WO2013067979A1

WO2013067979A1 - Method for determination of the concentration of uromodulin in serum and it's use as a serologic marker for the diagnosis of nephropathy

Info

Publication number: WO2013067979A1
Application number: PCT/CZ2012/000033
Authority: WO
Inventors: Viktor RŮŽIČKA
Original assignee: Biovendor - Laboratorní Medicína A.S.
Priority date: 2011-11-11
Filing date: 2012-04-16
Publication date: 2013-05-16
Also published as: CZ2011722A3; CZ304067B6

Abstract

This serologic method for the diagnosis of nephropathy is based on the determination of the concentration of human uromodulin in serum. The concentration of uromodulin in serum serves as an indicator of nephropathy and uromodulin is a serologic marker for the diagnosis of nephropathy.

Description

METHOD FOR DETERMINATION OF THE CONCENTRATION OF UROMODULIN IN SERUM AND IT'S USE AS A SEROLOGIC MARKER FOR THE DIAGNOSIS OF NEPHROPATHY

Field of technology

The invention covers a method for the determination of the concentration of uromodulin in serum and its use as a serologic marker for the diagnosis of nephropathy. Moreover, the invention concerns the immunoassay system for the determination of uromodulin level.

The field of application is medicine and medical diagnostics. Specifically, this invention belongs to the category of diagnostic markers for the detection of nephropathy:

State of the Art

Diagnosis and differential diagnosis of nephropathy is currently based on a combination of laboratory tests measuring a number of parameters (a so called multiplex analysis) and on imaging technologies (scintigraphy, ultrasound, CT, NMR). Less common is the use of invasive methods designed to discover defects iff both morphology of the kidney and its function (biopsy of the kidney, irivasive^rmethod§ estimating glomerular filtration) which are then used to determine the final diagnosis.

An optimal non invasive screening method for the determination of the function of the kidney is still not available. A combination of cystatin C and creatinine in various mathematical models is used for the detection of nephropathy but these measurements detect only more advanced forms of the disease. Various proteins and their combination in urine are often used to determine incipient damage of the kidney function. Proteinuria is one of the inseparable components of the differential diagnosis of kidney diseases. The causes of proteinuria include increased capillary permeability for proteins which exceeds the capacity of tubular absorption (glomerular proteinuria); insufficient tubular reabsorption of filtered protein- (tubular proteinuria); extreme elevation of a protein in blood (pre-renal proteinuria); or inflammatory and cytolytic processes in the urinary tract (post-renal proteinuria). The diagnosis of proteinuria is typically based on the determination of the protein/creatihine ratio in urine, electrophoretic examination of urine, determination of urinary albumin or urinary albumin/creatinine ratio, determination of proteinuria selectivity index, determination of a-1 -microglobulin, light immunoglobulin chains, or paraprotein in urine. A disadvantage of the above methods is that the diagnostic accuracy is insufficient and the results are often misleading.

The current practice of nephrology lacks, therefore, a diagnostic method of nephropathy with sufficient screening performance (robustness, diagnostic accuracy). For this and other reasons, there is an intense search for a diagnostic marker that would in a straightforward way enable diagnosis of nephropathy, and subsequent determination of etiology of nephropathy, at an early stage.

Tamm Horsfall protein (inhibitor of hemaglutination, THF) was isolated from human urine 50 years ago. Sikri et al (1981) localized THF in epithelial cells of the large ascending branches of the loop of Henle and the distal convoluted kidney tubules. Muchmore and Decker (1985) isolated the Tamm- Horsfall 85 kDa glycoprotein from the urine of pregnant females and proposed th name uromoduliri (referred to ^: as UMD in this text) because of its ability to inhibit antigen induced proliferation of T cells and monocytic cytotoxicity. The authors- showed thai! uromodulin (UMD) inhibits antigen-specific proliferation in vitro. Uromddulin is the most abundant protein in human urine; Sequencing of UMD conducted by Penhica et al a few years later showed that UMD is identical to THF and consists of 616 amino acids (86-90 kDa) containing 48 cystein residues connected by 24 disulfide bridges.

