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WO2018094021A1 - Résistance aux stéroïdes dans le syndrome néphrotique - Google Patents

Résistance aux stéroïdes dans le syndrome néphrotique Download PDF

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Publication number
WO2018094021A1
WO2018094021A1 PCT/US2017/061974 US2017061974W WO2018094021A1 WO 2018094021 A1 WO2018094021 A1 WO 2018094021A1 US 2017061974 W US2017061974 W US 2017061974W WO 2018094021 A1 WO2018094021 A1 WO 2018094021A1
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Prior art keywords
steroid resistance
steroid
biomarker
subject
protein
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Shipra Agrawal
William SMOVER
Jon B. Klein
Michael L. Merchant
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Nationwide Childrens Hospital Inc
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Nationwide Childrens Hospital Inc
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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
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/30—Unsupervised data analysis
    • 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
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding

Definitions

  • the presently-disclosed subject matter relates to methods for treating and identifying steroid resistance in subjects having nephrotic syndrome.
  • certain embodiments of the presently-disclosed subject matter relate to systems and methods for identifying steroid resistance in nephrotic syndrome that make use of biomarkers to predict treatment responsiveness and thereby reduce steroid-induced toxicity.
  • Glomerular disease is the third leading cause of end stage kidney disease in the U.S. and its related health care costs are approximately $4.1 billion annually.
  • Nephrotic syndrome (NS) is one of the most common forms of glomerular disease, and is a leading cause of end stage kidney disease (ESKD) in children.
  • EKD end stage kidney disease
  • Steroids are the primary therapy for NS, but 20-50% of patients fail to achieve a remission and are characterized as steroid resistant (SRNS).
  • SRNS steroid resistant
  • Currently, the only method of diagnosis is an invasive biopsy, which is typically not performed in children until first-line treatments fail. Unfortunately, in the absence of biomarkers to predict treatment responsiveness, many patients receive prolonged yet ineffective therapy, leaving them at high risk for both toxic side effects and disease progression.
  • the presently-disclosed subject matter is based, at least in part, on the identification of steroid resistance biomarkers through the use of mass spectrometry-based analyses of serial clinically phenotyped pediatric biological samples and on the subsequent identification of novel biomarkers that correlate-with/predict treatment response. In some embodiments, these biomarkers also aid in the identification of molecular targets for targeted nephrotic syndrome disease treatments.
  • the methods advantageously allow for point of care diagnostic testing and clinical reference laboratory assays to identify for subjects having glomerular disease, such as nephrotic syndrome, that require alternative treatment with non-steroidal methods such as administration of calcineurin inhibitors such as cyclosporine .
  • the methods allow for further applied research and drug development, such as to what may be useful to identify and phenotype pediatric patients that do not respond to steroid treatment such that these patients can be used in separate genomic studies or therapeutic clinical trials.
  • the methods allow for applied research and drug development that is useful to identify and characterize molecular regulators of steroid resistance or relapse.
  • the methods described herein are used to develop a strategy to identify other mechanisms to treat nephrotic syndrome in pediatric patients.
  • Figure 1 provides a scheme representing the "omic" analyses of paired (before and after 6-12 weeks of steroid therapy) SSNS and SRNS samples, which identified novel steroid resistance biomarkers and identified proteins involved in the molecular pathways of steroid resistance.
  • Figure 2 provides a scheme showing the proteomics workflow used by the inventors to identify biomarkers for steroid resistance and the genes and proteins involved in the mechanism of steroid resistance.
  • Figure 3 provides graphs showing the effects of steroid response on the relative abundance of the 13 steroid resistance biomarker proteins.
  • the graphs are plots of median iBAQ areas with interquartile ranges. All plots are for proteins significantly different at the pre-steroid treatment timepoint. Post time point comparator added for illustration purposes.
  • Figure 4 provides a heat map overview of differentially expressed proteins among SSNS and SRNS patients.
  • Figure 5 provides a scheme showing the Ingenuity Pathway analyses of steroid resistance biomarkers, suggesting five canonical pathways (p-value ⁇ 0.01) and one significant protein- protein network.
  • the inventors have described methods for identifying steroid resistance in subjects having glomerular disease such as nephrotic syndrome.
  • certain embodiments of the presently-disclosed subject matter includes methods for identifying steroid resistance in pediatric nephrotic syndrome that make use of biomarkers to predict treatment responsiveness and thereby reduce steroid-induced toxicity.
  • Treat", “treating”, and “treatment”, etc. refer to any action providing a benefit to a subject at risk for or afflicted with a condition or disease such as nephrotic syndrome, including improvement in the condition through lessening or suppression of at least one symptom, such as proteinuria or hypoalbuminemia, delay in progression of the disease, prevention or delay in the onset of the disease, etc.
  • the subject may be at risk due to the presence of a risk factor such as heart failure, hepatitis, diabetes, being genetically predisposed to nephrotic syndrome, and so on.
  • the term "in need of treatment” as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.
  • a "subject”, as used therein, can be a human or non-human animal, and is sometimes referred to as "the patient.”
  • Non-human animals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals, as well as reptiles, birds and fish.
  • the subject is a mammal, while in further embodiments the subject is human.
  • a subject can also be a pediatric human, which is a human having an age of 21 years or less. In some embodiments, the subject has an age of 17 years or less, while in further embodiments the subject has an age of 3 years or less.
  • diagnosis can encompass determining the nature of disease or condition in a subject, as well as determining the severity and probable outcome of disease or episode of disease or condition, and/or the prospect of recovery (prognosis).
  • diagnosis can also encompass diagnosis in the context of rational therapy, in which the diagnosis guides therapy, including initial selection of therapy, modification of therapy (e.g. , adjustment of dose and/or dosage regimen), and the like.
