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WO2012112589A2 - Procédés et compositions de détection du cholestérol dans l'urine comme biomarqueur de lésions rénales aigues - Google Patents

Procédés et compositions de détection du cholestérol dans l'urine comme biomarqueur de lésions rénales aigues Download PDF

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WO2012112589A2
WO2012112589A2 PCT/US2012/025100 US2012025100W WO2012112589A2 WO 2012112589 A2 WO2012112589 A2 WO 2012112589A2 US 2012025100 W US2012025100 W US 2012025100W WO 2012112589 A2 WO2012112589 A2 WO 2012112589A2
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cholesterol
aki
urine
subject
gene
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WO2012112589A9 (fr
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Richard A. ZAGER
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Fred Hutchinson Cancer Research Center
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/60—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • 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

  • sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification.
  • the name of the text file containing the sequence listing is: 38763_Sequence_Final_2012-02- 08.txt.
  • the file is 7KB; was created on February 8, 2012; and is being submitted via EFS-Web with the filing of the specification. FIELD OF THE INVENTION
  • the present invention relates to novel methods and reagents for detecting acute kidney injury based on detectable levels of cholesterol in the urine.
  • AKI acute kidney injury
  • a method for determining the presence of kidney injury in a mammalian subject. The method comprises (a) performing an assay to determine the presence or amount of cholesterol in a urine sample obtained from the subject; and (b) comparing the results of step (a) with a cholesterol reference standard. The presence or elevated level of cholesterol in step (a) as compared to the cholesterol reference standard indicates the presence of kidney injury in the subject.
  • the assay of step (a) determines the amount of cholesterol associated with a cellular component of the urine sample.
  • the cellular component comprises cell plasma membrane or cell organelle membranes.
  • the cellular component comprises components of proximal tubular cells, including intact cells.
  • the amount of cholesterol in the urine samples of step (a) is normalized using an internal control substance present in the cellular component of the urine samples.
  • the internal control substance comprises a phospholipid.
  • the method comprises obtaining a urine sample from the subject. In one embodiment, the method comprises processing the urine sample obtained from the subject. In one embodiment, the method comprises separating the cellular component from the urine. In one embodiment, the cellular component of the urine sample is separated from the urine by subjecting the urine sample to a sufficient centrifugal force to obtain a pellet containing cholesterol. In another embodiment, the cellular component of the urine sample is separated by filtering the urine sample to permit capture of cell components containing cholesterol.
  • the mammal is a rodent, rabbit, cow, horse, dog, cat, or human.
  • FIGURE 1 is a graphical representation of cholesterol levels in urinary pellets observed in control mice, mice with pre-renal azotemia, and mice with glycerol-mediated and maleate-mediated acute kidney injury (AKI) at 3 and 24 hours; the cholesterol levels were normalized with phospholipid phosphate (PLP) as an internal control; * signifies a significant difference (p ⁇ 0.01) as compared to normal controls, as described in Example 1;
  • FIGURE 2 is a graphical representation of urea-derived nitrogen levels per volume of blood, as determined by a blood urea nitrogen (BUN) analysis, for control mice, mice with pre-renal azotemia, and mice with glycerol-mediated and maleate- mediated acute kidney injury (AKI); * signifies a significant difference (p ⁇ 0.01) as compared to normal controls, as described in Example 1 ;
  • FIGURE 3 is a graphical representation of cholesterol levels detected in human urine pellet samples obtained from normal control individuals, intensive care unit (ICU) patients without AKI (“ICU / AKI-”), ICU patients with AKI (“ICU / AKI+”), and patients with chronic kidney disease (“CKD”); cholesterol levels are normalized using phospholipid phosphate (PLP) as a control; "NS” signifies a no significant difference as compared to normal controls and p values indicating significant differences are provided, as described in Example 2;
  • FIGURE 4 is a graphical representation of relative levels of RNA Polymerase II (Pol II) binding to the human 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) gene as determined in normal control individuals, ICU patients without AKI ("ICU / AKI-"), and ICU patients with AKI ("ICU / AKI+”); Pol II binding levels are normalized using Pol II binding to the ⁇ actin gene as an internal control; p values indicating significant differences in Pol II binding as compared to normal individuals are indicated, as described in Example 3; and
  • FIGURE 5 is a graphical representation of relative levels of trimethylated histone H3 at the lysine 4 position ("H3K4m3") at exon 1 of the human 3-hydroxy-3-methyl- glutaryl-CoA reductase (HMGCR) gene, as determined by ChIP analysis in normal control individuals, ICU patients without AKI ("ICU / AKI-”), and ICU patients with AKI ("ICU / AKI+”); H3K4m3 levels are normalized using the H3K4m3 levels to the ⁇ actin gene as an internal control; a p value indicating significant differences in H3K4m3 levels as compared to normal individuals is indicated, as described in Example 3.
