WO2012051463A2 - Profil métabolomique de l'urine en cas de néphropathie diabétique - Google Patents

Profil métabolomique de l'urine en cas de néphropathie diabétique Download PDF

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Publication number
WO2012051463A2
WO2012051463A2 PCT/US2011/056229 US2011056229W WO2012051463A2 WO 2012051463 A2 WO2012051463 A2 WO 2012051463A2 US 2011056229 W US2011056229 W US 2011056229W WO 2012051463 A2 WO2012051463 A2 WO 2012051463A2
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acid
level
subject
determined
organic
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PCT/US2011/056229
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English (en)
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WO2012051463A3 (fr
Inventor
Robert K. Naviaux
Kumar Sharma
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The Regents Of The University Of California
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Publication of WO2012051463A2 publication Critical patent/WO2012051463A2/fr
Publication of WO2012051463A3 publication Critical patent/WO2012051463A3/fr
Priority to US13/843,680 priority Critical patent/US20130276513A1/en
Priority to US14/963,138 priority patent/US20160178584A1/en

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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

Definitions

  • the technology relates in part to methods for identifying the presence of kidney disease, determining the level of kidney disease, or the progression of kidney disease, in a subject that has or has not been diagnosed with diabetes.
  • the technology further relates to methods for determining the targets for therapy for kidney disease, the efficacy of a treatment for kidney disease, and methods for determining the toxicity of a therapeutic in a subject with kidney disease.
  • the blood creatinine is thought to reflect the filtration rate of the nephron, but may be in the normal range, despite significant disease, and may not indicate progression or improvement.
  • the urine protein may indicate leakage of blood protein into the urine and may indicate kidney disease in some patients, but not all, and may also not indicate whether the disease is improving or deteriorating.
  • kidney disease therapy there is a need for methods of determining the likelihood or level of kidney disease in a patient. There is also a need for methods for determining the effectiveness or toxicity of a kidney disease therapy in a patient. Further, there is a need for method for identifying individual molecules or metabolites that have been found to distinguish patients with diabetic kidney disease from normal, non-diabetic subjects, or patients with diabetic kidney disease from patients with diabetes and no kidney disease, kidney disease of non-diabetic causes from diabetic kidney disease, and patients with diabetes and no kidney disease from normal, non-diabetic subjects.
  • the technology relates in part to methods for identifying the presence of kidney disease, determining the level of kidney disease, or the progression of kidney disease, in a subject that has or has not been diagnosed with diabetes.
  • the technology further relates to methods for determining the efficacy of a treatment for kidney disease, and methods for determining the toxicity of a therapeutic in a subject with kidney disease, or toxicity to the kidneys.
  • the technology further relates to the identification of biomarkers that indicate kidney disease, for example, diabetic kidney disease.
  • the biomarkers reflect mitochondrial function and the overall health of the organ.
  • the technology may, for example, also be used to identify a therapeutic.
  • a method is provided of identifying the presence or level of kidney disease in a subject, including determining the level of at least one organic acid of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in a subject.
  • a comparison is provided of the level of the at least one organic acid to a reference level of the at least one organic acid from a control or the same subject collected at a previous time. Detection can be either simultaneous or sequential and may be from the same biological sample or from multiple samples from the same or different subjects.
  • At least one organic acid may include any number up to all ten organic acids and combination of said acids alone or in combination with other small organic molecules with a molecular weight of less than 700 Daltons or metabolites such as, for example, blood creatinine or blood urea nitrogen.
  • the level of 5-oxoproline in a subject is determined.
  • the level of 5-oxoproline is determined from a reference level of 5-oxoproline.
  • the presence or level of kidney disease in the subject where the level of 5- oxoproline in the subject is increased when compared to the reference 5-oxoproline level is identified.
  • Other diseases so identified include diabetes or diabetic kidney disease.
  • the reference level of the organic acid or the 5-oxoproline is readily determined from a healthy patient or from a sample obtained from the subject at an earlier time.
  • the reference level of the organic acid or the 5-oxoproline is determined from an analysis of samples obtained from more than one healthy patient.
  • the level of the at least one organic acid is decreased at least 1 .5-, 2-, 3-, 4- fold or even lower compared to the reference level.
  • the level of 5-oxoproline is increased at least 1 .5-, 2-, 3-, 4-fold or even lower compared to the reference level.
  • the subject has not been diagnosed with diabetes.
  • the subject has kidney disease.
  • the level of the organic acid or the 5-oxoproline is determined using gas chromatography.
  • the level of the organic acid or the 5-oxoproline is determined using mass spectrometry.
  • the level of the organic acid is determined from a biological sample from the subject.
  • the sample contains urine, or a urine fraction, or blood or a blood fraction.
  • a method of determining the progression of kidney disease over time in a subject diagnosed with kidney disease, comprising determining the level of at least one organic acid of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in a subject; comparing the level of the at least one organic acid to the level of the at least one organic acid determined in a sample obtained from the subject at an earlier time point; and determining that the kidney disease has progressed in the subject where the at least one organic acid level in the subject is decreased when compared to the level determined in the sample obtained from the subject at the earlier time point.
  • the at least one organic acid is two such acids, three such acids, four such acids, five such acids, six such acids, 7 such, 8 such, 9 such or all 10 of such acids, alone or in combination with other small organic molecules with a molecular weight of less than 700 Daltons or metabolites such as creatinine
  • the method further comprises determining the level of 5-oxoproline in a subject, comparing the level to the level determined in a sample obtained from the subject at an earlier time point, and determining that kidney disease has progressed in the subject where the level of 5-oxoproline in the subject is increased when compared to the 5-oxoproline level in the sample obtained from the subject at the earlier time point.
  • the subject has diabetes.
  • the subject has diabetic kidney disease.
  • the level of the organic acid or acids is decreased at least 1 .5 fold compared to the level in the sample obtained from the subject at the earlier time point.
  • the level of the organic acid or acids is decreased at least 2 fold compared to the level in the sample obtained from the subject at the earlier time point.
  • the level of 5- oxoproline is increased at least 3 fold compared to the level in the sample obtained from the subject at the earlier time point.
  • the level of 5-oxoproline is increased at least 4 fold compared to the level in the sample obtained from the subject at the earlier time point.
  • the subject has not been diagnosed with diabetes.
  • the subject has kidney disease.
  • the level of the organic acid or the 5-oxoproline is determined using gas chromatography.
  • the level of the organic acid or the 5- oxoproline is determined using mass spectrometry.
  • the level of the organic acid is determined from a biological sample from the subject.
  • the sample contains urine or a urine fraction.
  • a method comprising: administering a therapeutic to a subject diagnosed with kidney disease; determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in the subject ; and determining whether the dosage of the therapeutic subsequently administered to the subject is adjusted based on the level of the at least one organic acid.
  • a method comprising determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in a subject diagnosed with kidney disease, wherein the subject has been
  • a method comprising determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in a subject diagnosed with kidney disease, wherein the subject has been administered a therapeutic; and determining whether the dosage of the therapeutic subsequently administered to the subject is adjusted based on the level of the at least one organic acid in the subject.
  • a method comprising receiving information comprising the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in a subject to whom a therapeutic has been administered; and maintaining a subsequent dosage of the therapeutic, or adjusting a subsequent dosage of the therapeutic to the subject based on the level of the at least one organic acid in the subject.
  • a method comprising determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in a subject diagnosed with kidney disease, wherein the subject has been
  • a method comprising determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in a subject diagnosed with kidney disease, wherein the subject has been
  • a method comprising administering a therapeutic to a subject diagnosed with kidney disease; determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in the subject;
  • the method comprises determining the levels of at least two organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least three organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid.
