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 the group consisting of 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 preclinical and / or early detection and / or diagnosis of kidney disease. The invention also 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 of a subject, and (b) comparing each concentration determined in step (a) to a control value, wherein the deviation of each concentration determined in step (a) from the control value is indicative of a preclinical and / or early kidney disease.

Description

Technical Field

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

[0002] Almost all kidney diseases begin without any symptoms and pain. Most kidney diseases progress undetectably for months / years before manifestation and diagnosis. Although these diseases can be prevented or even reversed during this period, there are no guidelines for preventive screening of preclinical kidney disease. Damage to the glomerular filtration apparatus causes leakage of plasma proteins into the primary urine. Currently, proteinuria or albuminuria is the earliest indicator of kidney damage (other than hematuria). However, this indicator indicates an already manifested disease, not its early stage. In addition, albuminuria triggers further progression of kidney damage itself. It is too late for clinically established kidney function indicators (such as creatinine in serum) to show pathological values, at which time 50% of kidney function is already disordered. Therefore, there is still an unresolved diagnostic dilemma.

[0003] Virtually all kidney diseases that progress to end-stage kidney disease (ESKD) requiring renal replacement therapy are treatable, and even curable, if detected and treated early. If kidney disease could be detected before albuminuria became measurable, treatment options would be available to slow, halt, or even reverse its progression.

[0004] For example, Alport syndrome (AS) is a genetic kidney disease 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). Most cases are X-linked due to mutations in the α5 chain of collagen (IV) (COL(IV)A5) (Hertz et al., 2012). Female carriers of X-linked AS can also be severely affected (Goka et al., 2021). Furthermore, there is considerable genetic and phenotypic variability (Savige et al., 2018). Preclinically, several alterations have been identified in the renal cortex of affected AS mice (Gessel et al., 2019; Dufek et al., 2016). Despite this, the specific pathogenic chain of events that leads to progressive renal fibrosis in AS due to collagen IV mutations remains unclear. Every healthy child is born with immature α1 / 2(IV) chains, but due to mutations, AS patients are unable to mature their GBMs with α3 / 4 / 5(IV) chains. Therefore, this defect, which sometimes manifests after birth, presents a diagnostic "window of opportunity." Recent expert guidelines recommend genetic testing for the diagnosis of Alport syndrome and for the identification of some modifications (Savige et al., 2019). However, application of these methods generally requires history and / or clinical evidence, such as hematuria (Gross et al., 2020). However, in previously undiagnosed families and due to de novo mutations in AS, this tool is not feasible for early diagnosis. Histological analysis is highly invasive, high-risk, and depicts relatively late progression events. To date, transplantation is the only cure for ESKD caused by AS (Savige et al., 2019). Despite this, the most effective and widely used treatment currently is inhibition of the renin-angiotensin-aldosterone system. In addition, several new approaches are being investigated to improve kidney function and extend life expectancy. Such approaches include the use of molecular chaperones, stem cells, stem cell extracellular vesicles, and exon skipping (see, for example, Yamamura et al., 2020). ACE inhibition has been shown to delay kidney failure for many years in a time-dependent manner (Kashtan et al., 2013; Noone et al., 2013). Specifically, the earlier treatment is initiated, the greater the beneficial effect. Generally, starting therapy in children before symptoms develops is most beneficial and optimally slows the progression of their kidney disease.

[0005] US2011 / 118127 A1 relates to gene expression triggered by drugs in specific tissues or cell lines and describes a method for screening drugs that cause toxicity, which utilizes a microarray containing probes for multiple genes. WO2018 / 094021A1 relates to steroid resistance in nephrotic syndrome and discloses a method for diagnosing steroid resistance in subjects with glomerular diseases (such as nephrotic syndrome), the method comprising determining the amount of at least one steroid resistance biomarker in a biological sample of the subject. These are examples of diagnosing drug cytotoxic effects and manifested kidney diseases.

[0006] Therefore, there is an urgent global need for early progressive biomarkers that indicate glomerular alarm signals before any clinical signs of glomerular injury to ensure early diagnosis, early initiation of treatment, monitoring of treatment, and development of new therapeutic strategies.

[0007] In addition, in developed countries, diabetic nephropathy (DN) is the main cause of end-stage renal disease (ESKD) and renal replacement therapy. However, the diagnostic tests established in clinical practice (i.e., serum creatinine, estimated glomerular filtration rate (eGFR) and albuminuria) indicate the rather late and irreversible stage of DN. In the scientific literature, there is no biomarker that can indicate the early stage DN in a preclinical and curable state before the onset of (micro) albuminuria in routine use. In addition, there is no indicator that distinguishes diabetic individuals who are more susceptible to nephropathy and renal failure from other individuals. DN is caused by changes in the endothelium and basement membrane of the glomerulus. The same is true for several other glomerulonephropathy. In addition, according to scientific literature, there is a slight consistency in the early molecular changes and tissue pathogenesis of most glomerulopathies, which are unrelated to their causes (see, for example, Hastings et al., 2021; Koopman et al., 2020) such as the deposition and activation of complement components, the deposition of other substances, basal thickening and podocyte disappearance. The filtration apparatus, including podocytes, endothelial cells, and mesangial cells, is uniformly damaged as in AS. Until then, these early processes could not be detected by established diagnostic methods, as discussed above. Subsequently, these changes progress to loss of the filtration barrier, proteinuria, tubular changes, and fibrosis. Summary of the Invention

[0008] According to the present invention, this object is achieved in that a combination of at least two proteins selected from the group consisting of:

[0009] Type XIII collagen (COLXIII),

[0010] Hyaluronan binding protein 2 (HABP2),

[0011] C4 binding protein (C4BP),

[0012] Complement factor H (CFH) and

[0013] Complement factor I (CFI).

[0014] According to the present invention, this object is achieved by a protein selected from the group consisting of:

[0015] Type XIII collagen (COLXIII),

[0016] Hyaluronan binding protein 2 (HABP2),

[0017] C4 binding protein (C4BP),

[0018] Complement factor H (CFH) and

[0019] Complement factor I (CFI)

[0020] and at least one additional protein selected from the group consisting of: for use as a biomarker in a method for preclinical and / or early detection and / or diagnosis of kidney disease in a mammal:

[0021] Fibrinogen,

[0022] Luminophore (LUM),

[0023] FMN,

[0024] vitronectin (VTN),

[0025] Gelsolin (GS),

[0026] Angiotensinogen (AGT),

[0027] Adiponectin (ADP),

[0028] Transforming growth factor β1 (TGF-β1),

[0029] Carboxyl-terminal propeptide of type I procollagen (PICP),

[0030] C-reactive protein (CRP),

[0031] α-1-acid glycoprotein (a1AGP),

[0032] Coagulation factor XIII (CFXIII),

[0033] Complement component 1q (C1q),

[0034] Complement component 9 (C9),

[0035] Fibronectin (FN),

[0036] Ficolin 1 (FIC),

[0037] Hemopexin (HPX),

[0038] Inter-α-trypsin inhibitor 4 (IATI4),

[0039] Leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0040] Retinol binding protein 4 (RBP4),

[0041] Serum amyloid A (SAA),

[0042] Talin 1 (Tal),

[0043] Kallikrein (KAL),

[0044] Dynein (DYN),

[0045] Fukutin(FUK),

[0046] Myosin IXA (MYIXA),

[0047] Kininogen 1,

[0048] α2-HS glycoprotein,

[0049] Complement C6,

[0050] Complement C7,

[0051] Complement C8,

[0052] APOH, beta-2-glycoprotein 1,

[0053] Serine protease inhibitor family A member 1,

[0054] transferrin,

[0055] α1-B glycoprotein,

[0056] Apo AI,

[0057] Apo D,

[0058] Alpha-1 microglobulin / double Kunitz inhibitor precursor,

[0059] Carboxypeptidase N subunit 2 and

[0060] Serine / arginine repeat matrix protein 3.

[0061] According to the present invention, this object is achieved in that a combination of at least two proteins selected from the group consisting of:

[0062] Type XIII collagen (COLXIII),

[0063] Hyaluronan binding protein 2 (HABP2),

[0064] C4 binding protein (C4BP),

[0065] Complement factor H (CFH),

[0066] Complement factor I (CFI),

[0067] Fibrinogen,

[0068] Luminophore (LUM),

[0069] FMN,

[0070] vitronectin (VTN),

[0071] Gelsolin (GS),

[0072] Angiotensinogen (AGT),

[0073] Adiponectin (ADP),

[0074] Transforming growth factor β1 (TGF-β1),

[0075] Carboxyl-terminal propeptide of type I procollagen (PICP),

[0076] C-reactive protein (CRP),

[0077] α-1-acid glycoprotein (a1AGP),

[0078] Coagulation factor XIII (CFXIII),

[0079] Complement component 1q (C1q),

[0080] Complement component 9 (C9),

[0081] Fibronectin (FN),

[0082] Ficolin 1 (FIC),

[0083] Hemopexin (HPX),

[0084] Inter-α-trypsin inhibitor 4 (IATI4),

[0085] Leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0086] Retinol binding protein 4 (RBP4),

[0087] Serum amyloid A (SAA),

[0088] Talin 1 (Tal),

[0089] Kallikrein (KAL),

[0090] Dynein (DYN),

[0091] Fukutin(FUK),

[0092] Myosin IXA (MYIXA),

[0093] Kininogen 1, α2-HS glycoprotein,

[0094] Complement C6,

[0095] Complement C7,

[0096] Complement C8, APOH, beta-2-glycoprotein 1, serine protease inhibitor family A member 1, transferrin, alpha 1-B glycoprotein, Apo AI, Apo D, alpha-1 microglobulin / double Kunitz inhibitor precursor, carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, complement C1s,

[0097] Complement C1r,

[0098] Complement C2,

[0099] Complement C3,

[0100] Complement C4,

[0101] Complement C5,

[0102] Complement factor B,

[0103] Haptoglobin, α2-macroglobulin, α-1-antitrypsin, ceruloplasmin,

[0104] Serine protease inhibitor family A member 3, Serine protease inhibitor family A member 5, Serine protease inhibitor family A member 7, Serine protease inhibitor family G member 1, Serine protease inhibitor family D member 1, Coagulation factor V,

[0105] Prothrombin,

[0106] Plasminogen, protein S,

[0107] Afamin,

[0108] Transthyretin, hemoglobin subunit α, fetuin B,

[0109] Insulin-like growth factor binding protein,

[0110] α-2-glycoprotein 1,

[0111] Paraoxonase 1,

[0112] Dipeptidyl peptidase 4,

[0113] C-type lectin domain family 3 member B,

[0114] Galectin 3 binding protein,

[0115] Apolipoprotein AIV (Apo AIV),

[0116] Apolipoprotein CI (Apo CI),

[0117] Apolipoprotein CII (Apo CII),

[0118] Apolipoprotein E (Apo E4),

[0119] Apolipoprotein M (Apo M),

[0120] Apolipoprotein B (Apo B),

[0121] Myosin IB,

[0122] Myosin XVIIIB,

[0123] Kinesin-like proteins,

[0124] CD109 molecule,

[0125] Maltase-glucoamylase,

[0126] Sacsin molecular chaperone,

[0127] Dispatch RND transporter family member 2,

[0128] DNA polymerase,

[0129] Transport protein particle complex subunit 2,

[0130] RB-related KRAB zinc finger,

[0131] TTC17-interacting proteins related to ciliogenesis,

[0132] Proteasome subunit α,

[0133] Regulates synaptic membrane exocytosis protein 2,

[0134] Extracellular matrix protein 1,

[0135] E-cadherin 5,

[0136] Coiled-coil domain-containing protein 178 and

[0137] Attracts protein.

