WO2008084331A2 - Biomarqeurs pour des troubles rénaux - Google Patents

Biomarqeurs pour des troubles rénaux Download PDF

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WO2008084331A2
WO2008084331A2 PCT/IB2007/004395 IB2007004395W WO2008084331A2 WO 2008084331 A2 WO2008084331 A2 WO 2008084331A2 IB 2007004395 W IB2007004395 W IB 2007004395W WO 2008084331 A2 WO2008084331 A2 WO 2008084331A2
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disease
sample
protein
kit
assay
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WO2008084331A3 (fr
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Solange Moll
Denis Hochstrasser
Pierre Lescuyer
Catherine Zimmermann
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Hopitaux Universitaires De Geneve
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Hopitaux Universitaires De Geneve
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • the present invention relates generally to the fields of molecular biology and medicine. More particularly, it concerns methods for the diagnosis, prediction and prevention of renal disorders including the rejection of transplanted kidney using certain biomarkers.
  • Diagnosis of glomerular and/or tubular kidney diseases and follow-up of the pathological process activity including the rejection of transplanted kidney are currently made respectively with renal biopsy and laboratory data (e.g., serum creatinine, proteinuria and urinary sediment).
  • Renal biopsies have significant clinical risks and limitations.
  • the renal biopsy allows appreciating the state of the lesions at a precise time point of the disease.
  • clinicians commonly choose not to perform a renal biopsy because of the high risks including bleeding, a unique kidney and damage to the kidney.
  • Usual laboratory data such as serum creatinine and urinary albumin levels, allow to some degree of analysis of the severity of renal function impairment.
  • they are indeed functional, not morphological parameters.
  • they are neither specific for each glomerular or tubular disorder nor sensitive enough to reflect the activity of the disease or the risk of kidney failure.
  • the present invention overcomes deficiencies in the art by providing specific biomarkers (e.g., glomerular podocytes and tubular cell markers) in urine, plasma and/or serum that could allow the diagnosis and follow-up evaluation of glomerular and/or tubular kidney diseases as well as detecting acute or chronic rejection of the transplanted kidney .
  • specific biomarkers e.g., glomerular podocytes and tubular cell markers
  • An aspect of the present invention relates to a method of assessing whether an individual has or is at risk for developing a renal disorder comprising the steps of: (a) obtaining a biological sample from the individual, (b) analyzing the sample to determine the presence, absence or amount of one or more biomarkers selected from the group consisting of the biomarkers of Table 1 and (c) assessing from said presence, absence or amount of the one or more biomarkers whether the individual has or is at risk for developing a renal disorder.
  • the analyzing may comprise determining the presence or amount of one or more of pigment epithelium-derived factor, Attractin, FGF 11 (fibroblast growth factor), nestin, Dermcidin, EGF-containing f ⁇ bulin-like matrix protein 1 (f ⁇ bulin 3), myosin Va (non muscle), neuroligin- 2, brain enriched hyaluronan binding protein, Ryanodine receptor 2, Ankyrin 3, serum paraoxonase/aryltransferase 1 (PONl), neuropilin-2 (NP-2), alpha2-macroglobulin or FERM domain containing protein 4A.
  • the sample may be a blood sample, a urine sample, a kidney biopsy sample, a serum sample or a plasma sample.
  • the analyzing of the sample may comprise performing one or more of an ELISA, an immunoassay, reverse transcription PCR (RT-PCR), a radioimmunoassay (RIA), an immunoradiometric assay, a fluoroimmunoassay, an immunoassay, a chemiluminescent assay, a bioluminescent assay, a gel electrophoresis, a Western blot analysis, a silver-stained gel analysis, an in situ hybridization assay, an immunohistochemistry assay, a protein biochip assay, a microfluidic chip-based immunoassay, a mass spectrometry assay, a GC/MS assay, a LC-MS/MS assay, a MALDI tof mass spectroscopy assay, SELDI tof mass spectroscopy assay, or a HPLC assay thereon.
  • an ELISA an immunoassay, reverse transcription PCR (RT-PCR), a radioimmunoa
  • the analyzing of the sample may comprise performing an immunoassay thereon.
  • the renal disorder may be acute renal failure.
  • the renal disorder is a glomerular disease, such as a minimal change disease, a focal or segmental glomerulosclerosis, a collapsing glomerulopathy, a membranous nephropathy, a membranoproliferative glomerulonephritis, a dense deposit disease, a cryoglobulinemia- associated glomerulonephritis, an IgA nephropathy, an Henoch-Sch ⁇ nlein disease, a postinfectious glomerulonephritis, a bacterial endocarditis, a pauci-immune crescentic glomerulonephritis, a Wegener granulomatosis, a microscopic polyangitis, a Churg-Strauss syndrome, an anti-GBM-antibidy mediated glomerulonephritis, a lupus
  • the glomerular disease may have organized deposits.
  • the glomerular disease may be a amyloidosis, a monoclonal immunoglobulin deposition disease, a fibrillary glomerulonephritis, or an immunotactoid glomerulopathy.