  • the method comprises determining the levels of at least four organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least five organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid.
  • the method comprises determining the levels of at least six organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least seven organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid.
  • the method comprises determining the levels of at least eight organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least nine organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the subject has diabetes.
  • the subject has not been diagnosed with diabetes.
  • the level of at least one organic acid is determined using gas chromatography.
  • the level of the organic acid is determined using mass spectrometry.
  • the level of the organic acid is determined from a biological sample from the subject.
  • the sample contains urine or a urine fraction.
  • a method for reducing toxicity of a treatment comprising; administering a therapeutic to a subject in need thereof; determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid in the subject; and maintaining a subsequent dosage of the therapeutic or adjusting a subsequent dosage of the therapeutic to the subject based on the level of the at least one organic acid in the subject.
  • the method comprises determining the levels of at least two organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least three organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid.
  • the method comprises determining the levels of at least four organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least five organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid.
  • the method comprises determining the levels of at least six organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least seven organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid.
  • the method comprises determining the levels of at least eight organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid. In some aspects, the method comprises determining the levels of at least nine organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, or azelaic acid.
  • the method comprises determining the level of the at least one organic acid in the subject before administering the therapeutic, and comparing the level in the subject before administering the therapeutic to the level in the subject after administering the therapeutic.
  • the level of the organic acid is determined from a biological sample from the subject.
  • the sample contains urine or a urine fraction.
  • the level of the organic acid is determined using mass spectrometry.
  • the level of the organic acid is determined from a biological sample from the subject.
  • methods for identifying the presence or level of kidney disease in a subject, comprising determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid in a sample obtained from the subject; comparing the level of the at least one organic acid with a reference level of the at least one organic acid, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of kidney disease in the subject where the at least one organic acid level in the subject is decreased when compared to the reference level of the at least one organic acid.
  • the level at least two organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least two reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least two organic acid levels in the subject are decreased when compared to the at least two reference organic acid levels.
  • the level of at least three organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least three reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least three organic acid levels in the subject are decreased when compared to the at least three reference organic acid levels.
  • the level of at least four organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least four reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least four organic acid levels in the subject are decreased when compared to the at least four reference organic acid levels.
  • the level of at least five organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least five reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least five organic acid levels in the subject are decreased when compared to the at least five reference organic acid levels.
  • the level of at least six organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least six reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least six organic acid levels in the subject are decreased when compared to the at least six reference organic acid levels.
  • the level of at least seven organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least seven reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least seven organic acid levels in the subject are decreased when compared to the at least seven reference organic acid levels.
  • the level of at least eight organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least eight reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least eight organic acid levels in the subject are decreased when compared to the at least eight reference organic acid levels.
  • the level of at least nine organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least nine reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least nine organic acid levels in the subject are decreased when compared to the at least nine reference organic acid levels.
  • the level of at least ten organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid, and azelaic acid are determined, compared to at least ten reference organic acids, and the presence or level of kidney disease in the subject is identified where the at least ten organic acid levels in the subject are decreased when compared to the at least ten reference organic acid levels.
  • the method further comprises determining the level of 5-oxoproline in a sample obtained from the subject, comparing the level to the level to a reference level of 5- oxoproline, and identifying the presence or level of kidney disease in the subject where the level of 5-oxoproline in the subject is increased when compared to the reference 5-oxoproline level.
  • the method also comprises determining the level of citrate in a sample obtained from the subject, comparing the level to the level to a reference level of citrate, and identifying the presence or level of kidney disease in the subject where the level of citrate in the subject is decreased when compared to the reference citrate level.
  • the level of the organic acid or acids is decreased at least 1 .5 fold compared to the reference level. In some embodiments, the level of the organic acid or acids is decreased at least 2 fold compared to the reference level. In some embodiments, the level of 5- oxoproline is increased compared to the reference level. In some embodiments, the level of citrate is increased compared to the reference level.
  • Also provided are methods of determining the progression of kidney disease over time in a subject diagnosed with kidney disease comprising determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, in a sample obtained from the subject; comparing the level of the at least one organic acid to the level of the at least one organic acid determined in a sample obtained from the subject at an earlier time point; determining that the kidney disease has progressed in the subject where the at least one organic acid level in the subject is decreased when compared to the level determined in the sample obtained from the subject at the earlier time point.
  • at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid
  • the method comprises determining the level of at least two organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, comparing the level of the at least two organic acids to the level of the at least two organic acids determined in a sample obtained from the subject at an earlier time point, and determining that the kidney disease has progressed in the subject where the at least two organic acid levels in the subject are decreased when compared to the levels determined in the sample obtained from the subject at the earlier time point.
  • the method comprises determining the level of at least three, four, five, six, seven, eight, nine, or ten organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, comparing the level of the at least two organic acids to the level of the at least three, four, five, six, seven, eight, nine, or ten organic acids, respectively, determined in a sample obtained from the subject at an earlier time point, and determining that the kidney disease has progressed in the subject where the at least three, four, five, six, seven, eight, nine, or ten, respectively, organic acid levels in the subject are decreased when compared to the levels determined in the sample obtained from the subject at the earlier time point.
  • the method further comprises determining the level of 5-oxoproline in a sample obtained from the subject, comparing the level to the level determined in a sample obtained from the subject at an earlier time point, and determining that kidney disease has progressed in the subject where the level of 5-oxoproline in the subject is increased when compared to the 5- oxoproline level in the sample obtained from the subject at the earlier time point.
  • the method further comprises determining the level of citrate in a sample obtained from the subject, comparing the level to the level determined in a sample obtained from the subject at an earlier time point, and determining that kidney disease has progressed in the subject where the level of citrate in the subject is increased when compared to the citrate level in the sample obtained from the subject at the earlier time point.
  • the level of the organic acid or acids is decreased at least 1 .5 fold compared to the level in the sample obtained from the subject at the earlier time point. In some embodiments, the level of the organic acid or acids is decreased at least 2 fold compared to the level in the sample obtained from the subject at the earlier time point.
  • the subject has diabetes. In certain embodiments, the subject has diabetic kidney disease. In certain embodiments, the subject has not been diagnosed with diabetes. In certain embodiments, the subject has kidney disease.
  • the level of the organic acid, the 5-oxoproline, or the citrate is determined using gas chromatography. In some embodiments, the level of the organic acid, the 5-oxoproline, or the citrate is determined using mass spectrometry. In some embodiments, the level of the organic acid is determined from a biological sample from the subject. In some embodiments, the sample contains urine or a urine fraction, or blood or a blood fraction.
  • Also provided are methods comprising administering a therapeutic to a subject diagnosed with kidney disease; determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, in a sample obtained from the subject; and determining whether the dosage of the therapeutic subsequently administered to the subject is adjusted based on the level of the at least one organic acid.
  • a method comprising determining the level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, in a sample obtained from a subject diagnosed with kidney disease, wherein the subject has been administered a therapeutic; and maintaining a subsequent dosage of the therapeutic or adjusting a subsequent dosage of the therapeutic administered to the subject based on the level of the at least one organic acid in the sample.
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, or ten organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, and maintaining a subsequent dosage of the therapeutic or adjusting a subsequent dosage of the therapeutic administered to the subject based on the levels of at least two, three, four, five, six, seven, eight, nine, or ten, respectively, organic acids in the sample.