  • polynucleotide refers to oligonucleotides, nucleotides, or to a fragment of any of these, to DNA or RNA (e.g., mRNA, rRNA, tRNA) of genomic or synthetic origin which may be single-stranded or double-stranded and may represent a sense or antisense strand, to peptide nucleic acids, or to any DNA-like or RNA-like material, natural or synthetic in origin, including, e.g. , iRNA, siRNAs, microRNAs, and ribonucleoproteins.
  • the term also encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides, as well as nucleic acid-like structures with synthetic backbones.
  • polypeptide and “polypeptide” are used interchangeably herein and refer to a compound made up of a chain of amino acid residues linked by peptide bonds.
  • An "active portion" of a polypeptide means a peptide that is less than the full length polypeptide, but which retains measurable biological activity and retains biological detection.
  • gene refers to a nucleotide sequence that can direct synthesis of an enzyme or other polypeptide molecule (e.g., can comprise coding sequences, for example, a contiguous open reading frame (ORF) which encodes a polypeptide) or can itself be functional in the organism.
  • ORF open reading frame
  • a gene in an organism can be clustered within an operon, as defined herein, wherein the operon is separated from other genes and/or operons by intergenic DNA. Individual genes contained within an operon can overlap without intergenic DNA between the individual genes.
  • the term "steroid" is used to encompass both corticosteroids and glucocorticoids.
  • the steroid can be chosen from but is not limited to beclomethasone, prednisolone, methylprednisolone, fluticasone, triamcinolone, budesonide, glucocorticoids, or dexamethasone, including equivalent drugs.
  • the term "therapeutically effective" is intended to qualify the amount of each agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies.
  • the therapeutically effective amount may be administered in one or more doses.
  • An effective dose is an amount sufficient to provide a certain effect, such as enzyme inhibition, but may or may not be therapeutically effective.
  • biomarkers provide a unique opportunity to define a novel set of serum proteins whose collective abundances change with disease and response to therapy. Additionally, the biomarkers provide prognostic and diagnostic information on response to therapy for pediatric patients suffering from nephrotic syndrome and, in certain embodiments, the biomarkers provide information to guide which patients might best need a biopsy.
  • the present invention provides a method for diagnosing steroid resistance in a subject having a glomerular disease such as nephrotic syndrome.
  • the method includes the steps of (a) providing a biological sample from the subject; (b) determining the amount of at least one steroid resistance biomarker selected from Table 1 in the biological sample; and (c) comparing the amount of the at least one steroid resistance biomarker to a control level of the at least one steroid resistance biomarker, wherein the subject is diagnosed as having steroid resistance or a risk thereof if there is a difference in the amount of the at least one steroid resistance biomarker in the sample as compared to the control level.
  • the inventors have identified a number of proteins and their corresponding genes that can be used to diagnose steroid resistance in a subject having glomerular disease (e.g., nephrotic syndrome). These proteins and their corresponding genes are referred to herein as steroid resistance biomarkers.
  • the steroid resistance biomarkers identified by the inventors are shown in Table 1. The ratios are expressed by comparing the levels in steroid sensitive nephrotic syndrome patients with the results steroid resistance nephrotic syndrome patients. The Log2 value provided in Table 1 also indicates if the biomarker decreases, in which case it shows a positive value, or increases, in which case it shows a negative value, in subject who have steroid resistance.
  • Table 1 Proteins whose plasma abundance are associated with steroid resistance in pediatric nephrotic syndrome patients (SSNS Pre vs. SRNS Pre)
  • Collagen alpha-3(VI) chain acts as a cell binding protein.
  • Insulin-like growth factor- binding protein 2 inhibits IGF-mediated growth and development rates and prolongs the half-life of IGFs.
  • 72 kDa type IV collagenase is a ubiquitous metalloproteinase that is involved in a variety of functions such as vasculature remodeling, tissue repair, and degradation of extracellular matrix proteins.
  • Apolipoprotein E mediates the binding, internalization, and catabolism of lipoprotein particles.
  • Adiponectin is an important adipokine involved in the control of fat metabolism and insulin sensitivity.
  • Sex hormone-binding globulin functions as an androgen transport protein and regulates the plasma metabolic clearance rate of steroid hormones by controlling their plasma concentration.
  • EGF-containing fibulin-like extracellular matrix protein 1 binds EGFR receptor, inducing EGFR autophosphorylation and the activation of downstream signaling pathways
  • inter-alpha-trypsin inhibitor heavy chain H4 is a type II acute- phase protein (APP)
  • vitamin D-binding protein is involved in vitamin D transport and the enhancement of the chemotactic activity of C5 alpha for neutrophils in inflammation and macrophage activation.
  • Antithrombin III is a serine protease inhibitor in plasma
  • zinc-alpha-2 glycoprotein stimulates lipid degradation in adipocytes
  • fetuin-B is a protease inhibitor.
  • the amount of a plurality of steroid resistance biomarkers is determined.
  • Examples of a plurality of steroid resistance biomarkers include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 steroid resistance biomarkers.
  • the steroid resistance biomarker is one that shows an increased level in subjects having steroid resistance. In other embodiments, the steroid resistance biomarker is one that shows a decreased level in subjects having steroid resistance.
  • the steroid resistance biomarker is selected from adiponectin, EGF-containing fibulin-like extracellular matrix protein 1, inter- alpha-tryp sin inhibitor heavy chain H4, hemopexin, antithrombin-III, and vitamin D-binding protein.
  • steroid resistant includes both acquired steroid resistance (Type I) and primary steroid resistance (Type II).
  • steroid unresponsiveness is a dynamic scale and there are varying degrees of unresponsiveness. From a clinical perspective, the diagnosis of steroid unresponsiveness is often based on the physician's judgment and treatment history with steroids. For example, in nephrotic syndrome, increasing the dose of steroids for a certain period of time without achieving symptomatic relief may be indicative of a possible state of steroid unresponsiveness in that individual.
  • the method includes the steps of providing a biological sample from the subject.
  • Biological samples include, but are not necessarily limited to bodily fluids such as urine and blood-related samples (e.g., whole blood, serum, plasma, and other blood-derived samples), urine, cerebral spinal fluid, bronchoalveolar lavage, and the like.