  • H3K4m3 trimethylated histone H3 at the lysine 4 position
  • HMGCR human 3-hydroxy-3-methyl- glutaryl-CoA reductase
  • azotemia refers to abnormally high levels of nitrogen-containing compounds in the blood. These elevated levels are often attributed to insufficient filtering of the compounds from the blood by the kidneys. Azotemia can generally be divided into three classifications: pre-renal, renal (or structural), and post-renal. Pre-renal azotemia is caused by restricted blood flow to the kidneys. Causes can include decreased cardiac output, restriction of the renal artery, and shock. There is no inherent damage to the kidneys that causes the elevated levels of nitrogen in the blood. Renal, or structural, azotemia is typically caused by damage or disease of the kidney that directly impairs the ability of the kidney to properly filter the blood. Such damage is referred to herein as acute kidney injury ("AKI").
  • AKI acute kidney injury
  • gene activation refers to the enhanced or increased transcription of the gene leading to increased levels of mRNA and, ultimately, increased levels of the corresponding protein.
  • cellular component refers to the portion of the urine that derives from cellular debris or whole cells that are sloughed off from the kidney tissue during the filtration process.
  • the cellular component can include proximal tubular cells or tubular "brush border” fragments that separate from the kidney tissue and enter tubular lumina.
  • HMG CoA reductase renal cortical 3-hydroxy-3-methyl-glutaryl-CoA reductase
  • HMGCR renal cortical 3-hydroxy-3-methyl-glutaryl-CoA reductase
  • this sequence of events is analogous to the so-called “heat shock” response: i.e., whereby a renal stress (i.e., heat shock) up-regulates cytoprotective molecules (i.e., heat shock proteins) which confer a cytoresistant state
  • cytoprotective molecules i.e., heat shock proteins
  • Gaudio, K.M., et al "Role of heat stress response in the tolerance of immature renal tubules to anoxia”
  • Am. J. Physiol. 274:F1029-1036 1998
  • Van Why, S.K. and N.J. Siegel "Heat shock proteins in renal injury and recovery," Curr. Opin. Nephrol. Hypertens. 7:407-412, 1998).
  • a morphologic correlate of AKI is sloughing of proximal tubular 'brush border' fragments and intact tubular cells into urine (Venkatachalam, M.A., et al, "Ischemic damage and repair in the rat proximal tubule: differences among the SI, S2, and S3 segments," Kidney Int. 4:31-49, 1978; Donohoe, J.F., et al, "Tubular leakage and obstruction after renal ischemia: structural-functional correlations," Kidney Int.
  • the inventor has discovered that evidence of increased HMGCR gene activity detectable in urine, including elevated cholesterol levels, elevated binding of RNA Polymerase II to the HMGCR gene promoter, and elevated chromatin modification corresponding to the HMGCR gene, correlate with AKI. Specifically, the inventor demonstrates herein that (1) acute tubular injury activates the HMGCR gene; (2) this results in increased cholesterol synthesis and proximal tubular cell cholesterol loading; and (3) upon release of tubular cells and tubular cell debris into the urinary space, increases in urinary pellet cholesterol content result. The inventor previously demonstrated that experimental chronic nephropathy does not activate these pathways (Johnson, A.C., et al, "Experimental glomerulopathy alters renal cortical cholesterol," Am. J. Pathol.
  • the present invention relates to the discovery that elevated levels of cholesterol detectable in the urine correlate with acute kidney injury (AKI). Elevated levels of cholesterol were detected in urinary pellets in two murine models of structural AKI, but not in a murine model of pre-renal azotemia. Furthermore, elevated levels of cholesterol were detected in urinary pellets from human intensive care unit (ICU) patients with AKI, but not in ICU patients without AKI or patients with chronic kidney disease.