[0138] According to the present invention, this object is achieved by a method for preclinical and / or early detection and / or diagnosis of kidney disease, comprising:

[0139] (a) determining the concentration of at least two proteins in a sample from a mammal,

[0140] wherein the at least two proteins are selected from

[0141] Type XIII collagen (COLXIII),

[0142] Hyaluronan binding protein 2 (HABP2),

[0143] C4 binding protein (C4BP),

[0144] Complement factor H (CFH) and

[0145] Complement factor I (CFI), or

[0146] wherein the at least two proteins are selected from the group consisting of:

[0147] A protein selected from the group consisting of:

[0148] Type XIII collagen (COLXIII),

[0149] Hyaluronan binding protein 2 (HABP2),

[0150] C4 binding protein (C4BP),

[0151] Complement factor H (CFH) and complement factor I (CFI),

[0152] and at least one additional protein selected from the group consisting of:

[0153] Fibrinogen,

[0154] Luminophore (LUM),

[0155] FMN,

[0156] vitronectin (VTN),

[0157] Gelsolin (GS),

[0158] Angiotensinogen (AGT),

[0159] Adiponectin (ADP),

[0160] Transforming growth factor β1 (TGF-β1),

[0161] Carboxyl-terminal propeptide of type I procollagen (PICP),

[0162] C-reactive protein (CRP),

[0163] α-1-acid glycoprotein (a1AGP),

[0164] Coagulation factor XIII (CFXIII),

[0165] Complement component 1q (C1q),

[0166] Complement component 9 (C9),

[0167] Fibronectin (FN),

[0168] Ficolin 1 (FIC),

[0169] Hemopexin (HPX),

[0170] Inter-α-trypsin inhibitor 4 (IATI4),

[0171] Leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4),

[0172] Serum amyloid A (SAA),

[0173] Talin 1 (Tal),

[0174] Kallikrein (KAL),

[0175] Dynein (DYN),

[0176] Fukutin(FUK),

[0177] Myosin IXA (MYIXA),

[0178] Kininogen 1,

[0179] α2-HS glycoprotein,

[0180] Complement C6,

[0181] Complement C7,

[0182] Complement C8,

[0183] APOH, beta-2-glycoprotein 1,

[0184] Serine protease inhibitor family A member 1,

[0185] transferrin,

[0186] α1-B glycoprotein,

[0187] Apo AI,

[0188] Apo D,

[0189] Alpha-1 microglobulin / double Kunitz inhibitor precursor,

[0190] Carboxypeptidase N subunit 2 and

[0191] Serine / arginine repeat matrix protein 3,

[0192] (b) comparing each concentration determined in step (a) with a control value,

[0193] wherein the deviation of each concentration determined in step (a) from the control value is indicative of a preclinical and / or

[0194] or early kidney disease.

[0195] According to the present invention, this object is achieved by a method for preclinical and / or early detection and / or diagnosis of kidney disease, comprising:

[0196] (a) determining the concentration of at least two proteins in a sample from a mammal,

[0197] wherein the at least two proteins are selected from

[0198] Type XIII collagen (COLXIII),

[0199] Hyaluronan binding protein 2 (HABP2),

[0200] C4 binding protein (C4BP),

[0201] Complement factor H (CFH),

[0202] Complement factor I (CFI),

[0203] Fibrinogen,

[0204] Luminophore (LUM),

[0205] FMN,

[0206] vitronectin (VTN),

[0207] Gelsolin (GS),

[0208] Angiotensinogen (AGT),

[0209] Adiponectin (ADP),

[0210] Transforming growth factor β1 (TGF-β1),

[0211] Carboxyl-terminal propeptide of type I procollagen (PICP),

[0212] C-reactive protein (CRP),

[0213] α-1-acid glycoprotein (a1AGP),

[0214] Coagulation factor XIII (CFXIII),

[0215] Complement component 1q (C1q),

[0216] Complement component 9 (C9),

[0217] Fibronectin (FN),

[0218] 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),

[0219] Kallikrein (KAL), dynein (DYN), Fukutin (FUK),

[0220] Myosin IXA (MYIXA), kininogen 1,

[0221] α2-HS glycoprotein,

[0222] Complement C6,

[0223] Complement C7,

[0224] Complement C8,

[0225] APOH, beta-2-glycoprotein 1, serine protease inhibitor family A member 1, transferrin,

[0226] α1-B glycoprotein, Apo AI,

[0227] Apo D,

[0228] α-1 microglobulin / double Kunitz inhibitor precursor, carboxypeptidase N subunit 2,

[0229] Serine / arginine repeat matrix protein 3, complement C1s,

[0230] Complement C1r,

[0231] Complement C2,

[0232] Complement C3,

[0233] Complement C4,

[0234] Complement C5,

[0235] Complement factor B,

[0236] Haptoglobin,

[0237] α2-macroglobulin, α-1-antitrypsin,

[0238] ceruloplasmin,

[0239] Serine protease inhibitor family A member 3, Serine protease inhibitor family A member 5, Serine protease inhibitor family A member 7, Serine protease inhibitor family G member 1, Serine protease inhibitor family D member 1, Coagulation factor V,

[0240] Prothrombin,

[0241] Plasminogen, protein S,

[0242] Afamin,

[0243] transthyretin,

[0244] Hemoglobin subunit α,

[0245] Fetuin B,

[0246] Insulin-like growth factor binding protein, alpha-2-glycoprotein 1,

[0247] Paraoxonase 1,

[0248] 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,

[0249] Myosin XVIIIB,

[0250] Kinesin-like protein, CD109 molecule,

[0251] Maltase-glucoamylase, Sacsin molecular chaperone,

[0252] Dispatch RND transporter family member 2, DNA polymerase,

[0253] Transport protein particle complex subunit 2, RB-related KRAB zinc finger, ciliogenesis-related TTC17 interacting protein, proteasome subunit α type, regulating synaptic membrane exocytosis protein 2, extracellular matrix protein 1,

[0254] E-cadherin 5,

[0255] Coiled-coil domain-containing protein 178 and

[0256] Attracts protein,

[0257] (b) comparing each concentration determined in step (a) with a control value,

[0258] wherein the deviation of each concentration determined in step (a) from the control value is indicative of a preclinical and / or

[0259] or early kidney disease.

[0260] DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

[0261] Before describing the present invention in more detail below, it should be understood that the present invention is not limited to the particular methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only used to describe the purpose of specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. For purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

[0262] Concentration, amount and other numerical data can be expressed or presented in range format herein.It should be understood that this range format is used only for convenience and brevity, and therefore should be flexibly interpreted as not only including the numerical values clearly listed as the limits of the range, but also including all individual numerical values or sub-ranges covered within the range, as if each numerical value and sub-range is clearly listed.As an illustration, the numerical range of "1 to 21" should be interpreted as not only including the values 1 to 21 clearly listed, but also including individual values and sub-ranges within the range.Therefore, included in this numerical range are individual values such as 1, 2, 3, 4, 5 ... 17, 18, 19, 20, 21 and sub-ranges such as 2 to 10, 8 to 15, etc. The same principle applies to the range of only listing one numerical value, such as "at least 90%".In addition, no matter how broad the described range or feature is, such an explanation should be applicable.

[0263] Preclinical and early-stage biomarkers for kidney disease

[0264] As summarized above, the present invention provides novel biomarkers for preclinical and / or early detection and / or diagnosis of kidney disease.

[0265] The biomarkers of the present invention are proteins disclosed herein. As used herein, the term "protein" also refers to / includes modified variants of the corresponding protein, fragments of the corresponding protein, protein chains, and complexes of the corresponding protein with another protein.

[0266] Furthermore, as used herein, the term "protein" refers to / includes all known identified chains that encapsulate the corresponding protein, modified variants of the corresponding protein, fragments of the corresponding protein, single protein chains, multiple protein chains, and complexes of the corresponding protein (chain) with additional proteins and protein chains, without being limited by their validation using currently available immunoassays (e.g., ELISA).

[0267] Stages have been defined to describe the course of chronic kidney disease (CKD) of varying origin (see Jerums et al., 2009):

[0268] Phase I: ultrafiltration;

[0269] Stage II: lesions with normoalbuminuria;

[0270] Stage III: microalbuminuria;

[0271] Stage IV: macroalbuminuria;

[0272] Stage V: End-stage renal failure.

[0273] In Alport syndrome (AS), the following stages describe renal damage and loss of function, with definitions based on those of Gross et al (2012):

[0274] Stage 0: Microhematuria without microalbuminuria (usually present at birth)

[0275] Stage I: microalbuminuria (30-300 mg albumin / g creatinine),

[0276] Stage II: Albuminuria >300 mg albumin / g creatinine,

[0277] Stage III: Normal renal function (creatinine clearance) decreased by >25%,

[0278] Stage IV: End-stage renal failure.

[0279] Diabetic kidney disease (DKD) is staged according to albuminuria and additionally taking into account eGFR as follows (see Boer et al., 2020):

[0280] eGFR (ml / min / 1.73m 2 );

[0281] G1 stage: ≥90;

[0282] G2: 60-89

[0283] G3a stage: 45-59

[0284] G3b stage: 30-44

[0285] G4 period: 15-29

[0286] G5 stage: <15

[0287] Albuminuria (mg / g creatinine)

[0288] A1 period: <30

[0289] A2 phase: 30-300

[0290] Phase A3: >300.

[0291] CKD of varying origin can also be staged based on estimated glomerular filtration rate (eGFR) and albuminuria levels as follows (see KDIGO 2012: Clinical practical guideline for the evaluation and management of chronic kidney disease):

[0292] Patients were categorized as G1-G5 based on eGFR and as A1-A3 based on ACR (albumin:creatinine ratio).

[0293] eGFR (ml / min / 1.73m 2 );

[0294] G1 stage: ≥90;

[0295] G2: 60-89

[0296] G3a stage: 45-59

[0297] G3b stage: 30-44

[0298] G4 period: 15-29

[0299] G5 stage: <15

[0300] ACR (mg / mmol)

[0301] Period A1: <3

[0302] Phase A2: 3-30

[0303] Phase A3: >30.

[0304] In the present invention, stages I and II (as defined for CKD of different origins), stages G1A1, G1A2, G2A2 and G3aA1 (as defined for DKD and CKD of different origins according to KDIGO 2012), and stages 0 and I (as defined for AS) are indicated by biomarkers. There is currently a lack of any recognized, applicable and specific clinical and laboratory signs to diagnose "(early) renal failure". During these stages, the disease persists but is asymptomatic. In contrast, in the literature, the term "early" mainly refers to the early stages of apparent renal damage that can already be detected by various commonly used biomarkers (such as KIM-1, NGAL, cystatin C and microalbuminuria).

[0305] As used herein, the "preclinical" stage of kidney disease refers to stage I (ultrafiltration) as defined for CKD of varying origin and / or stage G1A1 as defined (according to KDIGO 2012) for DKD and CKD of varying origin and / or stage 0 (microhematuria without microalbuminuria) as defined for AS.

[0306] Although both signs of hyperfiltration and microhematuria are used as indicators to define the preclinical stage of CKD, although they may indicate the disease, both signs also occur primarily under various physiological conditions such as physical activity, large fluid intake, or mild sports trauma, and therefore, these signs are not actually used as specific or even preclinical indicators of early kidney disease. Therefore, these signs are ignored by most physicians and pediatricians.

[0307] As used herein, the "early" stage of kidney disease refers to stage II (damage with normal albuminuria) as defined for CKD of various origins, stage G1A2, G2A2, G3aA1 as defined for DKD and CKD of various origins (according to KDIGO 2012), and / or stage I (microalbuminuria (30-300 mg albumin / g creatinine) as defined for AS. Thus, the early stage is characterized by slightly altered clinical and / or clinical chemical signs that are already in practical use. "Early" means that there are signs of incipient kidney changes and damage but severe kidney failure has not yet occurred.

[0308] The "preclinical" stage of kidney disease occurs earlier in time than (in other words: before) the "early" stage of kidney disease.

[0309] The present invention provides biomarkers for detecting and / or diagnosing preclinical and / or early stage renal disease. In other words, thanks to the biomarkers, it is possible to detect renal disease at a preclinical and / or early stage, which allows diagnosis of the development of renal disease in a subject / patient when a sample is collected at a preclinical and / or early stage.

[0310] Preferably, biomarkers are provided for the in vitro detection and / or in vitro diagnosis of kidney disease at a preclinical and / or early stage.

[0311] Preferably, the kidney disease is selected from the following chronic progressive kidney diseases:

[0312] Begins with glomerular damage (glomerulopathy),

[0313] Preferred are post-infectious glomerulopathy, IgA nephropathy, Henoch-Schoenlein-Purpura, focal segmental glomerulosclerosis, Alport syndrome, thin basement membrane nephropathy, and benign familial hematuria;

[0314] or

[0315] Begins elsewhere in the body with other dysregulations, and / or begins elsewhere in the kidneys with effects on the glomeruli (nephropathy),

[0316] Preferred are metabolic syndrome, hypertension, heart failure, drug nephrotoxicity, obesity, diabetes (diabetic nephropathy), nephrogenesis, renal dysplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract anomalies, renal duplication, and nephronophthisis.

[0317] The present invention provides a combination of at least two proteins selected from the group consisting of:

[0318] Type XIII collagen (COLXIII),

[0319] Hyaluronan binding protein 2 (HABP2),

[0320] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0321] Complement factor H (CFH) and

[0322] Complement factor I (CFI).

[0323] Therefore, the present invention provides the use of a combination of at least two proteins selected from the group consisting of:

[0324] Type XIII collagen (COLXIII),

[0325] Hyaluronan binding protein 2 (HABP2),

[0326] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0327] Complement factor H (CFH) and

[0328] Complement factor I (CFI).

[0329] As used herein, the term "combination" simply means that at least two biomarkers are used for detection and / or diagnosis. One biomarker is used together with at least one additional biomarker for detection and / or diagnosis.

[0330] In one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more biomarkers are used.

