ZA200309593B - Method for kidney disease detection by protein profiling. - Google Patents
Method for kidney disease detection by protein profiling. Download PDFInfo
- Publication number
- ZA200309593B ZA200309593B ZA200309593A ZA200309593A ZA200309593B ZA 200309593 B ZA200309593 B ZA 200309593B ZA 200309593 A ZA200309593 A ZA 200309593A ZA 200309593 A ZA200309593 A ZA 200309593A ZA 200309593 B ZA200309593 B ZA 200309593B
- Authority
- ZA
- South Africa
- Prior art keywords
- disease
- albumin
- protein
- renal
- urine
- Prior art date
Links
- 102000004169 proteins and genes Human genes 0.000 title claims description 123
- 108090000623 proteins and genes Proteins 0.000 title claims description 123
- 238000000034 method Methods 0.000 title claims description 112
- 208000017169 kidney disease Diseases 0.000 title claims description 71
- 238000001514 detection method Methods 0.000 title description 16
- 108010088751 Albumins Proteins 0.000 claims description 189
- 102000009027 Albumins Human genes 0.000 claims description 189
- 210000002700 urine Anatomy 0.000 claims description 88
- 239000012634 fragment Substances 0.000 claims description 60
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 50
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 45
- 206010012601 diabetes mellitus Diseases 0.000 claims description 42
- 201000010099 disease Diseases 0.000 claims description 40
- 239000000523 sample Substances 0.000 claims description 40
- 238000013467 fragmentation Methods 0.000 claims description 39
- 238000006062 fragmentation reaction Methods 0.000 claims description 39
- 210000003734 kidney Anatomy 0.000 claims description 30
- 108091005804 Peptidases Proteins 0.000 claims description 15
- 239000004365 Protease Substances 0.000 claims description 15
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 12
- 238000004587 chromatography analysis Methods 0.000 claims description 11
- 229940079593 drug Drugs 0.000 claims description 11
- 239000003814 drug Substances 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 238000001962 electrophoresis Methods 0.000 claims description 9
- 229940088598 enzyme Drugs 0.000 claims description 9
- 150000001413 amino acids Chemical class 0.000 claims description 7
- 102000006395 Globulins Human genes 0.000 claims description 6
- 108010044091 Globulins Proteins 0.000 claims description 6
- 108060003951 Immunoglobulin Proteins 0.000 claims description 6
- 238000010504 bond cleavage reaction Methods 0.000 claims description 6
- 102000018358 immunoglobulin Human genes 0.000 claims description 6
- 230000007017 scission Effects 0.000 claims description 6
- 206010018364 Glomerulonephritis Diseases 0.000 claims description 5
- 230000001154 acute effect Effects 0.000 claims description 5
- 208000035475 disorder Diseases 0.000 claims description 5
- 208000027866 inflammatory disease Diseases 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 4
- 201000001320 Atherosclerosis Diseases 0.000 claims description 4
- 102000003886 Glycoproteins Human genes 0.000 claims description 4
- 108090000288 Glycoproteins Proteins 0.000 claims description 4
- 102000004895 Lipoproteins Human genes 0.000 claims description 4
- 108090001030 Lipoproteins Proteins 0.000 claims description 4
- 208000034578 Multiple myelomas Diseases 0.000 claims description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 4
- 102100026383 Vasopressin-neurophysin 2-copeptin Human genes 0.000 claims description 4
- 201000010064 diabetes insipidus Diseases 0.000 claims description 4
- 208000014674 injury Diseases 0.000 claims description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 4
- 238000004949 mass spectrometry Methods 0.000 claims description 4
- 238000004062 sedimentation Methods 0.000 claims description 4
- 208000011580 syndromic disease Diseases 0.000 claims description 4
- 230000003612 virological effect Effects 0.000 claims description 4
- 208000009304 Acute Kidney Injury Diseases 0.000 claims description 3
- 208000010061 Autosomal Dominant Polycystic Kidney Diseases 0.000 claims description 3
- 208000002814 Autosomal Recessive Polycystic Kidney Diseases 0.000 claims description 3
- 208000017354 Autosomal recessive polycystic kidney disease Diseases 0.000 claims description 3
- 208000025760 Benign familial haematuria Diseases 0.000 claims description 3
- 102000008186 Collagen Human genes 0.000 claims description 3
- 108010035532 Collagen Proteins 0.000 claims description 3
- 206010010356 Congenital anomaly Diseases 0.000 claims description 3
- 208000024720 Fabry Disease Diseases 0.000 claims description 3
- 208000022461 Glomerular disease Diseases 0.000 claims description 3
- 206010018372 Glomerulonephritis membranous Diseases 0.000 claims description 3
- 206010018374 Glomerulonephritis minimal lesion Diseases 0.000 claims description 3
- 206010024229 Leprosy Diseases 0.000 claims description 3
- 208000004883 Lipoid Nephrosis Diseases 0.000 claims description 3
- 208000000175 Nail-Patella Syndrome Diseases 0.000 claims description 3