  • blood-related samples e.g., whole blood, serum, plasma, and other blood-derived samples
  • urine cerebral spinal fluid
  • bronchoalveolar lavage e.g., bronchoalveolar lavage
  • Another example of a biological sample is a tissue sample.
  • the levels of steroid resistance biomarkers in a biological sample taken from the subject can be assessed either quantitatively or qualitatively, usually quantitatively.
  • the levels of steroid resistance biomarkers can be determined either in vivo or in vitro.
  • the biological sample comprises blood, plasma, or serum.
  • the biological sample can be provided by immediately obtaining a biological sample from the subject, or a biological sample can be provided that was previously obtained.
  • a biological sample may be fresh or stored (e.g. blood or blood fraction stored in a blood bank). Samples can be stored for varying amounts of time, such as being stored for an hour, a day, a week, a month, or more than a month.
  • the biological sample may be a bodily fluid expressly obtained for the assays of this invention or a bodily fluid obtained for another purpose which can be subsampled for the assays of this invention.
  • the method also includes determining the amount of at least one steroid resistance biomarker in the biological sample.
  • the steroid resistance biomarker is a polynucleotide, while in other embodiments, the steroid resistance biomarker is a polypeptide.
  • the level of steroid resistance biomarker in a subject can be measured using an analytic device, which is a machine including a detector capable of identifying steroid resistance biomarkers and fragments thereof. The methods used to determine the amount of the steroid resistance biomarker will vary depending on whether proteins or gene expression is being detected.
  • the analytic device may be a spectrometric device, such as a mass spectrometer, an ultraviolet spectrometer, or a nuclear magnetic resonance spectrometer.
  • a spectrometer is a device that uses a spectroscopic technique to assess the concentration or amount of a given species in a medium such as a biological sample (e.g., a bodily fluid).
  • the analytic device used to measure the levels of steroid resistant biomarker can be either a portable or a stationary device.
  • the analytic device can also include additional equipment to provide purification (i.e. , physical separation) of analytes prior to analysis.
  • the analyte detector is a mass spectrometer, it may also include a high performance liquid chromatograph (HPLC) or gas chromatograph (GC) to purify the steroid resistance biomarkers before their detection by mass spectrometry, or it may be preferable to purify the protein using gel electrophoresis.
  • HPLC high performance liquid chromatograph
  • GC gas chromatograph
  • mass spectrometry-based methods can be used to assess levels of steroid resistance biomarkers in a biological sample.
  • Mass spectrometers include an ionizing source (e.g., electrospray ionization), an analyzer to separate the ions formed in the ionization source according to their mass-to-charge (m/z) ratios, and a detector for the charged ions.
  • ionizing source e.g., electrospray ionization
  • analyzer to separate the ions formed in the ionization source according to their mass-to-charge (m/z) ratios
  • detector for the charged ions e.g., m/z ratios
  • tandem mass spectrometry two or more analyzers are included.
  • Such methods are standard in the art and include, for example, HPLC with on-line electrospray ionization (ESI) and tandem mass spectrometry.
  • ESI on-line electrospray ionization
  • Polypeptide steroid resistance biomarkers may be determined by any of a variety of standard or novel recently developed protein analytic methods known in the art. These methods include absorbance, gel electrophoresis (e.g. , SDS-PAGE gel purification), a protein immunoblot (e.g. , western blot), chromatography (e.g. , size exclusion chromatography, ion exchange chromatography, and affinity chromatography), precipitation, ultracentrifugation, an immunoassay, such as an enzyme-linked immunosorbent assays (ELISA), and other common techniques known to one of ordinary skill in the art. Novel recently developed assays include bead based immunofluorescence assasys that can quantitatively measure different proteins in the same well with high sensitivity.
  • ELISA enzyme-linked immunosorbent assays
  • An immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g., a biomarker).
  • An immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, polyclonal antibodies raised to a biomarker from specific species such as rat, mouse, or human can be selected to obtain only those polyclonal antibodies that are specifically reactive with that biomarker and not with other proteins, except for polymorphic variants and alleles of the biomarker. This selection may be achieved by subtracting out antibodies that cross-react with the biomarker molecules from other species.
  • Immunoassays can be run using a variety of different formats, including competitive homogenous immunoassays, heterogeneous immunoassays, one-site non-competitive immunoassays, and two-site noncompetitive immunoassays.
  • Antibodies include polyclonal and monoclonal antibodies, as well as antibody fragments that contain the relevant antigen binding domain of the antibodies.
  • antibody refers to immunoglobulin molecules or other molecules which comprise at least one antigen-binding domain.
  • antibody as used herein is intended to include whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, multi- specific antibodies, single chain antibodies, epitope- binding fragments, e.g., Fab, Fab' and F(ab') 2 , Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, and totally synthetic and recombinant antibodies.
  • the antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • type e.g., IgG, IgE, IgM, IgD, IgA, and IgY
  • class e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2
  • subclass of immunoglobulin molecule e.g., immunoglobulin molecule.
  • Antibodies are designed for specific binding, as a result of the affinity of complementary determining region of the antibody for the epitope of the biological analyte.
  • An antibody “specifically binds” when the antibody preferentially binds a target structure, or subunit thereof, but binds to a substantially lesser degree or does not bind to a biological molecule that is not a target structure.
  • the antibody specifically binds to the target steroid resistance biomarker with a specific affinity of between 10 - " 8 M and 10 - " 11 M.
  • an antibody or antibody fragment binds to the steroid resistance biomarker with a specific affinity of greater than 10 "7 M, 10 "8 M, 10 “9 M, 10 “10 M, or 10 "11 M, between 10 "8 M - 10 "11 M, 10 “9 M - 10 "10 M, and 10 "10 M - 10 "11 M.
  • Specific activity can be measured using a competitive binding assay as set forth in Ausubel FM, (1994). Current Protocols in Molecular Biology. Chichester: John Wiley and Sons ("Ausubel”), which is incorporated herein by reference.