  • the method further comprises determining the level of 5-oxoproline in a sample obtained from the subject, and maintaining a subsequent dosage of the therapeutic or adjusting a subsequent dosage of the therapeutic administered to the subject based on the levels of 5-oxoproline in the sample. In some embodiments, the method further comprises determining the level of citrate in a sample obtained from the subject, and maintaining a subsequent dosage of the therapeutic or adjusting a subsequent dosage of the therapeutic administered to the subject based on the levels of citrate in the sample.
  • Also provided in some embodiments are methods for reducing toxicity of a treatment comprising: determining the pre-treatment level of at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, in a sample obtained from the subject;
  • a therapeutic administered to the subject; determining the post-treatment level of the at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, in the subject after; and lowering the subsequent dosage of the therapeutic where the post-treatment level of the at least one organic acid is decreased compared to the pre- treatment level of the at least one organic acid in the sample.
  • the post-treatment level of the at least one organic acid selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid
  • the method comprises determining the levels of at least two, three, four, five, six, seven, eight, nine, or ten organic acids selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, fumaric acid, oleic acid and azelaic acid, and lowering the subsequent dosage of the therapeutic where the post-treatment levels of the at least two, three, four, five, six, seven, eight, nine, or ten organic acids are decreased compared to the pre-treatment levels of the at least two, three, four, five, six, seven, eight, nine, or ten, respectively, organic acids in the sample.
  • the method further comprises determining the pre-treatment level of 5-oxoproline, in a sample obtained from the subject, determining the post-treatment level of 5-oxoproline, in the subject, and lowering the subsequent dosage of the therapeutic where the post-treatment level of 5-oxoproline is increased compared to the pre-treatment level of 5-oxoproline in the sample.
  • the method further comprises the pre-treatment level of citrate, in the subject, determining the post- treatment level of citrate, in a sample obtained from the subject, and lowering the subsequent dosage of the therapeutic where the post-treatment level of citrate is decreased compared to the pre-treatment level of citrate in the sample.
  • Also provided in some embodiments are methods of identifying the presence or level of diabetes related complications in a subject comprising determining the level of at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; comparing the level of the at least one metabolite with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of diabetes-related complications in the subject where the at least one metabolite level in the subject is decreased when compared to the reference level of the at least one metabolite.
  • the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites are determined, compared to at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve, reference metabolites, and the presence or level of diabetes related complications in the subject is identified where the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve, respectively, metabolite levels in the subject are decreased when compared to the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve, reference metabolite levels.
  • the diabetes related complication is a microvascular complication. In some embodiments, the diabetes related complication is a macrovascular complication.
  • Also provided in some embodiments are methods of determining the progression of a diabetes related complication over time in a subject diagnosed with a diabetes related complication, comprising determining the level at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; comparing the level of the at least one metabolite to the level of the at least one metabolite determined in a sample obtained from the subject at an earlier time point; and determining that the diabetes related complication has progressed in the subject where the at least one metabolite level in the subject is decreased when compared to the level determined in the sample obtained from the subject at the earlier time point.
  • the method comprises determining the progression of a diabetes related complication over time in a subject diagnosed with a diabetes related complication, comprising determining the level at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; comparing the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites to the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites, respectively determined in a sample obtained from the subject at an earlier time point; and determining that the diabetes related complication has progressed in the subject
  • Also provided in some embodiments are methods comprising: administering a therapeutic to a subject diagnosed with a diabetes related complication; determining the level of at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; and determining whether the dosage of the therapeutic subsequently administered to the subject is adjusted based on the level of the at least one metabolite.
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH- glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; and determining whether the dosage of the therapeutic subsequently administered to the subject is adjusted based on the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites, respectively.
  • Also provided in some embodiments are methods of identifying the presence or level of diabetes, cardiovascular disease, hypertension, or chronic kidney disease in an obese subject, comprising determining the level of at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; comparing the level of the at least one metabolite with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of diabetes-related complications in the subject where the at least one metabolite level in the subject is decreased when compared to the reference level of the at least one metabolite.
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; comparing the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites with a reference level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of diabetes-related complications in the
  • Also provided in some embodiments are methods of identifying the presence or level of diabetes, cardiovascular disease, hypertension, or chronic kidney disease in an obese subject comprising determining the level of at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; comparing the level of the at least one metabolite with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of diabetes, cardiovascular disease, hypertension, or chronic kidney disease in the subject where the at least one metabolite level in the subject is decreased when compared to the reference level of the at least one
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate in a sample obtained from the subject; comparing the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites with a reference level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of diabetes, cardiovascular disease, hyper
  • Also provided in some embodiments are methods of identifying the presence or level of hypertension in a subject comprising determining the level of at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH- glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citric acid in a sample obtained from the subject; comparing the level of the at least one metabolite with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of hypertension in the subject where the at least one metabolite level in the subject is decreased when compared to the reference level of the at least one metabolite.
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citric acid in a sample obtained from the subject; comparing the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites with a reference level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of hypertension in the subject
  • Also provided in some embodiments are methods of identifying the presence or level of liver disease in a subject having obesity, diabetes, or chronic kidney disease, comprising determining the level of at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citric acid in a sample obtained from the subject; comparing the level of the at least one metabolite with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of liver disease in the subject where the at least one metabolite level in the subject is decreased when compared to the reference level of the at least one metabolite.
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citric acid in a sample obtained from the subject; comparing the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites with a reference level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of liver disease in the subject
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, citric acid, and 5- oxoproline in a sample obtained from the subject; comparing the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or thirteen metabolites with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of joint involvement in the subject where the at least two, three, four, five, six
  • Also provided in some embodiments are methods of identifying the presence or level of sleep apnea, restrictive lung disease or obstructive lung disease in a subject having diabetes, obesity, or chronic kidney disease, comprising determining the level of at least one metabolite selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, citric acid, and 5-oxoproline in a sample obtained from the subject; comparing the level of the at least one metabolite with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of sleep apnea, restrictive lung disease or obstructive
  • the method comprises determining the level of at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites selected from the group consisting of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, citric acid, and 5-oxoproline in a sample obtained from the subject; comparing the level of the at least two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve metabolites with a reference level of the at least one metabolite, wherein the reference level has been determined from at least one sample collected from the same subject at a different time period; or the reference level has been determined from a sample or samples collected from one or more other subjects; and identifying the presence or level of sleep apnea, restrictive lung disease or obstructive lung disease
  • the subject has diabetes. In certain embodiments, the subject has diabetic kidney disease. In certain embodiments, the subject has not been diagnosed with diabetes. In certain embodiments, the subject has kidney disease.
  • the reference level of the organic acid, the metabolite, the 5-oxoproline, or the citrate is determined from a sample obtained from a healthy patient. In some embodiments, the reference level of the organic acid, the metabolite, the 5-oxoproline, or the citrate is
  • the reference level of the organic acid, the 5-oxoproline, or the citrate is determined from an analysis of samples obtained from more than one healthy patient.
  • the organic acid is selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, and uracil.
  • the level of the organic acid, the metabolite, the 5-oxoproline, or the citrate is determined using gas chromatography.
  • the level of the organic acid, the metabolite, the 5-oxoproline, or the citrate is determined using mass spectrometry.
  • the level of the organic acid, the 5-oxoproline, the citrate, or the metabolite is determined from a biological sample from the subject.
  • the sample contains urine, or a urine fraction, or blood or a blood fraction.
  • Figure 1 is a bar graph of citrate levels in patients with diabetic nephropathy who had been enrolled in a clinical trial and in normal volunteers.
  • P experimental drug
  • Z FDA approved drug
  • patients were not treated before their urines were evaluated.