- 206010028980 Neoplasm Diseases 0.000 claims description 3
- 206010029148 Nephrolithiasis Diseases 0.000 claims description 3
- 206010029164 Nephrotic syndrome Diseases 0.000 claims description 3
- 206010037596 Pyelonephritis Diseases 0.000 claims description 3
- 206010065427 Reflux nephropathy Diseases 0.000 claims description 3
- 208000033626 Renal failure acute Diseases 0.000 claims description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 3
- 102000005686 Serum Globulins Human genes 0.000 claims description 3
- 108010045362 Serum Globulins Proteins 0.000 claims description 3
- 206010052779 Transplant rejections Diseases 0.000 claims description 3
- 206010048302 Tubulointerstitial nephritis Diseases 0.000 claims description 3
- 201000011040 acute kidney failure Diseases 0.000 claims description 3
- 208000012998 acute renal failure Diseases 0.000 claims description 3
- 208000022185 autosomal dominant polycystic kidney disease Diseases 0.000 claims description 3
- 230000001580 bacterial effect Effects 0.000 claims description 3
- 229920001436 collagen Polymers 0.000 claims description 3
- 201000005206 focal segmental glomerulosclerosis Diseases 0.000 claims description 3
- 230000002068 genetic effect Effects 0.000 claims description 3
- 201000006334 interstitial nephritis Diseases 0.000 claims description 3
- 201000008350 membranous glomerulonephritis Diseases 0.000 claims description 3
- 231100000855 membranous nephropathy Toxicity 0.000 claims description 3
- 208000030761 polycystic kidney disease Diseases 0.000 claims description 3
- 238000012163 sequencing technique Methods 0.000 claims description 3
- LJRDOKAZOAKLDU-UDXJMMFXSA-N (2s,3s,4r,5r,6r)-5-amino-2-(aminomethyl)-6-[(2r,3s,4r,5s)-5-[(1r,2r,3s,5r,6s)-3,5-diamino-2-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-6-hydroxycyclohexyl]oxy-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl]oxyoxane-3,4-diol;sulfuric ac Chemical compound OS(O)(=O)=O.N[C@@H]1[C@@H](O)[C@H](O)[C@H](CN)O[C@@H]1O[C@H]1[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](N)C[C@@H](N)[C@@H]2O)O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)O[C@@H]1CO LJRDOKAZOAKLDU-UDXJMMFXSA-N 0.000 claims description 2
- 208000030507 AIDS Diseases 0.000 claims description 2
- 206010000599 Acromegaly Diseases 0.000 claims description 2
- 208000024985 Alport syndrome Diseases 0.000 claims description 2
- 206010002091 Anaesthesia Diseases 0.000 claims description 2
- 208000019838 Blood disease Diseases 0.000 claims description 2
- 108010075016 Ceruloplasmin Proteins 0.000 claims description 2
- 102100023321 Ceruloplasmin Human genes 0.000 claims description 2
- 208000005189 Embolism Diseases 0.000 claims description 2
- 208000017701 Endocrine disease Diseases 0.000 claims description 2
- 206010016207 Familial Mediterranean fever Diseases 0.000 claims description 2
- 108010049003 Fibrinogen Proteins 0.000 claims description 2
- 102000008946 Fibrinogen Human genes 0.000 claims description 2
- 206010018370 Glomerulonephritis membranoproliferative Diseases 0.000 claims description 2
- 206010018378 Glomerulonephritis rapidly progressive Diseases 0.000 claims description 2
- 108010015776 Glucose oxidase Proteins 0.000 claims description 2
- 239000004366 Glucose oxidase Substances 0.000 claims description 2
- 201000005569 Gout Diseases 0.000 claims description 2
- 206010072579 Granulomatosis with polyangiitis Diseases 0.000 claims description 2
- 108010051696 Growth Hormone Proteins 0.000 claims description 2
- 208000031886 HIV Infections Diseases 0.000 claims description 2
- 208000037357 HIV infectious disease Diseases 0.000 claims description 2
- 206010019280 Heart failures Diseases 0.000 claims description 2
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 claims description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 claims description 2
- 206010020772 Hypertension Diseases 0.000 claims description 2
- 208000010159 IgA glomerulonephritis Diseases 0.000 claims description 2
- 206010021263 IgA nephropathy Diseases 0.000 claims description 2
- 102000004877 Insulin Human genes 0.000 claims description 2
- 108090001061 Insulin Proteins 0.000 claims description 2
- 208000004451 Membranoproliferative Glomerulonephritis Diseases 0.000 claims description 2
- 102000016943 Muramidase Human genes 0.000 claims description 2
- 108010014251 Muramidase Proteins 0.000 claims description 2
- 108010062374 Myoglobin Proteins 0.000 claims description 2
- 102100030856 Myoglobin Human genes 0.000 claims description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 2
- 206010028851 Necrosis Diseases 0.000 claims description 2
- 108010061952 Orosomucoid Proteins 0.000 claims description 2
- 102000012404 Orosomucoid Human genes 0.000 claims description 2
- 206010033645 Pancreatitis Diseases 0.000 claims description 2
- 208000002774 Paraproteinemias Diseases 0.000 claims description 2
- 102000003982 Parathyroid hormone Human genes 0.000 claims description 2
- 108090000445 Parathyroid hormone Proteins 0.000 claims description 2