  • the renal disorder is a tubulo-interstitial disease, such as an ischemic tubular injury, a medication-induced tubulo-interstitial nephritis, a toxic tubulo- interstitial nephritis, an infectious tubulo-interstitial nephritis, a bacterial pyelonephritis, a viral infectious tubulo-interstitial nephritis which results from a polyomavirus infection or an HIV infection, a metabolic-induced tubulo-interstitial disease, a mixed connective disease, a cast nephropathy, a crystal nephropathy which may results from urate or oxalate or drug- induced crystals deposition, an acute cellular tubulo-interstitial allograft rejection, a tumoral infiltrative disease which result form a lymphoma or a post-transplant lymphoproliferative disease, or an obstructive disease of the
  • the renal disorder may be a vascular disease, such as a thrombotic microangiopathy, a nephroangiosclerosis, an atheroembolic disease, a mixed connective tissue disease, a polyarteritis nodosa, a calcineurin-inhibitor induced-vascular disease, an acute cellular vascular allograft rejection, or an acute humoral allograft rejection.
  • the renal disorder may result from a hereditary disease (e.g., Alport's syndrome, thin membrane disease, Fabry's disease, a polycystic kidney disease) or a renal disorder which results from a metabolic disease (e.g., a diabetic nephropathy).
  • the biomarker may be present or absent in the biological sample.
  • an increased amount of the biomarker in the biological sample relative to a control sample indicates that the individual has or is at risk of the renal disorder.
  • wherein the presence of the biomarker in the biological sample indicates that the individual has or is at risk of the renal disorder.
  • Another aspect of the present invention relates to a method for identifying a candidate biomarker for a renal disorder comprising: (a) obtaining a biological sample from an individual who has the renal disorder, (b) purifying a podocyte vesicle from the biological sample (e.g., urine); and (c) measuring the amount of the protein in the biological sample, wherein if the protein is present in the biological sample in an amount greater than is present in a control sample, then the protein is the candidate biomarker.
  • the candidate biomarker is greater than about 40 kDa or greater than about 60 kDa.
  • the candidate biomarker is a neuronal protein.
  • the purifying may comprise immunopurif ⁇ cation.
  • kits comprising: (a) a first antibody preparation that selectively binds immunologically to of one or more biomarkers selected from the group consisting of the biomarkers of Table 1, and (b) a suitable container means thereof.
  • the antibody selectively binds immunologically to pigment epithelium-derived factor, Attractin, FGF 11 (fibroblast growth factor), nestin, Dermcidin, EGF-containing f ⁇ bulin-like matrix protein 1 (f ⁇ bulin 3), myosin Va (non muscle), neuroligin- 2, brain enriched hyaluronan binding protein, Ryanodine receptor 2, Ankyrin 3, serum paraoxonase/aryltransferase 1 (PONl), neuropilin-2 (NP-2), alpha2-macroglobulin or FERM domain containing protein 4A.
  • the first antibody may be a monoclonal antibody or a polyclonal antibody.
  • the antibody preparation may be attached to a support, such as a polystyrene plate, test tube or a dipstick.
  • the kit may further comprise at least a second antibody preparation.
  • the second antibody preparation may comprise a detectable label (e.g. , a fluorescent tag, a chemiluminescent tag or an enzyme).
  • Said enzyme may be alkaline phosphatase or horseradish peroxidase.
  • the kit may further comprise a substrate for said enzyme.
  • the kit may further comprise a buffer or diluent and a suitable container means therefor.
  • the kit comprises an ELISA assay.
  • compositions of the invention can be used to achieve methods of the invention.
  • the term "about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
  • the present invention overcomes deficiencies in the prior art by providing methods to determine if a patient has or is at risk of developing a renal disorder comprising evaluating the presence, absence or amount of one or more protein biomarkers (e.g., a podocyte and/or tubular cell biomarker) produced by or present in the patient.
  • a biomarker e.g., a podocyte and/or tubular cell biomarker
  • a sample taken from the patient e.g., a urine sample or a blood or plasma sample
  • the present invention discloses the surprising discovery by the inventor that specific proteins, which are normally absent or present at very low concentrations in the urine of a healthy patient, can be found in higher concentrations in the urine and potentially in the blood, or one of its components such as serum or a RBC fraction, of patients who have specific renal disorders or are at risk of developing such disorders.
  • specific proteins which are normally absent or present at very low concentrations in the urine of a healthy patient, can be found in higher concentrations in the urine and potentially in the blood, or one of its components such as serum or a RBC fraction, of patients who have specific renal disorders or are at risk of developing such disorders.
  • biomarkers have been identified in the present invention as being indicative of a renal disorder.
  • the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20 or more of the biomarkers listed in Table 1 may be used to diagnose a renal disorder.
  • a fragment of a biomarker may also be used to diagnose or evaluate the progression of a renal disorder. For example, use of a fragment of a biomarker from listed in Table 1 may be used to diagnose a renal disorder.
  • the biomarker is selected from the group consisting of pigment epithelium-derived growth factor (PEDF), attractin, FGF 11, nestin, Dermcidin, fibulin 3, myosin Va, neuroligin-2, brain enriched hyaluronan binding protein, ryanodine receptor 2, ankyrin 3, FERM domain containing protein 4 A, serum paraoxonase/aryltransf erase, serum paraoxonase/aryltransferase 1 (PONl), neuropilin-2 (NP- 2), and alpha2-macroglobulin.
  • PEDF pigment epithelium-derived growth factor
  • the following primers may be used, e.g., in a PCR assay to detect human PONl : human PONl sense 5'-ATG GGA CTG GCA CTC TTC AG-3' (SEQ ID NO:1), human PON-I antisense 5'-TGA AAG CCA GTC CAT TAG GC-3' (SEQ ID NO:2).