  • Urine metabolomics was performed on each individual sample.
  • a cut-off p value of P ⁇ 0.00846 was chosen to have a false detection rate less than 0.05 to account for multiple testing.
  • the present technology provides relates to the identification of the presence of kidney disease, determination of the level of kidney disease, or the progression of kidney disease, in a subject that has or has not been diagnosed with diabetes.
  • the present technology also provides methods for determining the efficacy of a treatment for kidney disease, and methods for determining the toxicity of a therapeutic to a subject's kidneys.
  • the present technology further relates to the identification of biomarkers that indicate kidney disease, for example, diabetic kidney disease.
  • the biomarkers reflect mitochondrial function and the overall health of the organ.
  • the technology may, for example, also be used to identify a therapeutic for treating or preventing a kidney condition in the subject.
  • organic acids or 5-oxoproline readily obtained from blood, plasma, saliva, CSF, joint fluid, urine, as well as solid tissue biopsy. While urine is a sampling fluid in many embodiments of the technology owing to direct contact with the kidney, it is appreciated that other biological fluids have advantages in being sampled for other purposes and therefore allow for inventive determination of nephrological condition as part of a battery of tests performed on a single sample such as blood, plasma, serum, saliva, CSF, joint fluid or urine. It may be appreciated that the organic fluids may be detected using standard techniques, such as, for example, those presented herein, allowing for the varying methods for sample collection and initial processing.
  • a subject illustratively includes a dog, a cat, a horse, a cow, a pig, a sheep, a goat, a chicken, non-human primate, a human, a rat, and a mouse.
  • Subjects may, for example, include those suspected of having or at risk for developing diabetic kidney disease, diabetes, chronic kidney disease.
  • Marker refers to a small organic molecule or metabolite thereof which is differentially present in a sample taken from patients having kidney disease and/or or a proclivity for the disease as compared to a comparable sample taken from control subjects (e.g., a person with a negative diagnosis, normal or healthy subject) or from a historical value of the marker for the patient.
  • kidney disease indicates any disease or condition that affects the kidneys such as, for example, chronic kidney disease, acute kidney disease, congenital kidney disease, polycystic kidney disease, hypertensive kidney disease, inflammatory kidney disease, glomerulonephritis, tubulo-interstitial disease, and the like.
  • Chronic kidney disease often manifests in such a way that there are no detectable symptoms until there is irreversible damage to the kidneys.
  • patient a mammalian subject to be treated, for example, a human.
  • processes of the present technology find use in experimental animals, in veterinary application, and in the development of vertebrate models for disease, including, but not limited to, rodents including mice, rats, and hamsters; birds, fish reptiles, and primates.
  • normal subject and "healthy subject” refer to a mammalian subject, for example, a human,, that is not or has not suffered from kidney disease and does not have a history of past kidney disease.
  • Bio Sample includes polynucleotides, polypeptides, peptides, antibodies fragments and correlateable breakdown products and is a bodily fluid; a soluble fraction of a cell preparation, or media in which cells are grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint; skin; or hair; and fragments of the aforementioned.
  • diluent refers to, for example, any composition added to the biological sample so that the sample may be analyzed according to the methods of the current technology such as, for example gas chromatography or mass spectrometry. Appropriate diluents are discussed herein, and for example, in Hartmann, S., et al., Clin Chem. 2006 Jun;52(6):1 127-37. Epub 2006 Apr 13; and Barshop, BA, et al., Mol Genet Metab. 2000 Jan;69(1 ):64-8.
  • substrate refers to, for example, any material or composition to which the biological sample may be bound, such as, for example, beads, solid surfaces, microtiter plates, wafers, antibodies, filters, concentrators, and the like.
  • the panel of biomarkers discussed herein may be able to indicate the overall health of the kidney. It appears to be independent of the blood creatinine and the urine protein. The measurements of these metabolites may, for example, be used to identify patients with a reduction in kidney function, possibly at an earlier stage than the blood creatinine marker. In another example, the panel of metabolites may be used to determine whether a new or existing drug is harmful or beneficial for the kidney.
  • the level of one or more organic acids or metabolites may be compared to a reference level of the particular organic acids or metabolites.
  • the reference level may be determined from a biological sample obtained from the same subject at an earlier time period, for example, about 1 , 2, 3, 4, 5, 6, 7 days or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 years or more before the test biological sample is obtained.
  • two or more biological samples are obtained from a subject, and the levels of the organic acid or metabolite are determined in order to, for example, determining the progression of kidney disease, or another disease or condition discussed herein.
  • the two or more biological samples may be obtained independently, from, for example, about 1 , 2, 3, 4, 5, 6, 7 days or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, or 50 weeks, or 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 years or more apart.
  • the reference level may be determined from a biological sample obtained from a healthy patient, before, at the same time, or after the biological sample is obtained from the subject.
  • the reference level may be determined as an average of the levels of biological samples obtained from more than one healthy patient.
  • the reference level may be determined by the same entity that determines the level of the organic acid or metabolite in the biological sample obtained from the subject. Or, the reference level may be a level known, or published, by another entity. Optimized and Personalized Therapeutic Treatment
  • kidney disease for example, diabetic kidney disease
  • Treatment for kidney disease may be optimized by determining the concentration of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, or glutaric acid, 5-oxoproline or a panel of these biomarkers, during the course of treatment.
  • Different patients having different stages or types of kidney disease or diabetes may react differently to various therapies.
  • the response to treatment may be monitored by following the glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, 5-oxoproline or glutaric acid concentrations or levels in various body fluids or tissues.
  • the determination of the concentration, level, or amount of a metabolite may include detection by gas
  • Optimizing treatment for individual patients may help to avoid side effects as a result of overdosing, may help to determine when the treatment is ineffective and to change the course of treatment, or may help to determine when doses may be increased.
  • Technology discussed herein optimizes therapeutic methods for treating kidney disease, for example, diabetic kidney disease, by allowing a clinician to track a biomarker, such as, for example, glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, 5-oxoproline or glutaric acid, or a panel of these biomarkers, and determine whether a subsequent dose of a drug or vaccine for
  • a panel of biomarkers is meant at least two, three, four, five, six, seven, eight, nine, ten or eleven of the biomarkers selected from the group consisting of glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil, 5-oxoproline and glutaric acid.
  • the amount or concentration of certain biomarkers may change during the course of treatment of kidney disease.
  • Predetermined target levels of such biomarkers, or biomarker thresholds may be identified in normal subject, which allow a clinician to determine whether a subsequent dose of a drug administered to a subject in need thereof, such as a subject with a kidney disease, such as, for example, diabetic kidney disease. Based on this determination, the treatment may be increased, decreased or maintained.
  • a clinician can make such a determination based on whether the presence, absence or amount of a biomarker is below, above or about the same as a biomarker threshold, respectively, in certain embodiments.
  • determining that an over-represented biomarker level is significantly reduced and/or that an under-represented biomarker level is significantly increased after drug treatment or vaccination provides an indication to a clinician that an administered drug is exerting a therapeutic effect.
  • level is meant the concentration of the biomarker in a fluid or tissue, or the absolute amount in a tissue. Based on such a biomarker determination, a clinician could make a decision to maintain a subsequent dose of the drug or raise or lower the subsequent dose, including modifying the timing of administration.
  • drug or “therapeutic” includes traditional pharmaceuticals, such as small molecules, as well as biologies, such as nucleic acids, antibodies, proteins, polypeptides, modified cells and the like.
