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US20110301103A1 - Methods of Treatment - Google Patents

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Publication number
US20110301103A1
US20110301103A1 US13/152,169 US201113152169A US2011301103A1 US 20110301103 A1 US20110301103 A1 US 20110301103A1 US 201113152169 A US201113152169 A US 201113152169A US 2011301103 A1 US2011301103 A1 US 2011301103A1
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United States
Prior art keywords
angptl4
sialic acid
subject
sialylation
nephrotic syndrome
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US13/152,169
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English (en)
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Sumant S. Chugh
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Individual
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Individual
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Priority to US13/152,169 priority Critical patent/US20110301103A1/en
Priority to CA2801770A priority patent/CA2801770C/en
Priority to KR1020137000300A priority patent/KR20130132723A/ko
Priority to CN2011800380700A priority patent/CN103153301A/zh
Priority to CA3066017A priority patent/CA3066017C/en
Priority to JP2013513373A priority patent/JP2013533857A/ja
Priority to MX2017014807A priority patent/MX373643B/es
Priority to AU2011261268A priority patent/AU2011261268B2/en
Priority to EP11790460.7A priority patent/EP2575805A4/en
Priority to NZ703876A priority patent/NZ703876A/en
Priority to PCT/US2011/039058 priority patent/WO2011153429A2/en
Priority to MX2012014163A priority patent/MX359930B/es
Priority to KR1020187001031A priority patent/KR20180009382A/ko
Publication of US20110301103A1 publication Critical patent/US20110301103A1/en
Priority to IL223434A priority patent/IL223434A/en
Priority to US14/943,167 priority patent/US9827259B2/en
Priority to AU2017200933A priority patent/AU2017200933B2/en
Priority to US15/803,524 priority patent/US10624914B2/en
Priority to IL256026A priority patent/IL256026B/en
Assigned to NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR reassignment NATIONAL INSTITUTES OF HEALTH - DIRECTOR DEITR CONFIRMATORY LICENSE (SEE DOCUMENT FOR DETAILS). Assignors: UNIVERSITY OF ALABAMA AT BIRMINGHAM
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7012—Compounds having a free or esterified carboxyl group attached, directly or through a carbon chain, to a carbon atom of the saccharide radical, e.g. glucuronic acid, neuraminic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/10—Antioedematous agents; Diuretics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present disclosure is directed to methods for the treatment and prevention of nephrotic syndrome, diabetic conditions and conditions related thereto.
  • Podocytes are cells in the glomerular capillary loop in the kidneys.
  • the glomerulus filters blood, holding back large molecules such as proteins, and passing through small molecules such as water, salts, and sugar, as the first step in forming urine.
  • the long processes, or “foot projections,” of the podocytes wrap around the capillaries, and leave slits between them. Blood is filtered through these slits.
  • Kidneys affected by nephrotic syndrome have small pores in the podocytes which are large enough to permit proteins to transit, causing proteinuria.
  • Edema When protein is lost in the urine, its blood concentration decreases, allowing water to move into other areas of the body, which leads to swelling known as edema. Edema is commonly observed in the feet and legs, in the belly or abdomen (ascites), and around the eyes, but can occur anywhere, especially in response to gravity. Additionally, because of this extra fluid that stays in the body, people often gain weight, experience fatigue and may find that they urinate less often.
  • MCD minimal change disease
  • FGS focal segmental glomerulosclerosis
  • MN membranous nephropathy
  • MGN membranous glomerulonephritis
  • MPGN membranoproliferative glomerulonephritis
  • Hypertriglyceridemia may occur due to changes in the activity of enzymes that degrade triglycerides, such as lipoprotein lipase (2-4).
  • nephrotic syndrome The molecular basis of nephrotic syndrome is not known. Furthermore, the association of proteinuria and glucocorticoid sensitivity in nephrotic syndrome and the link between proteinuria and hypertriglyceridemia, two key components of nephrotic syndrome, have yet to be established. Therapy designed to reduce proteinuria further complicates the study of disease mechanisms. For example, glucocorticoids used to treat proteinuria in MCD independently raise plasma triglyceride levels (5), and normalization of plasma triglyceride levels lags behind the response of proteinuria to glucocorticoids in certain forms of nephrotic syndrome, such as MCD (6).
  • the present disclosure provides a disclosure of the biochemical basis of nephrotic syndrome (exemplified by a model of MCD) and provides an explanation for the observed proteinuria and other effects.
  • the present disclosure provides method for treating and/or preventing nephrotic syndrome, such as but not limited to, diabetic nephropathy, MCD, FSGS, MN/MGN, and MPGN, as well as methods of alleviating symptoms associated with nephrotic syndrome, including, but not limited to, proteinuria, hypercholesterolemia, hypertriglyceridemia, hypoalbuminemia and edema.
  • the present disclosure further provides methods for treating and preventing diabetic conditions and physiological effects thereof.
  • FIG. 1B shows glomerular Angptl4 mRNA expression was upregulated during nephrotoxic serum (NTS) induced heterologous phase proteinuria shown in FIG. 1A (n 4 rats/group); ***P ⁇ 0.001.
  • FIG. 1E shows by confocal imaging that Angptl4 in normal rat glomeruli co-localized with podocyte protein CD2AP, indicating expression in podocytes. Absorbing out reactivity from anti-Angptl4 antibody with recombinant Angptl4 abolished immunoreactivity.
  • FIG. 1F shows significant glomerular mRNA upregulation of Angptl4 was seen after injection of a single dose of puromycin aminonucleoside (PAN model) starting on Day 3 (peak up to 80 fold increase in different studies).
  • PAN model puromycin aminonucleoside
  • FIG. 1G shows glomeruli had increased Angptl4 expression (red) that overlapped partially (white arrows) with GBM heparan sulfate proteoglycans (white, intensity reduced in PAN) and podocyte nephrin (green) on day 6 after injection of a single dose of puromycin aminonucleoside (PAN model).
  • FIG. 2A show a transgenic (TG) rat model for podocyte specific over expression of Angptl4 in podocytes.
  • FIG. 2E shows that by age 5 months, diffuse foot process effacement was noted on EM in homozygous NPHS2-Angptl4 TG rats.
  • FIG. 2F shows immunogold EM analysis of these rats and revealed a progression from intact foot processes (FP) containing gold particle clusters and scattered GBM particles at age 1 month, to partial effacement with GBM gold particle clusters opposite to effaced foot processes (EFP) reaching up to the endothelial (ENDO) surface, and finally diffuse effacement with dense gold particle clusters in the GBM by age 5 months.
  • FP intact foot processes
  • EFP effaced foot processes
  • ENDO endothelial
  • FIG. 3 shows the relationship of Angptl4 overexpression with proteinuria.
  • FIG. 3E shows immunogold EM with anti-V5 antibody to specifically detect transgenic protein in 3 month heterozygous TG male rats and showed gold particles in effaced foot processes (EFP) and GBM in NPHS2-Angptl4 TG rats; EM 40,000 ⁇ .