  • ICU human intensive care unit
  • HMGCR 3-hydroxy-3- methyl-glutaryl-CoA reductase
  • mice were subjected to two models of AKI (rhabomyolysis; maleate toxicity) or to a model of pre-renal azotemia (indomethicin + surgical stress).
  • Urine samples were collected 3 or 24 hours later.
  • Experimental AKI, but not pre-renal azotemia, induced ⁇ 2- to 3-fold increases in urinary pellet cholesterol levels, and this occurred within 3 hours of AKI induction.
  • the urines were centrifuged, the pellets underwent lipid extraction, and the extracts were assayed for cholesterol and total membrane phospholipid content. The results were expressed as cholesterol/phospholipid ratios.
  • Clinical AKI demonstrated a doubling of pellet cholesterol levels.
  • neither critical illness without AKI, nor clinical CKD had this effect.
  • urinary protein AKI biomarkers e.g., NGAL, KIM-1, and MCP-1, are typically elevated with both acute as well as chronic kidney disease.
  • HMGCR gene activation in AKI+ patients was supported by findings of increased R A polymerase II binding to, and increased levels of a gene activating histone marker (H3K4m3) at, urinary fragments of the HMGCR gene (exon 1 ; chromatin immunoprecipitation). Because the degree of Pol Il-gene binding correlates with rates of gene transcription, this finding provides clinical support for the experimental observation that AKI increases HMGCR gene activity.
  • H3K4m3 a gene activating histone marker, H3K4m3, was also elevated at the HMGCR gene in the AKI+, but not in the AKI-, provides additional support that AKI induces HMGCR gene activation, and indicates that corresponding increases in urinary pellet cholesterol levels have utility for use as a biomarker for the presence of AKI.
  • cholesterol is highly stable in urine samples, in marked contrast to traditional protein biomarkers that are subject to enzymatic and non-enzymatic degradation.
  • cholesterol is a biomarker for cells that are recoverable in the urine.
  • Traditional urinary protein biomarkers of renal injury reflect a balance between what is generated in the kidney and what is generated outside the kidney with secondary urinary excretion. Thus, urinary protein biomarker levels may not reflect direct kidney events. This drawback is not an issue with cholesterol levels associated with the cell components of urinary samples because circulating cells do not gain access to urine by glomerular filtration (with the possible exception of red blood cells, which are readily detected).
  • cholesterol is the only known lipid biomarker of acute renal injury.
  • the current emphasis in the renal literature is to discover a panel of biomarkers to be used in concert, rather than relying on a single one.
  • the addition of a lipid biomarker to a protein biomarker panel has the potential to expand clinical utility.
  • Fourth, essentially all described protein biomarkers are elevated by both acute as well as chronic kidney disease (CKD).
  • CKD chronic kidney disease
  • the present disclosure provides a method for determining the presence of kidney injury in a mammalian subject.
  • the method comprises (A) performing an assay to determine the presence or amount of cholesterol in a urine sample obtained from the subject, and (B) comparing the results of step A with a cholesterol reference standard, wherein the presence or elevated level of cholesterol in step A compared to the reference standard indicates the presence of kidney injury in the subject.
  • the reference standard is a value derived from one or more subjects with no kidney injury.
  • the assay of step (A) measures cholesterol associated with a cellular component of the urine sample.
  • the cellular component can comprise cell plasma membranes or cell organelle membranes.
  • the cellular component comprises components of proximal tubular cells, including intact cells.
  • the method comprises obtaining a urine sample from the subject. It will be understood that the sample can be obtained from the subject directly by the practitioner of the method from the subject. The sample can also be obtained indirectly from the subject through an intermediary, such as a primary care provider or clinic technician.
  • the method comprises processing the urine sample.
  • the urine sample is processed to remove substantially all of the liquid from the sample.
  • the cellular component of the urine sample is separated from the urine in the sample.
  • the cellular component of the urine sample is separated from the urine by subjecting the urine sample to a sufficient centrifugal force to obtain a pellet containing cholesterol.
  • the cellular component of the urine sample is separated by filtering the urine sample to permit capture of cell components containing cholesterol.