Human urinary glycoprotein (HGP92) cleaved from THP was purified by Fontan et al in 1995. The authors proposed that the immunostimulatory properties of THF preparations are caused by its contamination with HGP92. Santambrogio et al (2008) described C-terminal cleavage of the mature protein secreted ^;rby the kidney: Jovine et al (2002) showed that the Zona Pellucida (ZP), a region that appears* in many eucaryotic extracellular proteins including UMD, enabled polymerization of proteins into filaments.

Recent research shows that UMD is produced by the ascending and descending branches of the loop of Henle and is excreted into urine at ah approximate concentration of 50mg/ml. UMD inhibits bacterial colonization of the urinary tract, formation of urinary stones, binds and activates leukocytes, and likely affects the permeability of water in the ascending branch of the loop of Hehle. UMD likely affects adhesion of pathogens (e.g. E. Coli) to urothelium, has signaling properties, and interacts with cytokines. At the same time, it appears that UMD inhibits the aggregation of the crystals of calcium oxalate in urine and affects the transport of ions (Na and CI) in the tubules.

Mutation of the UMD gene in experimental animal models is associated with the appearance of familial hypertrophic nephropathy (FJHN) or medullary kidney disease (MCFD2). Mice with deletion of UMD are susceptible to E. Coli infections which is considered a proof of the inhibitory function of UMD for E. Coli adhesioin to the urothelium. This is often accompanied by calcium oxalate urolithiasis and nephrocalcinosis (UMD binds Ca⁺² inhibiting crystallization of oxalate). The decrease of UMD in urine leads eventually to nephrocalcinosis.

Similar findings were demonstrated in human studies. Mutations of UMD lead to a decrease of the protein in urine, appearance of calcium oxalate urolithiasis, and even to kidney failure. It was simultaneously shown that UMD activates granulocytes which play a key role in the primary defense against infection. Decrease in UMD leads to urinary infections. Decrease in urinary UMD was associated with chronic renal insufficiency in some studies; however, these experimental results were^; not consistent between studies.

Recent studies showed an increased concentration of urinary UMD in connection with a common genetic polymorphism of the gene UMD which lead to a significant increase in the risk of chronic renal insufficiency (K5ttgen A, Hwang S-J, Larson MG, et al. Uromodulin levels associate with a common UMD variant and risk for incident CKD. J Am Soc Nephrol 2010;21 :337-344).

The document US 2011091915 describes a method for the testing of kidney diseases by the determination of UMD and immunoglobulin A in human urine. In addition, the document describes a testing kit including immunoglobulins recognizing UMD and immunoglobulin A. It was shown that urine of patients with kidney disease contains a higher concentration of UMD.

The goal of the presented discovery is early diagnosis of nephropathy and the subsequent monitoring of kidney diseases. The goal of the invention is to provide a method for early diagnosis and follow-up of nephropathy progression, A serologic marker for such diagnostic method and immunological tests which enable determination of a quantitative parameter which fulfills the role of a screening indicator for the determination of nephropathy and its risk and for the evaluation of disease progression. This is similar to the method and system of diagnosis of metabolic syndrome described in EP1904082. Summary of the invention

Based on extensive research and testing, we have determined that serum from healthy individuals contains UMD and the concentration is higher than the concentration of UMD in serum from individuals with nephropathy. The invention is based on this surprising fact which is in direct disagreement with the current knowledge and literature.

The invention consists of the method for diagnosis of nephropathy based on the determination of concentration of uromodulin in serum which is used as an indicator of nephropathy.