  • determining that an over- represented biomarker level is not significantly reduced and/or that an under-represented biomarker level is not significantly increased provides an indication to a clinician that an administered drug is not significantly exerting a therapeutic effect. Based on such a biomarker determination, a clinician could make a decision to increase a subsequent dose of the drug.
  • methods provided herein optimize therapeutic approaches as they provide the clinician with the ability to "dial in” an efficacious dosage of a drug and minimize side effects.
  • methods provided herein allow a clinician to "dial up” the dose of a drug to a therapeutically efficacious level, where the dialed up dosage is below a toxic threshold level.
  • treatment methods discussed herein enhance efficacy and reduce the likelihood of toxic side effects.
  • the methods discussed herein may be used to analyze the toxicity of a therapeutic not designed to treat kidney disease, but designed to treat another disease or condition. Using these methods, toxicity of the therapeutic to the kidney may be determined.
  • the presence, absence or amount of a biomarker can be determined within a subject (e.g., in situ) or outside a subject (e.g., ex vivo). In some embodiments, presence, absence or amount of a biomarker can be determined in cells (e.g., differentiated cells, stem cells), and in certain embodiments, presence, absence or amount of a biomarker can be determined in a substantially cell-free medium (e.g., in vitro).
  • the term "identifying the presence, absence or amount of a biomarker in a subject" as used herein refers to any method known in the art for assessing the biomarker and inferring the presence, absence or amount in the subject (e.g., in situ, ex vivo or in vitro methods).
  • a fluid or tissue sample often is obtained from a subject for determining presence, absence or amount of biomarker ex vivo.
  • Non-limiting parts of the body from which a tissue sample may be obtained include leg, arm, abdomen, upper back, lower back, chest, hand, finger, fingernail, foot, toe, toenail, neck, rectum, nose, throat, mouth, scalp, face, spine, throat, heart, lung, breast, kidney, liver, intestine, colon, pancreas, bladder, cervix, testes, muscle, skin, hair, tumor or area surrounding a tumor, and the like, in some embodiments.
  • a tissue sample can be obtained by any suitable method known in the art, including, without limitation, biopsy (e.g., shave, punch, incisional, excisional, curettage, fine needle aspirate, scoop, scallop, core needle, vacuum assisted, open surgical biopsies) and the like, in certain embodiments.
  • biopsy e.g., shave, punch, incisional, excisional, curettage, fine needle aspirate, scoop, scallop, core needle, vacuum assisted, open surgical biopsies
  • Examples of a fluid that can be obtained from a subject includes, without limitation, blood, cerebrospinal fluid, spinal fluid, lavage fluid (e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic), urine, interstitial fluid, feces, sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphatic fluid, bile, tears, sweat, breast milk, breast fluid, fluid from region of inflammation, fluid from region of muscle wasting and the like, in some embodiments.
  • lavage fluid e.g., bronchoalveolar, gastric, peritoneal, ductal, ear, arthroscopic
  • a sample from a subject may be processed prior to determining presence, absence or amount of a biomarker.
  • a blood sample from a subject may be processed to yield a certain fraction, including without limitation, plasma, serum, buffy coat, red blood cell layer and the like, and biomarker presence, absence or amount can be determined in the fraction.
  • a tissue sample e.g., tumor biopsy sample
  • an agent that visualizes a biomarker e.g., antibody
  • a tissue sample can be exposed to one or more of the following non-limiting conditions: washing, exposure to high salt or low salt solution (e.g., hypertonic, hypotonic, isotonic solution), exposure to shearing conditions (e.g., sonication, press (e.g., French press)), mincing, centrifugation, separation of cells, separation of tissue and the like.
  • high salt or low salt solution e.g., hypertonic, hypotonic, isotonic solution
  • shearing conditions e.g., sonication, press (e.g., French press)
  • mincing e.g., centrifugation, separation of cells, separation of tissue and the like.
  • a biomarker can be separated from tissue and the presence, absence or amount determined in vitro.
  • a sample also may be stored for a period of time prior to determining the presence, absence or amount of a biomarker (e.g., a sample may be frozen, cryopreserved, maintained in a preservation medium (e.g., formaldehyde)).
  • a preservation medium e.g., formaldehyde
  • a sample can be obtained from a subject at any suitable time of collection after a drug is delivered to the subject.
  • a sample may be collected within about one hour after a drug is delivered to a subject (e.g., within about 5, 10, 15, 20, 25, 30, 35, 40, 45, 55 or 60 minutes of delivering a drug), within about one day after a drug is delivered to a subject (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 hours of delivering a drug) or within about two weeks after a drug is delivered to a subject (e.g., within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14 days of delivering the drug).
  • a collection may be made on a specified schedule including hourly, daily, semi-weekly, weekly, bi-weekly, monthly, bi-monthly, quarterly, and yearly, and the like, for example. If a drug is administered continuously over a time period (e.g., infusion), the delay may be determined from the first moment of drug is introduced to the subject, from the time the drug administration ceases, or a point in-between (e.g.,
  • the presence, absence or amount of one or more biomarkers may be determined by any suitable method known in the art, and non-limiting determination methods are discussed herein.
  • Determining the presence, absence or amount of a biomarker sometimes comprises use of a biological assay.
  • a biological assay one or more signals detected in the assay can be converted to the presence, absence or amount of a biomarker. Converting a signal detected in the assay can comprise, for example, use of a standard curve, one or more standards (e.g., internal, external), a chart, a computer program that converts a signal to a presence, absence or amount of biomarker, and the like, and combinations of the foregoing.
  • An indication for adjusting or maintaining a subsequent drug dose can be based on the presence or absence of a biomarker. For example, when (i) low sensitivity determinations of biomarker levels are available, (ii) biomarker levels shift sharply in response to a drug, (iii) low levels or high levels of biomarker are present, and/or (iv) a drug is not appreciably toxic at levels of
  • presence or absence of a biomarker can be sufficient for generating an indication of adjusting or maintaining a subsequent drug dose.
  • An indication for adjusting or maintaining a subsequent drug dose often is based on the amount or level of a biomarker.
  • An amount of a biomarker can be a mean, median, nominal, range, interval, maximum, minimum, or relative amount, in some embodiments.
  • An amount of a biomarker can be expressed with or without a measurement error window in certain embodiments.
  • An amount of a biomarker in some embodiments can be expressed as a biomarker concentration, biomarker weight per unit weight, biomarker weight per unit volume, biomarker moles, biomarker moles per unit volume, biomarker moles per unit weight, biomarker weight per unit cells, biomarker volume per unit cells, biomarker moles per unit cells and the like. Weight can be expressed as
  • unit weight can be weight of subject or weight of sample from subject
  • unit volume can be volume of sample from the subject (e.g., blood sample volume)
  • unit cells can be per one cell or per a certain number of cells (e.g., micrograms of biomarker per 1000 cells).
  • an amount of biomarker determined from one tissue or fluid can be correlated to an amount of biomarker in another fluid or tissue, as known in the art.
  • An indication for adjusting or maintaining a subsequent drug dose often is generated by comparing a determined level of biomarker in a subject to a predetermined level of biomarker.
  • a predetermined level of biomarker sometimes is linked to a therapeutic or efficacious amount of drug in a subject, sometimes is linked to a toxic level of a drug, sometimes is linked to presence of a condition, sometimes is linked to a treatment midpoint and sometimes is linked to a treatment endpoint, in certain embodiments.
  • a predetermined level of a biomarker sometimes includes time as an element, and in some embodiments, a threshold is a time-dependent signature.
  • an organic acid level of about 0.2-fold less than a normal level, or less may indicate that the dosage of the drug or the frequency of administration may be increased in a subsequent administration.