  • the assay comprises determining the level of cholesterol in the urine, wherein an elevated amount of cholesterol in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject.
  • the assay to determine the level of cholesterol in the urine can comprise any assay for detection or quantification of cholesterol that are commonly known in the art. For example, as described in Example 1, the Amplex Red assay can be employed.
  • the cholesterol in the urine is associated with a cellular component derived at least in part from kidney proximal tubule cells. The cellular component can be isolated from the urine by methods such as, for example, centrifugation to form a pellet, or filtration.
  • the assay can further comprise normalizing the level of cholesterol in the urine to the level of an internal control substance in the cellular component of the urine samples.
  • Any internal control substances can be used, for example, that is known in the art to provide a comparison useful to infer differences or fluctuations in the amounts of cholesterol per volume of urine or relative to the amount of cellular component appearing in the urine.
  • internal control substance can comprise a phospholipid, as herein in the Examples.
  • the reference cholesterol standard is the level of cholesterol determined in one or more urine samples obtained from one or more mammalian subjects of the same species that do not have any known kidney injury or disease. In one embodiment, the reference standard is the level of cholesterol determined in one or more urine samples obtained from one or more mammalian subjects of the same species that do not have any detectable kidney injury or disease. In one embodiment, the reference standard is the level of cholesterol determined in one or more urine samples obtained from one or more mammalian subjects of the same species that do not have detectable AKI. In one embodiment, the level of cholesterol determined for the reference standard is determined with the same or similar assay used to determine the presence or amount of cholesterol in the subject.
  • the reference standard can be derived from one or more individuals of the same species as the subject, such as 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or more individuals, or any number between.
  • the reference standard is a numeric value of the cholesterol that represents a median or average level of cholesterol in a plurality of individuals of the same species of the subject that are known to not have AKI.
  • the reference standard is a value predetermined relative to the performance of the assay step on the urine sample obtained from the subject.
  • the results of step (A) are compared with a reference standard to determine the presence of AKI in the subject.
  • a determination is made when the amount of cholesterol in a urine sample obtained from the subject is elevated compared to the reference standard.
  • the presence of AKI in the subject is indicated when the amount of cholesterol in the urine sample obtained from the subject exceeds the value of reference standard by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, wherein percent refers to the reference standard.
  • a value of 75 exceeds a reference standard value of 50 by 50% of the reference standard value (i.e., 25).
  • the presence of AKI in the subject is determined when the comparison indicates a statistically significant elevation of cholesterol in a urine sample from the subject compared to the reference standard.
  • Statistically significant differences can be established according to standard statistical analytic methods familiar in the art. For example, as described in Example 2, t tests can be performed to compare values and ascertain statistical differences. Further, Bonferroni corrections can be used for multiple comparisons. Statistical significance can be established at any threshold accepted in the art. For example, as described below in Example 2, statistical significance was judged at a P value of ⁇ 0.05 when using unpaired t test comparisons.
  • the disclosure provides an additional method for determining the presence of kidney injury in a mammalian subject.
  • the method comprises: (A) performing an assay on the contents of a urine sample obtained from the subject to determine a relative activity level of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) gene in kidney proximal tubule cells; and (B) comparing the relative activity level of the HMGCR gene in step A to a reference standard, wherein an elevated level in step A compared to the standard indicates the presence of kidney injury in the subject.
  • the reference standard is a value derived from one or more subjects with no known kidney injury.
  • the method comprises the step of separating a cellular component from the urine in the sample.
  • the cellular component is separated from the urine in the sample by centrifugation or filtration.
  • the assay comprises determining the amount of 3-hydroxy-3- methyl-glutaryl-CoA reductase present in the urine sample, wherein an elevated amount of 3-hydroxy-3-methyl-glutaryl-CoA reductase in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject.
  • the 3-hydroxy-3-methyl-glutaryl-CoA reductase in the urine is associated with a cellular component of the urine sample.
  • the amount of 3- hydroxy-3-methyl-glutaryl-CoA reductase is determined by a method selected from the group consisting of mass spectrometry and Western blot employing an antibody or fragment thereof that specifically binds to 3-hydroxy-3-methyl-glutaryl-CoA reductase.