Our original goal was to find a serologic marker of kidney damage which originates strictly in the kidney and is not detectable in serum of healthy subjects under physiological conditions. The presence of such a marker in serum would indicate its release into circulation due to kidney damage. Surprisingly," we have found that uromodulin, a classic urinary protein not expected to be detectable in serum and therefore never measured, is present in serum of healthy subjects in measurable concentrations but that its concentration in serum is significantly decreased in patients with nephropathy. This is opposite of the expected increase in uromodulin concentration with disease.

- - The use of immunological tests for the quantification of uromodulin in serum are advantageous especially in combination with detection methods using optical or biosensor measurements. These tests allow rapid quantification of UMD in serum. There are many available methods for the determination of UMD, for example parallel or sequential concentration measurements, if a calibration based- on known concentrations of UMD can be achieved by the use of affinity reagents, immobilized on solid support and detecting UMD or its parts.

The most advantageous methods are those that include the sorbent materials bihdin^'g^'fhe reagents which are used for the detection of UMD in serum. Any sorbent material capable of biophysical immobilization of the detection reagent can be used. For the newly developed method, at least partially planar surfaces are especially useful (for example glass chips, plastic chips, or ELISA plates) because they allow a convenient and robust application of the method including detection based oh scanners or ELISA readers. Wells of ELISA plates are especially convenient surfaces for the method of immunologic detection of UMD.

Enzyme-Linked Immunoabsorbent Assay (ELISA) and its analogs, which use colorimetric detection with high sensitivity of detection, are therefore particularly suited for the determination of UMD concentration in serum. The support material with one or more immobilized sorbent substances, e.g. immunoglobulin with affinity for UMD and its derivatives, contacts serum, another biological fluid, or the assay solutions. This allows association of UMD or its derivatives present in the solution with the immunoglobulin. Serum can be diluted in an appropriate buffer, e.g. blocking buffer solution, to decrease interference and improve signal-to-noise ratio. Detection of UMD or its derivatives is achieved by the addition of another reagent; typically anti- UMD antibody, which has affinity to UMD and binds to it in the proximity of the immobilization surface.

Visualization of the bound detection substance can be achieved by many mechanisms. --The antibbdy recognizing UMD or its derivatives can be already labeled with a visualizable reagent, e.g. fluorescent dye or an enzyme that modifies the detection reagent of choice. The detection reagent, especially antibody, can also be labeled with a substance, e.g. biotin, that has affinity to another labeled substance used subsequently used for the visualization.

In case the primary antibody is not labeled, labeled secondary antibodies can be used to achieve detection. The secondary antibodies visualizing the primary complex are added at a convenient washing step which allows detection of the anti- UMD/substrate immobilized on the sorbent by a subsequent optical detection step.

The use of uromodulin as a serologic marker for the diagnosis of nephropathy is therefore also part of the invention.

We have developed ELISA tests for the detection of UMD in serum and in urine. Detection of uromodulin in serum (UMD-S) has excellent accuracy for the detection of nephropathy (better than other parameters currently in use).

Summary of the Figures

The invention is documented by several Figures. Figure 1 shows concentration of UMD in serum of healthy controls and patients with nephropathy in our first study. Figure 2 shows concentration of UMD in urine of healthy controls and patients with nephropathy in our first study. Figure 3 shows individual concentration values of UMD in serum of healthy controls and patients with nephropathy; the 125.4 g/l cutoff value was determined by ROC curve analysis. Figure 4 shows the ROC curve. Figure 5 ^"shows concentrations of UMD in serum of healthy controls and patients with nephropathy in our second study. Figure 6 shows uromodulin RD 72163100 on a 12% SDS-PAGE gel. Figure 7 shows the separation of anti-human uromodulin antibodies on 12% SDS-PAGE. Figure 8 shows relevant chromatograms from the purification. Figure 9 shows the distribution of UMD concentrations with age and gender of healthy adults.