  • Polynucleotide steroid resistance biomarkers may be determined by any of a variety of standard analytic methods known in the art.
  • the expression level of a biomarker gene (which is a polynucleotide) is determined by nucleic acid amplification of said gene or RNA produced by the gene using gene specific primer pairs, and quantifying the amplification results.
  • the nucleic acid amplification method used is realtime polymerase chain reaction (PCR) analysis.
  • RNA isolated from a sample is separated by agarose gel electrophoresis, and probed with a complementary DNA probe specific for the gene of interest.
  • PCR polymerase chain reaction
  • the total RNA isolated from the cell or tissue to be analyzed is reverse transcribed into first strand cDNA (RT-PCR), which is then used as a template to amplify a double stranded amplicon with target specific oligonucleotide primers.
  • RT-PCR first strand cDNA
  • detection is based on detectable labels, such as fluorescent dyes or radioactive isotopes.
  • DNA chips or microarrays are based on hybridization the target DNA to complementary target specific primers, washing out the unbound DNA and quantifying the bound target DNA.
  • Probes and primers used in the hybridization reactions may be designed based on the nucleotide sequence of the marker gene or amino acid sequence of the translated protein, corresponding to the marker gene.
  • a convenient quantitative hybridization method for determining variations in the amounts of expressed RNA is described in the international patent application WO 2002/055734.
  • biomarker gene expression may be quantified with real time PCR, also called quantitative real time PCR.
  • the method follows the general pattern of polymerase chain reaction, but the amplified region of the target DNA is quantified after each round of amplification by using fluorescent dyes, such as SYBR Green that intercalate with double- stranded DNA or modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
  • fluorescent dyes such as SYBR Green that intercalate with double- stranded DNA or modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
  • real time PCR is combined with reverse transcription to quantify low abundance mRNA.
  • the data can be analyzed by computer software, such as Applied Biosystems 7500 or 7500 Fast Real Time PCR Systems, to calculate relative gene expression between several samples, or mRNA copy number based on a standard curve.
  • Relative quantification is commonly used to compare expression levels of wild-type with mutated alleles or the expression levels of a gene in different tissues.
  • RQ determines the change in expression of a target gene in a test sample relative to the same sequence in a basal or calibrator sample (a sample used as the basis for comparative results).
  • the calibrator sample can be an untreated control or a sample at time zero in a time-course study.
  • housekeeping genes such as ⁇ -actin, glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) and ribosomal RNA (rRNA) are used as endogeneous controls, because their expression levels are relatively stable. Replicate reactions per sample and an endogenous control are needed to ensure statistical significance.
  • GPDH glyceraldehyde- 3-phosphate dehydrogenase
  • rRNA ribosomal RNA
  • Primer pairs are designed to hybridize or anneal to opposing strands of the DNA encoding the marker gene of interest such that through PCR amplification, a defined region of the marker gene is produced.
  • a "primer pair” thus refers to two primers, one having a forward designation and the other having a reverse designation relative to their respective orientations on a double- stranded DNA molecule, also called a sense and antisense sequence, such that under the PCR amplification conditions known to those skilled in the art, the forward primer anneals to and primes the amplification of the sense sequence and reverse primer anneals to and primes amplification of the antisense sequence.
  • Primers can be selected for use in the amplification reaction on the basis of, having minimal complementarity with other primers in the reaction (to minimize the formation of primer dimers) and having Tm values with the range of reaction temperatures appropriate for the amplification method, preferably PCR.
  • primers can be selected so as to anneal with specific regions of RNA template such that the resulting DNA amplification product ranges in size from 50 to 1000 base pairs, preferably from 50 to 300 base pairs in length and most preferably around 150 base pairs in length.
  • Primers typically from 10 to 100 nucleotides, preferably from 10 to 60 nucleotides in length, include naturally occurring or synthetically produced oligonucleotides capable of annealing to the target nucleic acids and acting as the point of initiation of nucleic acid synthesis under appropriate conditions, i.e., in the presence of nucleoside triphosphates, a polymerization agent, suitable temperature, pH and buffer.
  • oligonucleotide primers as well as performing the PCR and detection of the amplified DNA products are well known in the art.
  • Methods of the invention also include the step of comparing the amount of the at least one steroid resistance biomarker to a control level of the at least one steroid resistance biomarker.
  • the steroid resistance biomarker levels are compared with the control levels for the appropriate corresponding steroid resistance biomarkers; e.g., the level of vitamin D-binding protein in the biological sample is compared with the control level of vitamin D-binding protein.
  • Control levels of steroid resistance biomarkers represent the levels of the steroid resistance biomarkers in subjects who do not have significant steroid resistance.
  • the control levels may be already available in the literature, they may be determined by evaluating biomarker levels in a pool of relatively healthy subjects expected to exhibit normal responsiveness to steroids, or they may be determined in an individual prior to the development of steroid resistance.
  • the comparison can be conducted by any suitable method known to those skilled in the art.
  • the comparison can be carried out mathematically or qualitatively by an individual operating the analytic device or by another individual who has access to the data provided by the analytic device.
  • the steps of determining and comparing the levels of steroid resistance biomarkers can be carried out electronically (e.g., by an electronic data processor).
  • the invention provides a method treating a subject having glomerular disease such as nephrotic syndrome.
  • the method includes the steps of (a) providing a biological sample from the subject; (b) determining the amount of at least one steroid resistance biomarker selected from Table 1 in the biological sample; and (c) comparing the amount of the at least one steroid resistance biomarker to a control level of the at least one steroid resistance biomarker; (d) treating the subject for glomerular disease (e.g., nephrotic syndrome) using a therapy other than steroid treatment if there is a difference in the amount of the at least one steroid resistance biomarker in the sample as compared to the control level.