  • the expression of the biomarker may only be evaluated once in a patient, but preferably the expression of a biomarker evaluated in a subject more than once.
  • the presence or amount of a biomarker is analyzed in a patient 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times.
  • the time between the analyzing may be weeks ⁇ e.g., 1, 2, 3, 4, 5, 6, 7, 8 weeks), months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months) or years (e.g., 1 or more years).
  • the time between the analyzing will be decided by the patient's doctor and may vary depending on the individual patient and renal disease.
  • the present invention provides methods to determine if a patient has or is at risk of developing a renal disorder.
  • Renal disorders that may be diagnosed with the present invention include, but are not limited to, a glomerular disease, a tubulo-interstitial disease, a vascular disease and transplanted kidney rejection.
  • acute renal failure may be predicted and/or diagnosed early using biomarkers of the present invention.
  • Glomerular diseases which may be diagnosed via the present invention include a minimal change disease, a focal or segmental glomerulosclerosis, a collapsing glomerulopathy, a membranous nephropathy, a membranoproliferative glomerulonephritis, a dense deposit disease, a cryoglobulinemia-associated glomerulonephritis, an IgA nephropathy, an Henoch-Schonlein disease, a postinfectious glomerulonephritis, a bacterial endocarditis, a pauci-immune crescentic glomerulonephritis, a Wegener granulomatosis, a microscopic polyangitis, a Churg-Strauss syndrome, an anti-GBM-antibidy mediated glomerulonephritis, a lupus nephritis and a chronic allograft glomerulopathy.
  • the glomerular disease may also have organized
  • Tubulo-interstitial diseases which may be diagnosed via the present invention include an ischemic tubular injury, a medication-induced tubulo-interstitial nephritis, a toxic tubulo- interstitial nephritis, an infectious tubulo-interstitial nephritis, a bacterial pyelonephritis, a viral infectious tubulo-interstitial nephritis which results from a polyomavirus infection or an HIV infection, a metabolic-induced tubulo-interstitial disease, a mixed connective disease, a cast nephropathy, a crystal nephropathy which may results from urate or oxalate or drug- induced crystals deposition, an acute cellular tubulo-interstitial allograft rejection, a tumoral infiltrative disease which result form a lymphoma or a post-transplant lymphoproliferative disease and an obstructive disease of the kidney.
  • Vascular disease which may be diagnosed via the present invention include a thrombotic microangiopathy, a nephroangiosclerosis, an atheroembolic disease, a mixed connective tissue disease, a polyarteritis nodosa, a calcineurin-inhibitor induced-vascular disease, an acute cellular vascular allograft rejection and an acute humoral allograft rejection.
  • Renal disorders which result from a hereditary or metabolic disease may also be diagnosed via the present invention.
  • the hereditary disease may be Alport's syndrome, thin membrane disease, Fabry's disease, a polycystic kidney disease, a renal disorder which results from a metabolic disease may be a diabetic nephropathy.
  • Podocyte dysfunction is thought to be the starting point of segmental glomerulosclerosis (Gharahdaghi et al., 1999).
  • basic cell biological functions of the podocyte are not well known. The major reason of this incomplete knowledge is the difficulty to access and to study these cells.
  • the morphological expression of the biological dysfunction of podocytes is quite limited and uniform. Indeed, podocytic ultrastructural lesions are stereotypical. They include foot process effacement, hypertrophy with hyperplasia of organelles, reabsorption droplets, vacuolization of the cytoplasm, and, in most severe cases, podocyte drop out leading to the denudation of the glomerular basement membrane.
  • Albumin and other plasma proteins account for about 90% of this physiological proteinuria. The remainder is principally Tamm-Horsfall protein, secretory IgA and a small amount of small molecular weight proteins.
  • urinary excretion of small amounts of enzymes located in the cells of the renal tubules, and in particular in the brush border of the proximal tubules is found in normal individuals. Normal podocytes, or glomerular visceral epithelial cells, which are in direct contact with urinary space in the glomerulus, are known to release into urine small vesicles of various sizes lined by double membranes.
  • Exosomes are membrane vesicles generated by a process involving endocytosis, endosome sorting into perinuclear multivesicular bodies (MVB) and exocytosis of MVBs. Exosome formation into the urinary space has been described for numerous epithelial cells like renal tubule epithelial cells. Podocyte vesicles have been shown to express the transmembrane protein Complement Receptor 1 (CRl or CD35). The release of vesicle- bound CRl into urine was demonstrated to be specific for podocytes and constant over time.
  • CRl transmembrane protein Complement Receptor 1
  • This vesiculation process may be increased in pathological conditions.
  • Podocyte ultrastructural lesions especially incresead microvilli formation, are a constant feature of glomerulopathies characterized by increased proteinuria.
  • the data presented herein supports the hypothesis that these microvilli, localized in the urinary space as observed by electron microscopy analysis, may be excreted into urine as vesicles and represent a relevant part of the pathological proteinuria. Moreover, a modification of the protein pattern of these vesicles could reflect the physiopathological processes occurring in the damaged glomerulus. Since podocytes vesicles represent only a very small fraction of the physiological and pathological proteinuria, methods to enrich urinary samples in podocyte vesicles may be applied.
  • a biomarker nucleotide e.g., a biomarker mRNA
  • a tissue sample e.g., a blood, plasma or urine sample
  • methods known to those of skill in the art e.g. , gene chip analysis, reverse-transcription PCR or RT-PCR, Northern blot, etc.