  • FIG. 3H is a study of glomerular gene expression from the study in 3G, and shows that Angptl4 is a glucocorticoid sensitive gene. *P ⁇ 0.05; **P ⁇ 0.01
  • FIG. 4 shows the relationship of Angptl4 sialylation to proteinuria.
  • FIG. 4A shows 2D gel electrophoresis (200 ⁇ g protein/gel) and Western blot of protein from perfused glomeruli (upper panel, control; middle panel PAN; lower panel PAN plus the addition of glucocorticoid).
  • analysis revealed the presence of small amounts of fragments (red arrow; 1) and monomers (yellow arrow; 2) of Angptl4, and larger amounts of low order oligomers (pink arrows; 3) of Angptl4 migrating at neutral or high pI.
  • red arrow 1
  • monomers yellow arrow
  • pink arrows larger amounts of low order oligomers
  • FIG. 4B shows densitometry analysis of the Western blot from FIG. 4A ; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 4C shows 2D gel electrophoresis (150 ⁇ g protein/gel) and Western blot of proteins from Angptl4-HEK293 stable cell line incubated with control and probed with antibodies to MAA lecithin (top panel), anti Angplt4 antibodies (upper middle panel); Angptl4-HEK293 stable cell line incubated with sialic acid precursor ManNAc (25 mM) and probed with anti-Angptl4 antibodies (lower middle panel) and Angptl4-HEK293 stable cell line incubated with sialic acid precursor ManNAc (25 mM) and probed with antibodies to MAA lecithin (lower panel). Analysis of the concentrated supernatant revealed a shift from high pI forms (green arrow and line in upper middle panel) towards neutral pI forms (blue arrow in lower middle panel and lower panel), that were MAA reactive.
  • FIG. 4D shows that feeding NPHS2-Angptl4 TG rats with 1 mg/ml ManNAc in tap water for 12 days (d) caused a significant reduction in 18 hour albuminuria (nadir on Day 12 was 59.4 ⁇ 3.3% of baseline), which returned towards baseline values after 12 days of washout, and approached untreated control NPHS2-Angptl4 TG rat values on day 24 of washout (individual tracings and pilot study data in FIG. 6 ). In panel d, all*differences are with baseline values. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 5 shows characterization of recombinant Angptl4 produced by a HEK293 stable cell line.
  • FIG. 5A shows HEK293 cells stably transfected with a pcDNA-rat Angptl4-V5-His expression construct line showed 55,000 fold upregulation of Angptl4 mRNA expression compared to a control empty vector cell line. ***P ⁇ 0.001
  • FIG. 5B shows Angptl4-HEK293 cells secreted mostly intact protein into the supernatant under serum free conditions (as demonstrated by 2D gel electrophoresis and Western blot with pre-immune serum and anti-Angptl4 antibodies).
  • FIG. 6 shows administration of ManNAc reduced albuminuria in a rat model.
  • FIG. 6B shows individual tracings from the study group of NPHS2-Angptl4 TG rats treated with ManNAc (1 mg/ml) that completed the study. Two urine collections 12 days apart were conducted prior to the start of the study to ensure that these rats developed increasing albuminuria with time. The study revealed that ManNAc (1 mg/ml) reduced albuminuria in these animals.
  • Reproducible neutral pI Angptl4 spots are bordered by red oval (1), and high pI by green oval (2).
  • Neutral pI spots are also reactive with sialic acid binding lectin SNA I.
  • FIG. 6D shows densitometry of Angptl4 spots of FIG. 6C and indicates a significant increase in the percentage of neutral pI forms in ManNAC fed rats. ***P ⁇ 0.001.
  • FIG. 7 shows improvement in nephrotic syndrome parameters in ManNAc treated rats with puromycin aminonucleoside nephrosis (PAN model).
  • rats received a single intravenous injection of puromycin aminonucleoside (10 mg/100 gm), and were treated with ManNAC 80 mg/Kg body weight in tap water starting on Day 4, which coincides with the onset of proteinuria.
  • FIG. 7A shows a significant reduction in proteinuria in ManNAc treated rats on day 6 post administration of puromycin aminonucleoside. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 7B shows a significant improvement in plasma albumin levels in ManNAc treated rats on day 6 post administration of puromycin aminonucleoside. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 7C shows a significant reduction in hypercholesterolemia in ManNAc treated rats on day 6 post administration of puromycin aminonucleoside. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 7D shows a significant reduction in hypertriglyceridemia in ManNAc treated rats on day 6 post administration of puromycin aminonucleoside. *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • FIG. 8 shows the effect of ManNAc therapy on proteinuria in diabetic animals.
  • FIG. 8A shows 2D gel electrophoresis and Western blot analysis of glomeruli from diabetic db/db and control db/m mice using an anti-Angptl4 Ab.
  • the results show increased expression of Angptl4 in diabetic mouse glomeruli as compared to control, including the high pI forms that are involved in the pathogenesis of proteinuria.
  • FIG. 8B shows 2D gel electrophoresis and Western blot analysis of glomeruli from Zucker Diabetic Fatty rats and control normoglycemic rats using an anti-Angptl4 Ab.
  • the results show increased expression of Angptl4 in rat glomeruli from Zucker Diabetic Fatty rats as compared to control, including the high pI forms that are involved in the pathogenesis of proteinuria.
  • FIG. 9 shows the amino acid and cDNA sequences of Angptl4 from various species.
  • SEQ ID NOS. 1 and 2 show amino acid and cDNA sequence from human (Protein Variant 1 isoform a, long form; underlined amino acid sequences at a position 40 and 161-164);
  • SEQ ID NOS. 3 and 4 show amino acid and cDNA sequence from human (Protein Variant 3 isoform b, short form; underlined amino acid sequences at a position 40 and 161-164);
  • SEQ ID NOS. 5 and 6 show amino acid and cDNA sequence from rat;
  • SEQ ID NOS: 7 and 8 show amino acid and cDNA from mouse.
  • the present disclosure provides methods of treatment and/or prevention of nephrotic syndrome in a subject.
  • the nephrotic syndrome is characterized as minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN)/membranous glomerulonephritis (MGN) membranoproliferative glomerulonephritis (MPGN), and diabetic nephropathy.
  • MCD minimal change disease
  • FGS focal segmental glomerulosclerosis
  • MN membranous nephropathy
  • MGN membranous glomerulonephritis
  • MPGN membranoproliferative glomerulonephritis
  • the nephrotic syndrome is characterized as MCD.
  • the methods comprise the step of administering to a subject sialic acid or a sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • a therapeutically effective dose either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of nephrotic syndrome, thereby treating or preventing nephrotic syndrome in the subject.
  • the present disclosure provides methods of treatment and/or prevention of MCD in a subject.
  • the methods comprise the step of administering to a subject a sialic acid or sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of nephrotic syndrome, thereby treating or preventing nephrotic syndrome in the subject.
  • the present disclosure provides methods of alleviating one or more symptoms of nephrotic syndrome, such as, but not limited to, proteinuria, hypercholesterolemia, hypertriglyceridemia and edema.
  • nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the methods comprise the step of administering to a subject sialic acid or a sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of nephrotic syndrome, thereby alleviating one or more symptoms of nephrotic syndrome in the subject.
  • a polypeptide such as, but not limited to Angptl4
  • the present disclosure provides methods for reducing proteinuria in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the subject is suffering from a diabetic condition, such as, but not limited to, diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the proteinuria is caused by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • the methods comprise the step of administering to a subject sialic acid or a sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the induction of proteinuria thereby reducing proteinuria in the subject.
  • the present disclosure provides methods of reducing edema in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the edema is caused by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • the methods comprise the step of administering to a subject sialic acid or a sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • a therapeutically effective dose either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of the polypeptide, such as, but not limited to Angptl4, involved in the induction of edema thereby reducing edema in the subject.
  • the present disclosure provides methods of reducing hypercholesterolemia in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the hypercholesterolemia is caused, at least in part, by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • the methods comprise the step of administering to a subject sialic acid or a sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • a therapeutically effective dose either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of the polypeptide, such as, but not limited to Angptl4, involved in the induction of hypercholesterolemia thereby reducing hypercholesterolemia in the subject.
  • the present disclosure provides methods of reducing hypertriglyceridemia in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the hypertriglyceridemia is caused by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • the methods comprise the step of administering to a subject sialic acid or a sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • a therapeutically effective dose either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of the polypeptide, such as, but not limited to Angptl4, involved in the induction of hypertriglyceridemia thereby reducing hypertriglyceridemia in the subject.
  • the present disclosure provides methods of treatment and/or prevention of diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the foregoing conditions are caused by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • the methods comprise the step of administering to a subject sialic acid or a sialic acid precursor.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective dose, either alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such administration restores normal sialylation of the polypeptide, such as, but not limited to Angptl4, thereby treating or preventing the foregoing conditions in the subject.
  • the present disclosure provides a composition for use in the methods of the first through eight aspects.
  • the composition comprises sialic acid or one or more sialic acid precursors.
  • the sialic acid precursor is ManNAc.
  • the sialic acid precursor is a derivative of ManNAc.
  • Such composition may contain ManNAc and one or more derivatives of ManNAc as well as a secondary agent.
  • the present disclosure provides for methods of determining the status of a subject with respect to nephrotic syndrome or a diabetic condition.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the diabetic condition is diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease. In a specific embodiment, the diabetic condition is diabetic nephropathy.
  • such methods determine in the subject the level of a polypeptide associated with nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, the level of sialylation of a polypeptide associated with nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, or a combination of the foregoing.
  • the amount and/or level of sialylation of the polypeptide as determined from the subject may be compared to corresponding amounts and levels from a subject that is diagnosed as not suffering from nephrotic syndrome or a diabetic condition (control subject). Such amounts and levels may also be compared to a reference standard.
  • a decrease in the level of sialylation as compared to the control subject or reference standard indicates the subject is suffering from or at risk for, nephrotic syndrome or a diabetic condition; the level of sialylation may be determined with respect to the high pI form of the polypeptide (which is hyposialylated).
  • the present disclosure provides for methods of determining the efficacy of a treatment for nephrotic syndrome or a diabetic condition in a subject undergoing treatment for nephrotic syndrome or a diabetic condition.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the diabetic condition is diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the diabetic condition is diabetic nephropathy.
  • the amount and/or level of sialylation of the polypeptide as determined from the subject during treatment may be compared to corresponding amounts and/or levels from the subject prior to initiating treatment.
  • An increase in the level of sialylation in the subject undergoing treatment as compared to the level of sialylation determined prior to initiating treatment indicates the treatment is having the desired effect; the level of sialylation may be determined with respect to the high pI form of the polypeptide (which is hyposialylated).
  • the amount and/or level of the polypeptide as determined from the subject during treatment may be compared to corresponding amounts and/or levels a subject that is diagnosed as not suffering from nephrotic syndrome or a diabetic condition (control subject).
  • Such amounts and levels may also be compared to a reference standard.
  • a level of sialylation obtained from the subject during treatment that is equal to or approaching the level of sialylation from the control subject or reference indicates the treatment is having the desired effect; the level of sialylation may be determined with respect to the high pI form of the polypeptide (which is hyposialylated).
  • the present disclosure provides for methods of identifying a compound effective for treating or preventing nephrotic syndrome, a diabetic condition or a condition associated therewith.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the diabetic condition is diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the diabetic condition is diabetic nephropathy.
  • such screening methods comprises the steps of providing an assay system (as described in more detail below) that expresses a polypeptide involved in the etiology of nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, introducing into the assay system a test compound to be tested and determining whether the effect of the test compound on the level of sialylation of the polypeptide.
  • an assay system as described in more detail below
  • a test compound to be tested determining whether the effect of the test compound on the level of sialylation of the polypeptide.
  • NTS ⁇ 2-nephtotoxic serum
  • ZHX3 transcriptional factor zinc fingers and homeoboxes 3
  • ANGPTL4 The expression of the ANGPTL4 gene was analyzed in animal models of human glomerular disease, including puromycin nephrosis (PAN), a model of MCD, passive Heymann nephritis (PHN), a model of membranous nephropathy (MN), and anti-Thy1.1 nephritis, a model of mesangial injury.
  • PAN puromycin nephrosis
  • PPN passive Heymann nephritis
  • MN membranous nephropathy
  • anti-Thy1.1 nephritis a model of mesangial injury.
  • Angptl4 is a PPAR ⁇ (8) and PPAR ⁇ (9) target gene highly expressed in the liver and adipose tissue, strongly induced by fasting in white adipose tissue and liver, and is an apoptosis survival factor for vascular endothelial cells under normoxic conditions (10).
  • Angptl4 is a potent inhibitor of LPL (11), inducing significant hypertriglyceridemia following intravenous injection or adenovirus-mediated expression (12, 13).
  • a role of Angptl4 in proteinuria has not been previously reported.
  • the present disclosure shows a conclusive role of podocyte secreted Angptl4 in the etiology of in nephrotic syndrome and diabetic conditions.
  • the present disclosure demonstrates for the first time that a large part of podocyte secreted Angptl4 in experimental one form of nephrotic syndrome is hyposialylated and that improving sialylation dramatically reduces proteinuria, edema, hypercholesterolemia and hypertriglyceridemia and normalizes electrophoretic migration of Angptl4.
  • Angptl4 is the first glucocorticoid sensitive gene to be directly implicated in the pathogenesis of nephrotic syndrome and also the first direct link between proteinuria and hypertriglyceridemia in nephrotic syndrome.
  • Angptl4 amino acid and cDNA sequences from human Protein Variant 1 isoform a, long form and Protein Variant 3 isoform b, short form
  • rat and mouse are shown in FIG. 8 .
  • prevention refers to a course of action (such as administering a compound or pharmaceutical composition) initiated prior to the onset of a symptom, aspect, or characteristics of a disease or condition so as to prevent or reduce such symptom, aspect, or characteristics. Such preventing and suppressing need not be absolute to be useful.