  • an organic acid level of about 5% fold less than a normal level, or less may indicate that the dosage of the drug or the frequency of administration may be increased in a subsequent administration.
  • the term "dosage" is meant to include both the amount of the dose and the frequency of administration, such as, for example, the timing of the next dose.
  • Some treatment methods comprise (i) administering a drug to a subject in one or more
  • administrations e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses
  • determining the presence, absence or amount of a biomarker in or from the subject after (i) determining the presence, absence or amount of a biomarker in or from the subject after (i)
  • providing an indication of increasing, decreasing or maintaining a subsequent dose of the drug for administration to the subject and
  • presence, absence or amount of a biomarker is determined after each dose of drug has been administered to the subject, and sometimes presence, absence or amount of a biomarker is not determined after each dose of the drug has been administered (e.g., a biomarker is assessed after one or more of the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth dose, but not assessed every time after each dose is administered).
  • An indication for adjusting a subsequent drug dose can be considered a need to increase or a need to decrease a subsequent drug dose.
  • subsequent drug dose can be considered by a clinician, and the clinician may act on the indication in certain embodiments.
  • a clinician may opt not to act on an indication.
  • a clinician can opt to adjust or not adjust a subsequent drug dose based on the indication provided.
  • An indication of adjusting or maintaining a subsequent drug dose, and/or the subsequent drug dosage can be provided in any convenient manner.
  • An indication may be provided in tabular form (e.g., in a physical or electronic medium) in some embodiments.
  • a biomarker threshold may be provided in a table, and a clinician may compare the presence, absence or amount of the biomarker determined for a subject to the threshold. The clinician then can identify from the table an indication for subsequent drug dose.
  • an indication can be presented (e.g., displayed) by a computer after the presence, absence or amount of a biomarker is provided to computer (e.g., entered into memory on the computer).
  • presence, absence or amount of a biomarker determined for a subject can be provided to a computer (e.g., entered into computer memory by a user or transmitted to a computer via a remote device in a computer network), and software in the computer can generate an indication for adjusting or maintaining a subsequent drug dose, and/or provide the subsequent drug dose amount.
  • a subsequent dose can be determined based on certain factors other than biomarker presence, absence or amount, such as weight of the subject, one or more metabolite levels for the subject (e.g., metabolite levels pertaining to liver function) and the like, for example.
  • a clinician may administer the subsequent dose or provide instructions to adjust the dose to another person or entity.
  • a decision maker can be a computer or a displayed computer program output in some embodiments, and a health service provider may act on the indication or subsequent drug dose displayed by the computer.
  • a decision maker may administer the subsequent dose directly (e.g., infuse the subsequent dose into the subject) or remotely (e.g., pump parameters may be changed remotely by a decision maker).
  • a subject can be prescreened to determine whether or not the presence, absence or amount of a particular biomarker may be determined.
  • prescreens include identifying the presence or absence of a genetic marker (e.g., polymorphism, particular nucleotide sequence); identifying the presence, absence or amount of a particular metabolite.
  • a prescreen result can be used by a clinician in combination with the presence, absence or amount of a biomarker to determine whether a subsequent drug dose may be adjusted or maintained.
  • Example 1 Urine Metabolite Profiles of Diabetic Patients
  • the urine metabolite profile of diabetic patients with kidney disease was studied to help identify organic acid derangements that can serve as biomarkers.
  • the study population included 14 patients with a diagnosis of diabetic kidney disease with Chronic Kidney disease (D-CKD) 3-4 (Mean GFR 31 .79 +/- 7.329). This population was compared to a control of 23 healthy volunteers with no diabetes or kidney disease. The control group contained 6 (26%) and the diabetic kidney disease with Chronic Kidney disease group contained 5 (35%) females. Twenty-four hour urine was collected from both control and D-CKD subjects.
  • a composite quantitative urine organic acid estimation was done using the Agilent 5973 Glass Chromatography and Mass spectrometry. Seventy-six different organic acids were looked at per sample and the results were standardized per mmol of creatinine.
  • the two groups were compared for each of the seventy-six metabolites and found 1 1 significant metabolites using the unpaired t test.
  • a cut-off p value of P ⁇ 0.00846 was chosen to have a false detection rate less than 0.05 to account for multiple testing.
  • the panel of metabolites was reported as the amount of metabolite per standard amount of creatinine.
  • Glomerular filtration rate (GFR), albumin creatinine ratio (ACR) and protein creatinine ratio (PCR) of the Diabetes with Chronic Kidney Disease group were then correlated to the values of the eleven significant metabolites using linear regression analysis.
  • the organic acid profile of urine from Diabetes with Chronic Kidney Disease patients was significantly decreased in metabolites of intermediate pathway.
  • the following organic acids were significantly decreased in the Diabetes with Chronic Kidney Disease group, when compared to the healthy controls: glycolic acid, 3-OH isobutyric acid, 3-OH isovaleric acid, aconitic acid, homovanillic acid, citric acid, uracil.
  • the level of 5-oxoproline was significantly increased in the Diabetes with Chronic Kidney Disease group when compared to the controls. Of these metabolites, only the citric acid levels and 3-OH isovaleric acid correlated to albumin creatinine ratio but not to GFR.
  • Urine metabolomics may be used to identify biomarkers for predicting responses to a drug.
  • the testing and analysis of various drugs may be performed using methods similar to those provided in this example of the drug pirfenidone.
  • urine metabolomics could provide information on the function of a drug in a clinical trial, the following is presented.
  • Pirfenidone is an orally-available anti-fibrotic agent that has shown efficacy in animal models of diabetic nephropathy and human focal segmental glomerulsoclerosis.
  • a randomized double-blind, placebo-controlled exploratory trial of pirfenidone was performed in subjects with diabetic nephropathy with elevated albuminura and reduced glomerular filtration rate (eGFR) (20-75 ml/min per 1 .73m 2 ).
  • the pre-specified primary outcome was eGFR change after one year of therapy.
  • Diabetic nephropathy remains the leading cause of end-stage kidney disease (ESKD) in the US, accounting for over 40% of incident ESKD cases.
  • ESKD end-stage kidney disease
  • Diabetic nephropathy is characterized by inflammation, accumulation of mesangial matrix in established disease, marked tubulointerstitial fibrosis and vascular hyalinosis in advanced disease. Both mesangial matrix expansion and tubulointerstitial fibrosis correlate with progression of diabetic nephropathy to ESKD 1 -3.
  • RAS renin-angiotensin system
  • ACE-i angiotensin converting enzyme inhibitors
  • ARBs angiotensin II receptor blockers
  • Blood pressure-independent benefits of RAS inhibitors may contribute to renal protection, possibly via inhibiting pro-fibrotic factors, such as transforming growth factor-beta (TGF- ⁇ ) 7.
  • TGF- ⁇ transforming growth factor-beta
  • the intensive use of RAS inhibitors is often limited by severe hyperkalemia, further reduction in the systemic blood pressure, and decreased renal blood flow. Even when maximized, they may decrease rate of progression, but do not arrest or reverse diabetic nephropathy.
  • TGF- ⁇ system is activated and plays a pathogenetic role in diabetic kidney disease in animal models of both type 1 9 and type 2 diabetes 10.
  • TNF-alpha system has also been recently linked with human diabetic nephropathy based on circulating blood levels 13 and gene expression in kidneys from patients with diabetic nephropathy 14.
  • An orally bioavailable compound, pirfenidone has been found to inhibit TGF- ⁇ production and consequent matrix deposition in experimental animal models of lung and kidney disease 15,16. In animal models and cell culture studies, pirfenidone also reduces TNF-alpha production 17, 18.