- 208000018262 Peripheral vascular disease Diseases 0.000 claims description 2
- 201000004681 Psoriasis Diseases 0.000 claims description 2
- 102100038803 Somatotropin Human genes 0.000 claims description 2
- 201000009594 Systemic Scleroderma Diseases 0.000 claims description 2
- 208000004732 Systemic Vasculitis Diseases 0.000 claims description 2
- 206010042953 Systemic sclerosis Diseases 0.000 claims description 2
- 206010043561 Thrombocytopenic purpura Diseases 0.000 claims description 2
- 208000001435 Thromboembolism Diseases 0.000 claims description 2
- 102000004338 Transferrin Human genes 0.000 claims description 2
- 108090000901 Transferrin Proteins 0.000 claims description 2
- 208000027418 Wounds and injury Diseases 0.000 claims description 2
- 230000003187 abdominal effect Effects 0.000 claims description 2
- 238000012084 abdominal surgery Methods 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 208000009956 adenocarcinoma Diseases 0.000 claims description 2
- 206010002022 amyloidosis Diseases 0.000 claims description 2
- 230000037005 anaesthesia Effects 0.000 claims description 2
- 210000000481 breast Anatomy 0.000 claims description 2
- 208000029078 coronary artery disease Diseases 0.000 claims description 2
- 230000001054 cortical effect Effects 0.000 claims description 2
- 201000005637 crescentic glomerulonephritis Diseases 0.000 claims description 2
- 230000006378 damage Effects 0.000 claims description 2
- 201000001981 dermatomyositis Diseases 0.000 claims description 2
- 208000030172 endocrine system disease Diseases 0.000 claims description 2
- 229940012952 fibrinogen Drugs 0.000 claims description 2
- 208000030376 fibronectin glomerulopathy Diseases 0.000 claims description 2
- 229940116332 glucose oxidase Drugs 0.000 claims description 2
- 235000019420 glucose oxidase Nutrition 0.000 claims description 2
- 239000000122 growth hormone Substances 0.000 claims description 2
- 208000014951 hematologic disease Diseases 0.000 claims description 2
- 208000018706 hematopoietic system disease Diseases 0.000 claims description 2
- 208000003215 hereditary nephritis Diseases 0.000 claims description 2
- 239000005556 hormone Substances 0.000 claims description 2
- 229940088597 hormone Drugs 0.000 claims description 2
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 claims description 2
- 229940072221 immunoglobulins Drugs 0.000 claims description 2
- 230000006698 induction Effects 0.000 claims description 2
- 229940125396 insulin Drugs 0.000 claims description 2
- 210000004072 lung Anatomy 0.000 claims description 2
- 229960000274 lysozyme Drugs 0.000 claims description 2
- 239000004325 lysozyme Substances 0.000 claims description 2
- 235000010335 lysozyme Nutrition 0.000 claims description 2
- 201000001441 melanoma Diseases 0.000 claims description 2
- 208000010125 myocardial infarction Diseases 0.000 claims description 2
- 230000017074 necrotic cell death Effects 0.000 claims description 2
- 230000002956 necrotizing effect Effects 0.000 claims description 2
- 230000000414 obstructive effect Effects 0.000 claims description 2
- 229960001319 parathyroid hormone Drugs 0.000 claims description 2
- 239000000199 parathyroid hormone Substances 0.000 claims description 2
- 229960001639 penicillamine Drugs 0.000 claims description 2
- 201000006292 polyarteritis nodosa Diseases 0.000 claims description 2
- 208000007056 sickle cell anemia Diseases 0.000 claims description 2
- 201000002859 sleep apnea Diseases 0.000 claims description 2
- 150000003431 steroids Chemical class 0.000 claims description 2
- 238000001356 surgical procedure Methods 0.000 claims description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 2
- 230000001732 thrombotic effect Effects 0.000 claims description 2
- 239000012581 transferrin Substances 0.000 claims description 2
- 230000008733 trauma Effects 0.000 claims description 2
- 238000007631 vascular surgery Methods 0.000 claims description 2
- 238000000338 in vitro Methods 0.000 claims 2
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims 1
- 206010013654 Drug abuse Diseases 0.000 claims 1
- 201000004331 Henoch-Schoenlein purpura Diseases 0.000 claims 1
- 206010019617 Henoch-Schonlein purpura Diseases 0.000 claims 1
- 208000031814 IgA Vasculitis Diseases 0.000 claims 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 claims 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 claims 1
- 101710084021 Large envelope protein Proteins 0.000 claims 1
- 208000026911 Tuberous sclerosis complex Diseases 0.000 claims 1
- 201000011510 cancer Diseases 0.000 claims 1
- 239000013068 control sample Substances 0.000 claims 1
- 208000015446 immunoglobulin a vasculitis Diseases 0.000 claims 1
- 230000003211 malignant effect Effects 0.000 claims 1
- 208000023504 respiratory system disease Diseases 0.000 claims 1
- 208000017520 skin disease Diseases 0.000 claims 1
- 208000011117 substance-related disease Diseases 0.000 claims 1
- 208000009999 tuberous sclerosis Diseases 0.000 claims 1