[0331] The present invention provides at least two proteins selected from the group consisting of the following for use as biomarkers in a method for preclinical and / or early detection and / or diagnosis of kidney disease:

[0332] Type XIII collagen (COLXIII),

[0333] Hyaluronan binding protein 2 (HABP2),

[0334] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0335] Complement factor H (CFH) and

[0336] Complement factor I (CFI).

[0337] Therefore, the present invention provides the use of at least two proteins selected from the group consisting of:

[0338] Type XIII collagen (COLXIII),

[0339] Hyaluronan binding protein 2 (HABP2),

[0340] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0341] Complement factor H (CFH) and

[0342] Complement factor I (CFI).

[0343] In a preferred embodiment, COLXIII and C4BP, or COLXIII and C4BP and HABP2, or CFH and CFI are determined.

[0344] In one embodiment, COLXIII, HABP2, C4BP, CFH, and CFI are assayed.

[0345] The present invention provides a protein selected from the group consisting of:

[0346] Type XIII collagen (COLXIII),

[0347] Hyaluronan binding protein 2 (HABP2),

[0348] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0349] Complement factor H (CFH) and

[0350] Complement factor I (CFI)

[0351] and at least one additional protein selected from the group consisting of: for use as a biomarker in a method for preclinical and / or early detection and / or diagnosis of kidney disease:

[0352] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0353] Luminophore (LUM),

[0354] FMN,

[0355] vitronectin (VTN),

[0356] Gelsolin (GS),

[0357] Angiotensinogen (AGT),

[0358] Adiponectin (ADP),

[0359] Transforming growth factor β1 (TGF-β1), carboxyl-terminal propeptide of type I procollagen (PICP) and C-reactive protein (CRP),

[0360] α-1-acid glycoprotein (a1AGP),

[0361] Coagulation factor XIII (CFXIII),

[0362] Complement component 1q (C1q),

[0363] Complement component 9 (C9),

[0364] Fibronectin (FN),

[0365] Ficolin 1 (FIC),

[0366] Hemopexin (HPX),

[0367] Inter-alpha-trypsin inhibitor 4 (IATI4), leucine-rich alpha-2-glycoprotein 1 (LRGP1), retinol binding protein 4 (RBP4),

[0368] Serum amyloid A (SAA),

[0369] Talin 1 (Tal),

[0370] Kallikrein (KAL),

[0371] Dynein (DYN), preferably dynein heavy chain domain 1, Fukutin (FUK),

[0372] Myosin IXA (MYIXA),

[0373] Kininogen 1,

[0374] α2-HS glycoprotein,

[0375] Complement C6,

[0376] Complement C7,

[0377] Complement C8, preferably C8 alpha chain and / or C8 beta chain, APOH, beta-2-glycoprotein 1,

[0378] Serine protease inhibitor family A member 1, transferrin,

[0379] α1-B glycoprotein,

[0380] Apo AI,

[0381] Apo D,

[0382] α-1 microglobulin / double Kunitz inhibitor precursor, carboxypeptidase N subunit 2,

[0383] Serine / arginine repeat matrix protein 3, complement C1s,

[0384] Complement C1r,

[0385] Complement C2,

[0386] Complement C3,

[0387] Complement C4,

[0388] Complement C5,

[0389] Complement factor B,

[0390] Haptoglobin, α2-macroglobulin, α-1-antitrypsin, ceruloplasmin, serine protease inhibitor family A member 3, serine protease inhibitor family A member 5, serine protease inhibitor family A member 7, serine protease inhibitor family G member 1, serine protease inhibitor family D member 1, coagulation factor V,

[0391] Prothrombin,

[0392] Plasminogen, protein S, Afamin,

[0393] Transthyretin, hemoglobin subunit α, fetuin B,

[0394] Insulin-like growth factor binding protein, alpha-2-glycoprotein 1,

[0395] Paraoxonase 1,

[0396] 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,

[0397] Myosin XVIIIB, kinesin-like protein, CD109 molecule,

[0398] Maltase-glucoamylase,

[0399] Sacsin molecular chaperone,

[0400] Dispatch RND transporter family member 2,

[0401] DNA polymerase,

[0402] Transport protein particle complex subunit 2,

[0403] RB-related KRAB zinc finger,

[0404] TTC17-interacting proteins related to ciliogenesis,

[0405] Proteasome subunit α,

[0406] Regulates synaptic membrane exocytosis protein 2,

[0407] Extracellular matrix protein 1,

[0408] E-cadherin 5,

[0409] Coiled-coil domain-containing protein 178 and

[0410] Attracts protein.

[0411] Therefore, the present invention provides the use of a protein selected from the group consisting of:

[0412] Type XIII collagen (COLXIII),

[0413] Hyaluronan binding protein 2 (HABP2),

[0414] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0415] Complement factor H (CFH) and

[0416] Complement factor I (CFI).

[0417] The protein biomarkers of the present invention are shown in Table 1, which also shows the protein IDs and concentration deviations (average ratios of subfractions or overall trends) determined by mass spectrometry in diseased dogs compared to healthy controls. Protein IDs refer to the UniProt database of approximately 47,800 canine entries (date: October 26, 2017). In fact, the primary source of all identified proteins has generally been known for a long time, but they are also produced by several other organs and tissues, especially in the kidney. In addition to being produced from the liver, several acute phase proteins (including complement components) are widely produced by various cell types such as mesangial cells and other renal cells.

[0418] In urine from humans with kidney disease, the following proteins showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by mean ratios (mean ratios of subfractions or overall trend, mass spectrometry) and by ELISA:

[0419] Type XIII collagen (COLXIII) refers to a protein with the protein ID F1PJH3. Type XIII collagen is a homotrimer, or a protein composed of three identical peptide chains (monomers), each of which is called the α1 chain of type XIII collagen. Formally, these monomers are called type XIII collagen α-1 chains and are encoded by the COL3A1 gene in humans. Type XIII collagen is a type of fibrillar collagen with a long, inflexible triple-helical domain. COLXIII is a transmembrane protein expressed in glomerular endothelial cells.

[0420] In urine from humans with renal disease, COLXIII showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA. Figure 2 .

[0421] Hyaluronan-binding protein 2 (HABP2) refers to a 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 hyaluronan. It is involved in extracellular matrix organization and cell adhesion.

[0422] In urine from humans with renal disease, HABP2 showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA. Figure 2 .

[0423] C4 binding protein (C4BP), or complement component 4 binding protein, refers to a protein with the protein ID F1PGM9. C4b binding protein is involved in the regulation of the complement system. It is a multimeric protein composed of seven identical α chains, encoded by C4BPA, and a single β chain, encoded by C4BPB. In the kidney, it is produced by podocytes. The biomarker is C4BP, preferably the C4BP α chain (C4BPA).

[0424] In urine from humans with renal disease, C4BPA showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA. Figure 2 .

[0425] Complement factor H (CFH) refers to a protein with the protein ID F1PY40. Factor H is a member of the complement activation regulator family and a complement regulatory protein. It is a soluble glycoprotein that circulates in human plasma.

[0426] In urine from humans with renal disease, CFH showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA. Figure 2 .

[0427] Complement Factor I (CFI) refers to a protein with the protein ID J9NT17. Complement Factor I, also known as C3b / C4b inactivating factor, is a protein that, in humans, is encoded by the CFI gene. Complement Factor I (Factor I) is a protein of the complement system that regulates complement activation by cleaving cell-bound or fluid-phase C3b and C4b.

[0428] In urine from humans with kidney disease, CFI showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0429] Fibrinogen gamma chain (FGG) refers to a protein with the protein ID F1P8G0. The 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-derived 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).

[0430] In urine from humans with kidney disease, FGG showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0431] Luminocan (LUM) refers to a protein with the protein ID E2R416. Luminocan, also known as LUM, is an extracellular matrix protein that in humans is encoded by the LUM gene on chromosome 12.

[0432] In urine from humans with kidney disease, LUM showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0433] FMN 1 refers to the protein with protein ID J9P216. FMNs (fMN homologous proteins) are a group of proteins that participate in actin polymerization and associate with the fast-growing ends (barbed ends) of actin filaments.

[0434] In urine from humans with kidney disease, FMN showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0435] Vitronectin (VTN) refers to a protein with the protein ID Q2YF02. Vitronectin is a glycoprotein of the hemobindin family, 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 referred to as vitronectin (fragment).

[0436] In urine from humans with kidney disease, VTN showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0437] Gelsolin (GS) refers to a protein with the protein ID F6Y3P9. Gelsolin is an actin-binding protein and a key regulator of actin filament assembly and disassembly.

[0438] In urine from humans with kidney disease, GS showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0439] Angiotensinogen (AGT) refers to a protein with the protein ID F1PAL5. Angiotensinogen is a component of the renin-angiotensin system (RAS), a hormone system that regulates blood pressure and fluid balance.

[0440] In urine from humans with kidney disease, AGT showed a concentration deviation greater than or equal to (≥) 2 times the median of the standard concentration range as measured by ELISA.

[0441] 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 fatty acid breakdown. In humans, it is encoded by the ADIPOQ gene and is primarily produced in adipose tissue, but is also produced in muscle. In Table 1, it is referred to as adiponectin (fragment).

[0442] In urine from humans with kidney disease, ADP showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by ELISA.

[0443] Transforming growth factor β1 (TGF-β1) refers to a protein with protein ID P54831. Transforming growth factor β1 or TGF-β1 is a polypeptide member of the transforming growth factor β superfamily of cytokines. It is a secreted protein that performs many cellular functions, including controlling cell growth, cell proliferation, cell differentiation, matrix remodeling, and apoptosis. In humans, TGF-β1 is encoded by the TGFB1 gene. Since the concentration of TGF-β1 in our subfractions is very low, it is not identified by mass spectrometry (see 1.3.). Because we have identified many matrix proteins that indicate matrix remodeling, we measured TGF-β1 in human samples.

[0444] In urine from humans with kidney disease, TGF-β1 showed a concentration deviation greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0445] The carboxy-terminal propeptide (PICP) of type I procollagen refers to a protein fragment. PICP, derived from type I procollagen, has been identified as an indicator of type I collagen synthesis in bone matrix formation and skin regeneration. PICP is a fragment of the collagen I trimer, represented by collagen α1(I) chain (ID F1Q3I5), collagen α1(I) (fragment) (IDE5G723), and collagen α2(I) chain (ID F1PHY1) (listed in Table 1).

[0446] In urine from humans with kidney disease, PICP showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0447] C-reactive protein (CRP) or pentraxin refers to a protein with the protein ID T2KEN6. C-reactive protein (CRP) is a cyclic (ring-shaped) pentraxin present in blood plasma, whose circulating concentration increases in response to inflammation. It is a liver-derived acute phase protein 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 some types of bacteria) to activate the complement system via C1q.

[0448] In urine from humans with kidney disease, CRP showed a concentration deviation greater than or equal to (≥) 2 times the median of the standard concentration range as measured by ELISA.

[0449] Alpha-1-acid glycoprotein (a1AGP) refers to a protein with the protein ID F6Y713. Alpha-1-acid glycoprotein (α1AGp, AGP, or AAG), or orosomucoid mucin (ORM), is an acute phase protein present in plasma. It is an alpha-globulin glycoprotein regulated by two polymorphic genes. It is primarily synthesized in hepatocytes.

[0450] In urine from humans with renal disease, a1AGP showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA. Figure 2 .

[0451] Complement component 1q (C1q), composed of complement C1q A chain, complement C1q B chain, and complement C1q C chain, refers to proteins with protein IDs J9P4B4 (for A chain), J9P1G2 (for B chain), and E2RJC2 (for C chain) (as listed in Table 1). Complement component 1q (C1q) is a protein complex involved in the complement system. C1q, together with C1r and C1s, forms the C1 complex. C1q is composed of 18 polypeptide chains: 6 A chains, 6 B chains, and 6 C chains.

[0452] In urine from humans with kidney disease, C1q showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0453] Complement component 9 (C9) or complement C9 refers to the protein with protein ID J9P8Z6. Complement component 9 (C9) is a MACPF protein involved in the complement system. Once activated, approximately 12-18 C9 molecules polymerize to form pores in the target cell membrane, causing 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.

[0454] In urine from humans with kidney disease, C9 showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0455] Leucine-rich alpha-2-glycoprotein 1 (LRGP1) refers to a protein with the protein ID E2R833. In humans, leucine-rich alpha-2-glycoprotein 1 is encoded by the gene LRG1. The leucine-rich repeat (LRR) family of proteins, including LRG1, has been shown to be involved in protein-protein interactions, signal transduction, cell adhesion, and development. LRG1 is expressed during granulocyte differentiation.

[0456] In urine from humans with kidney disease, LRGP1 showed a concentration deviation greater than or equal to (≥) 2 times the median of the standard concentration range as determined by ELISA.

