Biomarkers for preclinical and / or early detection and / or diagnosis of kidney disease

Abstract

The present invention relates to biomarkers for the preclinical and / or early detection and / or diagnosis of kidney disease. In particular, a combination of at least two proteins selected from, for example, collagen type XIII (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), complement factor H (CFH), and complement factor I (CFI) is provided for use as a biomarker in a method for the preclinical and / or early detection and / or diagnosis of kidney disease. The present invention further relates to a method for the preclinical and / or early detection and / or diagnosis of kidney disease, comprising: (a) determining the concentrations of at least two proteins in a sample from a subject; and (b) comparing each concentration determined in step (a) with a control value, wherein deviation of each concentration determined in step (a) from the control value indicates preclinical and / or early kidney disease.

Description

[Technical Field]

[0001] The present invention relates to biomarkers for the preclinical and / or early detection and / or diagnosis of kidney disease. In particular, there is provided a combination of at least two proteins selected from, for example, collagen type XIII (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), complement factor H (CFH), and complement factor I (CFI), for use as a biomarker in a method for the preclinical and / or early detection and / or diagnosis of kidney disease. The present invention further relates to a method for the preclinical and / or early detection and / or diagnosis of kidney disease, comprising: (a) determining the concentrations of at least two proteins in a sample from a subject; and (b) comparing each of the concentrations determined in step (a) with a control value, wherein deviation of each of the concentrations determined in step (a) from the control value indicates preclinical and / or early kidney disease. [Background technology]

[0002] Almost all kidney diseases begin without any symptoms or pain. Most kidney diseases progress undetected for months or years before symptoms become apparent and diagnosis is made. While there are no preventative screening guidelines for preclinical kidney disease, it is possible to halt or even reverse the onset of kidney disease during this period. Damage to the glomerular filtration apparatus results in leakage of plasma proteins into the primary urine. Currently, proteinuria or albuminuria (other than hematuria) is the earliest parameter indicating kidney damage. However, this parameter indicates already manifest disease, not its early stages. Furthermore, albuminuria further accelerates kidney destruction itself. Clinically established kidney function parameters, such as serum creatinine, do not indicate pathological values ​​until 50% of kidney function has already been impaired, which is too late. Thus, the diagnostic dilemma remains unresolved.

[0003] Kidney disease that progresses to end-stage kidney disease (ESKD) requiring renal replacement therapy is virtually all treatable and even curable if detected and treated near the onset of the disease. Preemptive detection of nephropathy before measurable albuminuria would provide treatment options to slow, halt, or even reverse progression.

[0004] For example, Alport syndrome (AS) is a hereditary nephropathy characterized by hematuria and albuminuria, often leading to ESKD, hearing loss, and ocular changes (see, e.g., Hudson et al., 2003; Nagel et al., 2005). The majority of cases are caused by mutations in the α5 chain of collagen (IV) (COL(IV)A5) and are inherited via X-linkage (Hertz et al., 2012). Female carriers of X-linked AS can also be severely affected (Goka et al., 2021). Furthermore, there is considerable genotypic and phenotypic variability (Savige et al., 2018). Several alterations have been preclinically identified in the renal cortex of affected AS mice (Gessel et al., 2019; Dufek et al., 2016). Nevertheless, the specific pathogenic sequence of AS events, from type IV collagen mutations to progressive renal fibrosis, remains unclear. Although all children are born healthy with immature α1 / 2(IV) chains, due to mutations, AS patients are unable to mature GBMs with α3 / 4 / 5(IV) chains. Therefore, defects may manifest after birth, creating a diagnostic window. Recent expert guidelines recommend genetic testing for the diagnosis of Alport syndrome and the identification of several regulatory factors (Savige et al., 2019). Nevertheless, the application of these methods typically requires historical and / or clinical evidence, such as hematuria (Gross et al., 2020). However, in previously undiagnosed families and de novo AS mutations, early diagnosis with this tool is not feasible. Histological analysis is highly invasive, carries a high risk, and represents only a very late event in the progression of AS. To date, transplantation is the only curative treatment for ESKD due to AS (Savige et al., 2019). Nevertheless, the most effective and widely used treatment currently is inhibition of the renin-angiotensin-aldosterone system.In addition, several new approaches to improve kidney function and extend life expectancy are under investigation.Such approaches include the use of chaperones, stem cells, stem cell extracellular vesicles, and exon skipping (see, e.g., Yamamura et al., 2020). ACE inhibition has now been shown to delay renal failure by years in a time-dependent manner (Kashtan et al., 2013; Noone et al., 2013). Thus, the earlier treatment is initiated, the greater the benefit. In general, treatment initiation is most beneficial in asymptomatic patients, who can optimally slow the progression of kidney disease.

[0005] U.S. Patent Application Publication No. 2011 / 118127A1 relates to drug-induced gene expression in specific tissues or cell lines, and describes a method for screening for drugs that cause toxicity using a microarray containing probes for various genes. International Publication No. 2018 / 094021A1 relates to steroid resistance in nephrotic syndrome, and discloses a method for diagnosing steroid resistance in a subject with a glomerular disease such as nephrotic syndrome, comprising determining the amount of at least one steroid resistance biomarker in a biological sample from the subject. These are examples of the effects of cytotoxic drugs and the diagnosis of overt renal disease.

[0006] Therefore, early and progressive biomarkers that provide glomerular warning signals before any clinical signs of glomerular injury appear are urgently needed for early diagnosis, early treatment initiation, treatment monitoring, and even the development of novel therapeutic strategies.

[0007] Furthermore, in developed countries, diabetic nephropathy (DN) has become the leading cause of end-stage kidney disease (ESKD) and renal replacement therapy. However, established diagnostic tests in clinical practice, namely serum creatinine, estimated glomerular filtration rate (eGFR), and albuminuria, indicate the late and irreversible stages of DN. The scientific literature lacks routinely applicable, practical biomarkers that can indicate the onset of preclinical and potentially curable DN before the onset of (micro)albuminuria. Furthermore, there are no indicators to distinguish diabetic individuals predisposed to nephropathy and renal failure from other individuals. DN begins with alterations in the endothelium and basement membrane of the renal glomerulus, as is the case for several other glomerular nephropathies. Furthermore, the scientific literature reveals that the early molecular changes and histological abnormalities of most glomerulopathies, such as the deposition and activation of complement components, deposition of other substances, thickening of the basement membrane, and podocyte effacement, are only marginally consistent across all glomerulopathies, regardless of their cause (see, for example, Hastings et al., 2021; Koopman et al., 2020). The filtration apparatus, including podocytes, endothelium, and mesangial cells, is uniformly damaged, as in AS. Only then can these early processes be accessed through established diagnostic methods, such as those described above. These changes then progress to loss of the filtration barrier, albuminuria, tubular changes, and fibrosis. Summary of the Invention

[0008] According to the present invention, the object is to provide a combination for use as a biomarker in a method for the preclinical and / or early detection and / or diagnosis of kidney disease in a mammal, comprising: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI) The problem is solved by a combination of at least two proteins selected from:

[0009] According to the present invention, the object is to provide a protein for use as a biomarker in a method for the preclinical and / or early detection and / or diagnosis of kidney disease in a mammal, comprising: The protein is Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI) is selected from fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, and serine / arginine repeat matrix protein 3, a protein in combination with at least one further protein selected from is solved by

[0010] According to the present invention, the object is to provide a combination for use as a biomarker in a method for the preclinical and / or early detection and / or diagnosis of kidney disease in a mammal, comprising: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, Complement C1s, Complement C1r, Complement C2, Complement C3, Complement C4, Complement C5, Complement factor B, haptoglobin, α2-macroglobulin, alpha-1-antitrypsin, Ceruloplasmin, Serpin family A member 3, Serpin family A member 5, Serpin family A member 7, Serpin family G member 1, Serpin family D member 1, coagulation factor V, Prothrombin, plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit alpha, Fetuin B, insulin-like growth factor binding protein, alpha-2-glycoprotein 1, paraoxonase 1, dipeptidyl peptidase 4, C-type lectin domain family 3 member B, Galectin 3 binding protein, apolipoprotein AIV (Apo AIV), Apolipoprotein CI (Apo CI), Apolipoprotein CII (Apo CII), apolipoprotein E (Apo E4), Apolipoprotein M (Apo M), apolipoprotein B (Apo B), Myosin IB, Myosin XVIIIB, kinesin-like proteins, CD109 molecule, maltase-glucoamylase, succin molecular chaperone, Dispatched RND Transporter Family Member 2, DNA polymerase, Transport protein particle complex subunit 2, RB related KRAB zinc finger, ciliogenesis-associated TTC17-interacting protein, Proteasome subunit alpha type, Synaptic membrane exocytosis regulation 2, extracellular matrix protein 1, cadherin 5, coiled-coil domain-containing 178, and Attractin The problem is solved by a combination of at least two proteins selected from:

[0011] According to the present invention, the object is a method for the preclinical and / or early detection and / or diagnosis of kidney disease, comprising: (a) determining the concentrations of at least two proteins in a mammalian sample, The at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI), Selected from or or The at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI), and one protein selected from fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, and Serine / arginine repeat matrix protein 3 at least one further protein selected from and (b) comparing each concentration determined in step (a) with a control value; Including, Deviations from the control values ​​for each concentration determined in step (a) are resolved by the method as indicative of preclinical and / or early stage renal disease in the mammal.

[0012] According to the present invention, the object is a method for the preclinical and / or early detection and / or diagnosis of kidney disease, comprising: (a) determining the concentrations of at least two proteins in a mammalian sample, The at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, Complement C1s, Complement C1r, Complement C2, Complement C3, Complement C4, Complement C5, Complement factor B, haptoglobin, α2-macroglobulin, alpha-1-antitrypsin, Ceruloplasmin, Serpin family A member 3, Serpin family A member 5, Serpin family A member 7, Serpin family G member 1, Serpin family D member 1, coagulation factor V, Prothrombin, plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit alpha, Fetuin B, insulin-like growth factor binding protein, alpha-2-glycoprotein 1, paraoxonase 1, dipeptidyl peptidase 4, C-type lectin domain family 3 member B, Galectin 3 binding protein, apolipoprotein AIV (Apo AIV), Apolipoprotein CI (Apo CI), Apolipoprotein CII (Apo CII), apolipoprotein E (Apo E4), Apolipoprotein M (Apo M), apolipoprotein B (Apo B), Myosin IB, Myosin XVIIIB, kinesin-like proteins, CD109 molecule, maltase-glucoamylase, succin molecular chaperone, Dispatched RND Transporter Family Member 2, DNA polymerase, Transport protein particle complex subunit 2, RB related KRAB zinc finger, ciliogenesis-associated TTC17-interacting protein, Proteasome subunit alpha type, Synaptic membrane exocytosis regulation 2, extracellular matrix protein 1, cadherin 5, coiled-coil domain-containing 178, and Attractin and (b) comparing each concentration determined in step (a) with a control value; Including, Deviations from the control values ​​for each concentration determined in step (a) are resolved by the method as indicative of preclinical and / or early stage renal disease in the mammal. [Brief explanation of the drawings]

[0013] [Figure 1] Time course of disease progression and biomarker concentrations in renal disease. Several hypothetical biomarker (BM) types exist, which represent different targets for our approach. A: Preclinically applicable BM; its increase indicates changes that begin in the preclinical stage and persist even during progression. B: True preclinical BM; its increase indicates only preclinical changes. C: Early BM; its increase indicates changes that coincide with the onset of early clinical signs but are often overlooked or ignored (e.g., microhematuria in AS). D: Late BM; its increase indicates subsequent changes in overt disease. This type (e.g., AS (micro)albuminuria, decreased eGFR) is usually already applicable. This type was not the subject of this study. Types A and B were investigated in young AS mice (4 weeks old, see Muckova et al., 2015) and AS dogs (7 weeks old). This investigation also detects type C. Type B may be missed when applying human samples containing various time points during individual disease development. However, detected and verified BM of type A, B, and / or C should all be suitable for the very early diagnosis of renal injury. The same applies to the reverse concentration course, i.e., a decrease in concentration under altered conditions. [Figure 2] Box plots of single concentrations of preferred biomarker candidates determined by ELISA are shown. Left panel: samples from patients with X-linked Alport syndrome of different severity; right panel: other nephropathies. The order of the groups is the same for all three charts in each panel and is indicated at the bottom. Full results (p-values) are shown in Table 10; all other examples are not significant. [Figure 3]Box plots of preferred composite concentrations of candidate biomarkers obtained from ELISA using patient and control cohorts are shown. Complete results (p-values) are shown in Table 11. The order of the groups is the same for all charts and is indicated at the bottom. In Tables 10 and 11, groups that are significantly different as determined by between-group comparison (p-value less than 0.05 in post-hoc analysis) are shown in gray fill; all others are not significant. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention will now be described in more detail, but it will be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein, as these may vary. It will also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For purposes of the present invention, all references cited herein are incorporated herein by reference in their entirety.

