a p value of less than 0.05 calculated with Mann-Whitney U- test.

14. The method according to any one of the preceding claims, wherein the subject is a diabetic subject.

15. The method according to any one of the preceding claims, wherein the subject has ongoing SARS-COV-2 infection.

16. The method according to any one of the preceding claims, wherein the subject has, or is being tested for potentially having, one or more of: diabetic nephropathy, chronic kidney disease, acute kidney injury, Alport syndrome, Fabry disease, Focal Segmental Glomerulosclerosis, glomerulonephritis, IgA nephropathy, interstitial cystitis, kidney stones, kidney infection, polycystic kidney disease, pre-eclampsia.

17. A method of treatment, comprising: a. assessing kidney injury in a subject with method according to any one of the preceding claims; b. applying or adjusting a treatment given to the subject based on the results of the assessment.

18. The method according to claim 17, wherein the subject is diabetic, and upon detecting the presence of kidney injury, the patient is administered a further line of diabetes treatment compared to the currently administered line of treatment.

19. The method according to claim 18, wherein the further line of diabetes treatment entails adding to the patient's current treatment one or more of: metformin, insulin, a DPP-4 inhibitor, a GLP-1 receptor agonist, a SGLT2-inhibitor, sulfonylureas, a meglitinide.

20. The method according to claim 17, wherein the subject has one or more of the following conditions: diabetic nephropathy, chronic kidney disease, acute kidney injury, Alport syndrome, Fabry disease, Focal Segmental Glomerulosclerosis, glomerulonephritis, IgA nephropathy, interstitial cystitis, kidney stones, kidney infection, polycystic kidney disease, pre-eclampsia; and the subject is administered intensified therapy appropriate for the condition(s) the patient is suffering from, if progression of kidney injury is detected by the assessment.

21. A use of HPLBII-P in urine as a marker for kidney injury.

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