- 210000003712 lysosome Anatomy 0.000 description 44
- 230000001868 lysosomic effect Effects 0.000 description 44
- 238000003127 radioimmunoassay Methods 0.000 description 31
- 150000001875 compounds Chemical class 0.000 description 25
- 210000004027 cell Anatomy 0.000 description 17
- 230000000694 effects Effects 0.000 description 16
- 102000035118 modified proteins Human genes 0.000 description 16
- 108091005573 modified proteins Proteins 0.000 description 16
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 15
- 230000015556 catabolic process Effects 0.000 description 14
- 230000008859 change Effects 0.000 description 14
- 102000035195 Peptidases Human genes 0.000 description 13
- 230000002132 lysosomal effect Effects 0.000 description 13
- 208000007342 Diabetic Nephropathies Diseases 0.000 description 12
- 241000282414 Homo sapiens Species 0.000 description 12
- 208000033679 diabetic kidney disease Diseases 0.000 description 12
- 230000029142 excretion Effects 0.000 description 12
- 230000014759 maintenance of location Effects 0.000 description 10
- 230000008569 process Effects 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- 238000000926 separation method Methods 0.000 description 10
- 230000003213 activating effect Effects 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 239000002904 solvent Substances 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 239000000463 material Substances 0.000 description 8
- 241000894007 species Species 0.000 description 8
- 108090001069 Chymopapain Proteins 0.000 description 7
- XXAXVMUWHZHZMJ-UHFFFAOYSA-N Chymopapain Chemical compound OC1=CC(S(O)(=O)=O)=CC(S(O)(=O)=O)=C1O XXAXVMUWHZHZMJ-UHFFFAOYSA-N 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 238000001542 size-exclusion chromatography Methods 0.000 description 7
- 102000004142 Trypsin Human genes 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 6
- 239000000975 dye Substances 0.000 description 6
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 6
- 210000005084 renal tissue Anatomy 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 239000012588 trypsin Substances 0.000 description 6
- 206010027525 Microalbuminuria Diseases 0.000 description 5
- 230000033077 cellular process Effects 0.000 description 5
- 238000003018 immunoassay Methods 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000005227 gel permeation chromatography Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 229920002521 macromolecule Polymers 0.000 description 4
- 238000004513 sizing Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000008427 tissue turnover Effects 0.000 description 4
- 102000004506 Blood Proteins Human genes 0.000 description 3
- 108010017384 Blood Proteins Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- -1 V-8 Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000001028 anti-proliverative effect Effects 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 238000013399 early diagnosis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000000984 immunochemical effect Effects 0.000 description 3
- 230000016784 immunoglobulin production Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 210000004379 membrane Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 235000019419 proteases Nutrition 0.000 description 3
- 201000001474 proteinuria Diseases 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 230000002485 urinary effect Effects 0.000 description 3
- 102000005600 Cathepsins Human genes 0.000 description 2
- 108010084457 Cathepsins Proteins 0.000 description 2
- 102000008100 Human Serum Albumin Human genes 0.000 description 2
- 108091006905 Human Serum Albumin Proteins 0.000 description 2
- 101001018085 Lysobacter enzymogenes Lysyl endopeptidase Proteins 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 206010037549 Purpura Diseases 0.000 description 2
- 241001672981 Purpura Species 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 208000001647 Renal Insufficiency Diseases 0.000 description 2
- 208000034189 Sclerosis Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000005377 adsorption chromatography Methods 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 238000003491 array Methods 0.000 description 2
- RASZIXQTZOARSV-BDPUVYQTSA-N astacin Chemical compound CC=1C(=O)C(=O)CC(C)(C)C=1/C=C/C(/C)=C/C=C/C(/C)=C/C=C/C=C(C)C=CC=C(C)C=CC1=C(C)C(=O)C(=O)CC1(C)C RASZIXQTZOARSV-BDPUVYQTSA-N 0.000 description 2
- 239000012472 biological sample Substances 0.000 description 2
- 230000000747 cardiac effect Effects 0.000 description 2
- 239000002872 contrast media Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 238000003795 desorption Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000012156 elution solvent Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000001030 gas--liquid chromatography Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000001155 isoelectric focusing Methods 0.000 description 2
- 201000006370 kidney failure Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 238000004816 paper chromatography Methods 0.000 description 2