[0015] Concentrations, amounts, and other numerical data may be expressed or presented in range format herein. It is understood that such range format is used merely for convenience and brevity, and thus should be interpreted flexibly to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of example, a numerical range of "1 to 21" should be interpreted to include not only the explicitly recited values ​​1 to 21, but also each individual value and subrange within the specified range. Thus, included within this numerical range are individual values ​​such as 1, 2, 3, 4, 5...17, 18, 19, 20, 21, etc., and subranges such as 2 to 10, 8 to 15, etc. This same principle applies to ranges reciting only a single numerical value, such as "at least 90%." Furthermore, such interpretation should apply regardless of the breadth of the range or the characteristics recited.

[0016] Preclinical and early biomarkers of kidney disease As outlined above, the present invention provides novel biomarkers for the preclinical and / or early detection and / or diagnosis of kidney disease.

[0017] The biomarkers of the present invention are the proteins disclosed herein. As used herein, the term "protein" also refers to / includes engineered variants of the respective proteins, fragments of the respective proteins, protein chains, and complexes of the respective proteins with further proteins.

[0018] Furthermore, the term "protein" as used herein refers to / includes all known proteins, including identified chains of each protein, engineered variants of each protein, fragments of each protein, single protein chains, multiple protein chains, and complexes of each protein (chain) with further proteins and protein chains, regardless of validation using currently available immunoassays such as ELISA.

[0019] There are defined stages that describe the course of chronic kidney disease (CKD) of various origins (see Jerums et al., 2009): Stage I: Hyperfiltration; Stage II: functional impairment with normoalbuminuria; Stage III: Microalbuminuria; Stage IV: overt albuminuria; Stage V: End-stage renal failure.

[0020] In Alport syndrome (AS), the following stages of kidney damage and dysfunction have been described, as defined by Gross et al. (2012): Stage 0: Microhematuria without microalbuminuria (usually at birth); Stage I: Microalbuminuria (30-300 mg albumin / g Crea); Stage II: Albuminuria >300 mg albumin / gCrea; Stage III: >25% reduction in normal renal function (creatinine clearance); Stage IV: End-stage renal failure.

[0021] Diabetic kidney disease (DKD) is staged based on albuminuria and taking into account eGFR (see de Boer et al., 2020): eGFR(mL / min / 1.73m 2 ); Stage G1: ≥ 90; Stage G2: 60~89 Stage G3a: 45~59 Stage G3b: 30-44 Stage G4: 15~29 Stage G5:<15 Albuminuria (mg / g creatinine) Stage A1:<30 Stage A2:30~300 Stage A3:>300.

[0022] CKD of various origins can also be staged based on eGFR and levels of albuminuria as follows (see KDIGO 2012: Clinical Practice Guidelines for the Evaluation and Management of Chronic Kidney Disease):

[0023] Patients are classified into G1 to G5 based on eGFR and A1 to A3 based on ACR (albumin:creatinine ratio). eGFR(mL / min / 1.73m 2 ); Stage G1: ≥ 90; Stage G2: 60~89 Stage G3a: 45~59 Stage G3b: 30-44 Stage G4: 15~29 Stage G5:<15 ACR (mg / mmol) Stage A1:<3 Stage A2: 3~30 Stage A3:>30.

[0024] In the present invention, stages I and II (defined for CKD of various origins), stages G1A1, G1A2, G2A2, and G3aA1 (defined for DKD and CKD of various origins according to KDIGO 2012), and stages 0 and I (defined for AS) are addressed by biomarkers. There are no currently accepted, applicable, and specific clinical and laboratory signs for diagnosing "(early) renal failure." At these stages, the disease is advanced but asymptomatic. In contrast, in the literature, the term "early" mostly refers to the early stage of overt renal damage, which can already be detected by various commonly applied biomarkers such as KIM-1, NGAL, cystatin C, and microalbuminuria.

[0025] As used herein, "preclinical" stages of kidney disease refer to Stage I (hyperfiltration) as defined for CKD of various origins and / or Stage G1A1 as defined for DKD and CKD of various origins (according to KDIGO 2012), and / or Stage 0 (microhematuria without microalbuminuria) as defined for AS.

[0026] Although both signs of hyperfiltration and microhematuria are used as parameters to define the preclinical stage of CKD, although they may indicate disease, they do not function practically as specific or even preclinical indicators of early renal disease, mainly because both signs occur under various physiological circumstances, such as physical activity, excessive fluid intake, or even sterile sports injuries, and therefore these signs are ignored by the majority of physicians and pediatricians.

[0027] As used herein, "early" stages of kidney disease refer to stage II (failure with normal albuminuria) as defined for CKD of various origins, stages G1A2, G2A2, G3aA1 as defined for DKD and CKD of various origins (KDIGO 2012), and / or stage I (microalbuminuria (30-300 mg albumin / g creatinine)) as defined for AS. Thus, early stages are characterized by subtle changes in already operational clinical and / or clinical chemistry signs. "Early" means that there are signs of kidney changes and damage beginning, but severe kidney failure has not yet occurred.

[0028] The "preclinical" stage of kidney disease occurs earlier in time than (or, in other words, before) the "early" stage of kidney disease.

[0029] The present invention provides biomarkers for detecting and / or diagnosing kidney disease at its preclinical and / or early stages, in other words, the biomarkers make it possible to detect kidney disease at its preclinical and / or early stages, thereby enabling a subject / patient to be diagnosed as having kidney disease at its preclinical and / or early stages at the time the sample is taken.

[0030] Preferably, the biomarkers provide for the in vitro detection and / or in vitro diagnosis of preclinical and / or early stage kidney disease.

[0031] Preferably, the renal disease is A chronic progressive kidney disease that begins with damage to the glomeruli (glomerulopathy), Preferably, post-infectious glomerulopathy, IgA nephropathy, Henoch-Schönlein purpura, focal segmental glomerulosclerosis, Alport syndrome, thinning basement membrane nephropathy, benign familial hematuria, or Chronic progressive kidney disease that begins in other parts of the body and / or kidneys involving the glomeruli (nephropathy), accompanied by other regulatory disorders Preferably, metabolic syndrome, hypertension, heart failure, nephrotoxic drug therapy, adiposity, diabetes mellitus (diabetic nephropathy), renal agenesis, renal hypoplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract abnormalities, duplex kidneys, nephronophthisis is selected from.

[0032] The present invention provides a method for the detection and / or diagnosis of renal disease at a preclinical and / or early stage, comprising: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI) The present invention provides a combination of at least two proteins selected from:

[0033] Thus, the present invention provides a method for detecting and / or diagnosing kidney disease at a preclinical and / or early stage, as a biomarker. Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI) The present invention provides the use of a combination of at least two proteins selected from:

[0034] As used herein, the term "combination" means only that at least two biomarkers are used for detection and / or diagnosis, where one biomarker is used in conjunction with at least one additional biomarker for detection and / or diagnosis.

[0035] In one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20 or more biomarkers are used.

[0036] The present invention provides a method for the detection and / or diagnosis of renal disease at a preclinical and / or early stage, comprising: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI) The present invention provides at least two proteins selected from:

[0037] Thus, the present invention provides a method for detecting and / or diagnosing kidney disease at a preclinical and / or early stage, as a biomarker. Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI) The present invention provides the use of at least two proteins selected from:

[0038] In a preferred embodiment, COLXIII and C4BP, or COLXIII, C4BP and HABP2, or CFH and CFI are determined.

[0039] In one embodiment, COLXIII, HABP2, C4BP, CFH, and CFI are determined.

[0040] The present invention provides a method for the detection and / or diagnosis of renal disease at a preclinical and / or early stage, comprising: fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), and C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), Dynein (DYN), preferably dynein heavy chain domain 1; Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, preferably the C8 alpha chain and / or the C8 beta chain, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, Complement C1s, Complement C1r, Complement C2, Complement C3, Complement C4, Complement C5, Complement factor B, haptoglobin, α2-macroglobulin, alpha-1-antitrypsin, Ceruloplasmin, Serpin family A member 3, Serpin family A member 5, Serpin family A member 7, Serpin family G member 1, Serpin family D member 1, coagulation factor V, Prothrombin, plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit alpha, Fetuin B, insulin-like growth factor binding protein, alpha-2-glycoprotein 1, paraoxonase 1, dipeptidyl peptidase 4, C-type lectin domain family 3 member B, Galectin 3 binding protein, apolipoprotein AIV (Apo AIV), Apolipoprotein CI (Apo CI), Apolipoprotein CII (Apo CII), apolipoprotein E (Apo E4), Apolipoprotein M (Apo M), apolipoprotein B (Apo B), Myosin IB, Myosin XVIIIB, kinesin-like proteins, CD109 molecule, maltase-glucoamylase, succin molecular chaperone, Dispatched RND Transporter Family Member 2, DNA polymerase, Transport protein particle complex subunit 2, RB related KRAB zinc finger, ciliogenesis-associated TTC17-interacting protein, Proteasome subunit alpha type, Synaptic membrane exocytosis regulation 2, extracellular matrix protein 1, cadherin 5, coiled-coil domain-containing 178, and Attractin in combination with at least one additional protein selected from Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI) The present invention provides a protein selected from:

[0041] Thus, the present invention provides a method for the detection and / or diagnosis of kidney disease at a preclinical and / or early stage, comprising administering to the kidney a protein as disclosed above in combination with at least one further protein: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI) The present invention provides the use of a protein selected from

[0042] The protein biomarkers of the present invention are shown in Table 1, which also shows the protein ID and mass spectrometry-determined concentration deviations (mean ratios in subfractions or overall trends) in affected dogs compared to healthy controls. Protein IDs reference the UniProt database of approximately 47,800 dog entries (dated October 26, 2017).

[0043] Virtually all of the identified proteins have a generally ancient primary origin, but are also produced in several other organs and tissues, particularly the kidney. Some acute phase proteins, including complement components, are widely produced in various cell types beyond the liver, including mesangial cells and other kidney cells.

[0044] In the urine of people with renal disease, the following proteins exhibit concentrations that deviate by ≥ 2 times the median of the normal concentration range as determined by the mean ratio (subfraction mean ratio or overall trend, mass spectrometry) as well as by ELISA:

[0045] Collagen type XIII (COLXIII) refers to the protein with the protein ID F1PJH3. Type XIII collagen is a homotrimer, i.e., a protein composed of three identical peptide chains (monomers), each called the alpha-1 chain of type XIII collagen. Formally, this monomer is called type XIII collagen alpha-1 chain and is encoded by the COL3A1 gene in humans. Type XIII collagen is a fibrous collagen, and the protein has a long, inflexible triple-helical domain. COLXIII is a transmembrane protein expressed in glomerular endothelial cells.