[0457] The following biomarkers exhibited concentration deviations, at least in dogs with kidney disease, greater than or equal to (≥) 2 times the median of the standard concentration range as determined by mass spectrometry:

[0458] Coagulation factor XIII (CFXIII), which consists of the coagulation factor XIIIA chain and the coagulation factor XIIIB chain, refers to the protein with the protein IDs F1PKX3 (for the A chain) and F1Q041 (for the B chain) (as listed in Table 1). Factor XIII, or fibrin-stabilizing factor, also has extracellular matrix stabilizing activity and is a zymogen present in the blood of humans and some other animals. It is activated by thrombin to factor XIIIa. Factor XIIIa is an enzyme in the coagulation system that cross-links fibrin. Deficiency of XIII impairs clot stability and increases bleeding tendency. Human CFXIII is a heterotetramer. It consists of two enzymatic A peptides / chains and two non-enzymatic B peptides / chains. CFXIIIa is a dimer of activated A peptides / chains.

[0459] At least in dogs with kidney disease, CFXIII exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as measured by mass spectrometry.

[0460] Fibronectin (FN) refers to the protein with protein ID J9P8M2. Fibronectin (FN) is a multifunctional adhesion glycoprotein that plays an important role in tissue repair, regulation of cell adhesion and motility, and embryogenesis.

[0461] At least in dogs with renal disease, FN exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0462] Ficolin 1 (FIC) refers to a protein with the protein ID J9P7F7. Ficolin 1, also commonly known as M-ficolin, is encoded by the FCN1 gene in humans. Ficolin family proteins (ficolins) selectively recognize acetylated compounds. M-ficolin is primarily expressed in peripheral blood leukocytes and is hypothesized to function as a plasma protein with elastin-binding activity.

[0463] At least in dogs with renal disease, FIC showed concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0464] Hemopexin (HPX) refers to a protein with the protein ID F1PZR4. Hemopexin (or hemopexin; Hpx; Hx), also known as beta-1B-glycoprotein, is a glycoprotein encoded by the HPX gene in humans. Hemopexin is a plasma protein with the highest binding affinity for heme and possesses some enzymatic activity.

[0465] At least in dogs with renal disease, HPX exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by mass spectrometry.

[0466] Inter-alpha-trypsin inhibitor heavy chain 4 (IATIH4) refers to a protein with protein ID H9GWY3 (as listed in Table 1). Inter-alpha-trypsin inhibitors (IATI) are four plasma proteins (also listed in Table 1) composed of four different heavy chains selected from the groups IATIH1, IATIH2, IATIH3, and IATIH4 and one light chain selected from the groups AMBP or SPINT2. They act as protease inhibitors. The biomarker is inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4 (IATIH4).

[0467] At least in dogs with renal disease, IATI4 exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as measured by mass spectrometry.

[0468] 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. In humans, RBP4 is encoded by the RBP4 gene. It is primarily synthesized in the liver and circulates in the blood, where it binds to retinol and then forms a complex with transthyretin.

[0469] At least in dogs with kidney disease, RBP4 exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as measured by mass spectrometry.

[0470] Serum amyloid A (SAA) refers to a protein with protein ID J9NVE9. Serum amyloid A (SAA) proteins are a family of apolipoproteins associated with high-density lipoproteins (HDL) in plasma. Different isoforms of SAA are expressed at varying levels constitutively (constitutive SAA) or in response to inflammatory stimuli (acute phase SAA). These proteins are primarily produced by the liver.

[0471] At least in dogs with renal disease, SAA exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as measured by mass spectrometry.

[0472] Talin 1 (Tal) refers to a protein with the protein ID J9P5V6. Talin 1 is a protein encoded by the TLN1 gene in humans. Talin 1 is ubiquitously expressed and localizes to costameres in cardiac and skeletal muscle cells, and to focal adhesions in smooth muscle and non-muscle cells. The function of talin 1 is to mediate cell-cell adhesion via the connection of integrins to the actin cytoskeleton and to participate in the activation of integrins.

[0473] At least in dogs with kidney disease, Tal exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0474] Kallikrein (KAL) or Kallikrein B1 refers to a 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.

[0475] At least in dogs with renal disease, KAL exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as determined by mass spectrometry.

[0476] As listed in Table 1, dynein (DYN) or dynein heavy chain domain 1 refers to the protein with the protein ID F1PZK8. Dynamins are a family of cytoskeletal motor proteins that move along microtubules in cells. They convert chemical energy stored in ATP into mechanical work. Dynamins transport various cellular cargoes and provide the forces and displacements important in mitosis. Dynamins can be divided into two groups: cytoplasmic dyneins and axonemal dyneins. Dynamin heavy chain domain 1 (DNAH1) belongs to the latter group. The biomarker is dynein, preferably dynein heavy chain domain 1.

[0477] At least in dogs with kidney disease, DYN exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0478] Fukutin (FUK) refers to a protein with the protein ID E2R926. Fukutin is a eukaryotic protein essential for maintaining muscle integrity, cortical tissue development, 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, one of the most common autosomal recessive disorders in Japan. In humans, the protein is encoded by the FCMD gene (also known as FKTN).

[0479] At least in dogs with renal disease, FUK exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0480] Myosin IXa (MYIXA) refers to the protein with protein ID J9P 187. Myosin IXa (Myo9a, also previously known as myr7) is an actin-dependent motor protein of the unconventional myosin IX class.

[0481] At least in dogs with renal disease, MYIXA exhibited concentration excursions greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0482] Kininogen 1 refers to a protein with the protein ID E2R886. Kininogen 1 (KNG1), also known as α-2-thiol proteinase inhibitor, Williams-Fitzgerald-Flaujeac factor, or HMWK-kallikrein factor, is a protein encoded by the KNG1 gene in humans. Kininogen 1 is the precursor protein for high molecular weight kininogen (HMWK), low molecular weight kininogen (LMWK), and bradykinin.

[0483] At least in dogs with kidney disease, kininogen 1 exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0484] α2-HS glycoprotein refers to a protein with the protein ID E2QUV3. α-2-HS glycoprotein (AHSG, α-2-Heremans-Schmid 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.

[0485] At least in dogs with kidney disease, α2-HS glycoprotein exhibits concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0486] Complement C6 or complement component 6 refers to the protein with the protein ID E2RGT6. C6 is a member of the complement membrane attack complex (MAC), which also includes complement components C5b, C7, C8, and C9.

[0487] At least in dogs with kidney disease, C6 exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0488] Complement C7 or complement component 7 refers to the protein with protein ID E2RG01.

[0489] At least in dogs with kidney disease, C7 exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0490] Complement C8, or complement component 8, contains proteins with the protein IDs E2R109 (for the α chain), E2R141 (for the β chain), and F6XKC0 (for the γ chain) (as 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; it is composed of three different subunits. These subunits are called the C8 α chain, β chain, and γ chain, and are encoded by the genes C8A, C8B, and C8G, respectively. The biomarker is complement C8, preferably the C8 α chain and / or the C8 β chain.

[0491] At least in dogs with kidney disease, C8 exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0492] β-2-glycoprotein 1 (APOH) refers to a protein with protein ID P33703. β2-glycoprotein 1, also known as β-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 cardiolipin. β2-GP1 is also involved in complex coagulation processes.

[0493] At least in dogs with renal disease, APOH exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0494] 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. Its targets include elastase, plasmin, thrombin, trypsin, chymotrypsin, and plasminogen activator factor. This protein is produced in the liver and bone marrow by lymphocytes and monocytes in lymphoid tissues, as well as by Paneth cells in the intestine.

[0495] At least in dogs with renal disease, serpin family A member 1 exhibits concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0496] Transferrin refers to proteins with protein IDs J9P430 and F6V1W9. Transferrin is a glycoprotein found in vertebrates that contains two Fe 3+ ion binding site, binds and thus mediates the transport of iron (Fe) through the plasma (serum transferrin). In addition, transferrin and its fragments have many immunomodulatory activities. They are mainly produced in the liver, but are also produced in many other organs. Human transferrin is encoded by the TF gene.

[0497] At least in dogs with renal disease, transferrin exhibits concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0498] Alpha-1-B glycoprotein refers to a protein with the protein ID F1PCK2. In humans, alpha-1-B glycoprotein is encoded by the A1BG gene. The protein encoded by this gene is a plasma glycoprotein of unknown function. This protein displays sequence similarity to the variable regions of several members of the immunoglobulin supergene family.

[0499] At least in dogs with kidney disease, α1-B glycoprotein exhibits concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0500] Apo AI, or apolipoprotein AI, refers to a 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 specific role in lipid metabolism.

[0501] At least in dogs with renal disease, Apo AI exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0502] Apo D, or apolipoprotein D, refers to a protein with the protein ID E2RNM1. Apolipoprotein D (ApoD) is a protein encoded by the APOD gene in humans. ApoD is a member of the lipocalin family and is a small, hydrophobic molecule transporter.

[0503] At least in dogs with renal disease, Apo D exhibited concentration deviations greater than or equal to (≥) 2 times the median of the standard concentration range as measured by mass spectrometry.

[0504] Alpha-1 microglobulin / double Kunitz inhibitor precursor refers to a protein with protein ID J9NUI6. The protein AMBP is a protein that binds to IATI and is encoded by the AMBP gene in humans.

[0505] At least in dogs with renal disease, alpha-1 microglobulin / double Kunitz inhibitor precursor exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as determined by mass spectrometry.

[0506] Carboxypeptidase N subunit 2 refers to a protein with protein ID J9PAA4. Carboxypeptidase N subunit 2 is an enzyme that in humans is encoded by the CPN2 gene.

[0507] At least in dogs with kidney disease, carboxypeptidase N subunit 2 exhibited concentration deviations greater than or equal to (≥) 2-fold the median of the standard concentration range as measured by mass spectrometry.

[0508] 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).

[0509] At least in dogs with kidney disease, serine / arginine repeat matrix protein 1 exhibits a concentration deviation greater than or equal to (≥) 2-fold the median of a standard concentration range as measured by mass spectrometry.

[0510] Prior to mass spectrometry, all proteins had been denatured and digested by trypsin (as described in the LC-MS / MS analysis of Section 1.4 of Example 1 below) into characteristic tryptic peptides. Therefore, after analyzing all of these peptides and performing database searches, the candidate biomarkers identified by mass spectrometry were presented as protein chains, multiple variants and / or fragments (see Table 1 and Section 1.6 of Example 1 below). All biomarkers of the present invention derived from these discoveries were designated as proteins that appeared in non-denatured, real clinical samples suitable for diagnostic purposes. As discussed above, as used herein, the term "protein" refers to / includes all known proteins that encapsulate the identified chains of the corresponding protein, modified variants of the corresponding protein, fragments of the corresponding protein, single protein chains, multiple protein chains, and complexes of the corresponding protein (chain) with additional proteins and protein chains, without being limited to their validation using currently available immunoassays (such as ELISA).

[0511] In one embodiment, the at least one additional protein is preferably selected from:

[0512] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0513] Luminophore (LUM),

[0514] FMN,

[0515] vitronectin (VTN),

[0516] Gelsolin (GS),

[0517] Angiotensinogen (AGT),

[0518] Adiponectin (ADP),

[0519] Transforming growth factor β1 (TGF-β1),

[0520] Carboxyl-terminal propeptide of type I procollagen (PICP) and

[0521] C-reactive protein (CRP),

[0522] α-1-acid glycoprotein (a1AGP),

[0523] Coagulation factor XIII (CFXIII),

[0524] Complement component 1q (C1q),

[0525] Complement component 9 (C9),

[0526] Fibronectin (FN),

[0527] Ficolin 1 (FIC),

[0528] Hemopexin (HPX),

[0529] Inter-α-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4,

[0530] Leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0531] Retinol binding protein 4 (RBP4),

[0532] Serum amyloid A (SAA),

[0533] Talin 1 (Tal),

[0534] Kallikrein (KAL),

[0535] Dynein (DYN), preferably dynein heavy chain domain 1,

[0536] Fukutin (FUK) and

[0537] Myosin IXA (MYIXA),

[0538] More preferably, the at least one additional protein is selected from:

[0539] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0540] Luminophore (LUM),

[0541] FMN,

[0542] vitronectin (VTN),

[0543] Gelsolin (GS),

[0544] Angiotensinogen (AGT),

[0545] Adiponectin (ADP),

[0546] Transforming growth factor β1 (TGF-β1),

[0547] Carboxyl-terminal propeptide of type I procollagen (PICP) and

[0548] C-reactive protein (CRP).

[0549] In a preferred embodiment, the

[0550] C4BP and ADP, or

[0551] COLXIII and FMN, or

[0552] HABP2 and TGF-β1, or

[0553] HABP2 and FGG, or

[0554] CFH and GS, or

[0555] CFI and GS, or

[0556] CFI and VTN, or

[0557] CFH and FGG, or

[0558] CFI and FGG.