Examples of applications of the invention

As a non-limiting demonstration of the invention we show the following applications:

1. Use of an ELISA test for the determination of uromodulin (UMD) in body fluids as a marker of nephropathy. The ELISA test was used to examine 127 participants (66 males, 61 females). We have examined 15 healthy controls and 112 patients at the nephrologic outpatient clinic Prostejov, Czech Republic (DM nephropathy, pyelonephritis; CRI NS interstitial nephropathy) with the same distribution of age and gender. We have determined UMD in serum and urine, ApoH in urine, osteopontiri in urine, TFF1-3 in serum and urine, urea, creatinine, ions, protein and albumin in urine, A1 MG in urine and cystatin C in serum. Nephropathy was diagnosed by a urology specialist and defined as the presence of a minimum of three of the following markers: increased creatinin in serum, GF measured by MDRD or cystatin G < 1 ml/s, increased cystatin in serum, increased ratio of albumin/creatinin in urine, increased alpha-1 microglobulin in urine.

A subsequent comparison of the patients and healthy controls showed the following: a. range of UMD concentration in serum of healthy controls is significantly higher than the concentration in nephropathy patients (241 vs. 703 jjg/l, p<0.01). Figure 1 shows the detected concentration of UMD in serum. ^;

b. determination of UMD in urine showed that healthy controls have a significantly higher urinary concentration of UMD compared to the nephropathy patients (11,165 vs. 4,860 pg/l, p<0.01). Figure 1 shows the detected range of UMD concentrations in urine. Significant overlap of the concentration in the healthy and patient groups limits the prediction accuracy of the urinary test. We therefore describe only the serologic test in subsequent studies.

We have used ROC curve analysis for the determination of the cutoff value of UMD in serum for the determination of sensitivity and specificity of the measurement. We have found sensitivity of 92% at specificity of 100%, and AUC of 99% for UMD-S at the cutoff 125.4 ng/ml in the detection of kidney damage. Figure 3 shows the values and Figure 4 shows the ROC curve.

We have further used logistic (stepwise) regression (diagnosis yes/no, measured parameters) to select a model for the detection of nephropathy. Only UMD-S was selected for the model (p<0.01), ods 0.95, 96.1% patients included.

AU ROC 0.98 (0.965-0.99)

Observed {Expected Percent of correctly

, n^;;^.;y, . ^. '- _c|_assjfjed °' ^{:I s cuiorf a}'^ ^cf

,ψθ in ^ - Γ ίπ^« -::r _Λ v -;^■ -_er :: , ^' : r^rif:diy cf .e measurement.

Y^'l d^{a e i0' ;,}^^{¾i< : '::';r,' ' ' ''} 3 ^~ 80.00% ^ ^'Ί

Y = 1 ^{Wc :} -'^■■" ·-'^■ 110 ^: ·''^ί "^': 98.21% '- i- ^- ^. ^ diagnosis y½, no^.

Percent of correctly classified cases 96 06% ~ . , ·^{. .}·^,·

The data provide convincing evidence that the concentration of UMD in serum ϊβ significantly higher in healthy controls compared to the patients with nephropathy. The determination of uromodulin in serum has excellent prediction accuracy for the detection of nephropathy. The accuracy exceeds the accuracy of other parameters currently in use. The two by two table shows correct classification of more than 96% of the examined participants.

2. A similar study, smaller in scope, was carried out in collaboration with the First Faculty of Medicine, Prague, Czech Republic. We have examined 7 healthy controls and 7 nephropathy patients. The results were very similar to the results of the first study. Subsequently, we have compared the concentration of UMD in serum of patients with neuropathy. This study likewise showed a significantly higher concentration of uromodulin in healthy controls than in patients with neuropathy. There wasn't a single healthy person with concentration of UMD in serum lower than the patients (Figure 5).

Description of the protein used to generate the antibodies

We have used protein isolated from human urine (RD172163100, Biovendor) which is used in the ELI SA test.