[0046] In the urine of individuals with renal disease, COLXIII exhibits concentrations that deviate by more than two-fold from the median of the normal concentration range determined by ELISA (see also Figure 2).

[0047] Hyaluronan-binding protein 2 (HABP2) refers to the protein with the protein ID J9NV47. Hyaluronan-binding protein 2, also known as factor VII-activating protease (FSAP), is a protein encoded by the HABP2 gene in humans. The protein encoded by this gene is an extracellular serine protease that binds to hyaluronan. It is involved in extracellular matrix organization and cell adhesion.

[0048] In the urine of individuals with renal disease, HABP2 exhibits concentrations that deviate by more than two-fold from the median of the normal concentration range determined by ELISA (see also Figure 2).

[0049] C4-binding protein (C4BP) or complement component 4-binding protein refers to a protein with protein ID F1PGM9. C4b-binding protein is involved in regulating the complement system. It is a multimeric protein containing seven identical alpha chains encoded by C4BPA and one beta chain encoded by C4BPB. It is produced by podocytes in the kidney. The biomarker is C4BP, preferably the C4BP alpha chain (C4BPA).

[0050] In the urine of people with renal disease, C4BPA exhibits concentrations that deviate by more than (≥) 2-fold from the median of the normal concentration range determined by ELISA. See also Figure 2.

[0051] Complement factor H (CFH) refers to the protein with the protein ID F1PY40. Factor H is a member of the complement activation family of regulators and a complement control protein. It is a soluble glycoprotein that circulates in human plasma.

[0052] In the urine of people with renal disease, CFH exhibits concentrations that deviate by more than (≥) 2-fold from the median of the normal concentration range determined by ELISA. See also Figure 2.

[0053] Complement factor I (CFI) refers to the protein with the protein ID J9NT17. Complement factor I, also known as C3b / C4b inactivator, is a protein encoded by the CFI gene in humans. Complement factor I (factor I) is a protein in the complement system that regulates complement activation by cleaving cell-bound or fluid-phase C3b and C4b.

[0054] In the urine of humans with renal disease, CFI exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by ELISA.

[0055] Fibrinogen gamma chain (FGG) refers to a protein with protein ID F1P8G0. Fibrinogen gamma chain, encoded by the fibrinogen gamma gene (FGG), is a human gene found on chromosome 4. The protein encoded by this gene is the gamma component of fibrinogen, a blood-borne glycoprotein composed of three pairs of non-identical polypeptide chains. Fibrinogen is also a component of the glomerular basement membrane. The biomarker is fibrinogen, preferably fibrinogen gamma chain (FGG).

[0056] In the urine of people with renal disease, FGG exhibits a concentration deviation of more than (≥) 2 times the median of the normal concentration range determined by ELISA.

[0057] Lumican (LUM) refers to the protein with the protein ID E2R416. Lumican, also known as LUM, is an extracellular matrix protein that in humans is encoded by the LUM gene on chromosome 12.

[0058] In the urine of people with renal disease, LUM exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by ELISA.

[0059] Formin 1 (FMN) refers to a protein with the protein ID J9P216. Formins (formin homology proteins) are a group of proteins involved in actin polymerization and associated with the fast-growing ends (barbed ends) of actin filaments.

[0060] In the urine of people with renal disease, FMN exhibits concentrations that deviate by more than (≥) 2 times the median of the normal concentration range determined by ELISA.

[0061] Vitronectin (VTN) refers to the protein with the protein ID Q2YF02. Vitronectin is a glycoprotein of the hemopexin family that is abundant in serum, the extracellular matrix of several tissues including the glomerular basement membrane, and bone. In humans, it is encoded by the VTN gene. In Table 1, it is designated as vitronectin (fragment).

[0062] In the urine of people with renal disease, VTN exhibits concentrations that deviate by more than (≥) 2-fold from the median of the normal concentration range determined by ELISA.

[0063] Gelsolin (GS) refers to the protein with the protein ID F6Y3P9. Gelsolin is an actin-binding protein that is a key regulator of actin filament assembly and disassembly.

[0064] In the urine of people with renal disease, GS exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by ELISA.

[0065] Angiotensinogen (AGT) refers to the protein with the protein ID F1PAL5. Angiotensinogen is a component of the renin-angiotensin system (RAS), a hormonal system that regulates blood pressure and fluid balance.

[0066] In the urine of people with renal disease, AGT exhibits a concentration deviation of ≥2 times the median of the normal concentration range determined by ELISA.

[0067] Adiponectin (ADP) refers to a protein with the protein ID B5U1S6. Adiponectin (also known as GBP-28, apM1, AdipoQ, and Acrp30) is a protein hormone and adipokine involved in regulating glucose levels and the breakdown of fatty acids. In humans, it is encoded by the ADIPOQ gene and is produced primarily in adipose tissue, but also in muscle. In Table 1, it is referred to as adiponectin (fragment).

[0068] In the urine of people with renal disease, ADP concentration deviates by more than (≥) 2 times the median of the normal concentration range determined by ELISA.

[0069] Transforming growth factor-β1 (TGF-β1) refers to a protein with the protein ID P54831. Transforming growth factor beta 1, or TGF-β1, is a polypeptide member of the transforming growth factor beta superfamily of cytokines. It is a secreted protein responsible for many cellular functions, including cell growth, cell proliferation, cell differentiation, matrix remodeling, and the regulation of apoptosis. In humans, TGF-β1 is encoded by the TGFB1 gene. TGF-β1 was not identified by mass spectrometry because its concentration in subfractions was very low (see 1.3). Because many matrix proteins indicative of matrix remodeling have been identified, TGF-β1 was measured in human samples.

[0070] In the urine of people with renal disease, TGF-β1 exhibits a concentration deviation of ≥2 times the median of the normal concentration range determined by ELISA.

[0071] The carboxy-terminal propeptide of type I procollagen (PICP) is a protein fragment. Type I procollagen carboxy-terminal propeptide (PICP) derived from type I procollagen was identified as an indicator of type I collagen synthesis in bone matrix formation and skin repair. PICP is a fragment of the collagen I trimer, designated as collagen α1(I) chain (ID F1Q3I5), collagen α1(I) (fragment) (ID E5G723), and collagen α2(I) chain (ID F1PHY1) (Table 1).

[0072] In the urine of people with renal disease, PICP exhibits a concentration deviation of ≥2 times the median of the normal concentration range determined by ELISA.

[0073] C-reactive protein (CRP) or pentraxin refers to a protein with the protein ID T2KEN6. C-reactive protein (CRP) is a cyclic (ring-shaped) pentameric protein found in plasma, and its circulating levels increase in response to inflammation. It is an acute-phase protein of hepatic origin that increases after the secretion of interleukin-6 by macrophages and T cells. Its physiological role is to bind to lysophosphatidylcholine expressed on the surface of dead or dying cells (and certain bacteria) to activate the complement system via C1q.

[0074] In the urine of people with renal disease, CRP concentration deviates by more than (≥) 2 times the median of the normal concentration range determined by ELISA.

[0075] Alpha-1-acid glycoprotein (a1AGP) refers to the protein with the protein ID F6Y713. Alpha-1-acid glycoprotein (a1AGP, AGP, or AAG) or orosomucoid (ORM) is an acute-phase protein found in plasma. It is an alpha-globulin glycoprotein and is regulated by two polymorphic genes. It is primarily synthesized in hepatocytes.

[0076] In the urine of individuals with renal disease, a1AGP exhibits concentrations that deviate by more than two-fold from the median of the normal concentration range determined by ELISA (see also Figure 2).

[0077] Complement component 1q (C1q), consisting of complement C1q A chain, complement C1q B chain, and complement C1q C chain, refers to a protein with protein ID J9P4B4 (A chain), J9P1G2 (B chain), and E2RJC2 (C chain) (listed in Table 1). Complement component 1q (C1q) is a protein complex involved in the complement system. C1q forms the C1 complex together with C1r and C1s. C1q is composed of 18 polypeptide chains: six A chains, six B chains, and six C chains.

[0078] In the urine of people with renal disease, C1q exhibits a concentration deviation of ≥2 times the median of the normal concentration range determined by ELISA.

[0079] Complement component 9 (C9) or complement C9 refers to the protein with the protein ID J9P8Z6. Complement component 9 (C9) is a MACPF protein involved in the complement system. Upon activation, approximately 12-18 molecules of C9 polymerize to form pores in the target cell membrane, leading to lysis and cell death. C9 is a member of the complement membrane attack complex (MAC), which also includes complement components C5b, C6, C7, and C8.

[0080] In the urine of people with renal disease, C9 exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by ELISA.

[0081] Leucine-rich alpha-2-glycoprotein 1 (LRGP1) refers to a protein with the protein ID E2R833. Leucine-rich alpha-2-glycoprotein 1 is a protein encoded by the LRG1 gene in humans. The leucine-rich repeat (LRR) family of proteins, including LRG1, has been shown to be involved in protein-protein interactions, signal transduction, and cell adhesion and development. LRG1 is expressed during granulocyte differentiation.

[0082] In the urine of humans with renal disease, LRGP1 exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by ELISA.

[0083] The following biomarkers show concentration deviations in at least dogs with renal disease that are ≥ 2-fold greater than the median of the normal concentration range as determined by mass spectrometry:

[0084] Coagulation factor XIII (CFXIII), consisting of the coagulation factor XIII A chain and the coagulation factor XIII B chain, refers to a protein with the protein IDs F1PKX3 (A chain) and F1Q041 (B chain) (Table 1). Factor XIII, or fibrin-stabilizing factor, also has extracellular matrix stabilizing activity and is a zymogen found in the blood of humans and some other animals. It is activated by thrombin to become factor XIIIa. Factor XIIIa is an enzyme in the blood coagulation system that crosslinks fibrin. Factor XIII deficiency impairs clot stability and increases bleeding tendency. Human CFXIII is a heterotetramer consisting of two enzymatic A peptides / chains and two non-enzymatic B peptides / chains. CFXIIIa is a dimer of the activated A peptide / chain.

[0085] At least in dogs with renal disease, CFXIII exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0086] Fibronectin (FN) refers to the protein with the protein ID J9P8M2. Fibronectin (FN) is a multifunctional adhesive glycoprotein that plays an important role in tissue repair, regulating cell adhesion and motility, and embryogenesis.

[0087] At least in dogs with renal disease, FN exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0088] Ficolin 1 (FIC) refers to a protein with the protein ID J9P7F7. Ficolin-1, commonly referred to as M-ficolin, is a protein encoded by the FCN1 gene in humans. The ficolin family of proteins (ficolins) selectively recognizes acetylated compounds. M-ficolin is primarily expressed in peripheral blood leukocytes and is thought to function as a plasma protein with elastin-binding activity.

[0089] At least in dogs with renal disease, FIC shows a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0090] Hemopexin (HPX) refers to a protein with the protein ID F1PZR4. Hemopexin (also known as hemopexin; Hpx; Hx), also known as beta-1B-glycoprotein, is a glycoprotein encoded by the HPX gene in humans. Hemopexin is the plasma protein with the highest binding affinity for heme and several enzymatic activities.

[0091] At least in dogs with renal disease, HPX concentration deviates by more than two times the median of the standard concentration range determined by mass spectrometry.

[0092] Inter-alpha-trypsin inhibitor heavy chain 4 (IATIH4) refers to a protein with protein ID H9GWY3 (listed in Table 1). Inter-alpha-trypsin inhibitors (IATI) are four plasma proteins consisting of four different heavy chains selected from the IATIH1, IATIH2, IATIH3, and IATIH4 groups and one light chain selected from the AMBP or SPINT2 groups (also listed in Table 1). They function as protease inhibitors. The biomarker is inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4 (IATIH4).