  • Detecting a biomarker protein may be performed via many different techniques, as known in the art. Immunodetection methods may be used to measure increased or decreased expression of a biomarker (e.g., a podocyte biomarker) of the present invention in a biological sample from a patient. Examples of particular immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, immunohistochemistry, fluoroimmunoassay, chemiluminescent assay, electro-chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • the immunoassay readout could be a mass spectrometer.
  • proteins produced by podocytes may be detected in a biological sample from a patient using the methods described herein.
  • isolation of the podocytic vesicles of the whole urine may be performed using paramagnetic beads coated with a monoclonal antibody against CRl .
  • analysis of the protein pattern of these podocytic vesicles may be done via polyacrylamide gel electrophoresis. Patterns of the tubular vesicles (CRl -negative vesicles) and of the total proteins in urine (soluble proteins) may be compared. Specific proteins may also be identified using mass spectrometers (e.g., MALDI-TOF-TOF).
  • Tubular vesicles may be isolated during the same procedure and separated from the urinary soluble proteins using ultracentrifugation.
  • the enrichment in podocyte vesicles may be assessed by comparison with native urine (e.g., with or without concentration and/or immunoabsorption on magnetic beads, with Western-blot analyses using podocalyxin, a transmembrane protein specific of the apical surface of the podocyte, and/or with a semiquantitative evaluation of the detected bands by densitometry analysis).
  • the specificity of each sample may also be assessed with measurement of the CRl levels using a specific ELISA.
  • immunobinding methods involve measurement of the formation of immunocomplexes.
  • Other methods include methods for isolating and purifying the biomarker protein from an organelle, cell, tissue or organism's samples.
  • the antibody removes or binds to the antigenic biomarker protein or message from a sample.
  • the antibody will preferably be linked to a solid support, such as in the form of a column matrix, and the sample suspected of containing the message, protein, polypeptide and/or peptide antigenic component will be applied to the immobilized antibody.
  • the unwanted components will be washed from the column, leaving the antigen immunocomplexed to the immobilized antibody to be eluted.
  • the immunobinding methods also include methods for detecting and quantifying the amount of an antigen component in a sample and the detection and quantification of any immune complexes formed during the binding process.
  • detecting and quantifying the amount of an antigen component in a sample and the detection and quantification of any immune complexes formed during the binding process.
  • the biological sample analyzed may be any sample that is suspected of containing an antigen, such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, an organelle, separated and/or purified forms of any of the above antigen-containing compositions, or even any biological fluid that comes into contact with the cell or tissue, including blood, serum, plasma and/or a tissue sample.
  • an antigen such as, for example, a tissue section or specimen, a homogenized tissue extract, a cell, an organelle, separated and/or purified forms of any of the above antigen-containing compositions, or even any biological fluid that comes into contact with the cell or tissue, including blood, serum, plasma and/or a tissue sample.
  • the chosen biological sample with the antibody under effective conditions and for a period of time sufficient to allow the formation of immune complexes is generally a matter of simply adding the antibody composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with, i.e., to bind to, any biomarker antigens present.
  • the sample-antibody composition such as a tissue section, ELISA plate, dot blot or western blot, will generally be washed to remove any non-specifically bound antibody species, allowing only those antibodies specifically bound within the primary immune complexes to be detected.
  • the detection of immunocomplex formation is well known in the art and may be achieved through the application of numerous approaches.
  • biomarker antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specif ⁇ cally bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody, that has binding affinity for the antibody is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • One method of immunodetection uses two different antibodies.
  • a first step biotinylated, monoclonal or polyclonal antibody is used to detect the target antigen(s), and a second step antibody is then used to detect the biotin attached to the complexed biotin.
  • the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex.
  • the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
  • streptavidin or avidin
  • biotinylated DNA and/or complementary biotinylated DNA
  • the amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
  • This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • a conjugate can be produced which is macroscopically visible.
  • PCR Polymerase Chain Reaction
  • the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls.
  • the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
  • 2D gel electrophoresis as described in, e.g., Thongboonkerd et al.
  • cICAT cleavable isotope-coded affinity tags
  • iTRAQ isobaric tags for relative and absolute quantification
  • Western blot analysis is an established technique that is commonly employed for analyzing and identifying proteins.
  • the proteins are first separated by electrophoresis in polyacrylamide gel, then transferred ("blotted") onto a nitrocellulose membrane or treated paper, where they bind in the same pattern as they formed in the gel.
  • the antigen is overlaid first with antibody, then with anti-immunoglobulin or protein A labeled with a radioisotope, fluorescent dye, or enzyme.
  • a radioisotope fluorescent dye, or enzyme
  • immunoassays in their most simple and/or direct sense, are binding assays.
  • Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and/or radioimmunoassays (RIA) known in the art.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and/or the like may also be used. One of ordinary skill in the art would be familiar with use of ELISAs and other immunohistochemical assays.
  • a biomarker antibody of the present invention may also be used in conjunction with both fresh-frozen and/or formalin-fixed, paraffin-embedded tissue blocks.
  • the following methods for performing immunohistochemistry are provided as non-limiting descriptions of how the technique may be performed.
  • 5- ⁇ m histological sections are obtained from fresh-frozen biopsies. The sections are incubated with the primary antibody. Unbound antibody is removed by washing, and the bound antibody is detected either directly (if the primary antibody is labeled) or, indirectly using a fluorochrome-labeled secondary reagent. The staining is evaluated using fluorescence microscopy.