- 238000004810 partition chromatography Methods 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 235000019833 protease Nutrition 0.000 description 2
- 230000007420 reactivation Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- 208000006542 von Hippel-Lindau disease Diseases 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000007693 zone electrophoresis Methods 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 239000005541 ACE inhibitor Substances 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- ZHQQRIUYLMXDPP-SSDOTTSWSA-N Actinidine Natural products C1=NC=C(C)C2=C1[C@H](C)CC2 ZHQQRIUYLMXDPP-SSDOTTSWSA-N 0.000 description 1
- 102100022749 Aminopeptidase N Human genes 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 108010017640 Aspartic Acid Proteases Proteins 0.000 description 1
- 102000004580 Aspartic Acid Proteases Human genes 0.000 description 1
- 108090000658 Astacin Proteins 0.000 description 1
- 102000034498 Astacin Human genes 0.000 description 1
- 108010004032 Bromelains Proteins 0.000 description 1
- 108010049990 CD13 Antigens Proteins 0.000 description 1
- 108090000746 Chymosin Proteins 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- FMKGDHLSXFDSOU-BDPUVYQTSA-N Dienon-Astacin Natural products CC(=C/C=C/C=C(C)/C=C/C=C(C)/C=C/C1=C(C)C(=O)C(=CC1(C)C)O)C=CC=C(/C)C=CC2=C(C)C(=O)C(=CC2(C)C)O FMKGDHLSXFDSOU-BDPUVYQTSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 108010092208 Endothia aspartic proteinase Proteins 0.000 description 1
- 108010091443 Exopeptidases Proteins 0.000 description 1
- 102000018389 Exopeptidases Human genes 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 102000001399 Kallikrein Human genes 0.000 description 1
- 108060005987 Kallikrein Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 101001026869 Mus musculus F-box/LRR-repeat protein 3 Proteins 0.000 description 1
- 102000003729 Neprilysin Human genes 0.000 description 1
- 108090000028 Neprilysin Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102000016387 Pancreatic elastase Human genes 0.000 description 1
- 108010067372 Pancreatic elastase Proteins 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 101800001753 Protease Proteins 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 206010062237 Renal impairment Diseases 0.000 description 1
- 108090000783 Renin Proteins 0.000 description 1
- 102100028255 Renin Human genes 0.000 description 1
- 241000242678 Schistosoma Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108090001109 Thermolysin Proteins 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- XLYOFNOQVPJJNP-PWCQTSIFSA-N Tritiated water Chemical compound [3H]O[3H] XLYOFNOQVPJJNP-PWCQTSIFSA-N 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 241000223104 Trypanosoma Species 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- PAAZCQANMCYGAW-UHFFFAOYSA-N acetic acid;2,2,2-trifluoroacetic acid Chemical compound CC(O)=O.OC(=O)C(F)(F)F PAAZCQANMCYGAW-UHFFFAOYSA-N 0.000 description 1
- 108090000350 actinidain Proteins 0.000 description 1
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 150000004347 all-trans-retinol derivatives Chemical class 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 238000013103 analytical ultracentrifugation Methods 0.000 description 1
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 1
- 230000001093 anti-cancer Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 235000003676 astacin Nutrition 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019835 bromelain Nutrition 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000003560 cancer drug Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940080701 chymosin Drugs 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 230000036252 glycation Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000000076 hypertonic saline solution Substances 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 230000006525 intracellular process Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 230000007257 malfunction Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- GNOLWGAJQVLBSM-UHFFFAOYSA-N n,n,5,7-tetramethyl-1,2,3,4-tetrahydronaphthalen-1-amine Chemical compound C1=C(C)C=C2C(N(C)C)CCCC2=C1C GNOLWGAJQVLBSM-UHFFFAOYSA-N 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229960005489 paracetamol Drugs 0.000 description 1
- 230000003071 parasitic effect Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 231100000857 poor renal function Toxicity 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 229930002330 retinoic acid Natural products 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 108090000588 rhizopuspepsin Proteins 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 230000036325 urinary excretion Effects 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Description
METHOD FOR KIDNEY DISEASE DETECTION
BY PROTEIN PROFILING
. This application clans priority to U.S. Provisional Application Serial No. 60/301,251, filed June 28. 2001.