[0093] At least in dogs with renal disease, IATI4 shows a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0094] Retinol-binding protein 4 (RBP4) refers to a protein with the protein ID F1Q4D9. Retinol-binding protein 4, also known as RBP4, is a retinol transporter protein. In humans, RBP4 is encoded by the RBP4 gene. It is primarily synthesized in the liver and circulates in the blood in a complex with transthyretin, binding to retinol.

[0095] At least in dogs with renal disease, RBP4 exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0096] Serum amyloid A (SAA) refers to a protein with the protein ID J9NVE9. Serum amyloid A (SAA) protein is a family of apolipoproteins associated with high-density lipoprotein (HDL) in plasma. Different isoforms of SAA are expressed at different levels constitutively (constitutive SAA) or in response to inflammatory stimuli (acute-phase SAA). These proteins are primarily produced in the liver.

[0097] At least in dogs with renal disease, SAA concentration deviations are greater than or equal to two times the median of the standard concentration range determined by mass spectrometry.

[0098] Talin-1 (Tal) refers to a protein with the protein ID J9P5V6. Talin-1 is encoded by the TLN1 gene in humans. It is ubiquitously expressed and localized to costamere structures in cardiac and skeletal muscle cells and to focal adhesions in smooth muscle and non-muscle cells. Talin-1 mediates cell-cell adhesion through binding of integrins to the actin cytoskeleton and activation of integrins.

[0099] At least in dogs with renal disease, Tal exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0100] Kallikrein (KAL) or kallikrein B1 refers to the protein with the protein ID F1PNV5. KLKB1 is the gene that encodes the plasma kallikrein protein in humans. Kallikreins are a subgroup of serine proteases.

[0101] At least in dogs with renal disease, KAL shows a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0102] Dynein (DYN) or dynein heavy chain domain 1 refers to a protein with the protein ID F1PZK8, as shown in Table 1. Dyneins are a family of cytoskeletal motor proteins that move along microtubules within cells. They convert chemical energy stored in ATP into mechanical work. Dyneins transport various intracellular cargoes and provide important force and displacement in mitosis. Dyneins can be divided into two groups: cytoplasmic dyneins and axonemal dyneins. Dynein heavy chain domain 1 (DNAH1) belongs to the latter. The biomarker is dynein, preferably dynein heavy chain domain 1.

[0103] At least in dogs with renal disease, DYN exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0104] Fukutin (FUK) refers to a protein with the protein ID E2R926. Fukutin is a eukaryotic protein required for, for example, maintaining muscle integrity, cortical tissue formation, and normal eye development. Mutations in the fukutin gene have been shown to cause Fukuyama congenital muscular dystrophy (FCMD), a condition characterized by brain malformations, which is one of the most common autosomal recessive disorders in Japan. In humans, this protein is encoded by the FCMD gene (also known as FKTN).

[0105] At least in dogs with renal disease, FUK exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0106] Myosin IXA (MYIXA) refers to the protein with the protein ID J9P187. Myosin IXa (Myo9a, formerly known as myr7) is an actin-dependent motor protein of the unconventional myosin IX class.

[0107] At least in dogs with renal disease, MYIXA exhibits concentration deviations of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0108] Kininogen 1 refers to the protein having the protein ID E2R886. Kininogen-1 (KNG1), also known as alpha-2-thiol proteinase inhibitor, Williams-Fitzgerald-Fraudieck factor, or HMWK-kallikrein factor, is a protein encoded by the KNG1 gene in humans. Kininogen-1 is the precursor protein of high molecular weight kininogen (HMWK), low molecular weight kininogen (LMWK), and bradykinin.

[0109] At least in dogs with renal disease, kininogen 1 exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0110] Alpha-2-HS glycoprotein (α2-HS glycoprotein) refers to the protein with the protein ID E2QUV3. Alpha-2-HS glycoprotein (AHSG, alpha-2-Heremans-Schmidt glycoprotein), also known as fetuin A, is a protein encoded by the AHSG gene in humans. Fetuin A belongs to the fetuin class of plasma-binding proteins and is more abundant in fetal blood than in adult blood.

[0111] At least in dogs with renal disease, α-2-HS-glycoprotein exhibits a concentration deviation of ≥2 times the median of the normal concentration range determined by mass spectrometry.

[0112] Complement C6 or complement component 6 refers to the protein with protein ID E2RGT6. C6 is a member of the complement membrane attack complex (MAC), which also includes complement components C5b, C7, C8, and C9.

[0113] At least in dogs with renal disease, C6 exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0114] Complement C7 or complement component 7 refers to the protein with protein ID E2RG01.

[0115] At least in dogs with renal disease, C7 exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0116] Complement C8 or complement component 8 includes proteins with protein IDs E2R109 (alpha chain), E2R141 (beta chain), and F6XKC0 (gamma chain) (listed in Table 1). Complement component 8 is a protein involved in the complement system. It is part of the membrane attack complex (MAC). C8 is a heterotrimer and consists of three different subunits, called the C8 alpha chain, beta chain, and gamma chain, which are encoded by the C8A, C8B, and C8G genes, respectively. The biomarker is complement C8, preferably the C8 alpha chain and / or the C8 beta chain.

[0117] At least in dogs with renal disease, C8 exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0118] Beta2-glycoprotein 1 (APOH) refers to the protein with protein ID P33703. β2-glycoprotein 1, also known as beta-2 glycoprotein 1 and apolipoprotein H (Apo-H), is a multifunctional plasma protein encoded by the APOH gene in humans. One of its functions is to bind to cardiolipin. β2-GP1 is also intricately involved in aggregation.

[0119] At least in dogs with renal disease, APOH concentration deviates by more than two times the median of the standard concentration range determined by mass spectrometry.

[0120] Serpin family A member 1 refers to a protein with the protein ID F1PCE5. It is a serine protease inhibitor belonging to the serpin superfamily, whose targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator. This protein is produced in lymphoid and monocytic cells in the liver, bone marrow, and lymphoid tissues, as well as in Paneth cells of the intestine.

[0121] At least in dogs with renal disease, serpin family A member 1 exhibits a concentration deviation of 2-fold or more from the median of the standard concentration range determined by mass spectrometry.

[0122] Transferrin refers to a protein with the protein IDs J9P430 and F6V1W9. Transferrin contains two Fe 3+ Transferrin is a glycoprotein found in vertebrates that contains ion-binding sites and binds iron (Fe), thereby mediating its transport through plasma (serotransferrin). Furthermore, transferrin and its fragments possess numerous immunomodulatory activities. It is produced primarily in the liver, but also in many other organs. Human transferrin is encoded by the TF gene.

[0123] At least in dogs with renal disease, transferrin concentration deviates by more than two times the median of the standard concentration range determined by mass spectrometry.

[0124] Alpha-1B glycoprotein refers to the protein with the protein ID F1PCK2. Alpha-1B glycoprotein is a protein encoded by the A1BG gene in humans. The protein encoded by this gene is a plasma glycoprotein of unknown function. This protein shows sequence similarity to the variable regions of several immunoglobulin supergene family members.

[0125] At least in dogs with renal disease, α1-B glycoprotein exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0126] Apo AI or apolipoprotein AI refers to the protein with the protein ID J9P843. Apolipoprotein AI (Apo-AI) is a protein encoded by the APOA1 gene in humans. As a major component of HDL particles, it plays a unique role in lipid metabolism.

[0127] At least in dogs with renal disease, Apo AI exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0128] Apo D or apolipoprotein D refers to the protein with the protein ID E2RNM1. Apolipoprotein D (ApoD) is a protein that in humans is encoded by the APOA1 gene. ApoD is a member of the lipocalin family and is a transporter of small hydrophobic molecules.

[0129] At least in dogs with renal disease, Apo D exhibits a concentration deviation of ≥2 times the median of the standard concentration range determined by mass spectrometry.

[0130] The alpha-1-microglobulin / bikunin precursor refers to a protein with the protein ID J9NUI6. The protein AMBP is a protein that binds to IATI and is encoded by the AMBP gene in humans.

[0131] At least in dogs with renal disease, the alpha-1-microglobulin / bikunin precursor exhibits a concentration deviation of more than (≥) 2 times the median of the standard concentration range determined by mass spectrometry.

[0132] Carboxypeptidase N subunit 2 refers to the protein with the protein ID J9PAA4. In humans, carboxypeptidase N subunit 2 is encoded by the CPN2 gene.

[0133] At least in dogs with renal disease, carboxypeptidase N subunit 2 exhibits a concentration deviation of 2-fold or more of the median of the standard concentration range determined by mass spectrometry.

[0134] Serine / arginine repeat matrix protein 3 refers to the protein with protein ID F6Y120. Serine / arginine repeat matrix protein 1 is a protein encoded by the SRRM1 gene in humans. Serine / arginine repeat matrix protein 3 is listed in Table 1 as serine / arginine repeat matrix protein 3 (homolog).

[0135] At least in dogs with renal disease, serine / arginine repeat matrix protein 1 exhibits a concentration deviation of 2-fold or more from the median of the normal concentration range determined by mass spectrometry.

[0136] Prior to mass spectrometry analysis, all proteins were denatured and digested with trypsin (as described in Example 1, Section 1.4. LC-MS / MS Analysis, below) into characteristic tryptic peptides. Thus, after analyzing all of these peptides and searching databases, the biomarker candidates identified by mass spectrometry appear as protein chain(s), multiple variants, and / or fragments (see Table 1, and also Example 1, Section 1.6, below). From these findings, the biomarkers of the present invention are all represented as proteins present in real, non-denatured clinical samples suitable for diagnostic purposes. As noted above, the term "protein" as used herein refers to / includes all known proteins, including identified chains of each protein, engineered variants of each protein, fragments of each protein, single protein chains, multiple protein chains, and complexes of each protein (chain) with additional proteins and protein chains, regardless of validation using currently available immunoassays such as ELISA.

[0137] In one embodiment, the at least one further protein is preferably fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), and C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4; Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), Dynein (DYN), preferably dynein heavy chain domain 1; Fukuchin (FUK), and Myosin IXA (MYIXA) is selected from The at least one further protein is more preferably fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), and C-reactive protein (CRP) is selected from.

[0138] In a preferred embodiment, C4BP and ADP, or COLXIII and FMN, or HABP2 and TGF-β1, or HABP2 and FGG, or CFH and GS, or CFI and GS, or CFI and VTN, or CFH and FGG, or CFI and FGG is determined.

[0139] Furthermore, the present invention provides a method for the preclinical and / or early detection and / or diagnosis of kidney disease, comprising: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4 (IATIH4), Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), Dynein (DYN), preferably dynein heavy chain domain 1; Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, preferably the C8 alpha chain and / or the C8 beta chain, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, Complement C1s, Complement C1r, Complement C2, Complement C3, Complement C4, Complement C5, Complement factor B, haptoglobin, α2-macroglobulin, alpha-1-antitrypsin, Ceruloplasmin, Serpin family A member 3, Serpin family A member 5, Serpin family A member 7, Serpin family G member 1, Serpin family D member 1, coagulation factor V, Prothrombin, plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit alpha, Fetuin B, insulin-like growth factor binding protein, alpha-2-glycoprotein 1, paraoxonase 1, dipeptidyl peptidase 4, C-type lectin domain family 3 member B, Galectin 3 binding protein, apolipoprotein AIV (Apo AIV), Apolipoprotein CI (Apo CI), Apolipoprotein CII (Apo CII), apolipoprotein E (Apo E4), Apolipoprotein M (Apo M), apolipoprotein B (Apo B), Myosin IB, Myosin XVIIIB, kinesin-like proteins, CD109 molecule, maltase-glucoamylase, succin molecular chaperone, Dispatched RND Transporter Family Member 2, DNA polymerase, Transport protein particle complex subunit 2, RB related KRAB zinc finger, ciliogenesis-associated TTC17-interacting protein, Proteasome subunit alpha type, Synaptic membrane exocytosis regulation 2, extracellular matrix protein 1, cadherin 5, coiled-coil domain-containing 178, and Attractin The present invention provides at least two proteins selected from:

[0140] In other words, the present invention provides the use of at least two proteins selected from the proteins disclosed above as biomarkers for the preclinical and / or early detection and / or diagnosis of kidney disease.