[0559] Furthermore, the present invention provides at least two proteins selected from the group consisting of the following for use as biomarkers in a method for preclinical and / or early detection and / or diagnosis of kidney disease:

[0560] Type XIII collagen (COLXIII),

[0561] Hyaluronan binding protein 2 (HABP2),

[0562] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0563] Complement factor H (CFH),

[0564] Complement factor I (CFI),

[0565] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0566] Luminophore (LUM),

[0567] FMN,

[0568] vitronectin (VTN),

[0569] Gelsolin (GS),

[0570] Angiotensinogen (AGT),

[0571] Adiponectin (ADP),

[0572] Transforming growth factor β1 (TGF-β1),

[0573] C-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP),

[0574] α-1-acid glycoprotein (a1AGP),

[0575] Coagulation factor XIII (CFXIII),

[0576] Complement component 1q (C1q),

[0577] Complement component 9 (C9),

[0578] Fibronectin (FN),

[0579] Ficolin 1 (FIC),

[0580] Hemopexin (HPX),

[0581] Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4 (IATIH4), leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0582] Retinol binding protein 4 (RBP4),

[0583] Serum amyloid A (SAA),

[0584] Talin 1 (Tal),

[0585] Kallikrein (KAL),

[0586] Dynein (DYN), preferably dynein heavy chain domain 1, Fukutin (FUK),

[0587] Myosin IXA (MYIXA),

[0588] Kininogen 1,

[0589] α2-HS glycoprotein,

[0590] Complement C6,

[0591] Complement C7,

[0592] Complement C8, preferably C8 alpha chain and / or C8 beta chain, APOH, beta-2-glycoprotein 1,

[0593] Serine protease inhibitor family A member 1,

[0594] transferrin,

[0595] α1-B glycoprotein,

[0596] Apo AI,

[0597] Apo D,

[0598] Alpha-1 microglobulin / double Kunitz inhibitor precursor,

[0599] Carboxypeptidase N subunit 2,

[0600] Serine / arginine repeat matrix protein 3,

[0601] Complement C1s,

[0602] Complement C1r,

[0603] Complement C2,

[0604] Complement C3,

[0605] Complement C4,

[0606] Complement C5,

[0607] Complement factor B,

[0608] Haptoglobin, α2-macroglobulin, α-1-antitrypsin, ceruloplasmin, serine protease inhibitor family A member 3, serine protease inhibitor family A member 5, serine protease inhibitor family A member 7, serine protease inhibitor family G member 1, serine protease inhibitor family D member 1, coagulation factor V,

[0609] Prothrombin,

[0610] Plasminogen, protein S, Afamin,

[0611] Transthyretin, hemoglobin subunit α, fetuin B,

[0612] Insulin-like growth factor binding protein, alpha-2-glycoprotein 1,

[0613] Paraoxonase 1,

[0614] 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,

[0615] Myosin XVIIIB, kinesin-like protein,

[0616] CD109 molecule,

[0617] Maltase-glucoamylase,

[0618] Sacsin molecular chaperone,

[0619] Dispatch RND transporter family member 2,

[0620] DNA polymerase,

[0621] Transport protein particle complex subunit 2,

[0622] RB-related KRAB zinc finger,

[0623] TTC17-interacting proteins related to ciliogenesis,

[0624] Proteasome subunit α,

[0625] Regulates synaptic membrane exocytosis protein 2,

[0626] Extracellular matrix protein 1,

[0627] E-cadherin 5,

[0628] Coiled-coil domain-containing protein 178 and

[0629] Attracts protein.

[0630] In other words, the present invention provides the use of at least two proteins selected from the proteins disclosed above as biomarkers for preclinical and / or early detection and / or diagnosis of kidney disease.

[0631] The at least two proteins are preferably selected from the group consisting of

[0632] Type XIII collagen (COLXIII),

[0633] Hyaluronan binding protein 2 (HABP2),

[0634] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0635] Complement factor H (CFH),

[0636] Complement factor I (CFI),

[0637] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0638] Luminophore (LUM),

[0639] FMN,

[0640] vitronectin (VTN),

[0641] Gelsolin (GS),

[0642] Angiotensinogen (AGT),

[0643] Adiponectin (ADP),

[0644] Transforming growth factor β1 (TGF-β1),

[0645] Carboxyl-terminal propeptide of type I procollagen (PICP),

[0646] C-reactive protein (CRP),

[0647] α-1-acid glycoprotein (a1AGP),

[0648] Coagulation factor XIII (CFXIII),

[0649] Complement component 1q (C1q),

[0650] Complement component 9 (C9),

[0651] Fibronectin (FN),

[0652] Ficolin 1 (FIC),

[0653] Hemopexin (HPX),

[0654] Inter-α-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4 (IATIH4),

[0655] Leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0656] Retinol binding protein 4 (RBP4),

[0657] Serum amyloid A (SAA),

[0658] Talin 1 (Tal),

[0659] Kallikrein (KAL),

[0660] Dynein (DYN), preferably dynein heavy chain domain 1,

[0661] Fukutin (FUK) and

[0662] Myosin IXA (MYIXA).

[0663] In a preferred embodiment, the

[0664] ADP and PICP, or

[0665] AGT and GS, or

[0666] AGT and C9, or

[0667] GS and C9, or

[0668] GS and VTN, or

[0669] AGT and VTN, or

[0670] CFI and a1AGP, or

[0671] LRGP1 and C1q, or

[0672] CFH and C9, or

[0673] CRP and C1q.

[0674] Preferably, the concentration of each protein is determined in a sample from a mammal, preferably a human (subject).

[0675] The mammal is preferably

[0676] people,

[0677] Animals such as dogs, cats, rabbits, guinea pigs, hamsters, cows, pigs, horses, sheep, donkeys, goats, red deer or camels,

[0678] More preferably, the mammal is a human.

[0679] The sample is preferably a body fluid such as urine or blood, eg serum, plasma or whole blood.

[0680] More preferably, the body fluid is urine.

[0681] In one embodiment, when 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.

[0682] Preferably, the normalized concentration of each protein is compared to a control value.

[0683] Preferably, a control value for each protein is determined from healthy subjects, preferably a panel of healthy subjects, whose age and sex are matched to the subject at diagnosis. A "subject," or preferably a "human subject," is considered "healthy" if he or she is "renally healthy" at the time of sampling and remains "renally healthy" for a period of time corresponding to the conventional time during which kidney disease can be diagnosed by conventional means. For example, biobanks are suitable for obtaining samples from healthy subjects because these subjects are followed up after sampling. Preferably, the control value is the median of the standard concentration range.

[0684] In a preferred embodiment, a mathematical correlation of the concentrations of the biomarkers is used, such as the sum or preferably the product of the concentrations. 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.

[0685] Detection method

[0686] As discussed above, the present invention provides a method for preclinical and / or early detection and / or diagnosis of kidney disease, the method comprising:

[0687] (a) determining the concentration of at least two proteins in a sample from a mammal,

[0688] (b) comparing each concentration determined in step (a) with a control value,

[0689] wherein a deviation of each concentration determined in step (a) from said control value is indicative of preclinical and / or early stage renal disease.

[0690] Preferably, the kidney disease is selected from the following chronic progressive kidney diseases:

[0691] Begins with glomerular damage (glomerulopathy),

[0692] Postinfectious glomerulopathy, IgA nephropathy, Henoch-Schönlein purpura, focal segmental glomerulosclerosis, Alport syndrome, thin basement membrane nephropathy, and benign familial hematuria are preferred;

[0693] or

[0694] Begins elsewhere in the body with other dysregulations, and / or begins elsewhere in the kidneys with effects on the glomeruli (nephropathy),

[0695] Preferred are metabolic syndrome, hypertension, heart failure, drug nephrotoxicity, obesity, diabetes (diabetic nephropathy), nephrogenesis, renal dysplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract anomalies, renal duplication, and nephronophthisis.

[0696] —Step (a)

[0697] In step (a), the concentrations of at least two proteins in a mammalian sample are determined.

[0698] The mammal is preferably

[0699] people,

[0700] Animals such as dogs, cats, rabbits, guinea pigs, hamsters, cows, pigs, horses, sheep, donkeys, goats, red deer or camels,

[0701] More preferably, the mammal is a human.

[0702] The sample is preferably a body fluid such as urine or blood, eg serum, plasma or whole blood.

[0703] More preferably, the body fluid is urine.

[0704] In one embodiment, when 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.

[0705] The at least two proteins are selected from the biomarkers disclosed herein.

[0706] In a preferred embodiment, the at least two proteins are selected from

[0707] Type XIII collagen (COLXIII),

[0708] Hyaluronan binding protein 2 (HABP2),

[0709] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA),

[0710] Complement factor H (CFH) and

[0711] Complement factor I (CFI).

[0712] In a preferred embodiment, the at least two proteins are selected from the group consisting of:

[0713] - a protein selected from the group consisting of:

[0714] Type XIII collagen (COLXIII),

[0715] Hyaluronan binding protein 2 (HABP2),

[0716] C4 binding protein alpha (C4BP), preferably C4BP alpha chain (C4BPA), complement factor H (CFH) and

[0717] Complement factor I (CFI)

[0718] and at least one additional protein selected from the group consisting of:

[0719] -fibrinogen, preferably fibrinogen gamma chain (FGG),

[0720] - Luminophore (LUM),

[0721] - morphin 1 (FMN),

[0722] - vitronectin (VTN),

[0723] -Gelsolin (GS),

[0724] -Angiotensinogen (AGT),

[0725] -Adiponectin (ADP),

[0726] - Transforming growth factor β1 (TGF-β1),

[0727] - carboxyl-terminal propeptide of type I procollagen (PICP),

[0728] -C-reactive protein (CRP),

[0729] -α-1-acid glycoprotein (a1AGP),

[0730] - Coagulation factor XIII (CFXIII),

[0731] -Complement component 1q (C1q),

[0732] -Complement component 9 (C9),

[0733] - Fibronectin (FN),

[0734] -Ficolin 1 (FIC),

[0735] -Hemopexin (HPX),

[0736] - Inter-α-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4,

[0737] -Leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0738] -Retinol binding protein 4 (RBP4),

[0739] - Serum amyloid A (SAA),

[0740] -Talin 1 (Tal),

[0741] -Kallikrein (KAL),

[0742] - dynein (DYN), preferably dynein heavy chain domain 1,

[0743] -Fukutin(FUK),

[0744] -Myosin IXA (MYIXA),

[0745] -Kinogen 1,

[0746] -α2-HS glycoprotein,

[0747] -Complement C6,

[0748] -Complement C7,

[0749] - Complement C8, preferably C8 alpha chain and / or C8 beta chain,

[0750] -APOH, β-2-glycoprotein 1,

[0751] -Serine protease inhibitor family A member 1,

[0752] - transferrin,

[0753] -α1-B glycoprotein,

[0754] -Apo AI,

[0755] -Apo D,

[0756] -α-1 microglobulin / double Kunitz inhibitor precursor,

[0757] -Carboxypeptidase N subunit 2 and

[0758] -Serine / arginine repeat matrix protein 3.

[0759] In one embodiment, the at least one additional protein is preferably selected from:

[0760] -fibrinogen, preferably fibrinogen gamma chain (FGG),

[0761] - Luminophore (LUM),

[0762] - morphin 1 (FMN),

[0763] - vitronectin (VTN),

[0764] -Gelsolin (GS),

[0765] -Angiotensinogen (AGT),

[0766] -Adiponectin (ADP),

[0767] - Transforming growth factor β1 (TGF-β1),

[0768] - carboxyl-terminal propeptide of type I procollagen (PICP),

[0769] -C-reactive protein (CRP),

[0770] -α-1-acid glycoprotein (a1AGP),

[0771] - Coagulation factor XIII (CFXIII),

[0772] -Complement component 1q (C1q),

[0773] -Complement component 9 (C9),

[0774] - Fibronectin (FN),

[0775] -Ficolin 1 (FIC),

[0776] -Hemopexin (HPX),

[0777] - Inter-α-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4,

[0778] -Leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0779] -Retinol binding protein 4 (RBP4),

[0780] - Serum amyloid A (SAA),

[0781] -Talin 1 (Tal),

[0782] -Kallikrein (KAL),

[0783] - dynein (DYN), preferably dynein heavy chain domain 1,

[0784] -Fukutin (FUK) and

[0785] -Myosin IXA (MYIXA).

[0786] In a preferred embodiment, the at least one additional protein is selected from the group consisting of:

[0787] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0788] Luminophore (LUM),

[0789] FMN,

[0790] vitronectin (VTN),

[0791] Gelsolin (GS),

[0792] Angiotensinogen (AGT),

[0793] Adiponectin (ADP),

[0794] Transforming growth factor β1 (TGF-β1),

[0795] Carboxyl-terminal propeptide of type I procollagen (PICP) and

[0796] C-reactive protein (CRP).