  • the assay comprises determining the amount of mRNA corresponding to the 3-hydroxy-3-methyl-glutaryl-CoA reductase gene, wherein an elevated amount of mRNA in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject.
  • the assay comprises using reverse transcription polymerase chain reaction or Northern blot, using primers or probes specific for the 3-hydroxy-3-methyl-glutaryl-CoA reductase mRNA.
  • the assay comprises: (A) measuring the level of RNA Polymerase II (Pol II) bound to the 3-hydroxy-3-methyl-glutaryl-CoA reductase gene; and (B) normalizing the level of Pol II binding in step A to the level of Pol II binding to an internal control gene, wherein an elevated normalized amount of Pol II bound to the 3-hydroxy-3-methyl-glutaryl-CoA reductase gene measured in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject.
  • the assay comprises a chromatin immunoprecipitation (ChIP) step.
  • the assay comprises: (A) measuring the level of histone methylation in the chromatin structure corresponding to the 3-hydroxy-3-methyl-glutaryl- CoA reductase gene; and (B) normalizing the level of histone methylation in step A to the level of histone methylation in the chromatin structure corresponding to an internal control gene, wherein an elevated normalized amount of histone methylation measured in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject.
  • the assay comprises a chromatin immunoprecipitation (ChIP) step.
  • HMG CoA reductase renal cortical 3-hydroxy-3-methyl-glutaryl-CoA reductase
  • HMGCR renal cortical 3-hydroxy-3-methyl-glutaryl-CoA reductase
  • Urine samples were collected from mice that were subjected to two experimental models of AKI: glycerol induced rhabdomyolysis (Nath, K.A., et al, "Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat," J. Clin. Invest. 90:267-270, 1992; Nath, K.A., et al, "Renal response to repetitive exposure to heme proteins: chronic injury induced by an acute insult," Kidney Int.
  • mice 14 male CD-I mice were subjected to the glycerol model of AKI, and 13 male CD-I mice were subjected to the maleate model of AKI. Either 3 hours or 24 hours later (approximately half at each time point), the mice were anesthetized with a 50 mg/Kg intraperitoneal administration of pentobarbital, and urine was obtained by gentle pressure on the exposed urinary bladder. Urine extraction was followed by blood sample collection from the inferior vena cava for blood urea nitrogen (BUN) analysis.
  • BUN blood urea nitrogen
  • Urine samples were centrifuged at 12,000 rpm a to produce a pellet of the solid debris and cellular components of the urine. It will be understood that any centrifugation speed can be employed that produces a pellet.
  • the liquid urine was siphoned off.
  • Alternative methods to obtain and isolate the solid debris and/or cellular components of the urine sample include filtering the urine.
  • Lipid content within the centrifuged urine pellets was extracted in chloroform:methanol. Lipid fractions were assayed for cholesterol content using the Amplex Red assay (Stirewalt, D.L., et al, "Mevastatin can increase toxicity in primary AMLs exposed to standard therapeutic agents, but statin efficacy is not simply associated with ras hotspot mutations or overexpression," Leuk. Res. 27: 133-45, 2003; Li, H.Y., et al, "Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses," Blood 707:3628-34, 2003).
  • the Amplex Red assay (Molecular Probes, Eugene, Oregon) is a fluorometric technique that relies on the oxidation of cholesterol into a ketone and hydrogen peroxide.
  • the hydrogen peroxide reacts stoichiometrically with the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) in the presence of horseradish peroxidase to form the fluorescent compound resorufin.
  • a cholesterol standard curve can be determined for each plate using a cholesterol calibrator (Sigma) diluted at various concentrations in Amplex Red reaction buffer in lieu of cell lysates.
  • lipids obtained from a urine sample can be extracted in chloroform- methanol (1 :2) and dried under nitrogen.
  • samples are transferred to glass tubes containing an internal standard solution (stigmasterol, 100 ⁇ g/mL in ethyl acetate [EtOAc]; Sigma), dried under nitrogen, and reconstituted in 100 ⁇ ⁇ bis-(trimethylsilyl)trifluoroacetamide (BSTFA; Sigma) (25% vol/vol EtOAc). These samples are then sealed in an injection vial and heated for 1 hour at 60°C.