  • treatment refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a symptom, aspect, or characteristics of a disease or condition so as to eliminate or reduce such symptom, aspect, or characteristics.
  • Such treating need not be absolute to be useful.
  • in need of treatment refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.
  • in need of prevention refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a disease or condition that is preventable by a method or compound of the disclosure.
  • subject refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans.
  • mammals such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans.
  • the term may specify male or female or both, or exclude male or female.
  • terapéuticaally effective amount refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease or condition. Such effect need not be absolute to be beneficial.
  • therapeutically effective amount refers to an amount of sialic acid or the sialic acid precursor sufficient to restore normal sialylation patterns to a polypeptide, such as, but not limited to, Angltl4 or an amount of sialic acid or the sialic acid precursor sufficient to reduce proteinuria or another symptom of nephrotic syndrome in a subject.
  • pharmaceutically acceptable derivative means any pharmaceutically acceptable salt, ester, salt of an ester, solvate or other derivative of sialic acid or a sialic acid precursor of the present disclosure that, upon administration to a subject, is capable of providing (directly or indirectly) sialic acid or a sialic acid precursor of the disclosure or a metabolite or residue thereof.
  • Particularly favored derivatives are those that increase the bioavailability of sialic acid or the sialic acid precursor of the disclosure when such are administered to a subject (e.g., by allowing an orally administered compound to be more readily absorbed into the blood), enhance delivery of the sialic acid precursor to a given biological compartment, increase solubility to allow administration by injection, alter metabolism or alter rate of excretion.
  • the derivative is a prodrug.
  • salts of acidic or basic groups that may be present in sialic acid or the sialic acid precursor of the present disclosure.
  • the term “about” and “approximately” shall generally mean an acceptable degree of error or variation for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error or variation are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. For biological systems, the team “about” refers to an acceptable standard deviation of error, preferably not more than 2-fold of a give value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
  • the present disclosure provides methods of treatment and/or prevention of nephrotic syndrome.
  • the present disclosure further provides methods of treatment and/or prevention of MCD.
  • the present disclosure additionally provides methods of alleviating one or more symptoms of nephritic syndrome, such as, but not limited to, proteinuria, hypercholesterolemia, hypertriglyceridemia and edema.
  • the present disclosure provides methods for reducing proteinuria.
  • the present disclosure provides methods for reducing edema.
  • the present disclosure also provides methods for reducing hypercholesterolemia and hypertriglyceridemia
  • the present disclosure also provides methods for the treatment and/or prevention of a diabetic condition or a physiological condition associated therewith.
  • the present disclosure additionally provides for pharmaceutical compositions comprising sialic acid or one or more sialic acid precursors or combinations of the foregoing.
  • the teachings of the present disclosure provide for the treatment and/or prevention of nephrotic syndrome in a subject in need of such treatment or prevention.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor to the subject.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of nephrotic syndrome.
  • Such administration thereby treats and/or prevents nephrotic syndrome in the subject.
  • sialic acid or sialic acid precursor may be administered at a therapeutically effective amount. Furthermore, sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent. Such method may further comprise identifying a subject in need of such treatment and/or prevention.
  • the teachings of the present disclosure provide for the treatment and/or prevention of MCD in a subject in need of such treatment or prevention.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor to the subject.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of MCD.
  • Such administration thereby treats and/or prevents MCD in the subject.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective amount.
  • sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such method may further comprise identifying a subject in need of such treatment and/or prevention.
  • the teachings of the present disclosure provide for methods of alleviating one or more symptoms of nephrotic syndrome, such as, but not limited to, proteinuria, hypercholesterolemia, hypertriglyceridemia and edema.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephroapthy.
  • the nephrotic syndrome is characterized as MCD.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor to the subject. Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of nephrotic syndrome.
  • Such administration thereby alleviates one or more symptoms of nephrotic syndrome, such as, but not limited to, proteinuria and edema, nephrotic syndrome in the subject.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective amount.
  • sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such method may further comprise identifying a subject in need of such treatment and/or prevention.
  • the teachings of the present disclosure provide methods for reducing proteinuria in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephroapthy.
  • the nephrotic syndrome is characterized as MCD.
  • the subject is suffering from a disorder characterized by proteinuria, such as, but not limited to, diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the proteinuria is caused, at least in part, by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • a polypeptide such as, but not limited to, Angptl4.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor to the subject.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of proteinuria.
  • Such administration thereby reduces proteinuria in the subject.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective amount.
  • sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such method may further comprise identifying a subject in need of such reduction.
  • the teachings of the present disclosure provide methods for reducing edema in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the edema is caused, at least in part, by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor or a combination of the foregoing to the subject.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of proteinuria. Such administration thereby reduces proteinuria in the subject.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective amount.
  • sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such method may further comprise identifying a subject in need of such reduction.
  • the teachings of the present disclosure provide methods for reducing hypercholesterolemia in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the hypercholesterolemia is caused, at least in part, by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor to the subject.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of hypercholesterolemia. Such administration thereby reduces hypercholesterolemia in the subject.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective amount.
  • sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such method may further comprise identifying a subject in need of such reduction.
  • the teachings of the present disclosure provide methods for reducing hypertriglyceridemia in a subject.
  • the subject is suffering from nephrotic syndrome.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the hypertriglyceridemia is caused, at least in part, by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor to the subject.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of hypertriglyceridemia. Such administration thereby reduces hypertriglyceridemia in the subject.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective amount.
  • sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such method may further comprise identifying a subject in need of such reduction.
  • the teachings of the present disclosure provide methods for treatment and/or prevention of a diabetic condition in a subject or a physiological condition associated therewith.
  • the diabetic condition is diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the diabetic condition is diabetic nephropathy.
  • the physiological condition associated with the diabetic condition is proteinuria.
  • the diabetic condition is caused, at least in part, by hyposialylation of a polypeptide, such as, but not limited to, Angptl4.
  • Such method comprises the step of administering sialic acid or a sialic acid precursor to the subject.
  • Such administration restores normal sialylation of a polypeptide, such as, but not limited to Angptl4, involved in the etiology of the diabetic condition. Such administration thereby treats and/or prevents the diabetic condition in the subject.
  • the sialic acid or sialic acid precursor may be administered at a therapeutically effective amount.
  • sialic acid or the sialic acid precursor may be administered alone, as a part of a pharmaceutical composition or in combination with a secondary agent.
  • Such method may further comprise identifying a subject in need of such treatment and/or prevention.
  • the present disclosure also provides methods for determining the status of a subject with respect to nephrotic syndrome or a diabetic condition.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the diabetic condition is diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the diabetic condition is diabetic nephropathy.
  • such methods determine in the subject the level of a polypeptide associated with nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, the level of sialylation of a polypeptide associated with nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, or a combination of the foregoing.