  • 3- ⁇ m histological sections are obtained from formaldehyde fixed paraffin-embedded biopsies. These are deparaffmated and then endogenous peroxidase is blocked with 0.3% H 2 O 2 in methanol for 30 min. The sections are digested with 0.05% proteinase K in PBS for 2 min, and incubated with the primary antibody. Then they are incubated with the labeled, secondary binding ligand, or antibody. For detection, a substrate-chromogen system is used. Counterstaining is by hematoxylin Mayer, and dehydration by using decreasing alcohol concentrations. Stained sections are mounted with permanent mounting medium and the tissues are examined with a microscope.
  • IHC immunohistochemistry
  • frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -7O 0 C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections.
  • PBS phosphate buffered saline
  • OCT viscous embedding medium
  • Permanent-sections may be prepared by a similar method involving rehydration of the
  • a biomarker of the present invention may be analyzed using a protein array and/or a protein biochip.
  • Protein array technology allows high-throughput screening for gene expression and molecular interactions. Protein arrays appear as new and versatile tools in functional genomics, enabling the translation of gene expression patterns of normal and diseased tissues into protein product catalog. Protein function, such as enzyme activity, antibody specificity, and other ligand-receptor interactions and binding of nucleic acids or small molecules can be analyzed on a whole-genome level.
  • Glass slides are still widely used, since they are inexpensive and compatible with standard microarrayer and detection equipment. However, their limitations include multiple- based reactions, high evaporation rates, and possible cross-contamination.
  • Matrix slides offer a number of advantages, such as reduced evaporation and no possibility of cross-contamination, but they are expensive.
  • Nanochips for proteomics have the same advantages, in addition to reduced cost and the capability of multiple-component reactions.
  • a well-known protein chip is the ProteinChip by Ciphergen Biosystems Inc.
  • the ProteinChip is based on the surface-enhanced laser desorption and ionization (SELDI) process.
  • Known proteins are analyzed using functional assays that are on the chip.
  • chip surfaces can contain enzymes, receptor proteins, or antibodies that enable researchers to conduct protein-protein interaction studies, ligand binding studies, or immunoassays.
  • the ProteinChip system detects proteins ranging from small peptides of less than 1000 Da up to proteins of 300 kDa and calculates the mass based on time-of- flight (TOF).
  • TOF time-of- flight
  • the ProteinChip biomarker system is a protein biochip-based system that enables biomarker pattern recognition analysis to be done. This system allows researchers to address important clinical questions by investigating the proteome from a range of crude clinical samples (i.e., laser capture microdissected cells, biopsies, tissue, urine, and serum). The system also utilizes biomarker pattern software that automates pattern recognition-based statistical analysis methods to correlate protein expression patterns from clinical samples with disease phenotypes.
  • Some systems can perform biomarker discovery in days and validation of large sample sets within weeks.
  • a robotics system accessory automates sample processing, allowing hundreds of samples to be run per week and enabling a sufficient number of samples to be run, which provides high statistical confidence in comprehensive studies for marker discovery and validation.
  • Microfluidic Chip-Based Immunoassays Microfluidics is a very important innovation in biochip technology. Since microfluidic chips can be combined with mass spectrometric analysis, a microfluidic device has been devised in which an electrospray interface to a mass spectrometer is integrated with a capillary electrophoresis channel, an injector, and a protein digestion bed on a monolithic substrate (Wang et al., 2000). This chip thus provides a convenient platform for automated sample processing in proteomics applications.
  • Biosite manufactures the Triage protein chip that simultaneously measures 100 different proteins by immunoassays.
  • the Triage protein chip immunoassays are performed in a microfluidic plastic chip, and the results are achieved in 15 minutes with picomolar sensitivities.
  • Microfluidic fluid flow is controlled in the protein chip by the surface architecture and surface hydrophobicity in the microcapillaries.
  • the immunoassays utilize high-affinity antibodies and a near-infrared fluorescent label, which is read by a fluorometer.
  • tissue microarray technology provides a high-throughput approach for linking genes and gene products with normal and disease tissues at the cellular level in a parallel fashion.
  • tissue microarrays provide increased throughput in two ways: up to 1000 tissue specimens can be analyzed in a single experiment, either at the DNA, RNA, or protein level; and tens of thousands of replicate tissue microarrays can be generated from a set of tissues. This process provides a template for analyzing many more biomarkers than has ever been possible previously in a clinical setting, even using archival, formalin-fixed specimens. 4.
  • Such low protein yields and proteins that can only be isolated from limited source material can be subjected to nanoscale protein analysis: a nanocapture of specific proteins and complexes, and optimization of all subsequent sample-handling steps, leading to a mass analysis of peptide fragments.
  • This focused approach also termed targeted proteomics, involves examining subsets of the proteome (e.g., those proteins that are specifically modified, bind to a particular DNA sequence, or exist as members of higher- order complexes or any combination thereof). This approach is used to identify genetic determinants of cancer that alter cellular physiology and respond to agonists.
  • Multiphoton detection by Biotrace Inc. (Cincinnati), can quantify subzeptomole amounts of proteins and can be used for diagnostic proteomics, particularly for cytokines and other low-abundance proteins. Biotrace is also developing supersensitive protein biochips to detect concentrations of proteins as low as 5 fg/ml (0.2 attomole/ml), thereby permitting sensitivity that is 1000 times greater than current protein biochips.