[0797] In a preferred embodiment, the at least two proteins are selected from

[0798] Type XIII collagen (COLXIII),

[0799] Hyaluronan binding protein 2 (HABP2),

[0800] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA), complement factor H (CFH),

[0801] Complement factor I (CFI),

[0802] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0803] Luminophore (LUM),

[0804] FMN,

[0805] vitronectin (VTN),

[0806] Gelsolin (GS),

[0807] Angiotensinogen (AGT),

[0808] Adiponectin (ADP),

[0809] Transforming growth factor β1 (TGF-β1),

[0810] Carboxyl-terminal propeptide of type I procollagen (PICP),

[0811] C-reactive protein (CRP),

[0812] α-1-acid glycoprotein (a1AGP),

[0813] Coagulation factor XIII (CFXIII),

[0814] Complement component 1q (C1q),

[0815] Complement component 9 (C9),

[0816] Fibronectin (FN),

[0817] Ficolin 1 (FIC),

[0818] Hemopexin (HPX),

[0819] Inter-alpha-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4, leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0820] Retinol binding protein 4 (RBP4),

[0821] Serum amyloid A (SAA),

[0822] Talin 1 (Tal),

[0823] Kallikrein (KAL),

[0824] Dynein (DYN), preferably dynein heavy chain domain 1, Fukutin (FUK),

[0825] Myosin IXA (MYIXA),

[0826] Kininogen 1, α2-HS glycoprotein,

[0827] Complement C6,

[0828] Complement C7,

[0829] Complement C8, preferably C8 alpha chain and / or C8 beta chain, APOH, beta-2-glycoprotein 1, serine protease inhibitor family A member 1, transferrin, alpha 1-B glycoprotein, Apo AI, Apo D, alpha-1 microglobulin / double Kunitz inhibitor precursor, carboxypeptidase N subunit 2, serine / arginine repeat matrix protein 3, complement C1s,

[0830] Complement C1r,

[0831] Complement C2,

[0832] Complement C3,

[0833] Complement C4,

[0834] Complement C5,

[0835] Complement factor B,

[0836] Haptoglobin, α2-macroglobulin, α-1-antitrypsin, ceruloplasmin,

[0837] Serine protease inhibitor family A member 3, Serine protease inhibitor family A member 5, Serine protease inhibitor family A member 7, Serine protease inhibitor family G member 1, Serine protease inhibitor family D member 1, Coagulation factor V,

[0838] Prothrombin,

[0839] Plasminogen, protein S,

[0840] Afamin,

[0841] Transthyretin, hemoglobin subunit α, fetuin B,

[0842] Insulin-like growth factor binding protein,

[0843] α-2-glycoprotein 1,

[0844] Paraoxonase 1,

[0845] Dipeptidyl peptidase 4,

[0846] C-type lectin domain family 3 member B,

[0847] Galectin 3 binding protein,

[0848] Apolipoprotein AIV (Apo AIV),

[0849] Apolipoprotein CI (Apo CI),

[0850] Apolipoprotein CII (Apo CII),

[0851] Apolipoprotein E (Apo E4),

[0852] Apolipoprotein M (Apo M),

[0853] Apolipoprotein B (Apo B),

[0854] Myosin IB,

[0855] Myosin XVIIIB,

[0856] Kinesin-like proteins,

[0857] CD109 molecule,

[0858] Maltase-glucoamylase,

[0859] Sacsin molecular chaperone,

[0860] Dispatch RND transporter family member 2,

[0861] DNA polymerase,

[0862] Transport protein particle complex subunit 2,

[0863] RB-related KRAB zinc finger,

[0864] TTC17-interacting proteins related to ciliogenesis,

[0865] Proteasome subunit α,

[0866] Regulates synaptic membrane exocytosis protein 2,

[0867] Extracellular matrix protein 1,

[0868] E-cadherin 5,

[0869] Coiled-coil domain-containing protein 178 and

[0870] Attracts protein.

[0871] In one embodiment, the at least two proteins are preferably selected from:

[0872] Type XIII collagen (COLXIII),

[0873] Hyaluronan binding protein 2 (HABP2),

[0874] C4 binding protein (C4BP), preferably C4BPα chain (C4BPA), complement factor H (CFH),

[0875] Complement factor I (CFI),

[0876] Fibrinogen, preferably fibrinogen gamma chain (FGG),

[0877] Luminophore (LUM),

[0878] FMN,

[0879] vitronectin (VTN),

[0880] Gelsolin (GS),

[0881] Angiotensinogen (AGT),

[0882] Adiponectin (ADP),

[0883] Transforming growth factor β1 (TGF-β1),

[0884] Carboxyl-terminal propeptide of type I procollagen (PICP),

[0885] C-reactive protein (CRP),

[0886] α-1-acid glycoprotein (a1AGP),

[0887] Coagulation factor XIII (CFXIII),

[0888] Complement component 1q (C1q),

[0889] Complement component 9 (C9),

[0890] Fibronectin (FN),

[0891] Ficolin 1 (FIC),

[0892] Hemopexin (HPX),

[0893] Inter-α-trypsin inhibitor 4 (IATI4), preferably IATI heavy chain 4,

[0894] Leucine-rich alpha-2-glycoprotein 1 (LRGP1),

[0895] Retinol binding protein 4 (RBP4),

[0896] Serum amyloid A (SAA),

[0897] Talin 1 (Tal),

[0898] Kallikrein (KAL),

[0899] Dynein (DYN), preferably dynein heavy chain domain 1,

[0900] Fukutin (FUK) and

[0901] Myosin IXA (MYIXA).

[0902] In one embodiment, the

[0903] COLXIII and C4BP, or

[0904] COLXIII and C4BP and HABP2, or

[0905] Concentrations of CFH and CFI.

[0906] In one embodiment, the concentrations of COLXIII, HABP2, C4BP, CFH, and CFI are determined.

[0907] In one embodiment, the

[0908] C4BP and ADP, or

[0909] COLXIII and FMN, or

[0910] HABP2 and TGF-β1, or

[0911] HABP2 and FGG, or

[0912] CFH and GS, or

[0913] CFI and GS, or

[0914] CFI and VTN, or

[0915] CFH and FGG, or

[0916] Concentrations of CFI and FGG.

[0917] In one embodiment, the

[0918] ADP and PICP, or

[0919] AGT and GS, or

[0920] AGT and C9, or

[0921] GS and C9, or

[0922] GS and VTN, or

[0923] AGT and VTN, or

[0924] CFI and a1AGP, or

[0925] LRGP1 and C1q, or

[0926] CFH and C9, or

[0927] CRP and C1q concentrations.

[0928] In one embodiment, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more biomarkers are determined.

[0929] -Step (b)

[0930] In step (b), each concentration determined in step (a) is compared to a control value.

[0931] In one embodiment, when 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.

[0932] Preferably, the normalized concentration of each protein is compared to a control value.

[0933] Preferably, a control value for each protein is determined from healthy subjects, preferably a panel of healthy subjects, whose age and sex are matched to the subject at diagnosis. As discussed above, a subject is considered "healthy" if he or she is "renally healthy" at the time of sampling and remains "renally healthy" for a period of time corresponding to the conventional time period during which kidney disease can be diagnosed by conventional means. For example, biobanks are suitable for obtaining samples from healthy subjects because these subjects are followed up after sampling. Preferably, the control value is the median of the standard concentration range.

[0934] According to the present invention, a deviation of each concentration determined in step (a) from said control value is indicative of a preclinical and / or early stage kidney disease.

[0935] Preferably,

[0936] Deviations greater than or equal to (≥) 2 times the median of the standard concentration range are indicative of preclinical and / or early-stage renal disease; or

[0937] Deviations less than or equal to (≤) 0.5 times the median of the standard concentration range are indicative of preclinical and / or early-stage renal disease.

[0938] More preferably,

[0939] Deviations greater than or equal to (≥) 2 times the median of the standard concentration range are indicative of preclinical and / or early-stage renal disease.

[0940] Deviations greater than or equal to (≥) 2 times the median of the standard concentration range may range from 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.

[0941] In a preferred embodiment, a mathematical correlation of the concentrations of the biomarkers is used, such as the sum or preferably the product of the concentrations.

[0942] For example, the following products were constructed: ADP x C4BP, ColXIII x C4BP, ColXIII x FMN, HABP2 x TGF-β1, HABP2 x FGG, ColXIII x C4BP x HABP2.

[0943] Further Description of the Preferred Embodiments

[0944] Although several early cellular alterations have recently been discovered (see, e.g., Guo et al., 2019), to date no viable biomarkers exist for preclinical testing of Alport syndrome (AS).

[0945] For manifest AS, we have identified an expanded set of candidate biomarkers (BM) using our proteomic workflow and ELISA (Baum et al., 2008; Pohl et al., 2013). Unfortunately, similar studies of BM in the preclinical state (i.e., in toddlers) are not possible for ethical and practical reasons.

[0946] However, type IV collagen is highly conserved. Therefore, any mammal can develop AS, making animal models of AS ideal tools for studying pathogenesis and therapy (Kashtan et al., 2002). Mouse and canine models show a similar clinical course compared to humans. In addition, ACE inhibitors have been shown to be effective in COL4A5 before extensive evaluation in humans. - Dog (Grodecki et al., 1997) (AS dog) and COL4A3 - / -Disease progression has been delayed in both juvenile AS mice (Gross et al., 2003). These models allow for definitive diagnosis at preclinical stages of the disease through genetic means. Biomarkers indicative of disease progression can also be observed in these models. Therefore, our modified method (Wendler et al., 2013) has been applied to the serum of adolescent AS mice to generate several candidate BMs (Muckova et al., 2015).

[0947] To date, most of the candidate biomarkers described have been generated by hypothesis-driven studies. For preclinical processes, it is almost impossible to give a hypothesis. Therefore, the hypothesis-free search for reliable biomarkers is an opportunity to identify urgently needed novel biomarkers for diagnosis, prognosis and treatment monitoring of nephropathy. The search for preclinical biomarkers is highly challenging. These biomarkers can only be searched and evaluated in seemingly healthy but sick preclinical individuals who show predictable progression to the disease characteristics of nephropathy (see Figure 1 ).

[0948] A proteomic search for this early biomarker was performed for AS as a model genetic disease, see below. This search was performed in adolescent AS mice and AS dogs. Most of the candidate biomarkers identified can be considered "validated" because they overlap in two different species (see Table 1). In addition, some biomarkers show reversibility under treatment with ACE inhibitors (ramipril), and some of them have been evaluated by immunoassays in mice and dogs. We further validated a group of these candidate biomarkers in DM1 with expected preclinical DN and children with obesity, hypertension and metabolic syndrome (potential preclinical DM), as well as other kidney diseases in the early stages.

[0949] This specification discloses a methodologically complementary proteomic study that uses readily available serum to conduct a biomarker search in a well-defined preclinical stage of the model disease Alport syndrome (AS) and validation in blood and urine samples from pediatric patients who are at high risk of developing preclinical conditions of DN and in very early clinical stages of various kidney diseases.

[0950] To this end, an optimization and unbiased strategy is applied, which combines the following steps:

[0951] i. complex samples were collected from an animal model (dog) of the inherited glomerulopathy Alport syndrome (AS) for preclinical proteomic searching, and from children without and with diagnosed early-stage kidney disease for validation, ii. a comprehensive and precise workflow was applied to proteomic biomarker searching using dog samples from 7-week-old affected male and female dogs and their unaffected siblings, and iii. subsequent evaluation of biomarkers was performed using a human biobank of samples from 305 characterized children, including healthy controls and children with metabolic syndrome, hypertension, obesity, T2DM, T1DM, nephrogenesis, dysplasia, polycystic kidney dysplasia, urinary tract anomalies, renal duplications, ADPKD, ARPKD, nephronophthisis, post-infectious GN, vasculitis (IgAN, HSP), thin basement membrane nephropathy and benign familial hematuria, as well as AS.

[0952]

[0953]

[0954]

[0955]

[0956]

[0957] Legend for Table 1:

[0958] *Protein IDs refer to the UniProt database of approximately 47,800 canine entries (date: October 26, 2017).

[0959] Criteria for defining biomarkers:

[0960] For biomarker selection in mice, see Muckova et al., 2015.

[0961] After comparing data from diseased and non-diseased dogs, candidate BMs in dogs were selected from all identified protein chains in all analyzed 2D subfractions. Peptides supporting proteins identified in each 2D subfraction from AM were compared with peptides from the homologous 2D subfraction from NM by Compare, and the peptide from CF is compared with the peptide from NF.Quantity word " ratio " is relevant to concentration quotient (AM / NM, CF / NF), and " hit " is relevant to the amount of corresponding protein in the subfraction compared.The screening data of the protein about each identification are sorted into our separation matrix by internal macro, so that the chromatographic distribution of the protein variant of corresponding change is visualized.According to actual chromatographic distribution, when observing exclusive synergistic change, BM is limited to by " overall trend " (formula 1; Table 1, the 4th column and the 6th column) or by the mean value of all ratios> 2 times high concentration.Alternatively, using> 5 times high ratio mean values in different clusters of subfraction, i.e. different significantly elevated variants (Table 1, the 3rd column and the 5th column), BM is limited to a kind of fraction in whole protein family.