[0141] The at least two proteins preferably comprise: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4 (IATIH4), Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), Dynein (DYN), preferably dynein heavy chain domain 1; Fukuchin (FUK), and Myosin IXA (MYIXA). is selected from.

[0142] In a preferred embodiment, ADP and PICP, or AGT and GS, or AGT and C9, or GS and C9, or GS and VTN, or AGT and VTN, or CFI and a1AGP, or LRGP1 and C1q, or CFH and C9, or CRP and C1q is determined.

[0143] Preferably, the concentration of each protein is determined in a sample from a mammal, preferably a human subject.

[0144] The mammal is preferably Human, Animals such as dogs, cats, rabbits, guinea pigs, hamsters, cows, pigs, horses, sheep, donkeys, goats, red deer, or camels; More preferably, the mammal is a human.

[0145] The sample is preferably a body fluid such as urine or blood, for example serum, plasma, or whole blood.

[0146] The body fluid is more preferably urine.

[0147] In one embodiment, if the sample is a urine sample, the creatine concentration of the urine sample is also determined, and the concentration of the biomarker protein is then normalized to the creatine concentration.

[0148] Preferably, the normalized concentration of each protein is compared to a control value.

[0149] Preferably, control values ​​are determined for each protein from a group of healthy subjects, preferably healthy subjects matched with the subject to be diagnosed with regard to age and sex.

[0150] A "subject," or preferably a "human subject," is considered a "healthy subject" if they are "renally healthy" at the time the sample is taken and remain "renally healthy" for a period of time corresponding to the normal time during which kidney disease is diagnosable by conventional means. For example, biobanks are suitable sources of samples from healthy subjects because the subjects are followed up after the sample is taken.

[0151] Preferably, the control value is the midpoint of the standard concentration range.

[0152] In a preferred embodiment, a mathematical relationship of the concentrations of the biomarkers is used, such as a sum of the concentrations, or preferably a product of the concentrations.

[0153] For example, the following products are constructed: ADP x C4BP, ColXIII x C4BP, ColXIII x FMN, HABP2 x TGF-β1, HABP2 x FGG, ColXIII x C4BP x HABP2.

[0154] Detection Method As mentioned above, the present invention provides a method for preclinical and / or early detection and / or diagnosis of kidney disease, comprising: (a) determining the concentrations of at least two proteins in a mammalian sample; (b) comparing each concentration determined in step (a) with a control value; Including, The method further provides a method in which deviation of each concentration determined in step (a) from the control value is indicative of preclinical and / or early stage renal disease.

[0155] Preferably, the renal disease is A chronic progressive kidney disease that begins with damage to the glomeruli (glomerulopathy), Preferably, post-infectious glomerulopathy, IgA nephropathy, Henoch-Schönlein purpura, focal segmental glomerulosclerosis, Alport syndrome, thinning basement membrane nephropathy, benign familial hematuria, or Chronic progressive kidney disease that begins in other parts of the body and / or kidneys involving the glomeruli (nephropathy), accompanied by other regulatory disorders Preferably, metabolic syndrome, hypertension, heart failure, nephrotoxic drug therapy, adiposity, diabetes mellitus (diabetic nephropathy), renal agenesis, renal hypoplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract abnormalities, duplex kidneys, nephronophthisis is selected from.

[0156] -Step (a) In step (a), the concentrations of at least two proteins in a mammalian sample are determined.

[0157] The mammal is preferably Human, Animals such as dogs, cats, rabbits, guinea pigs, hamsters, cows, pigs, horses, sheep, donkeys, goats, red deer, or camels; More preferably, the mammal is a human.

[0158] The sample is preferably a body fluid such as urine or blood, for example serum, plasma, or whole blood.

[0159] The body fluid is more preferably urine.

[0160] In one embodiment, if the sample is a urine sample, the creatine concentration of the urine sample is also determined, and the concentration of the biomarker protein is then normalized to the creatine concentration.

[0161] The at least two proteins are selected from the biomarkers disclosed herein.

[0162] In a preferred embodiment, the at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI) is selected from.

[0163] In a preferred embodiment, the at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein alpha (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), and Complement factor I (CFI), One protein selected from and fibrinogen, preferably fibrinogen gamma chain (FGG), - Lumican (LUM), - Formin 1 (FMN), - vitronectin (VTN), - Gelsolin (GS), - angiotensinogen (AGT), - adiponectin (ADP), - Transforming growth factor-β1 (TGF-β1), - carboxy-terminal propeptide of type I procollagen (PICP), - C-reactive protein (CRP), - alpha-1-acid glycoprotein (a1AGP), - Coagulation factor XIII (CFXIII), - Complement component 1q (C1q), - Complement component 9 (C9), - fibronectin (FN), - Ficolin 1 (FIC), - Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4, - leucine-rich alpha-2-glycoprotein 1 (LRGP1), - retinol-binding protein 4 (RBP4), - Serum amyloid A (SAA), - Tallinn 1 (Tal), - Kallikrein (KAL), - dynein (DYN), preferably dynein heavy chain domain 1, - Fukuchin (FUK), - Myosin IXA (MYIXA). - kininogen 1, - α2-HS glycoprotein, - Complement C6, - Complement C7, complement C8, preferably the C8 alpha chain and / or the C8 beta chain, - APOH, beta-2-glycoprotein 1, - Serpin family A member 1, - transferrin, - α1-B glycoprotein, - Apo AI, - Apo D, - Alpha-1-microglobulin / bikunin precursor, - carboxypeptidase N subunit 2, and - Serine / Arginine Repeat Matrix Protein 3 at least one further protein selected from is selected from.

[0164] In one embodiment, the at least one further protein is preferably fibrinogen, preferably fibrinogen gamma chain (FGG), - Lumican (LUM), - Formin 1 (FMN), - vitronectin (VTN), - Gelsolin (GS), - angiotensinogen (AGT), - adiponectin (ADP), - Transforming growth factor-β1 (TGF-β1), - carboxy-terminal propeptide of type I procollagen (PICP), - C-reactive protein (CRP), - alpha-1-acid glycoprotein (a1AGP), - Coagulation factor XIII (CFXIII), - Complement component 1q (C1q), - Complement component 9 (C9), - fibronectin (FN), - Ficolin 1 (FIC), - Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4, - leucine-rich alpha-2-glycoprotein 1 (LRGP1), - retinol-binding protein 4 (RBP4), - Serum amyloid A (SAA), - Tallinn 1 (Tal), - Kallikrein (KAL), - dynein (DYN), preferably dynein heavy chain domain 1, - Fukuchin (FUK), and - Myosin IXA (MYIXA) is selected from.

[0165] In a preferred embodiment, the at least one further protein is fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), and C-reactive protein (CRP) is selected from.

[0166] In a preferred embodiment, the at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4; Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), Dynein (DYN), preferably dynein heavy chain domain 1; Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, preferably the C8 alpha chain and / or the C8 beta chain, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, Complement C1s, Complement C1r, Complement C2, Complement C3, Complement C4, Complement C5, Complement factor B, haptoglobin, α2-macroglobulin, alpha-1-antitrypsin, Ceruloplasmin, Serpin family A member 3, Serpin family A member 5, Serpin family A member 7, Serpin family G member 1, Serpin family D member 1, coagulation factor V, Prothrombin, plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit alpha, Fetuin B, insulin-like growth factor binding protein, alpha-2-glycoprotein 1, paraoxonase 1, dipeptidyl peptidase 4, C-type lectin domain family 3 member B, Galectin 3 binding protein, apolipoprotein AIV (Apo AIV), Apolipoprotein CI (Apo CI), Apolipoprotein CII (Apo CII), apolipoprotein E (Apo E4), Apolipoprotein M (Apo M), apolipoprotein B (Apo B), Myosin IB, Myosin XVIIIB, kinesin-like proteins, CD109 molecule, maltase-glucoamylase, succin molecular chaperone, Dispatched RND Transporter Family Member 2, DNA polymerase, Transport protein particle complex subunit 2, RB related KRAB zinc finger, ciliogenesis-associated TTC17-interacting protein, Proteasome subunit alpha type, Synaptic membrane exocytosis regulation 2, extracellular matrix protein 1, cadherin 5, coiled-coil domain-containing 178, and Attractin is selected from.

[0167] In one embodiment, the at least two proteins are preferably: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein (C4BP), preferably C4BP alpha chain (C4BPA), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (α1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4; Leucine-rich α-2-glycoprotein 1 (LRGP1), retinol-binding protein 4 (RBP4), serum amyloid A (SAA), Tallinn 1 (Tal), Kallikrein (KAL), Dynein (DYN), preferably dynein heavy chain domain 1; Fukuchin (FUK), and Myosin IXA (MYIXA) is selected from.

[0168] In one embodiment, COLXIII and C4BP, or COLXIII and C4BP and HABP2, or Determine CFH and CFI.

[0169] In one embodiment, the concentrations of COLXIII, HABP2, C4BP, CFH, and CFI are determined.

[0170] In one embodiment, C4BP and ADP, or COLXIII and FMN, or HABP2 and TGF-β1, or HABP2 and FGG, or CFH and GS, or CFI and GS, or CFI and VTN, or CFH and FGG, or The concentrations of CFI and FGG are determined.

[0171] In one embodiment, ADP and PICP, or AGT and GS, or AGT and C9, or GS and C9, or GS and VTN, or AGT and VTN, or CFI and a1AGP, or LRGP1 and C1q, or CFH and C9, or CRP and C1q concentrations are determined.

[0172] In one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, or more biomarkers are determined.

[0173] -Step (b) In step (b), each concentration determined in step (a) is compared with a control value.

[0174] In one embodiment, if the sample is a urine sample, the creatine concentration of the urine sample is also determined, and the concentration of the biomarker protein is then normalized to the creatine concentration.

[0175] Preferably, the normalized concentration of each protein is compared to a control value.

[0176] Preferably, control values ​​are determined for each protein from a group of healthy subjects, preferably healthy subjects matched with the subject to be diagnosed with regard to age and sex.

[0177] As noted above, a subject is considered a "healthy subject" if they are "renally healthy" at the time the sample is taken and remain "renally healthy" for a period of time corresponding to the typical time during which kidney disease is diagnosable by conventional means. For example, biobanks are a good source of samples from healthy subjects because the subjects are followed up after the sample is taken.

[0178] Preferably, the control value is the midpoint of the standard concentration range.

[0179] According to the present invention, deviation of each concentration determined in step (a) from said control value is indicative of preclinical and / or early stage renal disease.

[0180] Preferably, A deviation of ≥2-fold from the median of the normal concentration range indicates preclinical and / or early renal disease, or Deviations of less than or equal to 0.5 times the median of the normal concentration range indicate preclinical and / or early renal disease.

[0181] More preferably, A deviation of more than (≥) 2-fold from the median of the normal concentration range indicates preclinical and / or early renal disease.

[0182] A deviation of (≧) 2 times or more of the median of the standard concentration range can be in the range of 2 times to about 1.000 times, such as 2 times to 100 times, such as 2 times to 50 times or 2 times to 10 times.