  • the active local synthesis of biomarker protein, in the kidney, may be confirmed by the detection of mRNA using in situ hybridization.
  • PCR-product representing the biomarker protein cDNA is cloned into an expression vector.
  • the vector is linearized enzymatically, followed by phenol extraction and precipitation under RNAse free conditions.
  • Run-off digoxigenine (DIG) labelled sense- and anti-sense mRNA transcripts are transcribed using polymerase.
  • Paraffin-embedded sections are deparaff ⁇ nated, washed and rehydrated through the graded series of ethanol.
  • the sections are postfixed in 4% paraformaldehyde in TBS, washed in TBS and digested with proteinase K in TE buffer. After washing in TBS, the digestion is stopped by incubating the sections in TBS.
  • the sections are prehybridized in 4 x SSC and
  • the probes are diluted in hybridization buffer and incubated before adding to the sections. The sections are then incubated. After incubation, any unbound probe is washed off. The sections are incubated with anti-DIG-AP Fab fragments. Then the sections are washed in TBS and colour detection is performed. The sections are background stained with Mayer's hematoxylin. Negative controls include adjacent sections of normal renal tissue incubated with sense mRNA probe.
  • kits relating to the identification of a biomarker of the present invention preferably includes a container means and an antibody which selectively recognizes the biomarker.
  • an immunoassay kit such as an ELISA kit, may be produced to test for the presence or absence of a biomarker or the present invention. Kits of the present invention may be used to determine if a patient has or is at risk of a renal disorder.
  • Kits to differentiate tubulo-interstitial diseases from glomerular diseases e.g., a tubulo-interstitial nephritis test
  • kits to differenciate the different types of glomerular diseases e.g., a minimal change test, a focal and segmental glomerulosclerosis test, a membranous glomerulopathy test, etc.
  • kits to identify vascular diseases e.g., a thrombotic microangiopathy test
  • kits for allograft nephropathy e.g., an acute allograft rejection test
  • Urines were obtained from 19 healthy individuals (7 males and 12 females, age 37.9 ⁇
  • Protease inhibitor cocktail Complete mini protease inhibitor cocktail, Roche, Basel, Switzerland
  • antibiotics Pen-Strep, Gibco, Invitrogen, CA, USA
  • Urine specimens were centrifuged at 2500 g for 15 minutes at room temperature immediately after collecting to remove whole cells and cell debris. The supernatants were frozen at -80 0 C, and stored until concentration and analysis.
  • Each urine sample was spun at 3'500 g for 30 minutes at room temperature using a 10-kDA cutoff centrifugal column (Centricon plus-80, Amicon, Beverly, MA, USA) until approximately 1/100 of initial volume remained.
  • the concentrated urine was desalted and buffered with an equal volume of Tris-HCl 1OmM pH 8.5 in the same column at 3'500 g for 30 minutes.
  • the concentrated urines of the 19 healthy individuals were pooled into 7 samples of 2-3 individuals (control samples).
  • the protein amounts in urine concentrates were measured using a Biorad protein microassay based on the method of Lowry (1951).
  • the immunocoated magnetic beads were washed three times in three different wash buffers according to the manufacturer's instructions and immediately incubated with ImI concentrated and ultracentrifuged urine sample for 4 hours at 37°C with gentle mixing.
  • the CRl positive podocyte vesicles immunoabsorbed on the beads were then isolated with a magnetic bead separator and eluted in 1ml of lysis buffer (PBS pH 7.4 containing 5 niM EDTA pH 8.0, Triton 1%, SDS 0.1% and ImM PMFS) overnight at 4 0 C with gentle mixing.
  • the lysis buffer containing the CRl -positive podocyte lysate was then separated from the beads and centrifuged at 16'0OO g for 20 minutes at 4°C.
  • the supernatant containing the soluble proteins extracted from the podocyte vesicles was recovered and the protein concentration was measured using the Lowry method.
  • Podocyte vesicles enrichment was assessed using immunoblot analysis.
  • Original urines, samples obtained after concentration and samples obtained after concentration and immunoabsorption on magnetic beads were compared. An equal proportion of the original urine volume was loaded onto gels.
  • the proteins were separated by SDS-PAGE and transferred onto PVDF membranes as described by (Towbin et ah, 1979). After blocking with 5% milk (Ih), membranes were probed overnight with podocalyxin (monoclonal anti-human podocalyxin antibody; 0.2 ⁇ g/ml, R and D systems, Minneapolis, MN, USA).
  • podocalyxin monoclonal anti-human podocalyxin antibody
  • Peroxydase- conjugated, affinity-purified donkey anti-rabbit (1 :100 OOO) Jackson Immuno-Research, West Grove, PA, USA was used for 90 min at room temperature.
  • the antibody-antigen reactions were visualized by using enhanced chemiluminescence plus Western Blotting Kit (Roche, Basel, Switzerland) and light-sensitive film (Kodak Biomax XAR, Rochester, NY, USA). The density of the bands was quantified by National Institutes of Health ImageJ software.
  • Supernatant proteins (80-100 ⁇ g) obtained from CRl immunoabsorption were separated by SDS-PAGE on homemade 7.5% and 15% T Tris-Glycine gels (8 x 5 x 0.15 cm). After the migration, gels were stained with an MS-compatible silver stain (Blum et ah, 1987) and all the well-stained bands were cut. The cut bands were then destained with 15mM K3Fe(CN)6, 5OmM Na 2 S 2 O 3 and washed with Milli-Q water (Millipore, Billerica, MA) using standard protocols (Gharahdaghi, 1999). The gel pieces were dehydrated in 100% CH3CN and dried in a vacuum centrifuge.