  • EtOAc ethyl acetate
  • samples are applied to a chromatograph, e.g., Hewlett Packard 5890 Series II gas chromatograph, fitted with a flame ionization detector and a 30 m x 0.32 mm DB-5 (0.25 ⁇ ) column (e.g., J&W Scientific, Folsom, California).
  • a chromatograph e.g., Hewlett Packard 5890 Series II gas chromatograph, fitted with a flame ionization detector and a 30 m x 0.32 mm DB-5 (0.25 ⁇ ) column (e.g., J&W Scientific, Folsom, California).
  • the initial temperature (100°C) is maintained for 3 minutes, after which it is increased by 40°C per minute to 290°C and thereafter by 5°C per minute to 300°C for 5 minutes.
  • Cholesterol ethers are quantitated after elution from the gas chromatograph at 12.5 minutes.
  • Phospholipid phosphate (PLP) content can be assayed according to acceptable and known methods in the art; for example, as described in Van Veldhoven, P.P., and G.P Mannaerts, "Inorganic and Organic Phosphate Measurements in the Nanomolar Range,” Anal Biochem 161:45- 48, 1987.
  • the PLP content served as an internal standard in each sample assayed to normalize for the overall volume of cellular debris, specifically, cellular membranes found in the urine.
  • Alternatives to PLP for use as internal standards include other phospholipids, such as phosphatylcholine.
  • Blood urea nitrogen was assayed for the urine donors to confirm the occurrence of azotemia in the experimental AKI and pre-renal kidney azotemia models using standard, well-known techniques based on an autoanalyzer instrument.
  • the BUN analysis results were assayed from blood sample obtained at the 24 hour samples.
  • This Example describes the discovery that urinary pellet cholesterol levels are elevated in human patients with acute kidney injury (AKI), but not in critically ill intensive care unit (ICU) patients without AKI or patients with chronic kidney disease (CKD).
  • AKI acute kidney injury
  • ICU critically ill intensive care unit
  • CKD chronic kidney disease
  • Example 1 As described in Example 1, using multiple murine models, the presence of elevated cholesterol levels in urine pellets was correlated with structural AKI but not with pre-renal azotemia. Therefore, urine samples obtained from various human patients suffering from AKI or other diseases were similarly assessed to further ascertain the role of urinary pellet cholesterol levels as a biomarker for AKI.
  • AKI+ was defined as a > 50% (or >26.5 ⁇ / ⁇ ) increase in serum creatinine concentrations from baseline. Creatinine levels can be assayed according to standard methods known in the art.
  • the AKI- group was comprised of 15 critically ill, ICU-hospitalized patients who had comparable overall illness severity as the AKI+ group, as determined by APACHE II scores (Knaus, W.A., et al, "APACHE II: A Severity of Disease Classification System," Crit Care Med:.13 %-29, 1985) but who did not have AKI.
  • the AKI+ and AKI- populations were matched for age, race, gender, and sepsis status. Demographic information for these patients and the specifics of urine sample collection have been previously described (Ware, L.B., et al, "Renal cortical albumin gene induction and urinary albumin excretion in responses to acute kidney injury," Am. J. Physiol. 300:F628- F638, 201 1).
  • the CKD patient population consisted of six individuals with diabetic nephropathy, and nine individuals with non-diabetic CKD. These patients were enrolled in a study of CKD as part of a Seattle kidney study; Kidney Research Institute, Seattle, Washington. Subjects were eligible for the present study if they had a Modification of Diet in Renal Disease estimated GFR ⁇ 60 ml/min/ 1.73 m 2 , were not receiving dialysis, and were at least 18 years of age.
  • urinary pellet cholesterol levels for the chronic kidney disease (CKD) and ICU/AKI- patients were virtually identical to those of the normal volunteers, as denoted with "NS".
  • the ICU/AKI+ patients manifested a near-doubling of urine pellet cholesterol levels compared to the normal controls (p ⁇ 0.001) or the ICU/AKI- patients (p ⁇ 0.005).
  • the cholesterol levels were factored by the amount of PLP as an internal control for the cellular lipid present in the sample.
  • the levels of cholesterol represented in FIGURE 3 reflect the cholesterol levels present in the cells recovered in the urine, relative to other lipid constituents of the cells.