[0183] In a preferred embodiment, a mathematical relationship of the concentrations of the biomarkers is used, such as a sum of the concentrations, or preferably a product of the concentrations.

[0184] For example, the following products are constructed: ADP x C4BP, ColXIII x C4BP, ColXIII x FMN, HABP2 x TGF-β1, HABP2 x FGG, ColXIII x C4BP x HABP2.

[0185] Further Description and Preferred Embodiments Although several early cellular changes have recently been identified (see, e.g., Guo et al., 2019 ), to date, no practical biomarkers exist for the preclinical detection of Alport syndrome (AS).

[0186] In overt AS, we have identified a panel of potential biomarkers (BM) using proteomics workflows and ELISA (Baum et al., 2008; Pohl et al., 2013). Unfortunately, a similar exploration of BM in the preclinical state, i.e., in young children, is not possible for ethical and practical reasons.

[0187] However, type IV collagen is highly conserved. Therefore, any mammal can be affected by AS, making AS animal models ideal tools for studying pathogenesis and treatment (Kashtan et al., 2002). Mouse and dog models have shown similar clinical courses compared to humans. Furthermore, ACE inhibitors have been shown to inhibit COL4A5 prior to extensive evaluation in humans. - Dogs (Grodecki et al., 1997) (AS-dogs) and COL4A3 - / -The approach was shown to slow disease progression in both young AS mice (Gross et al., 2003) and AS mice (AS-mice). These models allow definitive diagnosis of the disease at a preclinical stage by genetic means. These models also allow the observation of biomarkers in body fluids that indicate disease progression. Thus, our improved approach (Wendler et al., 2013) has already been applied to the serum of young AS mice, resulting in the generation of several candidate BMs (Muckova et al., 2015).

[0188] To date, most of the biomarker candidates described have been derived from hypothesis-driven studies. Hypotheses are rarely generated during the preclinical phase. Therefore, a hypothesis-free search for reliable biomarkers is an opportunity to identify novel biomarkers for the diagnosis, prognosis, and treatment monitoring of nephropathy, and is urgently needed. Preclinical biomarker discovery is extremely challenging. These biomarkers could only be discovered and evaluated in seemingly healthy, but affected, preclinical individuals who exhibit disease characteristics predictive of progression to nephropathy (see Figure 1).

[0189] AS, as a model genetic disease, was subjected to such a proteomic search for early biomarkers. See below. This search was carried out using young AS mice and AS dogs. Most of the identified biomarker candidates can be considered "validated" because they overlap in the two different species (see Table 1). Furthermore, some were reversible with treatment with an ACE inhibitor (ramipril), and some of them were evaluated by immunoassays in mice and dogs. Furthermore, this panel of biomarker candidates was validated in DM1, a patient predictive of preclinical DN, as well as in children with obesity, hypertension, and metabolic syndrome (potential preclinical DM), and in the early stages of other nephropathies.

[0190] This paper describes a methodologically complementary proteomic study using readily accessible serum for biomarker discovery in the apparent preclinical stage of the model disease Alport syndrome (AS), and validation in blood and urine samples from pediatric patients with a high preclinical risk of developing DN and the very early clinical stages of various nephropathy.

[0191] To do so, we applied an optimized unbiased strategy that combined: i. To collect refined samples from an animal model (canine) of the inherited glomerulopathy Alport Syndrome (AS) for preclinical proteomic exploration and from puppies with or without a diagnosis of early nephropathy for validation. ii. Applying a comprehensive and precise workflow for proteomic biomarker discovery in samples from 7-week-old affected male and female dogs and their unaffected siblings; iii. Biomarkers will then be evaluated using a human biobank containing samples from 305 characterized children, including healthy controls, children with metabolic syndrome, hypertension, adiposity, T2DM, T1DM, renal aplasia, hypoplasia, polycystic renal dysplasia, urinary tract anomalies, duplication of kidneys, ADPKD, ARPKD, nephronophthisis, post-infectious GN, vasculitis (IgAN, HSP), basement membrane thinning nephropathy, and benign familial hematuria, and AS.

[0192] [Table 1-1]

[0193] [Table 1-2]

[0194] [Table 1-3]

[0195] [Table 1-4]

[0196] [Table 1-5]

[0197] Legend for Table 1: * Protein IDs refer to the UniProt database of approximately 47,800 dog entries (date: October 26, 2017).

[0198] Criteria to define a biomarker: For biomarker selection in mice, see Muckova et al., 2015.

[0199] Candidate BMs in dogs were selected from all identified protein chains in all analyzed 2D subfractions after comparing data from affected and unaffected dogs. Peptides supporting identified proteins in all 2D subfractions of AM were compared with those of the homologous 2D subfractions of NM using Sieve®, and those of CF were compared with those of NF. The quantifier "ratio" refers to the concentration quotient (AM / NM, CF / NF), and "hit" refers to the amount of each protein in the compared subfractions. To visualize the chromatographic distribution of altered protein variants, the sieving data for each identified protein was sorted into our separation matrix using an in-house macro. Depending on the actual chromatographic distribution, BMs were defined by a >2-fold higher concentration given by "Overall Trend" (Equation 1; Table 1, columns 4 and 6) or by the average of all ratios when exclusive synergistic changes were observed. Alternatively, ratios >5-fold higher in distinct clusters of subfractions, i.e., distinct, strongly elevated variants (Table 1, columns 3 and 5), were used to define BM as fractions excluded from the entire protein family.

[0200] Overall trend = (M i ×H i) / (H d / M d ) Equation 1 M i : average of all increased ratios H i : The number of all increased hits M d : average of all reduced ratios H d : the number of all reduced hits

[0201] In summary, 118 protein chains representing 103 proteins were elevated in affected compared to unaffected dogs (males and females), and 79 proteins fulfilled the criteria for BM candidates defined above.

[0202] na, no change; nf, not seen; 0, no cluster in this direction; x, overall trend higher in AM and / or CF; o, increased parts / protein variants higher in AM and / or CF. The term "DIV / 0" (division by zero, i.e., no peptides or hits seen in control samples) indicates an abnormally high value in diseased animals. Average ratio of all ratios for all subfractions with synergistic changes in proteins (AM / NM or CF / NF).

[0203] Raw spectra were analyzed using Proteome Discoverer 1.3 and the Sequest database search algorithm, applying a false positive rate of less than 0.5% and a UniProt database of approximately 47,800 dog entries (dated 2017 / 10 / 26).

[0204] Below, we show that after receiver operating characteristic (ROC) analysis, ColXIII, HaBP2, and C4BPA, as well as their combination, have an area under the ROC curve (AUC) significantly greater than 0.700. Furthermore, they exhibit reliable quality characteristics (high sensitivity, low false positive rate) both individually and in combination (Tables 2-9). This applies to the entire study group and to the subgroups of patients with elevated values ​​analyzed separately. Under conditions of inflammation (postinfectious glomerulopathy and vasculitis), acute-phase proteins (CRP, AGT, FGG) and complement components CFI and CFH also, and predictably, exhibit high AUC, sensitivity, and the lowest false positive rate.

[0205] [Table 2]

[0206] [Table 3]

[0207] [Table 4]

[0208] [Table 5]

[0209] [Table 6]

[0210] [Table 7]

[0211] [Table 8]

[0212] [Table 9]

[0213] Below are shown several significant differences between patient and control biomarker concentrations (Table 10) and combinations of biomarker concentrations (Table 11).

[0214] [Table 10-1]

[0215] [Table 10-2]

[0216] [Table 11-1]

[0217] [Table 11-2]

[0218] The following examples and figures are illustrative of the present invention but are not intended to be limiting thereof. [Example]

[0219] Example 1 Materials and Methods 1.1 Patients and samples Human samples: Two sets of patient samples were used: 1) real-world clinical samples from Alport patients (Table 12), prospective samples from pediatric patients with AS, type 1 diabetes mellitus (DM1), and various other nephropathies, and prospective samples from individuals without known nephropathy as controls (Table 13). The first set included longitudinal serum and urine samples from the early stages of AS progression. The second set was collected using standard operating procedures from healthy controls and patients with early nephropathy to evaluate the sensitivity and specificity of candidate biomarkers for targeted screening (ethic vote UHJ:2020-1800). Informed consent was obtained from all participants. All samples were stored at -80°C until use.

[0220] [Table 12]

[0221] Informed consent was obtained from all participants. Inclusion criteria were age >3 years and a clinical diagnosis of AS according to international criteria.

[0222] [Table 13-1]

[0223] [Table 13-2]

[0224] [Table 13-3]

[0225] A second set of participants was recruited, interviewed, and tested between September 2020 and November 2021, see Table 14.

[0226] The following standard operating procedures were applied: Blood samples from individuals were obtained in the morning. Blood was collected by venipuncture into 9 mL monovets (Sarstedt, Nuembroke, Germany). To obtain serum, blood was collected into serum monovets (02.1063.001). Unless otherwise specified, the clotting time was 90 minutes at room temperature. All samples were centrifuged at 1500 × g for 10 minutes at 20°C, immediately aliquoted, frozen at -20°C within 3 days, and then stored at -82°C until use. Plasma was obtained by collecting blood into EDTA-containing containers (02.1066.001). Unless otherwise specified, samples were immediately centrifuged, aliquoted, and stored as described above. Urine samples: Urine samples were obtained as spontaneous voiding close to venipuncture, aliquoted, frozen at -20°C within 3 days, and then stored at -82°C until use.

[0227] Thirty-two healthy controls were enrolled from subjects with no known signs of nephropathy, no history of nephropathy, and no signs of inflammation. Samples from these control individuals were obtained along with a second set. Seventy-two additional healthy controls were obtained from an independent cohort of pediatric control samples from the Leipzig Medical Biobank (Leipzig Research Center for Civilization Diseases, LIFE-child) obtained in 2014.

[0228] 1.2. Samples for proteomics exploration animal Four litters were obtained from a colony of X-linked AS (COL4A5-) mixed-breed dogs ( Lees, 2013 ).

[0229] Genotyping was performed using PCR on DNA isolated from blood. All animals were weaned at 6 weeks of age and maintained on established standard diet, housing, and socialization protocols.

[0230] Sampling Protocol A minimum of 4 mL of whole blood and 3 mL of urine (voided) were collected for serum and EDTA-plasma collection. For the sampling schedule, unaffected dogs were randomly paired with affected littermates. A complete standard urinalysis, UPC, and urinary albumin concentration were obtained. All samples were collected and processed using standard methods to minimize preanalytical variability. A minimum of two 0.5 mL aliquots of each sample type were stored at -80°C for future analysis and shipped on dry ice.

[0231] Samples used for proteomic exploration Equal volumes of three plasma samples from each group of animals (affected males (AM), carrier females (CF) at the preclinical stage (7 weeks), and healthy unaffected littermates (NM, NF, 7 weeks)) were combined, allowing for pre-fractionation of equal volumes and substantially equal amounts of protein into pools that were then analyzed (Table 14).

[0232] [Table 14]

[0233] 1.3. Proteomics Discovery EDTA-plasma pools from all four groups were separated, prepared, and analyzed by mass spectrometry in parallel.

[0234] Sample Fractionation: Our method, described in Rhode et al. (2019) and Wendler et al. (2013), combines native size exclusion (SEC, first dimension, 1D) chromatography followed by anion exchange (AEC, 2D) chromatography. After sequential 1D fractionation, all subsequent steps are performed in a microplate format. In this way, parallelization and automation are achieved, starting with 2D fractionation, followed by separation, spectrophotometric readout, temporary storage, hit picking, medium exchange, digestion, desalting, and finally application to an autosampler and subsequent LC-MS. 96 1D fractions are generated from each sample. Each 1D fraction is further separated into 43 2D fractions. This results in at least 4,128 2D subfractions containing 20–40 different proteins with total protein concentrations ranging from zero to 4.3 mg / mL. Fractions from all samples that show the same position (1D_2D) are referred to as homologous fractions. Because our separations are highly reproducible, we expect that components of each sample will be distributed to the same set of homologous fractions across all samples. Disease can alter the concentrations and / or post-translational modifications of sample components. Any changes can be detected by changes in the concentration ratios of homologous fractions. Furthermore, each single recovered protein is predicted to be present in a set of at least three consecutive fractions based on its chromatographic distribution. Because mass spectrometry requires significant time (see below), we selected a representative subset of fractions, i.e., every other chromatographic spot fraction showing sufficient protein content (>0.03 mg / mL), rather than all fractions, for analysis.