  • Peptides extracted following in-gel digestion were analyzed by LC-MS/MS as previously described (Burgess et al, 2006). Briefly, they were dissolved in 9 ⁇ l 5% CH 3 CN, 0.1% formic acid and 5 ⁇ l was loaded for LC-MS/MS analysis. A precolumn (100 ⁇ m inner diameter, 2-3.5 cm long) was connected directly to an analytical column (75 ⁇ m inner diameter, 9-10 cm long). Both columns were packed in-house with 5 ⁇ m, 3 A Zorbax Extend C- 18 (Agilent).
  • the eluate was sprayed directly into the nanoESI source of an LCQ DecaXP ion trap mass spectrometer (Thermo Finnigan, San Jose, CA) with a spray voltage of 1.8-2.2 kV.
  • Data dependant acquisition was used to automatically select 2 precursors for MS/MS from each MS spectrum (m/z range 400-1600).
  • MS/MS spectra were acquired with a normalized collision energy of 35%, an activation Q of 0.25 and an isolation width of 4 m/z. The activation time was 30 ms.
  • Dynamic exclusion was applied with a repeat count of 2, an exclusion time of 30 s, and an exclusion peak width of ⁇ 1.5 Da. Wideband activation was also applied. Maximum injection times of 50 ms and 200 ms were used for MS and MS/MS acquisitions, respectively, and the corresponding automatic gain control targets were set to 10 8 .
  • Peak lists were generated using Bioworks 3.1 software (Thermo Finnigan, San Jose,
  • the enrichment in podocyte vesicles was assessed by comparison of native urine, samples obtained after concentration and samples obtained after concentration and immunoabsorption on magnetic beads.
  • Western-blot analyses were performed using podocalyxin, a transmembrane protein specific of the apical surface of the podocyte, and a semiquantitative evaluation of the detected bands was done by densitometry analysis.
  • Podocalyxin which was not detectable in native urine sample, increased after concentration of normal urine samples and after concentration and immunoabsorption on magnetic beads. The increase after concentration and immunoabsoprtion was of about 13-20 fold in comparison with samples after concentration only.
  • the peak lists were searched against the UniProt combined Swiss-Prot and TrEMBL database restricted to human entries using Phenyx Virtual Desktop (Gene Bio, Switzerland) as previously described.
  • the acceptance criteria were more stringent than for the search of the Swiss Prot database alone (round 1 : AC score 16.0, peptide Z-score 8.0, peptide p-value 1 E-7; round 2: AC score 10.0, peptide Z-score 7.0, peptide p-value 1 E- 6).
  • PEDF pigment epithelium-derived factor, 46kDa
  • PEDF embryonic and neuroprotective factor, antiangiogenic factor, implicated in cell cycle and aging.
  • the protein is detected in normal kidney (human, murine, rat) and is immuno localized to the renal tubular epithelial cells of both cortex and medulla. In the rat kidney it is also detected in glomeruli with a staining suggestive of podocyte localization.
  • the protein was detected in normal human concentrated urine by Western blot analysis. An upregulation of the urinary protein levels was demonstrated in tubulo-interstitial nephritis (6/9) by Western blot analysis and ELISA. No or only slight modulation of the urinary protein levels was observed in glomerulonephritis (25/31). and in vascular disease
  • the inventors detected the protein in tubular cells (likely collecting tubes) by immunohistochemistry. No, or only a week, signal was detected in glomerular podocytes. In tubulo-interstitial nephritis, a decrease of the tubular immunostaining was observed (2/2). In glomerulopathies, the tubular immunostainign was similar to the normal controls (2/2). The hypothesis is that PEDF, in physiological condition, is filtered through glomerular barrier and reabsorbed by tubular cells. In case of tubular lesions, PEDF is nor more reabsorbed but excreted in high amount in urine.
  • FGF-I l Fibroblast growth factor 11 or FHF3 Fibroblast growth factor homologous factor 3, 25kDa
  • the protein was detected in normal human concentrated urine by Western blot analysis. No renal synthesis was observed with PCR .
  • Neuroligin 2 (9OkDa) is found to be expressed exclusively in the brain with no detectable specific signal in other tissues such as muscle (Scheiffele et al, 2000).
  • the protein was detected in normal human concentrated urine byWestern blot analysis. A renal synthesis was demonstrated with PCR analysis. A modulation of the urinary protein levels in glomerulopathies (FSGS and membranous GN) was observed by Western blot analysis. It is interesting to note that this modulation was similar to that observed with FGF-I l (for the same cases and in the same technical conditions).
  • Fibulin 3 (54 kDa), a protein with an important role in microfibrillar structures, is detected in the kidney around medium-sized vessels and some tubules, but only in restricted regions of the cortex (Giltay et ah, 1999).
  • the protein was detected in normal human concentrated urine by Western blot analysis. A renal synthesis was demonstrated with PCR analysis. A modulation of the urinary protein levels in glomerulopathies (FSGS and membranous GN) was observed by Western blot analysis.
  • PONl Serum paraoxonase/aryltransferase 1
  • PON-I was detected in only one urine sample. This urine was obtained from a patient with severe lupus nephritis and renal insufficiency. In order to confirm the presence of PONl in this pathological urine and to asses the possible modulation of the protein levels in case of renal disease, Western-blot analysis was performed on pathological and normal urines.