  • the level of the high pI form of the polypeptide is determined. This form has been shown to be hyposialylated and indicative of nephrotic syndrome or a diabetic condition.
  • the amount and/or level of sialylation of the polypeptide as determined from the subject may be compared to corresponding amounts and levels from a subject that is diagnosed as not suffering from nephrotic syndrome or a diabetic condition (control subject). Such amounts and levels may also be compared to a reference standard. A decrease in the level of sialylation as compared to the control subject or reference standard indicates the subject is suffering from or at risk for, nephrotic syndrome or a diabetic condition; as discussed above, the level of sialylation may be determined with respect to the high pI form of the polypeptide (which is hyposialylated).
  • the present disclosure further provides methods for determining the efficacy of a treatment for nephrotic syndrome or a diabetic condition in a subject undergoing treatment for nephrotic syndrome or a diabetic condition.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy.
  • the nephrotic syndrome is characterized as MCD.
  • the diabetic condition is diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the diabetic condition is diabetic nephropathy.
  • such methods determine in the subject the level of a polypeptide associated with nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, the level of sialylation of a polypeptide associated with nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, or a combination of the foregoing.
  • the level of the high pI form of the polypeptide is determined. This form has been shown to be hyposialylated and indicative of nephrotic syndrome or a diabetic condition.
  • the amount and/or level of sialylation of the polypeptide as determined from the subject during treatment may be compared to corresponding amounts and/or levels from the subject prior to initiating treatment.
  • An increase in the level of sialylation in the subject undergoing treatment as compared to the level of sialylation determined prior to initiating treatment indicates the treatment is having the desired effect; as discussed above, the level of sialylation may be determined with respect to the high pI form of the polypeptide (which is hyposialylated).
  • the amount and/or level of the polypeptide as determined from the subject during treatment may be compared to corresponding amounts and/or levels a subject that is diagnosed as not suffering from nephrotic syndrome or a diabetic condition (control subject).
  • Such amounts and levels may also be compared to a reference standard.
  • a level of sialylation obtained from the subject during treatment that is equal to or approaching the level of sialylation from the control subject or reference indicates the treatment is having the desired effect; as discussed above, the level of sialylation may be determined with respect to the high pI form of the polypeptide (which is hyposialylated).
  • Any method known in the art for determining protein levels and levels of sialylation may be used in such methods, including, but not limited to, any methods described herein.
  • FIG. 5C shows the utility of this approach.
  • This figure demonstrates that only patients with MCD relapse had circulating hyposialylated form of Angptl4 polypeptide (this 55-70 kDa pI 8-8.5 form of the Angptl4 polypeptide is indicated by the oval); this polypeptide was absent in patients with MCD remission.
  • Increased circulating neutral pI monomers and oligomers were noted in MCD patients in relapse (arrow), and monomers only in MN (arrow).
  • the present disclosure also relates to a method for identifying a compound effective for treating or preventing nephrotic syndrome, a diabetic condition or a condition associated therewith, such as, but not limited to, proteinuria, hypercholesterolemia, hypertriglyceridemia or edema.
  • the nephrotic syndrome is characterized as MCD, FSGS, MN or MGN.
  • the nephrotic syndrome is characterized as MCD.
  • the diabetic condition is diabetic nephropathy, diabetes mellitus, lupus nephritis or primary glomular disease.
  • the diabetic condition is diabetic nephropathy.
  • the methods include determining the level of sialylation of a polypeptide involved in the etiology of nephrotic syndrome, such as, but not limited to, Angptl4.
  • such screening methods comprises the steps of providing an assay system (as described in more detail below) that expresses a polypeptide involved in the etiology of nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, introducing into the assay system a test compound to be tested and determining whether the effect of the test compound on the level of sialylation of the polypeptide.
  • the methods involve the identification of candidate or test compounds or agents (polypeptides, functional nucleic acids, carbohydrates, antibodies, small molecules or other molecules) which effect the level of sialylation of the polypeptide. Such compounds may then be further tested in appropriate systems (such as, but not limited to, the animal models systems described herein) to determine the activity of the identified compounds.
  • Candidate compounds are identified using a variety of assays, such as, but not limited to, assays that employ cells which express a polypeptide involved in the etiology of nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4 or in assays with isolated polypeptides.
  • the various assays can employ a variety of variants of such polypeptides (e. g., full-length, a biologically active fragment, or a fusion protein which includes all or a portion of the desired polypeptide).
  • polypeptides can be derived from any suitable mammalian species (e. g., human, rat or murine); in a specific embodiment, the polypeptide is derived from a human.
  • the cell may either naturally express a polypeptide involved in the etiology of nephrotic syndrome or a diabetic condition, such as, but not limited to, Angptl4, or may be modified to express the same.
  • cells can be modified to express a desired polypeptide through conventional molecular biology techniques, such as by infecting the cell with a virus comprising such polypeptide.
  • the cell can also be a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding such polypeptide.
  • full length polypeptides, fragments or fusion proteins containing at least a part of such polypeptide may be used. Exemplary assay systems are described in the current specification.
  • the various screening assays may be combined with an in vivo assay entailing measuring the effect of the test compound on the symptoms the disease states and conditions discussed herein.
  • the compounds may be evaluated to determine if they impact a parameter associated with nephrotic syndrome or a diabetic condition or a condition related thereto, such as, but not limited to, proteinuria hypercholesterolemia, hypertriglyceridemia or edema.
  • Such parameters include, but are not limited to, determining 1) the level of sialylation of a polypeptide involved in the etiology of hypercholesterolemia, reduces hypertriglyceridemia or related conditions, such as, but not limited to Angptl4 and 2) the amount of high pI forms a polypeptide involved in the etiology of hypercholesterolemia, reduces hypertriglyceridemia or related conditions, such as, but not limited to Angptl4 (these forms are hyposialylated); 3) determining the level of protein excretion, either total or with regard to specific components; and 4) determining the impact of the test compound on kidney morphology, such as, but not limited, the podocyte.
  • such a screening assay can be performed, for example, by determining the level of sialylation of a polypeptide involved in the etiology of hypercholesterolemia, reduces hypertriglyceridemia, such as, but not limited to, Angptl4 and detecting a difference in the level of sialylation of such polypeptide in the presence of as compared to the absence of a test compound.
  • the high pI forms of such polypeptide are specifically examined (this form is hyposialylated).
  • Such screening assay may be in vitro, in vivo or ex vivo and may be cell culture based (either with whole cells or lysates) or may be based on an animal model. Any assay of the present disclosure may be used in the foregoing method.
  • test compounds for use in the screening methods can be obtained from any suitable source, such as conventional compound libraries.
  • the test compounds can also be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries, spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the “one-bead one-compound” library method and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds. Examples of methods for the synthesis of molecular libraries can be found in the art. Libraries of compounds may be presented in solution or on beads, bacteria, spores, plasmids or phage.
  • the high pI forms of Angptl4 refer to a polypeptide migrating at 55-70 kDa and having a pI of 8-8.5.