[0962] Overall trend = (M i *H i ) / (H d / M d )Formula 1

[0963] M i : The average of all increasing ratios

[0964] H i : count of all hits added

[0965] M d : The average of all reduced ratios

[0966] H d : Count of all hits that were reduced

[0967] In total, 118 protein chains indicated that 103 proteins were more abundant in affected dogs than in unaffected dogs (male and female), and 79 proteins met the criteria for candidate BM defined above.

[0968] na: not changed; nf: not found; 0: no cluster in that direction; x: general trend higher in AM and / or CF; o: increased fraction / protein variant higher in AM and / or CF. The term "DIV / 0" (divided by zero, i.e., no peptide or hit found in the control sample) indicates a value that is abnormally high in diseased animals. The average ratio of all ratios for all subfractions with coordinated protein changes (AM / NM or CF / NF) is shown.

[0969] The raw spectra were analyzed using Proteome Discoverer 1.3 and the Sequest database search algorithm (applying a false discovery rate of <0.5%) and the UniProt database of approximately 47,800 canine entries (date: 10 / 26 / 2017).

[0970] Below, we demonstrate, following receiver operating characteristic (ROC) analysis, that for ColXIII, HaBP2, and C4BPA, as well as their combination, the area under the ROC curve (AUC) is well above 0.700. They also exhibit reliable quality characteristics (high sensitivity, low false-positive rate) individually and in combination (Tables 2 to 9). This applies to the entire study group as well as to the subgroups of patients analyzed individually with higher values. In inflammation (post-infectious glomerulopathy and vasculitis), acute phase proteins (CRP, AGT, FGG) and complement components CFI and CFH additionally and as expected yield high AUC, sensitivity, and the lowest false-positive rate.

[0971] Table 2 ROC analysis, comparison of values from all patients and controls

[0972]

[0973]

[0974] Table 3 ROC analysis, comparison of values from patients with obesity, hypertension, metabolic syndrome and T2DM with controls

[0975]

[0976] Table 4 ROC analysis, comparison of values from patients with T1DM and controls

[0977]

[0978] Table 5: ROC analysis of subgroups of patients with nephrogenesis, dysplasia, and polycystic kidney dysplasia compared with all controls

[0979]

[0980] Table 6 ROC analysis, comparison of the subgroup of patients with urinary tract anomalies and renal duplications with all controls

[0981]

[0982] Table 7 ROC analysis, comparison of patient subgroups with ADPKD and ARDKD with all controls

[0983]

[0984] Table 8 ROC analysis, comparison of subgroups of patients with post-infectious glomerulopathy and vasculitis (IgAN, HSP) with all controls

[0985]

[0986]

[0987] Table 9 ROC analysis, comparison of subgroups of patients with AS, thin basement membrane nephropathy, and familial hematuria with all controls

[0988]

[0989]

[0990]

[0991]

[0992] The following examples and figures illustrate the invention without, however, limiting it. BRIEF DESCRIPTION OF THE DRAWINGS

[0993] Figure 1 .Time course of disease evolution and biomarker concentrations in renal disease.

[0994] There are several putative biomarker (BM) types that are differently targeted for detection by our method.

[0995] A: Preclinically applicable BM; its increase indicates changes that started in the preclinical stage and continued during progression.

[0996] B: True preclinical BM; its increase only indicates preclinical changes.

[0997] C: Early BM; its increase indicates changes that coincide with the development of early clinical signs and are often overlooked or under-monitored (eg, in AS microhematuria).

[0998] D: Advanced BM; its increase indicates subsequent changes in the disease that are manifest. This type (e.g., in AS (micro)albuminuria, decreased eGFR) has usually been used. This type is not the subject of the present invention.

[0999] A search was conducted in adolescent AS mice (4 weeks, see Muckova et al., 2015) and AS dogs (7 weeks) for types A and B. This search also detected type C.

[1000] Evaluation using human samples encompassing individual points in disease progression may miss type B. However, for all BMs tested and validated, types A, B and / or C should be suitable for very early diagnosis of kidney damage.

[1001] The same is true for an inverse concentration process, where the concentration decreases during the change.

[1002] Figure 2Box plots of single concentrations of preferred candidate biomarkers measured by ELISA are shown. Left panel: samples from patients with X-linked Alport syndrome of varying severity; right panel: other renal diseases. The order of the groups is the same in all three graphs of each figure and is shown at the bottom. The complete results (p values) are given in Table 10; all other cases were non-significant.

[1003] Figure 3 Box plots of the preferred combination concentrations of candidate biomarkers obtained from ELISA of patient and control cohorts are shown. The complete results (p values) are given in Table 11. The order of the groups is the same in all graphs and is shown at the bottom. Groups that were significantly different (p values less than 0.05 in post hoc analysis) as determined by group-by-group comparison are indicated by gray fill in Tables 10 and 11, and all other cases are not significant. DETAILED DESCRIPTION

[1004] Example

[1005] Example 1 Materials and Methods

[1006] 1.1 Patients and samples

[1007] Human samples:

[1008] Two groups of patient samples have been applied: 1) actual clinical samples derived from Alport patients (Table 12), prospective samples from pediatric patients with AS, type 1 diabetes (DM1) and various other kidney diseases, and from individuals without known kidney disease as a control group (Table 13). The first group included longitudinal serum and urine samples during the early stages of AS progression. The second group was generated using standard operating procedures to provide samples from healthy controls and patients with early-stage kidney disease to evaluate the sensitivity and specificity of our candidate biomarkers for target screening (ethical vote UHJ: 2020-1800). Informed consent was obtained from all participating subjects. All samples were stored at -80°C until use.

[1009] Table 12: Data on samples from 35 male X-linked Alport patients

[1010]

[1011]

[1012] Informed consent was obtained from all participating subjects. Inclusion criteria were age > 3 years and clinical diagnosis of AS according to international criteria.

[1013]

[1014]

[1015]

[1016] A second group of participants was recruited and examined between September 2020 and November 2021, see Table 14.

[1017] The following standard operating procedures were applied: blood samples were obtained from individuals in the morning. Blood was drawn into a 9 mL monovette (Salstätt, Ninbrecht, Germany) by venipuncture. To obtain serum, blood was collected into a serum monovette (02.1063.001). If not otherwise stated, the clotting time was 90 minutes at room temperature. All samples were centrifuged at 1500 x g for 10 minutes at 20°C, immediately aliquoted and frozen at -20°C for no more than 3 days, and then stored at -82°C until use. Plasma was obtained by drawing blood into a container (02.1066.001) containing EDTA. If not otherwise stated, the sample was immediately centrifuged, aliquoted and stored as described above. Urine samples were obtained by spontaneous urination before venipuncture, aliquoted and frozen at -20°C for no more than 3 days, and then stored at -82°C until use.

[1018] Thirty-two healthy controls were recruited from subjects without any signs or history of known renal disease or inflammatory signs. Samples from these control individuals were obtained along with the second cohort. In addition, 72 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 Children) obtained in 2014.

[1019] 1.2. Samples used for proteome search

[1020] animal

[1021] Four litters have been generated from a mixed breed dog population of dogs with X-linked AS (COL4A5-) (Lees, 2013).

[1022] Genotyping was performed using PCR on DNA isolated from blood. All animals were weaned at 6 weeks of age and maintained on an established standard diet, husbandry, and socialization regimen.

[1023] Sampling plan

[1024] A minimum of 4 mL of whole blood and 3 mL of urine (excreted) were collected, with whole blood used to collect serum and EDTA plasma. Unaffected dogs were randomly paired with their infected littermates for the sampling plan. Complete standard urinalysis, UPC, and urine albumin concentrations were determined. All samples were collected and processed by standard methods to minimize pre-analysis variations. A minimum of two 0.5 mL aliquots of each sample type were taken, stored at -80°C for future analysis, and transported on dry ice.

[1025] Samples for proteome searching

[1026] Equal volumes of three plasma samples from each group of animals (affected males (AM), female carriers (CF), and nonaffected normal offspring (NM, NF, week 7) in the preclinical phase (week 7)) were combined. Thus, equal volumes and nearly equal amounts of protein from each mixture were pre-fractionated and subsequently analyzed (Table 14).

[1027] Table 14 Data for mixed dog samples used for proteome search

[1028]

[1029] Samples were obtained at 7 weeks of age; Tr: trace, N: negative.

[1030] 1.3. Proteome Search

[1031] Throughout the process, EDTA-plasma pools from all four groups were separated, prepared, and analyzed by mass spectrometry in parallel.

[1032] Sample fractionation: Our method combines native size exclusion (SEC, first dimension, 1D) and subsequent anion exchange (AEC, 2D) chromatography as described by Rhode et al. (2019) and Wendler et al. (2013). After continuous 1D fractionation, all further procedures were performed in microplate format. Thus, starting from 2D fractionation, parallelization and automation were achieved for separation, spectrophotometric readout, temporary storage, hit picking, medium exchange, digestion, desalting, and finally application to the autosampler followed by LC-MS. 96 1D fractions were generated from each sample. Each 1D fraction was further separated into 43 2D fractions. Thus, at least 4128 2D subfractions were generated, containing 20-40 different proteins and with a total protein concentration ranging from zero to 4.3 mg / mL. Fractions showing the same position (1D_2D) in all samples were called homologous fractions. Because the separation of the inventors is highly reproducible, it is expected that every sample component is assigned to one group of identical homologous fractions in all samples. Disease can change the concentration and / or post-translational modification of sample components. Both of these changes can be detected by changing the concentration ratio of the homologous fractions. In addition, due to their chromatographic distribution, it is expected that every single protein to be recovered is in the group of at least three continuous fractions. Since mass spectrometry analysis requires a lot of time (vide infra), it is not necessary to select all fractions but to select the representative part of the fraction, i.e., every two fractions (>0.03mg / mL) that show enough protein content in all chromatographic spots to analyze.

[1033] LC-MS / MS analysis

[1034] Separation of tryptic peptides before mass spectrometry was performed on an Accela 1250 UHPLC system on a Hypersil Gold UHPLC column (1.9 μm, 50 mm × 1.0 mm) (both from Thermo Fisher Scientific, USA). A binary gradient elution using a mobile phase of 0.1% formic acid in (A) water and (B) acetonitrile was used at a flow rate of 150 μL / min (0 min-1 min 5% B, 21 min 30% B, 24 min 40% B, 25 min 90% B, 25.1 min-26 min 90% B, 26.1 min-30 min 5% B) was used. Tandem mass spectrometry (MS / MS) measurements were performed on an LTQ Orbitrap Discovery (Thermo Fisher Scientific, USA) by positive ion heated electrospray ionization (H-ESI) at an evaporator temperature of 200°C. The ion spray was dried using a sheath gas flow rate (30.0) and an auxiliary gas flow rate (10.0) (both nitrogen flow rates are arbitrary units). The ionization voltage and temperature of the ion transfer tube were set to 4.5 kV and 275 ° C, respectively. The MS / MS system was operated in data-dependent TOP10 mode using 1 microscan. To this end, ions were monitored at m / z 350-1700 in a full scan centroid mode in the LTQ ion trap. The ten most dense ions were run by collision-induced dissociation (CID) for further orbital trap high resolution (30,000) analysis (profile data type). Broadband activation was used. The automatic gain control (AGC) target value of the Orbitrap mass analyzer in full scan mode was 1.0×106. LC-MS / MS was operated via the graphical interface of Xcalibur software 2.1. Fractions were run in duplicate. After each repetition, a blank (i.e., a water sample) was applied to avoid carryover. For quality control and inspection, a digest of 50 μg / mL transferrin (human (Holo), SERVA Electrophoresis GmbH, 36756) was analyzed in quadruplicate per microplate.

[1035] Protein identification and data analysis

[1036] Protein identification: MS / MS spectra were analyzed using Proteome Discoverer 1.3 and Sequest database searches (an iteratively updated human Fasta database with a false discovery rate of 0.01) downloaded from www.uniprot.org (10 / 26 / 2017).