[0235] 1.4. LC-MS / MS analysis Tryptic peptide separation prior to mass spectrometry analysis was performed on a Hypersil Gold UHPLC column (1.9 μm, 50 × 1.0 mm) using an Accela 1250 UHPLC system (both Thermo Fisher Scientific, USA) with a binary gradient elution of the mobile phase: (A) water and (B) acetonitrile with 0.1% formic acid, at a flow rate of 150 μL / min throughout (0–1 min: 5% B, 21 min: 30% B, 24 min: 40% B, 25 min: 90% B, 25.1–26 min: 90% B, 26.1–30 min: 5% B). Tandem mass spectrometry (MS / MS) measurements were performed on an LTQ Orbitrap Discovery (Thermo Fisher Scientific, USA) using positively heated electrospray ionization (H-ESI) with a vaporizer temperature of 200 °C. The ion spray was dried using a sheath gas flow (30.0) and auxiliary gas flow (10.0) (both nitrogen gas flows, arbitrary units). The ionization voltage and ion tube temperature were set to 4.5 kV and 275 °C, respectively. The MS / MS system was operated in data-dependent TOP10 mode using one microscan. For this purpose, ions were monitored in full-scan centroid mode from m / z 350 to 1700 in the LTQ ion trap. The 10 most intense ions were subjected to collision-induced dissociation (CID) and further Orbitrap high-resolution (30,000) analysis (profile data type). Broadband activation was used. The automatic gain control (AGC) target value for the Orbitrap mass spectrometer in full-scan mode was 1.0 × 106. LC-MS / MS was operated via the graphical interface of Xcalibur software 2.1. Fractions were performed in duplicate. After each duplicate, a blank (i.e., a water sample) was applied to avoid carryover. For quality control and testing, 50 μg / mL of the digest of transferrin (Holo, SERVA Electrophoresis GmbH, 36756) was analyzed in quadruplicate per microplate.

[0236] 1.5. Protein identification and data analysis Protein identification: MS / MS spectra were analyzed using Proteome Discoverer 1.3 and Sequest database searching (a continuously updated Human Fasta database, downloaded from www.uniprot.org, 2017 / 10 / 26) applying a false positive rate of 0.01.

[0237] Pairwise analysis: To detect differences between two samples (diseased vs. control, technical duplicates), analysis was performed using SIEVE 2.0 (software for label-free differential analysis, Thermo Fisher Scientific, USA). MS data (raw files created with XCALIBUR) were imported and analyzed as "two-sample differential analysis" for corresponding pairs of fractions. Results were filtered by charge-dependent Xcorr (Xcorr 1.5, 2.0, 2.25, and 2.5 for charges 1, 2, 3, and >3, respectively) and a p-value of 0.05. Results are expressed as fold-change ratios between diseased and control. The resulting protein list was exported to Excel, and fraction locations (1D_2D) were labeled. All proteins from all fractions were then merged, sorted according to protein ID, and macros were applied to 1D and 2D locations. In this way, clusters of peptides associated with different proteins with similar ratios and orientations could be identified. The clustering process dynamically adapted to the distribution of peptide numbers: two to six consecutive 1D and / or 2D fractions were included as a cluster. However, fractions with opposite ratios were not considered clusters. Proteins with ratios >4 and <0.25 were considered strictly up-regulated and down-regulated, respectively, if they were present in at least two adjacent chromatographic fractions, despite a lower (synergistic) ratio. Protein clusters were assigned to three categories: proteins with only positive or negative ratios, reflecting changes in the total concentration of the respective protein in the sample, and proteins with both up-regulated and down-regulated clusters. For the latter proteins, changes in post-translational modifications may drive changes in distribution along the chromatography. All categories were considered indicative changes. Finally, proteins were selected into a single result list (see Table 1) based on ratio and number of hits.

[0238] 1.6. Validation of biomarker candidates The protein clusters mentioned in Section 1.5 represent different variants (proteoforms) of each protein family that are present at different levels in the blood of diseased but preclinical animals compared to their respective controls. The specific characteristics of these variants (i.e., PTMs, complexes, and fragments) are unknown to date, as are their tissue origin and their occurrence in urine, a highly practical specimen for (pediatric) screening purposes.

[0239] Therefore, for preliminary evaluation, 27 biomarker candidates and their variants were immunoquantified in serum, EDTA plasma, and urine from a small number of individuals from all dog groups (NM, AM, NF, CF) using a sample set and commercially available ELISA kits separate from those selected for proteomic exploration (see Table 15A).

[0240] A stepwise procedure was then carried out in humans. First, 28 biomarker candidates were evaluated in 6–8 sets of characteristic blood and urine samples from patients and control individuals. All assays were performed in duplicate according to the manufacturer's instructions. Promising biomarker candidates that showed different mean values ​​between patients and controls were then quantified in the maximum number of samples from the biobank. The commercially available ELISA kits used are summarized in Table 15B, and most of them required long incubation times to achieve the required high sensitivity.

[0241] Analysis of ELISA results: Data were analyzed using Sigma Plot software version 14.5. First, the Shapiro-Wilk test was used to analyze the normal distribution of the dataset. For data that did not follow a normal distribution, a Kruskal-Wallis one-way analysis of variance based on ranks was used. Post-hoc analysis was performed using pairwise U tests. Statistical significance was set at p<0.05. Next, correlation analysis of the immunoreactivity of biomarker proteins was performed using Pearson and Spearman methods using the SPSS program (version 27). In this way, proteins with positive and negative correlations could be identified and combined by multiplication.

[0242] [Table 15-1]

[0243] [Table 15-2]

[0244] A. Measurements on dog samples: When dog-specific or reliable kits were not available, other species-specific assays were applied due to the high sequence similarity. Of the kits examined, those marked with a yes (y) showed reliable antigen concentrations. Other kits either could not detect naturally occurring proteoforms in our samples or were not sensitive enough.

[0245] B. Measurements on human samples: The kit was performed according to the supplier's instructions with the following modifications: all Cloud-Clone and CUSBIO assays were performed with double the incubation time. Samples were preincubated with 0.5% Tween 20 before gelsolin measurement. For urinary PICP, TGF-β1, and CRP quantification, longer incubation times or concentrated specimens were applied. Therefore, samples were concentrated approximately 10- to 60-fold using Amicon® Ultra-15 Centrifugal filters (3K, UFC900324), depending on the starting volume available.

[0246] The features disclosed in the above description, in the claims and / or in the accompanying drawings may, both individually and in any combination thereof, be material for realizing the invention in diverse forms thereof.

[0247] References Baum, A. et al. Searching biomarker candidates in serum using multidimensional native chromatography. II Method evaluation with Alport syndrome and severe inflammation. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 876, 31-40 (2008). I. H. de Boer, M. L. Caramori, J. C. N. Chan, H. J. L. Heerspink, C. Hurst, K. Khunti, et al., Executive summary of the 2020 KDIGO Diabetes Management in CKD Guideline: evidence-based advances in monitoring and treatment, Kidney Int 98 (4): 839-848 (2020). Dufek, B. et al. Endothelin A receptor activation on mesangial cells initiates Alport glomerular disease. Kidney international 90, 300-310 (2016). Gessel, M.M. et al. Two Specific Sulfatide Species Are Dysregulated during Renal Development in a Mouse Model of Alport Syndrome. Lipids 54, 411-418 (2019). Goka, S., Copelovitch, L. & Levy Erez, D. Long-term outcome among females with Alport syndrome from a single pediatric center. Pediatric nephrology 36, 945-951 (2021). Grodecki, K.M. et al. Treatment of X-linked hereditary nephritis in Samoyed dogs with angiotensin converting enzyme (ACE) inhibitor. Journal of comparative pathology 117, 209-225 (1997). Gross, O. et al. Preemptive ramipril therapy delays renal failure and reduces renal fibrosis in COL4A3-knockout mice with Alport syndrome. Kidney Int 63, 438-446 (2003). Gross, O. et al. Safety and Efficacy of the ACE-Inhibitor Ramipril in Alport Syndrome: The Double-Blind, Randomized, Placebo-Controlled, Multicenter Phase III EARLY PRO-TECT Alport Trial in Pediatric Patients. ISRN pediatrics 2012, 436046 (2012) Gross, O. et al. Advances and unmet needs in genetic, basic and clinical science in Alport syndrome: report from the 2015 International Workshop on Alport Syndrome. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association - European Renal Association 32, 916-924 (2017). Guo, J. et al. Dysregulated Expression of microRNA-21 and Disease-Related Genes in Human Patients and in a Mouse Model of Alport Syndrome. Hum Gene Ther 30, 865-881 (2019). Hertz, J.M., Thomassen, M., Storey, H. & Flinter, F. Clinical utility gene card for: Alport syndrome. Eur J Hum Genet 20 (2012). Hudson, B.G., Tryggvason, K., Sundaramoorthy, M. & Neilson, E.G. Alport's syndrome, Goodpasture's syndrome, and type IV collagen. N Engl J Med 348, 2543-2556 (2003). Jerums G, Panagiotopolous S, Premaratne E, MacIsaac RJ. Integrating albuminurea and GFR in the assessment of diabetic nephropathy. Nat Rev Nephrol 5(7), 397-406 (2009). Accession No. 19556994 DOI: 10.1038 / nrneph.2009.91 Kashtan, C.E. Animal models of Alport syndrome. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association - European Renal Association 17, 1359-1362 (2002). Kashtan, C.E. et al. Clinical practice recommendations for the treatment of Alport syndrome: a statement of the Alport Syndrome Research Collaborative. Pediatr Nephrol 28, 5-11 (2013). Kidney Disease: Improving Global Outcomes (KDIGO) 2012 clinical practical guideline for the evaluation and management of chronic kidney disease. Kidney International Suppl. 3, 1-150 (2013). Koopman , JJE , van Essen , MF , Rennke , HG , de Vries , APJ & van Kooten , C. Deposition of the Membrane Attack Complex in Healthy and Diseased Human Kidneys . Front Immunol 11, 599974 (2020). Kromeyer-Hauschild , K. , Wabitsch , M. , Kunze , D. , Geller , F. , Geisz , HC , Hesse , V. , & Hebebrand , J. Monatsschr . Kinderheilk.149 (2001). Lees, GE Kidney Diseases Caused by Glomerular Basement Membrane Type IV Collagen Defects in Dogs. J Vet Emerg Crit Care 23, 184–193. Muckova, P. et al. Preclinical Alterations in the Serum of COL(IV)A3(-) / (-) Mice as Early Biomarkers of Alport Syndrome. J Proteome Res 14, 5202–5214. Nagel , M. , Nagorka , S. & Gross , O. Novel COL4A5 , COL4A4 , and COL4A3 mutations in Alport syndrome . Hum Mutation 26, 60 (2005). Noone , D. & Licht , C. An update on the pathomechanisms and future therapies of Alport syndrome. Pediatr Nephrol 28, 1025–1036. Pohl, M. et al. Diagnosis of Alport syndrome--search for proteomic biomarkers in body fluids. Pediatric nephrology 28, 2117-2123 (2013). Rhode, H., Muckova, P., Buchler, R., Wendler, S., Tautkus, B., Vogel, M. et al. A next generation setup for pre-fractionation of non-denatured proteins reveals diverse albumin proteoforms each carrying several post-translational modifications. Sci Rep 2019 Vol. 9 Issue 1 Pages 11733. Accession Number: 31409882 DOI: 10.1038 / s41598-019-48278-y. Roccella EJ et al. (National High Blood Pressure Education Program Working Group on High Blood Pressure in Children and Adolescents). The fourth report on the diagnosis, evaluation, and treatment of high blood pressure in children and adolescents. Pediatrics. 114 (S2) 555-576 (2004). Savige, J. et al. Expert consensus guidelines for the genetic diagnosis of Alport syndrome. Pediatr Nephrol (2018). Savige, J. et al. Expert consensus guidelines for the genetic diagnosis of Alport syndrome. Pediatric nephrology 34, 1175-1189 (2019). Wendler, S., Tautkus, B., Nemitz, S., Pesek, J., Kruger, T., Opitz, S., Bartz, M., Richter, S., Oehme, H., Haenel, T., Moore, T., Kreusch, S., Hanf, B., Schmidt, L., Rhode, H. in Automations Systems of the 21st Century. (ed. D. Arent, Freebush, M.) 1-50 (nova publishers, New York; 2013). Yamamura, T. et al. Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5. Nat Commun 11, 2777 (2020).