  • kits for carrying out any method of the present disclosure which can contain any of the compounds and/or compositions disclosed herein or otherwise useful for practicing a method of the disclosure.
  • the present disclosure provides for various uses of sialic acid (N-acetylneuraminic acid) or a sialic acid precursor.
  • the sialic acid precursor is N-acetylmannosamine (ManNAc; also referred to as 2-Acetamido-2-deoxy-D-mannose or N-acetyl-D-mannosamine).
  • ManNAc N-acetylmannosamine
  • the structure of ManNAc is shown in formula I below.
  • the sialic acid precursor is defined by the formula Ia below.
  • X and/or Y are independently a C1 to C5 alkyl.
  • X and/or Y are propyl; in a further embodiment, X and/or Y are butyl.
  • At least one of B, C, D, E or G is not H or OH. In a further embodiment, at least two of B, C, D, E or G is not H or OH. In still a further embodiment, at least three of B, C, D, E or G is not H or OH. In yet another embodiment, none of B, C, D, E or G is not H or OH.
  • G is CH 3 , A is CH 2 , and at least one of B, C, D or E is not H or OH. In a further particular embodiment, G is CH 3 , A is CH 2 , and at least two of B, C, D or E are not H or OH. In still a further particular embodiment, G is CH 3 , A is CH 2 , and at least three of B, C, D or E are not H or OH. In yet a further particular embodiment, G is CH 3 , A is CH 2 , and all of B, C, D or E are not H or OH. In the foregoing, in one embodiment, B, C and D may each independently be CO—X.
  • Representative derivatives falling under the formula I include, but are not limited to, Bu4ManNAc, 3,4,6-O-Bu3ManNAc or 1,3,4-O-Bu3ManNAc (such compounds are described in Aich, et al, Glycoconj J. DOI 10.1007/s10719-010-9292-3 accepted Apr. 14, 2010).
  • sialic acid precursors include N-levulinoyl sialic acid (SiaLev) and N-levulinoylmannosamine (ManLev) (Charter et al. Glycobiology. 2000 October; 10(10):1049-56).
  • compositions of the present disclosure comprise one or more compounds useful in the treatment and prevention methods of the present disclosure, such as, but not limited to, sialic acid, sialic acid precursors, those compounds identified in the present disclosure or identified by a screening method of the present disclosure.
  • such compounds increase the sialylation of a polypeptide, such as, but not limited to, Angptl4.
  • the compounds are sialic acid precursors.
  • Exemplary sialic acid precursors include, but are not limited to, ManNAc and ManNAc derivatives.
  • compositions disclosed may comprise one or more of such compounds, in combination with a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier examples of such carriers and methods of formulation may be found in Remington: The Science and Practice of Pharmacy (20 th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor).
  • To form a pharmaceutically acceptable composition suitable for administration, such compositions will contain an therapeutically effective amount of compound.
  • compositions of the disclosure may be used in the treatment and prevention methods of the present disclosure. Such compositions are administered to a subject in amounts sufficient to deliver a therapeutically effective amount of the compound(s) so as to be effective in the treatment and prevention methods disclosed herein.
  • the therapeutically effective amount may vary according to a variety of factors such as, but not limited to, the subject's condition, weight, sex and age. Other factors include the mode and site of administration.
  • the compositions of the present disclosure may be administered only one time to the subject or more than one time to the subject. Furthermore, when the compositions are administered to the subject more than once, a variety of regimens may be used, such as, but not limited to, one per day, once per week or once per month.
  • compositions may also be administered to the subject more than one time per day.
  • the therapeutically effective amount and appropriate dosing regimens may be identified by routine testing in order to obtain optimal activity, while minimizing any potential side effects.
  • co-administration or sequential administration of other agents may be desirable.
  • Daily doses of a sailic acid precursor may range from about 0.1 g/day to about 75 g/day, from about 0.5 g/day to about 50 g/day, from about 1 g/day to about 10 g/day, from about 0.1 g/day to about 5 g/day, from about 0.1 g/day to about 3 g/day, and from about 0.1 g/day to about 1 g/day.
  • compositions may be provided to the subject in any method known in the art.
  • routes of administration include, but are not limited to, oral, subcutaneous, rectal, parenteral, subcutaneous, intramuscular, intraperitoneal, intravenous, topical, epicutaneous, intraosseous, intramuscular, dermal, transdermal, intrathoracic, intrapulmonary, intranasal or pulmonary routes
  • compositions of the present disclosure may further comprise agents which improve the solubility, half-life, absorption, etc. of the compound(s). Furthermore, the compositions of the present disclosure may further comprise agents that attenuate undesirable side effects and/or or decrease the toxicity of the compounds(s). Examples of such agents are described in a variety of texts, such a, but not limited to, Remington: The Science and Practice of Pharmacy (20 th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor).
  • compositions of the present disclosure can be administered in a wide variety of dosage forms for administration.
  • the compositions can be administered in forms, such as, but not limited to, tablets, capsules, sachets, lozenges, troches, pills, powders, granules, elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments, creams, pastes, emulsions, or solutions for intravenous administration or injection.
  • Other dosage forms include administration transdermally, via patch mechanism or ointment. Any of the foregoing may be modified to provide for timed release and/or sustained release formulations.
  • the pharmaceutical compositions may further comprise a pharmaceutically acceptable carriers include, but are not limited to, vehicles, adjuvants, surfactants, suspending agents, emulsifying agents, inert fillers, diluents, excipients, wetting agents, binders, lubricants, buffering agents, disintegrating agents and carriers, as well as accessory agents, such as, but not limited to, coloring agents and flavoring agents (collectively referred to herein as a carrier).
  • the pharmaceutically acceptable carrier is chemically inert to the active compounds and has no detrimental side effects or toxicity under the conditions of use.
  • the pharmaceutically acceptable carriers can include polymers and polymer matrices. The nature of the pharmaceutically acceptable carrier may differ depending on the particular dosage form employed and other characteristics of the composition.
  • the compound(s) may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier, such as, but not limited to, inert fillers, suitable binders, lubricants, disintegrating agents and accessory agents.
  • suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthum gum and the like.
  • Tablet forms can include one or more of the following: lactose, sucrose, mannitol, corn starch, potato starch, alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, calcium stearate, zinc stearate, stearic acid as well as the other carriers described herein.
  • Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
  • a flavor usually sucrose and acacia or tragacanth
  • pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acadia, emulsions, and gels containing, in addition to the active ingredient, such carriers as are known in the art.
  • the nucleic acid molecules of the present disclosure can be dissolved in diluents, such as water, saline, or alcohols.
  • the oral liquid forms may comprise suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like.
  • suitable and coloring agents or other accessory agents can also be incorporated into the mixture.