[1037] Paired analysis: To detect differences between two samples (diseased group vs control, technical replicates), analysis was performed using SIEVE2.0 (software for label-free differential analysis, Thermo Fisher Scientific, USA). MS data (raw files generated by XCALIBUR) were imported and analyzed as "two-sample differential analysis" using corresponding fraction pairs. The results were filtered by charge dependency Xcorr (Xcorr for charges 1, 2, 3, and >3 were 1.5, 2.0, 2.25, and 2.5, respectively) and a p-value of 0.05. The results are expressed as the fold change between the diseased group and the control. The subsequent protein list was exported to Excel and labeled with fraction position (1D_2D). Thereafter, all proteins from all fractions were merged and sorted according to their protein IDs, followed by 1D and 2D localization using macros. Thus, we were able to identify peptide clusters associated with different proteins with similar ratios and orientations. The clustering process was dynamically adjusted based on the distribution of peptide counts: two to six consecutive 1D and / or 2D fractions were grouped into clusters. The protein of the present invention is to select the protein of the present invention according to the present invention.However, related fractions with opposite ratios are not considered to be clusters. Proteins with ratios>4 and<0.25 are considered to be strictly up-regulated and down-regulated respectively when they appear in at least two positions, regardless of the lower (cooperative) ratio in the adjacent chromatographic fractions. Protein clusters are divided into three categories: proteins with only positive or negative ratios reflecting the changes in the total concentration of the corresponding protein in the sample, and proteins with up-regulated and down-regulated clusters. For the latter protein, changes in post-translational modifications can promote changes in chromatographic distribution. All categories are considered to be indicative changes. Finally, proteins are selected in a single result list (see Table 1) based on ratios and hit numbers.

[1038] Validation of candidate biomarkers

[1039] The protein clusters mentioned in Section 1.5. represent different variants (proteoforms) of the corresponding protein families that are present at varying concentrations in the blood of diseased preclinical animals compared to corresponding controls. The specific characteristics of these variants (i.e., PTMs, complexes, and fragments), as well as their tissue origin and the extent of their occurrence in urine, are currently unknown for (pediatric) screening purposes.

[1040] Therefore, for preliminary evaluation, 27 candidate biomarkers and their variants were immunoquantified in serum, EDTA plasma and urine of a small number of individuals from all groups of dogs (NM, AM, NF, CF) using another sample set different from that selected for the proteome search and commercially available ELISA kits (see Table 15A).

[1041] Thereafter, a step-by-step procedure was performed in humans. First, 28 candidate biomarkers were evaluated in 6 to 8 sets of characteristic blood and urine samples from individuals in the patient and control groups. All assays were performed in duplicate according to the manufacturer's manual. Second, potential candidate biomarkers that showed different mean values between patients and controls were quantified using the maximum sample size of our biobank. The commercially available ELISA kits used are summarized in Table 15B, with most kits using extended incubation times to achieve the required high sensitivity.

[1042] ELISA result analysis: Data were analyzed by Sigma Plot software version 14.5. First, the normal distribution of the data set was analyzed by the Shapiro-Wilk test. For data that did not conform to the normal distribution, the Kruskal Wallis single-factor ANOVA rank sum test was used. Post hoc analysis was performed by paired (U test). Statistical significance was set to p < 0.05. Secondly, the immunoreactivity of the biomarker proteins to each other was analyzed according to Pearson and Spearman using the SPSS program (version 27). Therefore, positively and negatively correlated proteins can be identified and combined by amplification.

[1043] Table 15 Specifications of ELISA test kits for dog and human samples

[1044] Table 15A Measurements in Dog Samples

[1045]

[1046]

[1047] Table 15B Measurements in Human Samples

[1048]

[1049]

[1050] A. Measurements in Dog Samples: When no dog-specific or reliable kits were available, we used other species-specific assays due to high sequence similarity. Of the kits examined, those marked with yes (y) produced reliable antigen concentrations. Other kits either did not detect the protein form naturally present in our samples or were not sensitive enough.

[1051] B. Measurements in human samples: The kits were performed according to the supplier's instructions with the following modifications: All assays from Cloud-Clone and CUSBIO were performed with twice the incubation time. Samples were pre-incubated with 0.5% Tween 20 before measuring gelsolin. For quantification of PICP, TGF-β1, and CRP in urine, the incubation time was extended or concentrated samples were used. For this purpose, samples were pre-incubated in 1% Tween 20, depending on the available starting volume. The cells were concentrated by about 10-fold to about 60-fold using an Ultra-15 centrifugal filter (3K, UFC900324).

[1052] The features disclosed in the preceding description, in the claims and / or in the drawings may, alone or in any combination thereof, be material for realizing the invention in its various forms.

[1053] References

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Claims

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

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

3. A combination of a protein and at least one further 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, The protein is selected from the group consisting of: Type XIII collagen (COLXIII), Hyaluronan binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH) and Complement factor I (CFI), The at least one additional protein is selected from the group consisting of: Fibrinogen, Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), Carboxyl-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (a1AGP), Coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), Fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-α-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), Myosin IXA (MYIXA), Kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1, Serine protease inhibitor family A member 1, transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1 microglobulin / double Kunitz inhibitor precursor, Carboxypeptidase N subunit 2 and Serine / arginine repeat matrix protein 3, The at least one additional protein is preferably selected from Fibrinogen, Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), Carboxyl-terminal propeptide of type I procollagen (PICP) and C-reactive protein (CRP).

4. The combination for use according to claim 3, wherein 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.

5. A combination of at least two proteins for use as biomarkers in a method for preclinical and / or early detection and / or diagnosis of kidney disease in a mammal, said at least two proteins being selected from Type XIII collagen (COLXIII), Hyaluronan binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), Fibrinogen, Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), Carboxyl-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (a1AGP), Coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), Fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-α-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), Myosin IXA (MYIXA), Kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1, Serine protease inhibitor family A member 1, transferrin, α1-B glycoprotein, Apo AI, Apo D, α-1 microglobulin / double Kunitz inhibitor 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, Serine protease inhibitor family A member 3, Serine protease inhibitor family A member 5, Serine protease inhibitor family A member 7, Serine protease inhibitor family G member 1, Serine protease inhibitor family D member 1, Coagulation factor V, Prothrombin, Plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit α, 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, Sacsin molecular chaperone, Dispatch RND transporter family member 2, DNA polymerase, Transport protein particle complex subunit 2, RB-related KRAB zinc finger, Ciliogenesis-related TTC17 interacting protein, proteasome subunit α type, Regulates synaptic membrane exocytosis protein 2, Extracellular matrix protein 1, E-cadherin 5, Coiled-coil domain-containing protein 178 and attractor protein, The at least two proteins are preferably selected from type XIII collagen (COLXIII), Hyaluronan binding protein 2 (HABP2), C4 binding protein α (C4BP), Complement factor H (CFH), Complement factor I (CFI), Fibrinogen, Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), Carboxyl-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (a1AGP), Coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), Fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-α-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).

6. The combination for use according to claim 5, wherein 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.

7. The combination for use according to any one of claims 1 to 6, wherein the kidney disease is selected from the following chronic progressive kidney diseases: Begins with glomerular damage (glomerulopathy), Postinfectious glomerulopathy, IgA nephropathy, Henoch-Schönlein purpura, focal segmental glomerulosclerosis, Alport syndrome, thin basement membrane nephropathy, and benign familial hematuria are preferred; or Begins elsewhere in the body with other dysregulations, and / or begins elsewhere in the kidneys with effects on the glomeruli (nephropathy), Preferred are metabolic syndrome, hypertension, heart failure, drug nephrotoxicity, obesity, diabetes (diabetic nephropathy), nephrogenesis, renal dysplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract anomalies, renal duplication, and nephronophthisis.

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

9. The combination for use according to any one of claims 1 to 8, wherein the sample is a body fluid, preferably urine or blood, such as serum, plasma, whole blood, wherein more preferably the sample is urine, And / or wherein 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, wherein preferably the mammal is a human.

10. A method for preclinical and / or early detection and / or diagnosis of kidney disease, comprising (a) determining the concentration of at least two proteins in a sample from a mammal, wherein the at least two proteins are selected from Type XIII collagen (COLXIII), Hyaluronan binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH) and Complement factor I (CFI) or wherein the at least two proteins are selected from the group consisting of: A protein selected from the group consisting of: Type XIII collagen (COLXIII), Hyaluronan binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH) and Complement factor I (CFI) and at least one additional protein selected from the group consisting of: Fibrinogen, Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), C-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (a1AGP), Coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), Fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-α-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), Myosin IXA (MYIXA), Kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1, Serine protease inhibitor family A member 1, transferrin, α1-B glycoprotein, Apo AI, Apo D, Alpha-1 microglobulin / double Kunitz inhibitor precursor, Carboxypeptidase N subunit 2 and Serine / arginine repeat matrix protein 3, said at least one additional protein is preferably selected from Fibrinogen, preferably fibrinogen gamma chain (FGG), Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), Carboxyl-terminal propeptide of type I procollagen (PICP) and C-reactive protein (CRP), (b) comparing each of said concentrations determined in step (a) with a control value, wherein a deviation of each of said concentrations determined in step (a) from said control value is indicative of preclinical and / or early stage renal disease in said mammal.

11. A method for preclinical and / or early detection and / or diagnosis of kidney disease, comprising (a) determining the concentration of at least two proteins in a sample from a mammal, wherein the at least two proteins are selected from Type XIII collagen (COLXIII), Hyaluronan binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), Fibrinogen, Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), Carboxyl-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-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), Myosin IXA (MYIXA), Kininogen 1, α2-HS glycoprotein, Complement C6, Complement C7, Complement C8, APOH, beta-2-glycoprotein 1, Serine protease inhibitor family A member 1, transferrin, α1-B glycoprotein, Apo AI, Apo D, α-1 microglobulin / double Kunitz inhibitor 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, Serine protease inhibitor family A member 3, Serine protease inhibitor family A member 5, Serine protease inhibitor family A member 7, Serine protease inhibitor family G member 1, Serine protease inhibitor family D member 1, Coagulation factor V, Prothrombin, Plasminogen, Protein S, Afamin, transthyretin, Hemoglobin subunit α, 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, Sacsin molecular chaperone, Dispatch RND transporter family member 2, DNA polymerase, Transport protein particle complex subunit 2, RB-associated KRAB zinc finger, Ciliogenesis-related TTC17 interacting protein, proteasome subunit α type, Regulates synaptic membrane exocytosis protein 2, Extracellular matrix protein 1, E-cadherin 5, Coiled-coil domain-containing protein 178 and attractor protein, wherein the at least two proteins are preferably selected from Type XIII collagen (COLXIII), Hyaluronan binding protein 2 (HABP2), C4 binding protein (C4BP), Complement factor H (CFH), Complement factor I (CFI), Fibrinogen, Luminophore (LUM), FMN, vitronectin (VTN), Gelsolin (GS), Angiotensinogen (AGT), Adiponectin (ADP), Transforming growth factor β1 (TGF-β1), Carboxyl-terminal propeptide of type I procollagen (PICP), C-reactive protein (CRP), α-1-acid glycoprotein (a1AGP), Coagulation factor XIII (CFXIII), Complement component 1q (C1q), Complement component 9 (C9), Fibronectin (FN), Ficolin 1 (FIC), Hemopexin (HPX), Inter-α-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), (b) comparing each of said concentrations determined in step (a) with a control value, wherein a deviation of each of said concentrations determined in step (a) from said control value is indicative of preclinical and / or early stage renal disease in said mammal.

12. The method according to claim 10 or 11, wherein the kidney disease is selected from the following chronic progressive kidney diseases: Begins with glomerular damage (glomerulopathy), Postinfectious glomerulopathy, IgA nephropathy, Henoch-Schönlein purpura, focal segmental glomerulosclerosis, Alport syndrome, thin basement membrane nephropathy, and benign familial hematuria are preferred; or Begins elsewhere in the body with other dysregulations, and / or begins elsewhere in the kidneys with effects on the glomeruli (nephropathy), Preferred are metabolic syndrome, hypertension, heart failure, drug nephrotoxicity, obesity, diabetes (diabetic nephropathy), nephrogenesis, renal dysplasia, polycystic renal dysplasia, autosomal dominant polycystic disease, autosomal recessive polycystic disease, urinary tract anomalies, renal duplication, and nephronophthisis.

13. The method according to any one of claims 10 to 12, wherein the sample is a body fluid, preferably urine or blood, such as serum, plasma, whole blood, wherein more preferably the sample is urine, And / or wherein 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, wherein preferably the mammal is a human.

14. The method according to any one of claims 10 to 13, wherein COLXIII and C4BP, or COLXIII and C4BP and HABP2, or Concentrations of CFH and CFI, or The concentrations of COLXIII, HABP2, C4BP, CFH, and CFI were measured.

15. The method according to any one of claims 10 to 14, wherein 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, or It was determined 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.

16. The method according to any one of claims 10 to 15, wherein the control value for each protein from a healthy subject is determined, and wherein the control value is preferably a standard concentration range, and / or wherein a deviation of said concentration determined in step (a) from the median of said standard concentration range is indicative of preclinical and / or early stage renal disease, Preferably Deviations greater than or equal to (≥) 2 times the median of the standard concentration range are indicative of preclinical and / or early-stage renal disease; or Deviations less than or equal to (≤) 0.5 times the median of the standard concentration range are indicative of preclinical and / or early-stage renal disease, More preferably Deviations greater than or equal to (≥) 2 times the median of the standard concentration range are indicative of preclinical and / or early-stage renal disease.

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