  • Oral pirfenidone administered to db/db mice after the onset of established diabetic kidney disease was effective in reducing glomerulosclerosis 19.
  • eGFR estimated glomerular filtration rate
  • Urine albumin/creatinine ratio was evaluated as a secondary outcome. There were no significant differences among study groups in change in ACR from baseline to the end of study
  • Urine TGF-beta levels increased by 1 .4 pg/mg creatinine (95% CI 0.2, 6.6) over 12 months in the placebo group, numerically increased by 0.3 pg/mg (95% CI -1 .6 , 5.6) in the pirfenidone 1200 mg group, and numerically declined by 0.1 pg/mg (95% CI -3.4, 3.7) in the pirfenidone 2400 mg group (all P values >0.05).
  • diastolic blood pressure was higher in the pirfenidone 2400 mg group and plasma albumin was higher in the pirfenidone 1200 mg group (Table 1 ).
  • the diastolic blood pressure was not associated with eGFR change in any treatment group or in the total study population. Multiple linear regression models, however, demonstrated that higher baseline plasma albumin was associated with eGFR change among all groups (p ⁇ 0.001 ) (Table 3).
  • Novel treatment approaches for diabetic nephropathy that reduce the rate of renal function decline are urgently needed, as the number of patients with ESKD attributed to diabetes continues to increase.
  • the available approaches to reduce the rate of renal function decline primarily work in an indirect manner via reducing hyperglycemia or blood pressure.
  • the eGFR improvement in the pirfenidone 1200 mg group was noted as early as 6 months after treatment initiation and was maintained through the end-of-study. It is possible that the early increase in eGFR may be due to a hemodynamic effect. Whether the benefit on eGFR is due to a reduction of matrix expansion in the glomerulus or tubulointerstitial compartments remains unknown as biopsies were not performed in this study. The significant improvement in eGFR suggests that treatment to reduce renal fibrosis may confer some degree of regression of the disease process in diabetic nephropathy.
  • Novel non-invasive biomarkers that correlate with progression and/or regression may be suitable as surrogate markers for demonstrating regression and would be of great assistance in future studies evaluating anti-fibrotic therapies.
  • a candidate list of biomarkers within this study was selected. None of the selected biomarkers significantly changed with therapy, or predicted response to therapy. Whether these results reflect low statistical power due to the relatively small sample size within each treatment arm, or whether other markers may reflect treatment response more precisely is presently unknown, and an important area for future research. Of great interest, however, was that several biomarkers were strongly associated with eGFR at baseline.
  • inflammatory biomarkers validate the results of the prior study and suggest that these factors may signify the ongoing inflammation occurring systemically, and potentially influencing progression within the kidney. Further support for inflammatory mechanisms in diabetic nephropathy is provided from gene expression data and renal biopsies from patients with diabetic nephropathy 14. In these studies using isolated glomeruli, markers of the inflammatory network were strongly up-regulated.
  • Albuminuria is the classic biomarker for diabetic nephropathy and reduction is thought to be beneficial. A reduction in albuminuria has been found to correlate with reducing the rate of renal function decline with ARB treatment 28. Notably, albuminuria did not decrease with pirfendione treatment. This data is similar to what was observed in a recent open-label clinical study of pirfenidone in patients with advanced FSGS, as pirfenidone treatment was associated with reduction of the rate of renal function decline without attenuating albuminuria 20. Similarly, in an animal model pirfenidone conferred benefit to glomerular histology and reduced gene expression of matrix molecules in the diabetic db/db mouse, without lowering albuminuria 19.
  • Urine levels of TGF-beta have been found to be increased in patients with diabetic nephropathy 29-31 and may reflect ongoing renal production 31 .
  • urine levels of TGF- was not significantly affected by pirfenidone; this may be due to the wide variability of urine levels in patients and/or the small sample size evaluated here.
  • Pirfenidone binding to albumin may impact the pharmacokinetic profile, although how increased albumin binding of pirfenidone may be beneficial is unclear.
  • the present study also highlights the need to identify new markers of renal disease progression, apart from albuminuria, that could be used as surrogate markers of anti-fibrotic therapies.
  • Pirfenidone is a pyridone derivative that has both anti-fibrotic and anti-inflammatory properties.
  • IPF idiopathic pulmonary fibrosis
  • Two phase III studies of pirfenidone for IPF in the North America, Europe, and Australia reported equivocal statistical outcomes, but overall reduced disease severity. At present, the drug has not been approved by the FDA for clinical use in the United States.
  • the study protocol was approved by the institutional review boards of the participating centers and all patients provided written consent. Trial sites were Thomas Jefferson University, Philadelphia, PA; the Mayo Clinic, Rochester, MN; and the NIH Clinical Center, Bethesda, MD. At the pre-study evaluation, the potential study candidates provided a medical history and underwent a complete physical examination, including clinical laboratory determinations and the measurement of vital signs. Entry criteria included a history of type 1 or type 2 diabetes, eGFR 20-75 ml/min/1 .73 m2, microalbuminuria or overt proteinuria, blood pressure ⁇ 140/90 mm Hg on an a ACEi, ARB or the combination, if tolerated. Exclusion criteria included other causes of kidney disease, history of photosensitivity rash, and liver disease.
  • a stratified block randomization scheme was used to maximize the balance of patients with type 1 and type 2 diabetes assigned to each treatment group, using randomization blocks of size 4.
  • Eligible subjects were randomly assigned to receive pirfenidone 1200 mg daily, pirfenidone 2400 mg daily, or placebo for 54 weeks.
  • Subjects received two 400 mg capsules of pirfenidone three times, one capsule of pirfenidone and one capsule of placebo three times daily), or two capsules of placebo three times daily.
  • Subjects who developed nausea, heartburn or reflux, epigastric pain, or severe fatigue that persisted for more than one week had a dose reduction of 25% and then gradually brought back to their intended dose. If symptoms persisted, the dose was kept at the lowest tolerable dose (but not less than 50% of original dose) if the subject desired to continue in the protocol.
  • the primary endpoint was eGFR change from baseline to end of study.
  • Baseline eGFR was determined from two serum creatinine measurements within 3 weeks prior to starting study drug.
  • End of study eGFR was determined from two serum creatinine measurements at week 52 and week 54, prior to stopping study medication.
  • Serum creatinine was measured by the Jaffe method separately at each clinic site laboratory.
  • eGFR was determined by the 4-variable Modification of Diet in Renal Disease Study equation 37.
  • Secondary endpoints included the change in urine ACR and the change in urine TGF- / urine creatinine.
  • Biomarkers were analyzed in plasma and urine from samples collected at both baseline visits and both end-of-study visits, with the two results averaged. Plasma biomarkers were measured by MesoScale Discovery (MSD) platform at the UCSD Clinical Translational Research Institute (CTRI) facility. The MSD platform uses electrochemiluminescence tags on specific antibodies that emit light when electrochemically stimulated.
  • MSD MesoScale Discovery
  • the specific blood biomarkers measured were interferon-y (IFNy), interleukin 1 (IL-1 ), tumor necrosis factor (TNF), soluble TNF receptor 1 (S TNF R1 ), YKL40 (also called chitinase like protein or human cartilage glycoprotein-39), brain natriuretic peptide (BNP), and TGF-beta1 .
  • Fibroblast growth factor 23 (FGF23) was measured by a C-terminal ELISA assay (Immutopics, San Clemente, CA).
  • Urine biomarkers included urine TGF-beta1 (measured by Quantikine, R&D Biosystems, as previously described 29). Urine albumin was measured by nephelometry and urine creatinine was measured by the Jaffe method.