[01] The invention relates to improved methods of detecting an early stage of renal disease and/or renal complications of a disease, particularly diabetes.
[02] The appearance of excess protein such as albumin in the urine is indicative of kidney disease. Diabetic nephropathy is such a disease.
[03] The applicant has found that proteins, including albumin, are normally excreted as a mixture of native protein and fragments that are specifically produced during renal passage
Osicka T.M. et al., Nephrology, 2:199-212 (1996)). Proteins are heavily degraded during renal passage by post-glomerular (basement membrane) cells that may include tubular cells.
Lysosomes in renal tubular cells may be responsible for the breakdown of proteins excreted during renal passage. FIG. 1 illustrates the progress of filtered intact albumin into tubular cells and breakdown of albumin to provide excreted albumin fragments. The breakdown products are excreted into the tubular lumen. In normal individuals, most of the albumin in the urine is fragmented.
[04] When lysosome activity or intracellular processes directing substrates to lysosomes is reduced, more of the high molecular weight, and substantially full length albumin appears in the urine. This reflects an imbalance in the cellular processes in the kidney tissue.
[05] The applicant has discovered that when proteins, including major plasma proteins such as . albumin and immunoglobulin, are filtered by the kidney, they are subsequently degraded by cells in the kidney prior to the material being excreted (see, PCT published application WO ) 00/37944). 1t is likely that filtered proteins are taken up by tubular c lls. Tubular cells lie beyond the kidney filter and come in direct contact with the primary filtrate. When proteins are internalized by the tubular cells, they are directed towards the lysosomes, where they are partially degraded to various size fragments. and then regurgitated to outside the cell. These regurgitated fragments, of which there may be at least 60 different fragments generated from any . one particular type of protein. are then excreted into the urine.
[06] The applicant has discovered that in renal disease fragmentation of proteins is inhibited. . This means that substantially full-length filtered proteins are excreted in a person suffering from renal disease. This transition from fragmentation to inhibition of fragmentation of excreted proteins is a basis for the development of new drugs and diagnostic assays. For example, initial changes that occur with the onset of renal complications in diabetes are associated with a change in the fragmentation profile of excreted albumin. This leads to an apparent microalbuminuria that 1s synonymous with the development of diabetic nephropathy. It is likely that this is due to an inhibition in the lysosomal activity of tubular cells in diabetes. Thus, drugs can be formulated to turn on lysosomal activity in diabetes where renal complications are occurring. The drugs may also be useful in other renal diseases where lysosomal activities are affected, or in diabetes without renal complications in situations where lysosomal activity is turned off in non-renal tissues. Such drugs include antiproliferative drugs, such as anti cancer drugs. [07) However, by the time the excess albumin is detected, kidney disease has progressed, possibly to a stage where 1t 1s irreversible and treatment has little effect. Therefore there is a continuing need in the art to provide a test that is more sensitive than the currently known radioimmunoassay to detect such a disease as early as possible so that the disease can be either prevented or a treatment protocol commenced early on in the disease.
[08] However, previous attempts to use urinary protein profiles for diagnostic purposes have been rather disappointing with respect to their clinical validity, in part because of the insufficient reproducibility, sensitivity, and rapidity of available techniques. Thus, there exists a continuing need for an improvement in methods for improved methods of detecting an early stage of renal disease and/or renal complications of a disease, particularly diabetes
. (09] In one embodiment, the invention provides improved methods of detecting an early stage of renal disease and/or renal complicatinns of a disease. narticularly diabetes. A fisgmentation ) profile is determined in terms of the size, and sequence of particular fragments derived from intact filtered proteins together with the position where enzyme scission occurs along the protein polypeptide chain. The fragmentation profile is characteristic of the discascd state of the kidney.
Accordingly. methods of detecting early signs of a disease, including kidney discasc. . determining a patient's propensity for the discasc, preventing the onset of the discase. and treating the disease at the earliest stage possible are some of the objects of the invention. ig [10] The method involves taking urine from a subject, and separating all the fragments. [na particular embodiment. the separation is bv HPLC (single dimensional or two dimensional or three dimensional electrophoresis and/or chromatography), then sizing the fragments by mass spectrometry and using amino acid sequencing to determine the peptide sequence and where enzyme scission occurred.