  • glomerular disease e.g., nephrotic syndrome
  • Glomerular disease affect the function of the kidneys and the glomeruli, which are small units within the kidney where blood is cleaned. Glomerular diseases include many conditions with a variety of genetic and environmental causes, but they fall into a few major categories: Minimal Change Disease, which describes effacement of podocyte foot processes, that serves as a filter; Glomerulonephritis, which describes the inflammation of the membrane tissue in the kidney that serves as a filter, separating wastes and extra fluid from the blood, and glomerulosclerosis, which describes the scarring or hardening of the tiny blood vessels within the kidney.
  • Minimal Change Disease which describes effacement of podocyte foot processes, that serves as a filter
  • Glomerulonephritis which describes the inflammation of the membrane tissue in the kidney that serves as a filter, separating wastes and extra fluid from the blood
  • glomerulosclerosis which describes the scarring or hardening of the tiny blood vessels within the kidney.
  • glomerulonephritis and glomerulosclerosis have different causes, they can both lead to kidney failure.
  • examples of glomerular disease include nephrotic syndrome, minimal change disease, diabetic nephropathy, and other conditions known to those skilled in the art.
  • Glomerular diseases damage the glomeruli, letting protein and sometimes red blood cells leak into the urine. Glomerular disease can also interfere with the clearance of waste products by the kidney, so they begin to build up in the blood. Furthermore, loss of blood proteins like albumin in the urine can result in a fall in their level in the bloodstream. When albumin leaks into the urine, the blood loses its capacity to absorb extra fluid from the body. Fluid can accumulate outside the circulatory system in the face, hands, feet, or ankles and cause swelling. Symptoms of glomerular disease include albuminuria, hematuria, reduced glomerular filtration rate, proteinuria, and edema.
  • Nephrotic syndrome is a general term that refers to the loss of protein in the urine (proteinuria), hyperlipidemia (hypercholesterolemia and hypertriglyceridemia), and edema. Nephrotic syndrome involves changes in the pathology of cells in the kidney, such as podocytes. Many conditions are categorized as nephrotic syndromes, including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) (also called membranous glomerulonephritis, MGN), and membranoproliferative glomerulonephritis (MPGN).
  • MCD minimal change disease
  • FGS focal segmental glomerulosclerosis
  • MN membranous nephropathy
  • MGN membranous nephropathy
  • MGN membranous glomerulonephritis
  • MPGN membranoproliferative glomerulonephritis
  • nephrotic syndrome A variety of signs and symptoms are known to be associated with nephrotic syndrome. These include respiratory tract infection, allergy, macrohematuria, symptoms of infection, hypotension, respiratory distress, tachypnea, seizure, anorexia, irritability, fatigue, and disarrhea. Nephrotic syndrome can be diagnosed using urinalysis, urine protein quantification, serum albumin quantification, and a lipid panel. With the advent of electron microscopy, the changes now known as the hallmarks for the disease include diffuse loss of podocyte foot processes, vacuolation of the podocyte foot processes, and growth of microvilli on the visceral epithelial cells. Diabetic nephropathy is the most common cause of nephrotic syndrome.
  • Nephrotic syndrome is defined by the presence of nephrotic -range proteinuria, edema, hyperlipidemia, and hypoalbuminemia. While nephrotic-range proteinuria in adults is characterized by protein excretion of 3.5 g or more per day, in children it is defined as protein excretion of more than 40 mg/m fh or a first-morning urine protein/creatinine of 2-3 mg/mg creatinine or greater. In some embodiments, the nephrotic syndrome is pediatric nephrotic syndrome, which is the occurrence of pediatric nephrotic syndrome in a child.
  • the methods of treatment described herein include determining whether the subject has steroid resistance by determining the levels of steroid resistance biomarkers in the subject and comparing them with the corresponding control values.
  • the steroid resistance biomarker can be a protein (or its corresponding gene) including one or more of the biomarkers selected from collagen alpha-3(VI) chain, insulin-like growth factor-binding protein 2, 72 kDa type IV collagenase, apolipoprotein E, adiponectin, sex hormone-binding globulin, EGF-containing fibulin-like extracellular matrix protein, inter-alpha-trypsin inhibitor heavy chain H4, hemopexin, vitamin D-binding protein, antithrombin-III, zinc-alpha-2-glycoprotein, and fetuin-B.
  • the method includes determining the amount of a plurality of steroid resistance biomarkers.
  • the level of the steroid resistance biomarker is increased in subjects having steroid resistance, while in other embodiments the level of the steroid resistance biomarker is decreased in subjects having steroid resistance. Whether or not an increase or decrease in the level of the steroid resistance biomarker is associated with steroid resistance depends on the particular biomarker being evaluated. [0056] Determining that a subject is steroid resistance can be used to guide treatment of the subject.
  • Patients with steroid resistance have a more progressive disease course and related poor outcomes when compared with patients that are steroid-sensitive.
  • glomerular disease e.g., nephrotic syndrome
  • a variety of non-steroid methods for treating nephrotic syndrome are known. See Tune B. and Mendoa S., J Am Soc Nephrol., 8(5), 824-32 (1997), the disclosure of which is incorporated herein by reference.
  • Examples of therapy other than steroid treatment include nutritional therapy, administration of a diuretic, and administration of a non-steroidal immunosuppressant.
  • Non-steroidal immunosuppressants suitable for treatment of nephrotic syndrome include cytotoxic drugs such as cyclophosphamide and chlorambucil, calcineurin inhibitors such as cyclosporine and tacrolimus, inosine monophosphate dehydrogenase (IMPDH) inhibitors such as mycophenolate mofetil and mizoribine, and the anti-CD20 antibody rituximab.
  • Other non-steroidal methods for treating nephrotic syndrome include administration of the immuno stimulant levamisole and non-immunological methods such as administration of vitamin E.
  • Diuretics such as furosemide can be used to control edema in nephrotic syndrome.
  • Nutritional therapy based on a diet with the correct energy intake and balance of proteins that will be used in synthesis processes and not as a source of calories that minimizes protein intake can also be used.