Figure 6 shows the RD172163100 uromodulin on a 12% SDS-PAGE. The first column represents molecular weight markers (14, 21 , 31 , 45, 66, 97 kDa); second column represents reduced protein denatured by heating (5pg/lane); third column represents native non-reduced protein (5pg/lane). -^{Vi i '} - ^¾ :

Manufacturing Process

The protein was purified from human urine as described (Santambrogio et al., 2008). Briefly, 400 ml of urine of several men was mixed with an appropriate volume of 1.6M NaCI and mixed for 16 hours at 3-9 °C. Supernatant was separated by centrifugation and the precipitate was resuspended in 100ml 0.58M NaCI and centrifuged again immediately. The resulting solid fraction was dissolved in de-ionized water and dialyzed several times against dH₂0. Subsequently, the protein was freeze-dryed from dH20, 0.3ml/vial, at an initial concentration of 0.3mg/ml.

Generation of antibodies and their processing

Polyclonal anti-uromodulin antibodies were generated and affinity purified by immunization of sheep. Two sheep were immunized according to the following scheme:

Day 1. 1x subcutaneous 300 pg antigen + CFA, 1 :1

Day 14. 1x subcutaneous 300 pg antigen + ICFA, 1 :1

Day 28. tx subcutaneous 300 pg antigen + ICFA, 1 :1

Day 56. 1x subcutaneous 300 pg antigen + ICFA, 1 :1

Day 66. blood collection 500 ml blood

Day 67-127. waiting period

Day 128. 1x subcutaneous 300 pg antigen + ICFA, 1 :1

Day 138. blood collection 500 ml blood CFA - complete Freund adjuvant (Sigma)

ICFA - incomplete Freund adjuvant (Sigma)

Generation of serum

Sheep blood was centrifuged 20 minutes at 2400G and 4 °C. Serum was stored at - 20 °C.

Affinity Purification

An- 0.5 g affinity column Poros AL (Applied Biosystems) was packed with 1 mg uromodulin (Biovendor) in agreement with manufacturer's suggestions. Prurification was carried out in two steps:

1. Affinity purification: the antibodies were bound to the affinity column in 0.1 M PBS, pH 7.4 and eluted with 0.1 M PBS, 13 mM HCI, 0.15 mM NaCI.

2. Final purification was carried out on a protein G column (Applied Biosystems).¹ The affinity purified antibodies were bound to the protein G column in 0.1 M PBS, pH 7.4 and eluted with 0.1 M PBS, 13 mM HCI, 0.15 mM NaCI.

Testing of the antibodies: Purified antibodies were separated by a 12%- SDS-PAGE and stained with Coomassie Blue. r i τ,^! . ^;; οΓί

Titration using indirect ELISA: Microtitration plate (Nunc) was filled with UMD at 25mg/well. Purified antibodies were distributed at an initial concentration 1mg/ml and diluted 1 :3 in series. The titre was defined as the solution of the antibody with absorbance lower than 1.5.

Sheep titre: 30,000 · ^{'' "■■■ ■} ^::-t ^G S^ V.?- T

Figure^J7- shows the separation of anti-human uromodulin antibodies on 12% SDS- PAGE as follows:

Lane 1. MW standard (97, 66, 45, 31 , 21 , 14 kDa) : S r - ' v ^ Lane 2. Anti-human uromodulin immunoglobulin under reducing conditions, 2.5 pg/lane

Figure 8 shows chromatograms of the purification: a. Affinity purification; b. Protein G column.

EtllSA development

Filling of the microtitration plate:

The microtitration plate was filled with affinity purified sheep antibody (BioVendor, 2 pg/ml in 0.1 M carbonate buffer) and stored overnight at 4 °C. Following 16-20 hours of filling overnight at 4 °C, the plates were washed with TBS- Twen. Unsaturated sites were blocked with TBS - 0.5 % BSA 4 % saccharose ahd incubated for 1 hour at RT.