[11] Although not limited to any particular disease, according to the method of the invention, the disease sought to be diagnosed includes nephropathy, diabetes insipidus, diabetes type I, diabetes II, renal disease (glomerulonephritis, bacterial and viral glomerulonephritides, IgA nephropathy and Henoch-Schénlein Purpura, membranoproliferative glomeruloneplhritis, membranous nephropathy, Sjégren's syndrome, nephrotic syndrome (minimal change disease, focal glomerulosclerosis and related disorders), acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, GU tract inflammatory disease, Pre-clampsia, renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis), genetic renal disease (medullary cystic, medullar sponge, polycystic kidney disease (autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tuborous sclerosis), von Hippel-Lindau disease, familial thin-glomerular basement membrane disease, collagen III glomerulopathy, fibronectin glomerulopathy, Alport's syndrome, Fabry's disease, Nail-Patella Syndrome, congenital urologic anomalies), monoclonal gammopathies (multiple myeloma, amyloidosis and related disorders), febrile illness (familial Mediterranean fever, HIV infection -AIDS), inflammatory disease (systemic vasculitides (polyarteritis nodosa, Wegener's granulomatosis, polyarteritis, necrotizing and crescentic glomerulonephritis), polymyositis-dermatomyositis, pancreatitis, rheumatoid arthritis, systemic lupus erythematosus, gout), blood disorders (sickle cell disease, thrombotic thrombocytopenia purpura, hemolytic-uremic syndrome, acute cortical necrosis. renal . thromboembolism), trauma and surgery (extensive injury, burns, abdominal and vascular surgery, induction of anesthesia), drugs (penicillamine, steroids) and -'~°g abuse --~'~-ant ' disease (epithelial (lung, breast), adenocarcinoma (renal), melanoma, lymphoreticular, multiple myeloma), circulatory disease (myocardial infarction, cardiac failure, peripheral vascular disease, hypertension. coronary heart disease, non-atherosclerotic cardiovascular disease, atherosclerotic cardiovascular disease), skin discase (psoriasis, systemic sclerosis), respiratory . discase (COPD, obstructive sleep apnoea. hypoia at high altitude) and endocrine disease (acromegaly, diabetes mellitus, diabetes insipidus). Specific proteinuria, and in particular, - albuminuna (micro- and macro-), is a marker of these disease.
[12] In asecond embodiment, the invention provides improved methods of detecting non- renal diseases. With the recognition that filtered proteins are degraded during renal passage, the methods described in this application can also detect protein fragments derived from proteins generated by non-renal discase. Non-renal diseases, such as cancers. generate increased levels of proteins into the circulation. The urinary analysis of these filtered proteins would currently not detect the intact form of these proteins. Therefore a method as described below to detect and analyze fragments resulting from degradation during renal passage that will be able to detect the seriousness of the disease.
[13] Both embodiments can use non-antibody technology, by separating a desired protein and its fragments from urine samples in a three-dimensional fashion; isolating the fragments; and determining the sequence of the protein and its fragments. This assay is repeated over a period of time. A change in the fragmentation profile over time indicates early stage of a particular disease. A change in the size of the fragments, as determined by sequence analysis, can indicate which type of renal disease the subject has a propensity to develop.
[14] These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto. ‘BRIEF DESCRIPTION OF THE DRAWINGS
[15] FIG. I illustrates the progress of filtered intact albumin into tubular cells and breakdown of albumin to provide excreted albumin fragments.
[16] FIG. 2 (2a and 2b) illustrate a representative profile of CH) HSA in (a) urtne and (b) : plasma collected from normal, healthy volunteers by size exclusion chromatography. Urine conteins mostly fragmented albumin. And plasma contains mostly intact albumin.
[17] FIG. 3 illustrates urine from normal, healthy volunteer showing a fragmented albumin peak. but no intact albumin peak from size exclusion chromatography.
[18] FIG. 4 illustrates urine from a diabetic patient showing both ntact and fragmented . albumin peaks from size exclusion chromatography.
[19] FIG. 5 illustrates a HPLC profile of albumin alone. } [20] FIG. 6 illustrates the HPLC profile of plasma from normal. healthy volunteer showing albumin peaks. [211 FIG. 7 shows the HPLC profile of urine from normal, healthy volunteer with fragmented products of albumin but no intact albumin peak. (22] FIG. 8 shows the HPLC profile of a urine sample from a normoalbuminuric diabetic patient showing albumin breakdown products and a small-modified albumin peak at approximately 39-44 minutes retention time.
[23] FIG. 9 shows the H{PLC profile of urine from a normoalbuminuric diabetic patient showing signs of kidney failure and the presence of the characteristic spiked albumin peak at approximately 39-44 minutes retention time.
[24] FIG. 10 illustrates a HPLC profile of a normoalbuminuric diabetic patient showing signs of kidney failure and the presence of the characteristic spiked modified albumin peak at approximately 39-44 minutes retention time.