  • kits comprising one or more biomarker- specific probes and a package for holding the probes.
  • probes include antibodies and polynucleotide primers.
  • Suitable packages include, for example, bottles, vials or syringes. The package may be formed from a variety of materials such as glass or plastic.
  • the container holds a composition that may be effective for treating the condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierce-able by a hypodermic injection needle).
  • the label or package insert may indicate that the composition may be used for identifying steroid resistance in subjects having glomerular disease such as nephrotic syndrome.
  • the kit comprises specific primer pairs for enabling in vitro analysis of expression level of at least one biomarker gene and instructions for use.
  • the kit comprises means for carrying out hybridization or amplification assay.
  • the kit comprises antibodies that specifically bind to at least one steroid resistance biomarker protein, and instructions for use.
  • the kit comprises means for carrying to the specific binding, and detection of binding.
  • the antibody or fragment thereof that specifically binds to a steroid resistance biomarker protein can be any of the antibodies described herein.
  • the antibody is selected from the group of antibodies consisting of Fab2, Fab4, and Fab6, or variants thereof including only conservative sequence modifications.
  • the reagents may be supplied in a solid (e.g., lyophilized) or liquid form.
  • the kits of the present invention may optionally comprise different containers (e.g., vial, ampoule, test tube, flask or bottle) for each individual buffer, cell type and/or reagent. Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the disclosed methods may also be provided.
  • the individual containers of the kit are preferably maintained in close confinement for commercial sale.
  • the kit can include a carrier for the various components of the kit.
  • the carrier can be a container or support in the form of, e.g., a bag, box, tube or rack, and is optionally compartmentalized.
  • the carrier may define an enclosed container for safety purposes during shipment and storage.
  • the kit further includes instructions for using the kit to detect steroid resistance in a subject having glomerular disease (e.g., nephrotic syndrome). While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions" can include the address of an internet site that provides the instructions.
  • the inventors have also identified a number of proteins that appear to be involved in the molecular mechanism of steroid resistance.
  • Table 2 lists sixty six (66) proteins that change levels coordinately between SSNS and SRNS with steroid treatment. Twenty-four (24) proteins have discoordinated changes in serum abundance with steroid treatment including 14 that increase in SSNS and 10 that decrease in SSNS by 7-weeks post corticosteroid therapy.
  • Fibulin-2 (EGF-containing fibulin-like extracellular matrix
  • IGFBP2 Insulin-like growth factor-binding protein 2 IGFBP2 -/-
  • the proteins and their associated genes can be targeted to either suppress or increase their expression (as appropriate) in order to provide new methods for decreasing steroid resistance in glomerular disease such as nephrotic syndrome.
  • the effectiveness of candidate drugs can be evaluated either by determining their effect on the target protein, or by evaluating their effect on steroid resistance.
  • Chemical candidates are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds.
  • embodiments of the invention provide a method for screening any library of chemical candidates, including the wide variety of known combinatorial chemistry-type libraries. The method can be used to identify a wide variety of different types of drugs capable of decreasing steroid resistance.
  • candidate drugs can include polypeptides, polynucleotides, or a small organic compounds, as described herein.
  • chemical candidates are synthetic compounds.
  • a number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides.
  • other aspects use libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.
  • the chemical candidates are proteins
  • they may be naturally occurring proteins or fragments of naturally occurring proteins.
  • cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be tested.
  • libraries of prokaryotic and eukaryotic proteins may be made for screening against any number of compositions.
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
  • the chemical candidates are peptides ranging in size from about 2 to about 50 amino acids, with from about 5 to about 30 amino acids being preferred, and from about 8 to about 20 being particularly preferred.
  • the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
  • the term "randomized” is intended to mean that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
  • the library should provide a sufficiently structurally diverse population of randomized agents to effect a probabilistically sufficient range of diversity to allow interaction with a particular immune component, such as an ICI molecule. Accordingly, an interaction library must be large enough so that at least one of its members will have a structure that interacts with a immune component. Those skilled in the art would understand how to best construct a sufficiently large and diverse library.
  • nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
  • the chemical candidates are nucleic acids.
  • nucleic acid or “oligonucleotide” used herein means at least two nucleotides covalently linked together.
  • Embodiments composed of nucleic acids will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages.
  • Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones.
  • nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. As will be appreciated by those in the art, all of these nucleic acid analogs may find use in various inventive embodiments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made. [0073] The present invention is illustrated by the following example. It is to be understood that the particular example, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
  • the present discovery study used a combination of high abundant serum protein immunodepletion and 1-dimensional liquid chromatography-high mass accuracy proteomic method to study serum proteins from children with steroid sensitive and resistant nephrotic syndrome. Importantly this study used paired serum samples from steroid-naive patients and -4- 6 weeks post-corticosteroid treatment.
  • Figure 2 provides a scheme showing the proteomics workflow used by the inventors to identify biomarkers for steroid resistance and the genes and proteins involved in the mechanism of steroid resistance.
  • Hypothesis 1- The serum proteome of steroid-naive pediatric NS patients can be used to predict corticosteroid response. (Predictor candidate biomarkers); Hypothesis 2- The serum proteome of SSNS and SRNS pediatric patients can be used to define markers of corticosteroid resistance. (Explanatory candidate biomarkers)
  • a hierarchical clustering method with heatmap visualization was used to mine the proteomic data and determine patterns of serum protein abundance as a function of steroid resistance.
  • the relative abundance across patient samples and time points was converted to a fractional abundance (0 to 1) and plotted in a heatmap.
  • the largest cluster contained the largest number of identified proteins largely clustered around or slightly below the average fractional abundance value.
  • the remaining three branches comprising proteins having dynamic changes between patient cohorts and treatment times.
  • the dominant abundance feature for the second (magenta) cluster branch were higher pre- versus post- steroid fractional abundances that were generally greatest in pre-SSNS samples.