Conjugate: Affinity purified sheep antibody (BioVendor) was conjugated with Biotin^_LC-LC-NHS-sulfo (cn 21338) according to the manufacturer's instructions and used at a final concentration of 0.25 pg/ml. We used the streptavidin-HRP conjugate (Roche, cn 11089153).

Calibrant: Human uromodulin isolated from urine was lyophilized and used as a standard at an initial concentration of 320 ng/ml. _y : : J

The following test-characteristics were used: ^{; :}— - -- ·^α' a. Typical standard curve for human uromodulin showed a lower detection limit of less than 0.12 ng/ml. ^¾ c , : .; : : b. Linearity (Table 1)

c) Yield of the additive (Table 2)

Sample Measured Expected Yield

(ng/ml) (ng/ml) N/0 (%)

1 94.45 - -

480.60 494.45 97.2

282.95 294.45 96.1

187.90 194.45 96.6

2 104.35 - -

506.40 504.35 100.4

281.20 304.35 92.4

186.55 204.35 91.3 d) Intralaboratory precision (Table 3)

e) Interlaboratory precision (Table 4)

f) Influence of gender on the concentration of uromodulin (Table 5)

The distribution of uromodulin concentrations with gender and age in healthy controls are listed in Figure 9. The concentration of uromodulin is evenly distributed and is not affected by either gender or age. Industrial applicability

This invention will find a broad application field in medicine, specifically in medical diagnostics. Use of the method for the determination of uromodulin concentration in serum as a marker of nephropathy and a method for immunological testing for determination of the uromodulin concentration will facilitate an immediate detection of nephropathy in patients which will enable a timely administration of corrective measures.

Literature

1. Sikri, K.L., Foster, C.L, MacHugh, N. & Marshall, R.D. Localization of Tarhm- Horsfall glycoprotein in the human kidney using immuno-fluorescence and immuno-electron microscopical techniques. J. Anat 132, 597-605 (1981). ^;

2. Santambrogio, S. et al. Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage. Biochem. Bibphys. Res. Commun 370, 410-413 (2008).

3. Muchmore, A.V. & Decker, J.M. Uromodulin: a unique 85-kilodalton immunosuppressive glycoprotein isolated from urine of pregnant women. Science 229, 479-481 (1985).

4. Jovine, L., Qi, H., Williams, Z., Litscher, E. & Wassarman, P.M. The ZP domain is a conserved module for polymerization of extracellular proteins. Nat Cell Biol 4, 457-461 (2002).

5. Fontan, E., Jusforgues-Saklani, H., Briend, E. & Fauve, R.M; Purification of a 92 kDa human immunostimulating glycoprotein obtained from the Tamm-Horsfall glycoprotein. J. Immunol. Methods 187, 81-84 (1995).

Claims

Patent Claims

1. Method for diagnosis of nephropathy characterised in that concentration of human uromodulin is determined in serum that is subsequently used as a diagnostic indicator of nephropathy.

2. Method according to claim 1 , characterised in that a system for immunological testing is used for determining the concentration of human uromodulin in serum.

3. Method according to claim 2, characterised in that a sorbent material with immobilized substances that have affinity to human uromodulin or its parts is used.

4. Method according to claim 3, characterised in that the immobilized substances which have affinity to UMD or its parts are immunoglobulins.

5. Method according to claim 3, characterised in that at least part of the sorbent support material is formed as a planar surface.

6. Method according to claims 3 and 4, characterised in that the sorbent materials are part of a plate for an ELISA detection method.

7. Method according to claims 2 to 5, characterised in that an ELISA method is a system for immunologic testing.

8. Method according to claim 7, characterised in that ELISA consists of an anti-UMD antibody, secondary detection antibody, sorbent support materials, and solutions for incubation, washing, and detection of UMD.

9. Method according to claims 2 to 9, characterised in that a scanner or ELISA reader is used for evaluation.

10. Use of uromodulin as a serologic marker for the diagnosis of nephropathy.