  • Protein samples from concentrated urines were separated by SDS-PAGE. Urines obtained from 3 normal individuals and from 3 patients with biopsy-proven renal diseases were analyzed. Proteins were transferred to PVDF membranes as described by Towbin et al. (1979). After blocking with 5% milk (Ih), membranes were probed overnight with polyclonal rabbit anti-human PON-I diluted 1/1000 (Atlas Antibodies AB, Sweden). Peroxydase-conjugated, affinity-purified donkey anti-rabbit (1 :100 OOO) (Jackson Immuno-Research, West Grove, PA, USA) was used for 90 min at room temperature.
  • the antibody-antigen reactions were visualized by using enhanced chemiluminescence plus Western Blotting Kit (Roche, Basel, Switzerland) and light-sensitive film (Kodak Biomax XAR, Rochester, NY, USA). Whole kidney tissue extract (40 ⁇ g) was also analysed. Liver (40 ⁇ g) was used as positive control.
  • Normal renal tissue was obtained from normal parts of four nephrectomies performed for renal carcinoma. Histological sections (3 ⁇ m) were obtained from formaldehyde-fixed paraffin-embedded tissues. Sections were deparaffinized and endogeneous peroxidase was blocked with a peroxydase blocking solution (DAKO, Gostrup, Denmark) for 10 minutes at RT. The sections were treated for antigen retrieval with microwave irradiation in citrate buffer saline for 10 min , and incubated with the primary polyclonal rabbit anti-human PON- 1 antibody (Atlas Antibodies AB, Sweden) at a 1 :200 dilution overnight at 4 0 C.
  • DAKO peroxydase blocking solution
  • Negative controls included adjacent sections of normal renal tissue incubated with rabbit IgG fraction, bovine serum albumin and buffer alone.
  • PON-I protein was detected exclusively at three different locations in the kidney: the glomeruli, the tubules and the vessels.
  • PON-I was detected in podocytes.
  • some staining was detected in parietal epithelial cells of the Bowman's capsule as well as in the urinary Bowman's space, likely corresponding to the primitive urine as suggested by the positive staining observed in the tubular pole of the glomerulus.
  • Glomerular endothelial and mesangial cells were negative.
  • PON-I was detected in the cytoplasm of distal tubules, both in cortex and in medulla..
  • a luminal positive staining was noted in some of the cortical and medullary tubules, most likely corresponding to urine.
  • PON-I protein was detected in the arteries, in the smooth muscle cells of the media. Large and small arteries as well as arterioles of both cortex and medulla were positive.
  • a positive but weaker staining was also detected in the endothelial cells of the cortical and medullary arteries.
  • a positive staining was detected in the lumen of vessels, likely corresponding to the serum components.
  • No staining was detected in the endothelial cells of veins and peritubular capillaries.
  • the other structures in the cortex and medulla including the interstitial cells were negative. No glomerular, tubular or vascular staining was detected with rabbit immunoglobulin (Ig) G used a as a negative control.
  • Normal human renal tissue was obtained from normal parts of 3 nephrectomies performed for renal cell carcinoma. In addition, for one nephrectomy, cortex was separated from medulla. Tissues were disrupted with a Tissue Taeror (Biospec Products, Racine, WI).
  • Primer pairs were obtained from Clontech (La Jolla, CA, USA). Mock reactions devoid of cDNA served as negative controls. PCR products were analyzed by agarose gel electrophoresis followed by staining with ethidium bromide.
  • Neuropilin-2 (NP-2) is a cell surface transmembrane protein originally characterized as a receptor for the type 3 semaphorins, and more recently for a number of vascular endothelial growth factor (VEGF) isoforms (Cohen et al, 2002).
  • VEGF vascular endothelial growth factor
  • NP-2 expression was detected in the CNS, lung, mesenteric, muscular, and submucosal plexuses of the intestine and in bone and cartilage (Chen et al, 1997; Giger et al, 1998).
  • the expression pattern of NP-2 in adult mice or in human tissue outside the CNS is not well documented.
  • NP-2 was reported to be expressed by a subset of serotonin-expressing neuroendocrine cells in the stomach and small and large intestines and in pancreas in the islet periphery in co-localization with glucagons-expressing (alpha) cells (Cohen et al, 2001; Cohen et al, 2002).
  • X 2 macroglobulin ( ⁇ 2 M) is a 720 kDA glycoprotein which is mainly synthesized in the liver (de Sain-van der Velden, 1998). Increased levels of ⁇ 2 M are well described in patients with nephrotic syndrome regardless of the primary disease (Vazirdi, 1994). (X 2 M, which was identified in human urine, has been suggested to be a marker of glomerular diseases.
  • the protein was detected in normal human concentrated urine by Western blot analysis. A modulation of the urinary protein levels in glomerulopathies was observed by Western blot analysis.

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Abstract

La présente invention porte sur des procédés pour déterminer si un patient a ou est à risque de développer un trouble rénal. Dans certains modes de réalisation, des biomarqueurs spécifiques sont fournis, lesquels peuvent être utilisés pour déterminer si un patient a ou est à risque de développer un trouble rénal (par exemple, une insuffisance rénale aiguë).
PCT/IB2007/004395 2006-06-21 2007-06-20 Biomarqeurs pour des troubles rénaux Ceased WO2008084331A2 (fr)

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