  • Membership in the second cluster included candidate predictor proteins ADIPOQ, APOE, COL6A3, EFEMP1, IGFBP2, ITIH4, MMP and SHBG.
  • the dominant abundance feature for the third cluster branch were higher pre- versus post-steroid fractional abundances that were generally greatest in pre-SRNS.
  • Membership in the third cluster included predictor proteins AZPG1, FETUB, GC, HPX, and SERPINC1.
  • the last cluster included two proteins, SERPINA6 and TGFBI, whose relative abundance was largely detectable only in post-treatment SSNS samples. The results of the heat map analysis are shown in Figure 4.
  • Ingenuity Pathway Analysis (IPA) analyses of predictor gene names suggested five canonical pathways (p-value ⁇ 0.01) and one significant protein-protein network ( Figure 5).
  • the top five canonical pathways are (1) famesoid X receptor (FXR)/RXR activation, (2) liver X receptor (LXR) /retinoid X receptor (RXR) activation, (3) airway pathology in chronic obstructive pulmonary disease, (4) acute phase response signaling, (4) hepatic fibrosis/hepatic stellate cell activation, and the extrinsic prothrombin activation pathway.
  • the principal network for the predictor candidate bio markers was built using 8 of 13 submitted gene names including ADIPOQ, APOE, EFEMP1, HPX, IGFBP2, MMP2, SERPINC1, and SHBG. As shown in Figure 3, five proteins (SHBG, EFEMP1, HPX, IGFBP2, and SERPINC1) were positioned at network edges while MMP2, ADIPOQ and APOE were position as network nodes with all nodes under the indirect regulation (dashed lines) of the cytokine tumor necrosis factor (TNF).
  • TNF tumor necrosis factor
  • the top five canonical pathways identified by IPA analyses of explanatory candidate marker gene names and in order of statistical significance were: (1) liver X receptor (LXR) /retinoid X receptor (RXR) activation, (2) famesoid X receptor (FXR)/RXR activation, (3) acute phase response signaling, (4) coagulation system, and (5) atherosclerosis signaling.
  • LXR liver X receptor
  • RXR retinoid X receptor
  • FXR famesoid X receptor
  • RXR famesoid X receptor
  • RXR famesoid X receptor
  • RXR famesoid X receptor
  • RXR famesoid X receptor
  • coagulation system coagulation system
  • atherosclerosis signaling The top three networks assemble by IPA overlapped and had common disease/functional attributions of metabolic disease, hematological system development and function, inflammatory response, cell death and survival.
  • the major pathways known in NS and resistance based on the inventors studies are insulin signaling, glucocorticoid signaling, dyslipidemia, complement factors, hormone/vitamin/steroid binding proteins, and other pathways (e.g., hemopexin, MMP2 etc.).
  • Hierarchical clustering analysis suggests differentially abundant proteins fall within three clusters including groups of predictor candidate markers increased in SSNS and decreased in SSNS prior to steroid therapy and increased in SSNS following steroid therapy.
  • Pathways analysis results suggest that for SSNS and SRNS, the response to steroid therapy may involve canonical pathways including (a) LXR/RXR activation, (b) acute phase response signaling, and (c) FXR/RXR activation.
  • Protein-protein interaction mapping using StringDB suggests two proteins forming a unique cluster that increase in SSNS patients post-steroid therapy are involved with corticosteroid transport and corticosteroid signaling.
  • NS-specific plasma proteomic differences exist and these differences may be useful as candidate prognostic biomarkers and may assist in defining the different NS pathophysiologies.
  • the proteomics workflow ( Figure 2) addressed the label free comparison of high mass accuracy data sets developed from patient plasma samples using an FPLC-antibody based method to immune-depleted of the 20 most common plasma proteins prior to trypsinization. These data were filtered for three categories of plasma proteins including proteins whose abundance (a) differentiating SSNS versus SSRS samples collected pre-steroid treatment, (b) differentiating SSNS versus SSRS samples collected post-steroid treatment, and (c) differentiating proteins whose abundance changes correspond to steroid treatment independent of nephrotic syndrome resolution.
  • the data filtering approaches were directed by absolute or relative differences in the protein abundance, unbiased statistical or pathways approaches and lastly expert review of the proteomic data.
  • EDTA-plasma samples from study enrollment and 1-year follow-up were immune- depleted of the 20 most abundant plasma proteins using the ProteoPrep20 LC column (Sigma, St. Louis, MO) and an AKTA PureLl FPLC (GE Healthcare, Pittsburgh, PA).
  • the non-retained flow through containing low abundant proteins (LAP) was concentrated using ultrafiltration concentrators (Amicon-4 with 5kDa Ultracel-3 membranes, Millipore, Billerica, MA), and the protein recovered using two 1 mL rinses of 0.5mM EDTA/lOmM HEPES pH 7.6. LAP recovery yields were estimated using fluorometric quantitation and the Qubit system (ThermoFisher, Waltham, MA).
  • Peptides were separated with a 170 min, 2-78% acetonitrile gradient in 0.1% formic acid and one dimensional liquid chromatography using an EASY n-LC UHPLC system (ThermoFisher Scientific) with a 2-cm Dionex Acclaim PepMap 100 (CI 8, 3 ⁇ , 100 A) trap column (Dionex, Sunnyvale, CA), and a 50-cm Dionex Acclaim PepMap RSLC (CI 8, 2 ⁇ , 100 A) separating column (Dionex) and tandem mass spectrometry data generated by nanospray ionization into an LTQ Orbitrap ELITE mass spectrometer (Thermo-Fisher Scientific).
  • EASY n-LC UHPLC system ThermoFisher Scientific
  • 2-cm Dionex Acclaim PepMap 100 CI 8, 3 ⁇ , 100 A
  • trap column Dionex, Sunnyvale, CA
  • 50-cm Dionex Acclaim PepMap RSLC
  • the lock mass option was enabled (0% lock mass abundance) using the 371.101236 m/z polysiloxane peak as an internal calibrant.