Claims

1. 1. A combination for use as a biomarker in a method for preclinical and / or early detection and / or diagnosis of kidney disease in a mammal, comprising: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI) A combination of at least two proteins selected from:

2. COLXIII and C4BP, or COLXIII and C4BP and HABP2, or Determine CFH and CFI, or The combination for use according to claim 1, wherein COLXIII, HABP2, C4BP, CFH, and CFI are measured.

3. 1. A protein for use as a biomarker in a method for preclinical and / or early detection and / or diagnosis of kidney disease in a mammal, comprising: The protein is Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI) is selected from The protein is fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), alpha-1-acid glycoprotein (a1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4), Serum amyloid A (SAA), Talin 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, and serine / arginine repeat matrix protein 3, in combination with at least one further protein selected from The at least one further protein is preferably fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), and C-reactive protein (CRP) A protein selected from

4. C4BP and ADP, or COLXIII and FMN, or HABP2 and TGF-β1, or HABP2 and FGG, or CFH and GS, or CFI and GS, or CFI and VTN, or CFH and FGG, or CFI and FGG The combination for use according to claim 3, wherein the following is determined:

5. 1. A combination for use as a biomarker in a method for preclinical and / or early detection and / or diagnosis of kidney disease in a mammal, comprising: The combination is Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), alpha-1-acid glycoprotein (a1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4), Serum amyloid A (SAA), Talin 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, Complement C1s, Complement C1r, Complement C2, Complement C3, Complement C4, Complement C5, Complement factor B, haptoglobin, α2-macroglobulin, alpha-1-antitrypsin, Ceruloplasmin, Serpin family A member 3, Serpin family A member 5, Serpin family A member 7, Serpin family G member 1, Serpin family D member 1, coagulation factor V, Prothrombin, plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit alpha, Fetuin B, insulin-like growth factor binding protein, alpha-2-glycoprotein 1, paraoxonase 1, Dipeptidyl peptidase 4, C-type lectin domain family 3 member B, Galectin 3 binding protein, apolipoprotein AIV (Apo AIV), Apolipoprotein CI (Apo CI), Apolipoprotein CII (Apo CII), Apolipoprotein E (Apo E), Apolipoprotein M (Apo M), Apolipoprotein B (Apo B), Myosin IB, Myosin XVIIIB, kinesin-like proteins, CD109 molecule, maltase-glucoamylase, succin molecular chaperone, Dispatched RND transporter family member 2, DNA polymerase, Transport protein particle complex subunit 2, RB related KRAB zinc finger, ciliogenesis-associated TTC17-interacting protein, Proteasome subunit alpha type, Regulation of synaptic membrane exocytosis 2, extracellular matrix protein 1, cadherin 5, coiled-coil domain-containing 178, and Attractin A combination of at least two proteins selected from The at least two proteins preferably comprise: Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4-binding protein alpha (C4BP), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), alpha-1-acid glycoprotein (a1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4), Serum amyloid A (SAA), Talin 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukutin (FUK), and Myosin IXA (MYIXA) A combination selected from:

6. ADP and PICP, or AGT and GS, or AGT and C9, or GS and C9, or GS and VTN, or AGT and VTN, or CFI and a1AGP, or LRGP1 and C1q, or CFH and C9, or CRP and C1q The combination for use according to claim 5, wherein the following is determined:

7. The kidney disease is A chronic, progressive kidney disease that begins with damage to the glomeruli (glomerulopathy), Preferably, the present invention is directed to post-infectious glomerulopathy, IgA nephropathy, Henoch-Schönlein purpura, focal segmental glomerulosclerosis, Alport syndrome, thinning basement membrane nephropathy, benign familial hematuria, or Chronic progressive kidney disease that begins in other parts of the body and / or kidneys involving the glomeruli (nephropathy), accompanied by other dysregulations; Preferably, metabolic syndrome, hypertension, heart failure, nephrotoxic drug therapy, adiposity, diabetes mellitus (diabetic nephropathy), renal agenesis, renal hypoplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract abnormalities, duplex kidneys, nephronophthisis The combination for use according to any one of claims 1 to 6, selected from:

8. the concentration of each of said proteins is determined in a mammalian sample; The combination for use according to any one of claims 1 to 7, wherein said concentration is preferably compared with a control value.

9. The sample is a body fluid, preferably urine or blood, e.g., serum, plasma, whole blood, more preferably, the sample is urine; and / or the mammal is a human or an animal such as a dog, cat, rabbit, guinea pig, hamster, cow, pig, horse, sheep, donkey, goat, red deer or camel, preferably wherein the mammal is a human.

10. 1. A method for preclinical and / or early detection and / or diagnosis of kidney disease, comprising: (a) determining the concentrations of at least two proteins in a mammalian sample, The at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI) Selected from or or The at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), and Complement factor I (CFI), one protein selected from fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), alpha-1-acid glycoprotein (a1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4), Serum amyloid A (SAA), Talin 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, and serine / arginine repeat matrix protein 3, at least one further protein selected from is selected from The at least one further protein is preferably fibrinogen, preferably fibrinogen gamma chain (FGG), Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), and C-reactive protein (CRP), and (b) comparing each concentration determined in step (a) with a control value; Including, A method wherein deviation of each concentration determined in step (a) from said control value is indicative of preclinical and / or early renal disease in said mammal.

11. 1. A method for preclinical and / or early detection and / or diagnosis of kidney disease, comprising: (a) determining the concentrations of at least two proteins in a mammalian sample, The at least two proteins are Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), alpha-1-acid glycoprotein (a1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4), Serum amyloid A (SAA), Talin 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukuchin (FUK), Myosin IXA (MYIXA), kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1; Serpin family A member 1, Transferrin, α1-B glycoprotein, Apo AI, Apo D, alpha-1-microglobulin / bikunin precursor, Carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, Complement C1s, Complement C1r, Complement C2, Complement C3, Complement C4, Complement C5, Complement factor B, haptoglobin, α2-macroglobulin, alpha-1-antitrypsin, Ceruloplasmin, Serpin family A member 3, Serpin family A member 5, Serpin family A member 7, Serpin family G member 1, Serpin family D member 1, coagulation factor V, Prothrombin, plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit alpha, Fetuin B, insulin-like growth factor binding protein, alpha-2-glycoprotein 1, paraoxonase 1, Dipeptidyl peptidase 4, C-type lectin domain family 3 member B, Galectin 3 binding protein, apolipoprotein AIV (Apo AIV), Apolipoprotein CI (Apo CI), Apolipoprotein CII (Apo CII), apolipoprotein E (Apo E4), Apolipoprotein M (Apo M), Apolipoprotein B (Apo B), Myosin IB, Myosin XVIIIB, kinesin-like proteins, CD109 molecule, maltase-glucoamylase, succin molecular chaperone, Dispatched RND transporter family member 2, DNA polymerase, Transport protein particle complex subunit 2, RB related KRAB zinc finger, ciliogenesis-associated TTC17-interacting protein, Proteasome subunit alpha type, Regulation of synaptic membrane exocytosis 2, extracellular matrix protein 1, cadherin 5, coiled-coil domain-containing 178, and Attractin is selected from The at least two proteins are preferably Type XIII collagen (COLXIII), hyaluronic acid binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), fibrinogen, Lumican (LUM), Formin 1 (FMN), Vitronectin (VTN), Gelsolin (GS), angiotensinogen (AGT), adiponectin (ADP), Transforming growth factor-β1 (TGF-β1), Carboxy-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), alpha-1-acid glycoprotein (a1AGP), coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-alpha-trypsin inhibitor 4 (IATI4), leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4), Serum amyloid A (SAA), Talin 1 (Tal), Kallikrein (KAL), dynein (DYN), Fukutin (FUK), and Myosin IXA (MYIXA), and (b) comparing each concentration determined in step (a) with a control value; Including, A method wherein deviation of each concentration determined in step (a) from said control value is indicative of preclinical and / or early renal disease in said mammal.

12. The kidney disease is A chronic, progressive kidney disease that begins with damage to the glomeruli (glomerulopathy), Preferably, the present invention is directed to post-infectious glomerulopathy, IgA nephropathy, Henoch-Schönlein purpura, focal segmental glomerulosclerosis, Alport syndrome, thinning basement membrane nephropathy, benign familial hematuria, or Chronic progressive kidney disease that begins in other parts of the body and / or kidneys involving the glomeruli (nephropathy), accompanied by other dysregulations; Preferably, metabolic syndrome, hypertension, heart failure, nephrotoxic drug therapy, adiposity, diabetes mellitus (diabetic nephropathy), renal agenesis, renal hypoplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract abnormalities, duplex kidneys, nephronophthisis 12. The method of claim 10 or 11, wherein the

13. The sample is a body fluid, preferably urine or blood, e.g., serum, plasma, whole blood, more preferably, the sample is urine; and / or wherein said mammal is a human or an animal such as a dog, cat, rabbit, guinea pig, hamster, cow, pig, horse, sheep, donkey, goat, red deer or camel, preferably wherein said mammal is a human.

14. COLXIII and C4BP, or COLXIII and C4BP and HABP2, or Determine CFH and CFI concentrations, or The method of any one of claims 10 to 13, wherein the concentrations of COLXIII, HABP2, C4BP, CFH, and CFI are determined.

15. C4BP and ADP, or COLXIII and FMN, or HABP2 and TGF-β1, or HABP2 and FGG, or CFH and GS, or CFI and GS, or CFI and VTN, or CFH and FGG, or Determine the concentrations of CFI and FGG, or ADP and PICP, or AGT and GS, or AGT and C9, or GS and C9, or GS and VTN, or AGT and VTN, or CFI and a1AGP, or LRGP1 and C1q, or CFH and C9, or The method according to any one of claims 10 to 14, wherein the concentrations of CRP and C1q are determined.

16. the control values ​​are determined for each protein in healthy subjects, the control values ​​preferably being in a standard concentration range; and / or deviation of the concentration determined in step (a) from the median of said standard concentration range is indicative of preclinical and / or early renal disease. Preferably, a deviation of ≥ 2-fold from the median of said standard concentration range is indicative of preclinical and / or early renal disease; or A deviation of less than or equal to 0.5 times the median of the standard concentration range indicates preclinical and / or early renal disease. More preferably, 16. The method of any one of claims 10 to 15, wherein a deviation of ≥ 2 times the median of the standard concentration range is indicative of preclinical and / or early renal disease.

Images