  • statin therapy which is common in patients with CKD, could theoretically be a confounding variable in interpreting urinary pellet cholesterol levels.
  • statins do not alter renal cortical cholesterol content (Zager, R.A., et al, "The mevalonate pathway during acute tubular injury: selected determinants and consequences," Am. J. Pathol. 767:681-692, 2002).
  • This Example describes the discovery that patients with acute kidney injury (AKI) exhibit increased levels of RNA polymerase II (Pol II) and of histone H3 lysine 4 trimethylation (H3K4m3) at exon 1 of HMG CoA reductase (HMGCR) gene in urine chromatin samples.
  • AKI acute kidney injury
  • Polymerase II Polymerase II
  • H3K4m3 histone H3 lysine 4 trimethylation
  • HMGCR HMG CoA reductase
  • AKI Experimental acute kidney injury
  • HMGCR HMG CoA reductase
  • chromatin immunoprecipitation assay can be successfully deployed for measuring Pol II - gene binding, using sheared, formalin fixed, urinary chromatin samples (Ware, L.B., et al, "Renal cortical albumin gene induction and urinary albumin excretion in responses to acute kidney injury," Am. J. Physiol.
  • H3K4m3 a notable example of this altered histone profile is an increase in the amount of trimethylated histone H3 at the lysine 4 position, yielding H3K4m3 (Naito, M. et al, "Renal ischemia-induced cholesterol loading: Transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene," Am. J. Pathol. 774:54-62, 2009).
  • the up-regulation of the HMGCR gene was investigated for a correlation with the occurrence of AKI. Specifically, levels of Pol II binding to exon 1 of the HMGCR gene were assayed in the context of ICU/AKI+, ICU/AKI-, and normal control patients. Similarly, the trimethylation levels of histone H3 at lysine 4 position (yielding H3K4m3) of the chromatin corresponding to exon 1 of the HMGCR gene were assayed in the context of ICU / AKI +, ICU / AKI-, and normal control patients.
  • the pellets were resuspended in 1 ml of IP buffer (containing the following inhibitors: 0.5 mmol/L dithiothreitol, 10 ⁇ g/ml leupeptin, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 30 mmol/L p-nitrophenyl phosphate, 10 mmol/L NaF, 0.1 mmol/L a 3 V04, 0.1 mmol/L a 2 Mo04, and
  • Sheared chromatin were aliquoted in wells of 96-well polystyrene high-binding capacity microplates (No. 9018; Corning, Corning, NY). The wells were washed once with 200 ⁇ of PBS per well, and were incubated overnight with 0.2 ⁇ g of protein A (No. P7837; Sigma, St. Louis, Missouri) in 100 ⁇ of PBS per well. After washing (200 ⁇ of PBS per well), well walls were blocked with 200 ⁇ of blocking buffer (15 to 60 minutes, 22°C). The wells were cleared and 0.25 ⁇ g of monoclonal antibody specific for RNA Polymerase II ("Pol II CTD 4h8"; No.
  • GTX25408, Gene Tex, Irvine, California were added with 100 ⁇ of blocking buffer per well (60 minutes, 22°C).
  • Chromatin samples 5.0- ⁇ 1 chromatin preparations/ 100 ⁇ of blocking buffer
  • plates were floated in an ultrasonic water bath (60 minutes, 4°C) to accelerate protein-antibody binding.
  • the wells were washed three times with 200 ⁇ of IP buffer and one time with 200 ⁇ of TE buffer.
  • Wells were incubated with 100 ⁇ of elution buffer (15 minutes at 55°C, followed by 15 minutes at 95°C).
  • Total DNA (input) was isolated using the same plate and concurrently with immunoprecipitated DNA by suspending 5.0 ⁇ of chromatin in 100 ⁇ of elution buffer (15 minutes at 55°C, followed by 15 minutes at 95°C). DNA samples were stored (-20°C).