  • IFNy interferon-y
  • IL-1 interleukin 1
  • TNF tumor necrosis factor
  • S TNF R1 soluble TNF receptor 1
  • BNP brain
  • the pre-specified primary endpoint analysis was the eGFR change, compared between each pirfenidone treatment group with placebo group using the Student t-test. Secondary endpoints (ACR change and urine TGF-beta1 changes were similarly evaluated. When one of the two visits was missing, a single observation was used. Due to missing data the assumption that the data points arose from an identical distribution (equal variance) was put in question. Therefore in addition to the two-sample t-tests, we also performed permutation tests with 10,000 permutations of the treatment assignments, as well as an analysis of covariance (ANCOVA) 38 with iterated re- weighted least-squares (IWLS) 39 as confirmatory analyses. Data are presented as mean ⁇ SD for normally distributed variables, and medians and interquartile ranges for skewed variables.
  • Fibroblast growth factor-23 is associated with parathyroid hormone and renal function in a population-based cohort of elderly men. Eur J Endocrinol 158, 125-9 (2008).
  • Diabetes Mellitus type 2 n,% 16 (76%) 13 (76%) 11 (79%) 0.99
  • Diabetes duration (years), mean ⁇ SD 24 ⁇ 13 18 ⁇ 10 19 ⁇ 9 0.25
  • HbA1c (g/dL), mean ⁇ SD 7.3 ⁇ 1.4 7.4 ⁇ 1.2 7.1 ⁇ 1.4 0.81
  • HDL cholesterol (mg/dL), mean ⁇ SD 45 ⁇ 11 44 ⁇ 9 43 ⁇ 12 0.86
  • LDL cholesterol (mg/dL), mean ⁇ SD 96 ⁇ 28 108 ⁇ 39 104 ⁇ 24 0.53
  • Triglycerides (mg/dL), median (IQR) * 104 (70, 171) 116 (76, 188) 109 (88, 157) 0.83
  • Serum albumin (g/dl) mean ⁇ SD 4.4 ⁇ 0.3 4.6 ⁇ 0.4 4.2 ⁇ 0.4 0.04 eGFR (ml/min/1.73m 2 ), mean ⁇ SD 39 ⁇ 13 38 ⁇ 13 39 ⁇ 13 0.95
  • Placebo Arm 21 0.4 (-0.2, 1 .1 ) 0.21
  • Urine metabolomics was performed in subjects enrolled in this study at baseline and at 6 months after enrollment. A panel of metabolites based on a specific ratio added predictive value to the outcome on the drug. Additionally two metabolites (azelaic acid and citric acid) were affected by the drug and correlated with outcomes. Similar approaches can be employed with other clinical trials to identify which patients will respond to drug, which patients will not, and identify potential new mechanisms.
  • metabolite profiles could predict adverse events in patients that are ongoing or will develop in the future.
  • Example 3 Prediction of medical complications in patients with diabetes.
  • diabetes-related complications such as microvascular complications (for example, nephropathy, neuropathy, retinopathy) or macrovascular complications
  • cardiovascular peripheral vascular or cerberovascular disease
  • kidney disease Often, once they develop evidence of kidney disease, their risk for these other complications increases.
  • patients having or at risk for kidney disease may be identified before the onset of severe kidney disease. Those patients identified as having or being at risk for kidney disease may then be identified as being likely to develop of microvascular and macrovascular complications. Also, the methods provided herein for identifying kidney disease may also be used to identify patients at risk for developing diabetes-related complications, such as microvascular or macrovascular complications.
  • the patients are identified determined based on the levels of metabolites such as, for example, lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH- isobutyric acid, palmitic acid, and citrate found in urine or blood samples from the subject. ( Figures 2-4).
  • metabolites such as, for example, lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH- isobutyric acid, palmitic acid, and citrate found in urine or blood samples from the subject.
  • Example 4 Prediction of diabetes, cardiovascular disease, hypertension, and CKD in individuals with obesity.
  • a profile including lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citrate found in urine or blood samples from the subject predicts underlying organ dysfunction that portends diabetes, cardiovascular disease, hypertension or CKD in specific individuals of asymptomatic members of the group.
  • Example 5 Prediction of diabetes, cardiovascular disease, hypertension, and CKD in individuals with obesity.
  • Example 4 The procedure of Example 4 is repeated with a blood sample collected in concert with the urine sample.
  • the blood sample trends parallel those for urine samples when compared to blood sample controls.
  • Example 6 Prediction of hypertension and complications of hypertension. High blood pressure is found in many individuals and puts patients at risk of cardiovascular and kidney complications. Through the measurement of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citric acid found in urine or blood samples from the subject , one can identify subtle signs of organ dysfunction in patients with hypertension and be able to monitor organ dysfunction in response to drug or non-drug therapies.
  • liver disease Many individuals with obesity, diabetes or CKD have liver disease.
  • the methods provided herein provide an easier method of detecting liver disease through measurement of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, and citric acid found in urine or blood samples from the subject.
  • liver function is determined. The results may provide sensitive clues as to underlying liver function and its response to drug and non-drug therapies, such as diet, exercise, or lifestyle changes through repeated testing of samples as function of time, alone or in combination with conventional liver enzyme
  • Example 8 Ancillary Joint involvement.
  • the methods provide herein provide an easier method of detecting joint involvement.
  • Using the methods provided herein through measurement of lactic acid, glycolic acid, fumaric acid, malic acid, adipic acid, 2-OH-glutaric acid, aconitic acid, homovanillic acid, stearic acid, 3-OH-isobutyric acid, palmitic acid, citric acid, and 5-oxoproline found in urine or blood samples from the subject, joint involvement is determined.
  • results provide sensitive clues as to underlying joint involvement and its response to drug and non-drug therapies, such as diet, exercise, or lifestyle changes through repeated testing of samples as function of time, alone or in combination with conventional ultrasound and MRI measurements of joints.
  • drug and non-drug therapies such as diet, exercise, or lifestyle changes through repeated testing of samples as function of time, alone or in combination with conventional ultrasound and MRI measurements of joints.
  • Other metabolites in the urine may also provide informational value to understand the development and severity of joint disease.
  • Example 9 Ancillary Lung disease.
  • lung involvement such as sleep apnea, restrictive lung disease or obstructive lung disease.
  • lung involvement is determined.
  • the results provide sensitive clues as to response to drug and non-drug therapies, such as diet, exercise, or lifestyle changes through repeated testing of samples as function of time, alone or in combination with conventional pulmonary measurements.
  • Other metabolites in the urine may also provide informational value to understand the development and severity of lung disease.
  • a or “an” can refer to one of or a plurality of the elements it modifies (e.g., "a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described.
  • the term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., "about 1 , 2 and 3" refers to about 1 , about 2 and about 3).
  • a weight of "about 100 grams” can include weights between 90 grams and 1 10 grams.

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Abstract

La technologie concerne en partie des procédés d'identification de la présence d'une néphropathie, de détermination du degré de la néphropathie ou de l'évolution de la néphropathie, chez un sujet chez lequel a été ou non diagnostiqué un diabète. La technologie concerne en outre des procédés pour déterminer les cibles de la thérapie de la néphropathie, l'efficacité d'un traitement de la néphropathie, et des procédés de détermination de la toxicité d'une substance thérapeutique chez un sujet atteint d'une néphropathie.
PCT/US2011/056229 2010-10-14 2011-10-13 Profil métabolomique de l'urine en cas de néphropathie diabétique WO2012051463A2 (fr)

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