  • Proteome Discoverer v2.0.0.802 was used to analyze the data collected by the mass spectrometer with SequestHT searches performed using the 7/7/2015 version of the UniprotKB Homo sapiens reference proteome canonical and isoform sequences appended with the nonhuman sequences from the 1/1/2012 version of thegpm.org cRAP database.
  • the resulting files from Proteome Discoverer were loaded into Scaffold Q+S v4.4.5 and the peptide false discovery rate (FDR) was calculated using the Scaffold Local FDR algorithm.
  • FDR peptide false discovery rate
  • the resulting protein lists were annotated with human gene ontology information from the Gene Ontology Annotations Database.
  • protein lists were curated by removing all proteins with a prevalence of less than 75% in at any sample cohort (SSNS-pre, SSNS-post, SRNS-pre, SRNS-post).
  • predictor candidate biomarkers were established using a Wilcoxon test of medians and interquartile ranges retaining those proteins achieving a statistical significance at a p-value ⁇ 0.05.
  • Explanatory candidate biomarkers were selected by comparing post - pre median values for SSNS and SRNS iBAQ values retaining those proteins achieving a Mann- Whitney litest p-value of ⁇ 0.05.
  • proteomic data (differentially regulated gene products with the Log2 fold change of SRNS to SSNS for both the pre-steroid treatment sample set and the post-steroid treatment sample set) were also qualitatively assessed to provide approaches to functional annotation of data by submitting lists of identified proteins and expression patterns for pathways analysis using Ingenuity Pathways Analysis (IPA) software.
  • IPA Ingenuity Pathways Analysis
  • the goals for the IPA analysis was to focus on protein networks formed by candidate proteins and the canonical pathways impacted by differential protein abundance with direction of the canonical pathway activation.
  • StringDB was used to supplement protein-protein interaction (PPI) networks not for proteins not uniquely identified within IPA networks.

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Abstract

L'invention concerne un procédé de diagnostic de la résistance aux stéroïdes chez un sujet présentant une maladie glomérulaire telle que le syndrome néphrotique. Le procédé comprend les étapes consistant : à fournir un échantillon biologique provenant du sujet ; à déterminer la quantité d'un ou de plusieurs biomarqueurs de résistance aux stéroïdes dans l'échantillon biologique ; et à comparer la quantité desdits biomarqueurs de résistance aux stéroïdes à un niveau de contrôle desdits biomarqueurs de résistance aux stéroïdes. Le sujet est diagnostiqué comme présentant une résistance aux stéroïdes ou un risque de cette dernière s'il y a une différence dans la quantité desdits biomarqueurs de résistance aux stéroïdes dans l'échantillon par comparaison au niveau de contrôle. La détermination de la résistance du sujet aux stéroïdes peut être utilisée de manière à guider le traitement du sujet.
PCT/US2017/061974 2016-11-16 2017-11-16 Résistance aux stéroïdes dans le syndrome néphrotique Ceased WO2018094021A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020068506A1 (fr) * 2018-09-24 2020-04-02 President And Fellows Of Harvard College Systèmes et procédés de classification de tumeurs
EP4397976A1 (fr) * 2023-01-09 2024-07-10 x-kidney diagnostics GmbH Biomarqueurs pour la détection préclinique et/ou en stade précoce et/ou le diagnostic de maladies rénales
CN118534127A (zh) * 2024-04-12 2024-08-23 郑州大学 补体h因子相关蛋白作为糖尿病肾病诊断的生物标志物的应用
WO2025045894A1 (fr) * 2023-08-28 2025-03-06 Institut National de la Santé et de la Recherche Médicale Méthodes et trousses de diagnostic de cause du syndrome néphrotique et d'orientation de la thérapie

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Publication number Priority date Publication date Assignee Title
WO2009145831A1 (fr) * 2008-04-02 2009-12-03 Geisinger Clinic Traitement des maladies à spectre de syndrome néphrotique idiopathique à l'aide de basiliximab
WO2015066640A1 (fr) * 2013-11-01 2015-05-07 The Research Institute At Nationwide Children's Hospital Kit et procédé pour identifier la sensibilité individuelle à la thérapie stéroïde du syndrome néphrotique
US20160061845A1 (en) * 2014-08-29 2016-03-03 Children's Hospital Medical Center Compositions and methods for treating steroid resistant nephrotic syndrome and/or steroid sensitive nephrotic syndrome

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009145831A1 (fr) * 2008-04-02 2009-12-03 Geisinger Clinic Traitement des maladies à spectre de syndrome néphrotique idiopathique à l'aide de basiliximab
WO2015066640A1 (fr) * 2013-11-01 2015-05-07 The Research Institute At Nationwide Children's Hospital Kit et procédé pour identifier la sensibilité individuelle à la thérapie stéroïde du syndrome néphrotique
US20160061845A1 (en) * 2014-08-29 2016-03-03 Children's Hospital Medical Center Compositions and methods for treating steroid resistant nephrotic syndrome and/or steroid sensitive nephrotic syndrome

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020068506A1 (fr) * 2018-09-24 2020-04-02 President And Fellows Of Harvard College Systèmes et procédés de classification de tumeurs
EP4397976A1 (fr) * 2023-01-09 2024-07-10 x-kidney diagnostics GmbH Biomarqueurs pour la détection préclinique et/ou en stade précoce et/ou le diagnostic de maladies rénales
WO2024149767A3 (fr) * 2023-01-09 2024-08-22 x-kidney diagnostics GmbH Biomarqueurs pour la détection et/ou le diagnostic pré-cliniques et/ou précoces de maladies rénales
WO2025045894A1 (fr) * 2023-08-28 2025-03-06 Institut National de la Santé et de la Recherche Médicale Méthodes et trousses de diagnostic de cause du syndrome néphrotique et d'orientation de la thérapie
CN118534127A (zh) * 2024-04-12 2024-08-23 郑州大学 补体h因子相关蛋白作为糖尿病肾病诊断的生物标志物的应用

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