  • phosphate-buffered saline PBS
  • TE buffer 10 mmol/L Tris, 1 mmol/L ethylene diamine tetraacetic acid, pH 7.0
  • immunoprecipitation (IP) buffer 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 5 mmol/L ethylene diamine tetraacetic acid, NP-40 (0.5% v/v), Triton X-100 (1.0% v/v); blocking buffer: 5% bovine serum albumin, 100 ⁇ g/ml sheared salmon sperm DNA in IP buffer; elution buffer: 25 mmol/L Tris base, 1 mmol/L ethylene diamine tetraacetic acid, pH 9.8, 200 ⁇ g/ml proteina
  • DNA samples obtained from chromatin immunoprecipitation were assayed for the HMGCR exon 1 and ⁇ -actin by quantitative PCR.
  • the reaction mixture contained 2.5 ⁇ of optimized PCR buffer with dye, DNA polymerase, dNTPs with dUTP, and buffer (2* SYBR® Green PCR master mix (SensiMix, Quantace)), 2.3 ⁇ of DNA template, and 0.2 ⁇ of primers (10 ⁇ /L) in 5- ⁇ 1 final volume in a 384-well optical reaction plate (Applied Biosystems, Foster City, California).
  • Amplification three step, 40 cycles
  • data acquisition, and analyses were done using the 7900HT real-time PCR system and SDS Enterprise Database (Applied Biosystems). Primers for the reactions are listed below in Table 2.
  • ChIP analysis was performed on urinary pellet samples from AKI- and AKI+ patients and from normal volunteers to assess trimethylation levels of histone H3 at lysine 4 position (yielding H3K4m3) corresponding to exon 1 of the HMGCR gene. Briefly, ChIP analysis was performed as described above in regard to the assessment of Pol II binding. The chromatin was immunoprecipitated using 0.5 ⁇ g rabbit polyclonal antibody, specific for H3K4m3 (No. Ab8580, Abeam, Cambridge, MA).
  • the present invention is not limited to the above-described approaches to assess HMGCR activity from the urine. Additional embodiments are contemplated in which relative levels of HMGCR gene product are assayed. For instance, the presence of HMG CoA reductase protein in the urine pellets can be assayed directly using mass spectrometry or Western blot staining employing antibodies, or fragments thereof, that are specific for the protein. In a further embodiment, mRNA corresponding to the HMGCR gene can be quantified from urine samples using well-known techniques such as quantitative reverse transcription PCR ("RT PCR"). Exemplary primers for the RT PCR assays are listed in Table 2 above, which are specific for Exon 1 of the MHGCR gene.
  • RT PCR quantitative reverse transcription PCR
  • Additional primers employed in an RT PCR assay can be synthesized to specifically prime the reaction for the HMGCR mRNA template, which is set forth below in Table 3 and is listed in the GenBank database as accession number NM_000859.2 (SEQ ID NO: l).
  • the intermediate level of Pol II-HMGCR binding in the AKI- patients stems from the fact that many critically ill patients sustain sub-clinical renal injury (23, 25) that is insufficient in degree to induce either clinically overt AKI (i.e., as denoted by azotemia), or renal tubular cholesterol loading.
  • a doubling of the H3K4m3 levels at exon 1 of the HMGCR gene was observed in the AKI+ patients, compared to either the AKI- cohort or the normal volunteers (p ⁇ 0.03). There was no significant difference between the AKI- and normal control patients. This data is consistent with the concept that AKI induces HMGCR gene activation, as evidenced by histone modification, culminating in increased renal tubular, and ultimately, urinary pellet cholesterol levels.

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Abstract

La présente invention concerne des procédés et des tests de diagnostic utiles pour détecter la présence de lésions rénales aigues chez un sujet. La présence du cholestérol ou la quantité de celui-ci est déterminée dans un échantillon d'urine prélevé chez le sujet. La présence ou la quantité de cholestérol déterminée est comparée à un standard de référence, la présence ou la quantité élevée de cholestérol dans l'échantillon d'urine prélevé chez le sujet indiquant la présence d'une lésion rénale chez le sujet.
PCT/US2012/025100 2011-02-14 2012-02-14 Procédés et compositions de détection du cholestérol dans l'urine comme biomarqueur de lésions rénales aigues WO2012112589A2 (fr)

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US11406490B2 (en) 2012-01-24 2022-08-09 Alcon Inc. Modular intraocular lens designs and methods

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* Cited by examiner, † Cited by third party
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US11406490B2 (en) 2012-01-24 2022-08-09 Alcon Inc. Modular intraocular lens designs and methods

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