  • Other dispersing agents include glycerin and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the patient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the compound(s) may be administered in a physiologically acceptable diluent, such as a sterile liquid or mixture of liquids, including water, saline, aqueous dextrose and related sugar solutions, an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols, such as propylene glycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, a fatty acid, a fatty acid ester or glyceride, or an acetylated fatty acid glyceride with or without the addition of a pharmaceutically acceptable surfactant, such as, but not limited to, a soap, an oil or a detergent, suspending agent, such as, but not limited to, pectin, carbomers, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose
  • Oils which can be used in parenteral formulations, include petroleum, animal, vegetable, or synthetic oils. Specific examples of oils include peanut, soybean, sesame, cottonseed, corn, olive, petrolatum, and mineral.
  • Suitable fatty acids for use in parenteral formulations include polyethylene sorbitan fatty acid esters, such as sorbitan monooleate and the high molecular weight adducts of ethylene oxide with a hydrophobic base, formed by the condensation of propylene oxide with propylene glycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are examples of suitable fatty acid esters.
  • Suitable soaps for use in parenteral formulations include fatty alkali metal, ammonium, and triethanolamine salts
  • suitable detergents include (a) cationic detergents such as, for example, dimethyldialkylammonium halides, and alkylpyridinium halides, (b) anionic detergents such as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergents such as, for example, fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers, (d) amphoteric detergents such as, for example, alkylbeta-aminopropionates, and 2-alkylimidazoline quaternary ammonium salts, and (e) mixtures thereof.
  • compositions may contain one or more nonionic surfactants having a hydrophile-lipophile balance (HLB) of from about 12 to about 17.
  • HLB hydrophile-lipophile balance
  • the quantity of surfactant in such formulations ranges from about 5% to about 15% by weight.
  • Topical dosage forms such as, but not limited to, ointments, creams, pastes, emulsions, containing the nucleic acid molecule of the present disclosure, can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations. Inclusion of a skin exfoliant or dermal abrasive preparation may also be used. Such topical preparations may be applied to a patch, bandage or dressing for transdermal delivery or may be applied to a bandage or dressing for delivery directly to the site of a wound or cutaneous injury.
  • carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, all
  • the compound(s) of the present disclosure can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines. Such liposomes may also contain monoclonal antibodies to direct delivery of the liposome to a particular cell type or group of cell types.
  • the compound(s) of the present disclosure may also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include, but are not limited to, polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • a drug for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
  • sialic acid or sialic acid precursors disclosed herein can be administered as a food supplement or incorporated into food or drink items.
  • Angptl4 mRNA expression is upregulated (70-fold) in rat glomeruli at the peak of complement- and leukocyte-independent heterologous phase proteinuria 24 hours after injection of NTS ( FIGS. 1A and 1B ).
  • Injection of NTS into Angptl4 ⁇ / ⁇ mice caused significant reduction in proteinuria ( FIG. 1C ) and foot process effacement ( FIG. 1D ), suggesting a key role for Angptl4 in glomerular disease.
  • Normal rat glomeruli express Angptl4 in a capillary loop pattern that co-localized with podocyte protein CD2AP ( FIG. 1E ).
  • FIG. 1F Angptl4 mRNA expression did not change in anti-Thy1.1 nephritis, or in collapsing focal and segmental glomerulosclerosis ( FIG. 1F ) induced in rats by injection of sera from patients with this disease (18).
  • EM immunogold electron microscopy
  • FIG. 1I Biopsies from patients with glucocorticoid sensitive MCD and age and sex-matched controls revealed a faint podocyte pattern in control kidney biopsies, and increased expression in the podocyte with additional GBM overlap and trace spotty overlap with the endothelium at the margins.
  • FIG. 2A A transgenic rat models for podocyte specific Angptl4 overexpression was developed and is shown in FIG. 2A (NPHS2-Angptl4 TG). Analysis of mRNA expression in organs that normally express Angptl4 confirmed specificity of expression ( FIG. 2B ). Histological assessment of 3 month old heterozygous male NPHS2-Angptl4 TG rats revealed normal appearing glomeruli with prominent podocytes ( FIG. 2C ) on light microscopy and increased podocyte Angptl4 expression by confocal imaging ( FIG. 2D ). Electron microscopy of 5 month old homozygous and most heterozygous TG rats ( FIG. 2E ) revealed diffuse foot process effacement. Immunogold EM of homozygous TG rats revealed a correlation between accumulation of Angptl4 in the GBM and progressive development of foot process effacement between age one to five months ( FIG. 2F ).
  • NPHS2-Angptl4 TG rats develop more proteinuria ( FIG. 3F ) than wild type littermates after induction of PAN. Blood pressure was significantly lower in proteinuric heterozygous NPHS2-Angptl4 rats compared to wild type controls (data not shown).
  • proteinuria in PAN is partially glucocorticoid sensitive on day 6 ( FIG. 3G ), and some of this glucocorticoid sensitivity is related to Angptl4 ( FIG. 3H ).
  • Glomerular protein Western blot typically underestimates Angptl4 production, since the protein is rapidly secreted.
  • Angptl4 was analyzed by 2D electrophoresis and Western blotting ( FIG. 4A ) in glomeruli from control (upper panel), a PAN model at day 6 (middle panel) and a PAN model at day 6 with glucocorticoid coadministration (lower panel).
  • FIG. 5A shows HEK293 cells stably transfected with a pcDNA-rat Angptl4-V5-His expression construct line showed 55,000 fold upregulation of Angptl4 mRNA expression compared to a control empty vector cell line. ***P ⁇ 0.001
  • FIG. 5B shows Angptl4-HEK293 cells secreted mostly intact protein into the supernatant underserum free conditions (as demonstrated by 2D gel electrophoresis and Western blot with pre-immune serum and anti-Angptl4 antibodies).
  • Angptl4-HEK293 stable cell line Sialylation of Angptl4 was studied in vitro ( FIG. 4C ) using the Angptl4-HEK293 stable cell line.
  • HEK293 cells normally express no or little Angptl4 polypeptide; after transfection with the pcDNA-rat Angptl4-V5-His expression construct, this cell line showed a 55,000 fold upregulation of Angptl4 mRNA expression compared to a control empty vector cell line ( FIG. 5A ).
  • the Angptl4 polypeptide expressed by the HEK293-Angptl4 cell line is normally secreted by this cell line is only trace MAA reactive at the low pI end ( FIG.
  • FIG. 6A shows representative tracing of albuminuria from pilot studies with heavily albuminuric rats receiving increasing doses of ManNAc. The study revealed that ManNAc reduced albuminuria in these animals. However, a large dose requirement to reduce albuminuria in these rats was required. In order to conduct a more efficient and indicative study, rats with 15-25 fold higher albuminuria than wild type rats were used for further analysis.
  • FIG. 4D shows individual tracings from the study group of NPHS2-Angptl4 TG rats treated with ManNAc (1 mg/ml and which went through the complete study. Two urine collections 12 days apart were conducted prior to the start of the study to ensure that these rats developed increasing albuminuria with time. The study revealed that ManNAc (1 mg/ml) reduced albuminuria in these animals.
  • FIGS. 6C and 6D Densitometry analysis of Western blots for glomerular Angptl4 from rats euthanized on Day 12 ( FIGS. 6C and 6D ) showed an increase in the neutral pI Angptl4 fraction from 48.3+5% in control rats to 72.9+1.4% in ManNAc treated rats ( FIG. 6D ).
  • the neutral pI fraction was also reactive with sialic acid binding lectin Sambucus nigra (SNA I), confirming increased sialylation of Angptl4 in ManNAc treated rats ( FIG. 6C ).
  • FIG. 7 shows improvements in nephrotic syndrome parameters in ManNAc treated rats with PAN, a model of MCD.
  • Rats received a single intravenous injection of puromycin aminonucleoside (10 mg/100 gm), and were treated with ManNAC 80 mg/Kg body weight in tap water starting on Day 4, which coincides with the onset of proteinuria.
  • Data from Day 6 is shown.
  • Significant reduction in proteinuria FIG. 7A
  • improvement in plasma albumin levels FIG. 7B
  • reduction in cholesterol FIG. 7C
  • reduction in triglycerides FIG. 7D
  • Proteinuria including albuminuria
  • Angptl4 has been noted in other diseases as discussed herein, including diabetes mellitus.
  • the role of Angptl4 in diabetes mellitus was also analyzed using the db/db mouse.
  • the db/db was identified initially in 1966 in Jackson Labs as an obese mouse that was hyperphagic soon on weaning.
  • the diabetic gene (db) is transmitted as an autosomal recessive trait.
  • the db gene encodes for a G-to-T point mutation of the leptin receptor, leading to abnormal splicing and defective signaling of the adipocyte-derived hormone leptin.
  • albumin excretion rates are higher by 8- to 62-fold in db/db mice beginning at the age of 8 wk.
  • the range of albuminuria is between 68 and 303 ⁇ g/24 h in the db/db male mouse, whereas it is between 4 and 21 ⁇ g/24 h in the age-matched heterozygous littermate.
  • FIGS. 8A-C show the effect of ManNAc therapy on proteinuria in diabetic animals.
  • FIG. 8A shows 2D gel electrophoresis and Western blot analysis of glomeruli from diabetic db/db and control db/m mice. This analysis showed increased expression of Angptl4 in diabetic db/db mouse glomeruli, including the high pI hyposialylated forms that are involved in the pathogenesis of proteinuria. These high pI hyposialylated forms of Angptl4 are sensitive to sialic acid precursor therapy, including ManNAc and ManNAc derivatives.
  • FIG. 8B shows 2D gel electrophoresis and Western blot analysis of glomeruli from Zucker diabetic fatty rats. This analysis showed increased expression of Angptl4 in Zucker diabetic fatty rat glomeruli, including the high pI hyposialylated forms that are involved in the pathogenesis of proteinuria. These high pI hyposialylated forms of Angptl4 are sensitive to sialic acid precursor therapy, including ManNAc and ManNAc derivatives.
  • the present disclosure shows a key role of podocyte secreted Angptl4 in the pathogenesis of nephrotic syndrome, exemplified using a model of MCD and the role of Angptl4 in conditions related to nephrotic syndrome, such as, but not limited to, proteinuria.
  • the present disclosures shows increased podocyte expression of Angptl4 in human kidney biopsies, selective proteinuria with over 500-fold increase in albuminuria in NPHS2-Angptl4 TG rats, glucocorticoid sensitivity of the Angptl4 gene, and light and electron microscopic findings consistent with human MCD.
  • the secretion of high pI Angptl4 by the podocyte in MCD is likely to facilitate the tethering of Angptl4 to the GBM, and prior studies have shown the binding of Angptl4 to heparan sulfate proteoglycans (21). This correlates well with reduced GBM charge, a hallmark of MCD, in rodents with TG expression of Angptl4 from the podocyte. It is likely that charge facilitates the transit of high pI Angptl4 across the GBM against the direction of fluid flow.
  • podocyte or endothelial cell secreted factors probably work synergistically with Angptl4 in the pathogenesis of MCD.
  • podocyte secreted Angptl4 remains tethered to the glomerular capillary loop or escapes into the urinary space, where it is taken up by the proximal tubule.
  • PAN peripheral blood pressure
  • podocyte secreted Angptl4 escapes into the circulation.
  • the lack of additional permeabilizing factors in NPHS2-Angptl4 TG rats is also likely to also explain the more gradual onset of proteinuria than in PAN or patients with MCD.
  • a sialic acid precursor restores normal sialylation of Angplt4 polypeptide and reduces physiological abnormalities associated nephrotic syndrome (including, but not limited to, MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy), diabetes mellitus, lupus nephritis or primary glomular disease.
  • physiological abnormalities associated nephrotic syndrome including, but not limited to, MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy
  • diabetes mellitus lupus nephritis or primary glomular disease.
  • the present disclosure provides a treatment for such conditions.
  • the E. Coli expressed purified full length protein was used to generate a polyclonal antibody in rabbits (Proteintech group, Inc. Chicago Ill. USA) that was tested by ELISA and Western blot. Antibody reactive bands were excised from GelCode blue stained gels, trypsin digested and presence of Angptl4 peptide sequences confirmed by MALDI-TOF/TOF. Part of the antiserum was affinity purified to the antigen. Unless otherwise specified, all studies described used this antibody. An additional polyclonal antibody against the N-terminal part of rat Angptl4 (amino acids 7-86 excluding signal peptide) was similarly raised in rabbits.
  • rats were injected intravenously with 10 units/100 gm weight of porcine heparin 15 minutes prior to euthanasia, and activity measured using an assay from Roar Biomedical, Inc (New York N.Y.). Serum triglycerides were measured in the fasting state.
  • ELISA Bethyl laboratories, Montgomery Tex. USA
  • NPHS2-Angptl4 TG rats were generated as follows: The vector pTRE-tight was digested with StuI and EcoRI to remove the minimum CMV promoter between by 278 and 324, the 5′ overhangs blunt ended with T4 DNA polymerase, and re-ligated to generate pTRE-tight MP (minus promoter). For podocyte specific expression, a rat Angptl4 cDNA construct (including the signal sequence) with a C-terminal V5 tag was placed upstream of a SV40 polyA tail.
  • the human NPHS2 promoter was cloned upstream by PCR using a published human NPHS2 promoter construct as template (gi22652661) without DMSO to exclude a naturally occurring loop between bps 2343 and 2568 to improve expression.
  • Transgenic rats were generated by microinjection of the digested DNA constructs into fertilized Sprague Dawley eggs (conducted at University of Michigan), implantation into pseudopregnant host Sprague Dawley females, and the resulting offsprings were genotyped by routine PCR and TaqMan genomic DNA real time PCR strategy using construct specific and control genomic prolactin primer and probe combinations. Two founder lines for podocyte specific expression were generated. Data from NPHS2-Angpt14 TG rat line 740 (5 copies of the transgene) and were stable over 4 generations, are presented. Urinary total protein was assessed using the Bradford method (Biorad laboratories, Hercules Calif. USA), and albuminuria by ELISA (Bethyl laboratories, Montgomery Tex. USA).

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