[25] FIG. 11 illustrates a HPLC of a macroalbuminuric diabetic patient showing high levels of the normal albumin as well as the characteristic spiked appearance at approximately 39-44 minutes retention time.
[26] FIG. 12 illustrates a longitudinal study of a patient in which the modified protein was detected at a time prior to onset of diabetic nephropathy, indicating predisposition to diabetic nephropathy, and the delay in treatment caused by relying on conventional RIA methods. (27] FIG. 13 illustrates a longitudinal study of a patient in which the modified protein was detected at a time prior to onset of diabetic nephropathy, indicating predisposition to diabetic nephropathy, and the delay in treatment caused by relying on conventional RIA methods. (28] FIG. 14 illustrates a longitudinal study of a patient in which the modified protein was . detected at a time prior to onset of diabetic nephropathy, indicating predisposition to diabetic nephropathy, and the delay in treatment cause? ™ r~! ying on conventional RIA methods.
[29] FIG. 15 shows the HPLC chromatogram used as a criterion of purity of the modified albumin of Example 4.
[30] FIG. 16 is a schematic diagram tlustrating the manner in which an intact filtered protein } may be degraded by normal functioning kidneys and diseased kidneys.
[31] FIG. 17 illustrates the HPLC profile of a trypsin digested sample of albumin that has been - filtered through a 30,000 molecular weight cut-off membrane. The filtrate yields many peaks eluting between 2 to 30 minutes.
[32] FIG. 18 illustrates the HPLC profile of a control, normal subject showing many fragments in the cluting range of 10 to 30 minutes. The HPLC profile of a diabetic patient with macroalbuminuna (1457 microgram per minute) shows a significantly different fragment profile in the range of 10 -30 minutes.
[33] FIG. 19 illustrates the HPLC profile of a subject with renal disease. As compared with
FIG. 18, the fragmentation process of filtered proteins is inhibited. The number of fragments 1s decreased and the size of the fragments is increased.
[34] The applicant has discovered that when proteins, including major plasma proteins such as albumin and immunoglobulin, are filtered by the kidney they are subsequently degraded by cells in the kidney prior to the material being excreted. It is likely that filtered proteins are taken up by tubular cells. Tubular cells lie beyond the kidney filter and come in direct contact with the primary filtrate. When proteins are internalized by the tubular cells, they are directed towards the lysosomes, where they are partially degraded to various size fragments, and then regurgitated to outside the cell. These regurgitated fragments, of which there may be at least 60 different fragments generated from any one particular type of protein, are then excreted into the urine. (35] The applicant has discovered that in renal disease fragmentation of proteins is inhibited.
This means that substantially full-length filtered proteins will be excreted in a person suffering from renal disease. This transition from fragmentation to inhibition of fragmentation of excreted proteins is a basis for the development of new drugs and diagnostic assays. For example, initial . changes that occur with the onset of renal complications in diabetes are associated with a change in the fragmentation profile of excreted albumin. This leads to an apparent micro» tbuminuria,
which is synonymous with the development of diabetic nephropathy. It is likely that this is due to an inhibition in the lysosomal activity of tubular cells in diabetes.
[36] Thus, drugs can be formulated to turn on lysosomal activity in diabetes where renal ’ complications are occurring. The drugs may also be useful in other renal diseases where lysosomal activities are affected. or in diabetes without renal complications in situations where lysosomal activity is turned off in non-renal tissues. Such drugs include antiproliferative drugs, such as ant cancer drugs or antibodies to neutralize TGF-beta. [371 The applicant has discovered a unique assay for detecting protein fragment arrays of specific proteins, which are detected in the urine of subjects. Detection of the protein fragment array and changes to the protein fragment array are predictive of a predisposition to renal disease. {38] The principle of the protein fragment array is shown in FIG. 16. The intact protein is represented by a series of regions representing specific amino acid sequences within the protein.
All proteins have these specific primary structures. When such a protein from plasma, like albumin or immunoglobulin is filtered it is filtered intact. However, after the protein is filtered it may be taken up by renal cells, such as early proximal tubular cells, and be degraded, by enzymes within lysosomes, to many fragments (FIG. 16). These fragments are excreted in urine.
For normal functioning kidneys, the fragmentation process is maximal with small fragments derived from many individual filtered proteins being produced and ultimately excreted. FIG. 17 illustrates a fragmentation profile from the trypsin digest of albumin. A similar profile is seen in the unne of a control, normal volunteer (FIG. 18). In terms of the number of fragments produced from each protein and the nature of the peptide splitting (i.e., the position along the protein where scission occurs), the fragmentation profile is specific. The size and sequence characteristic of the individual fragments will be characteristic of the specificity and activity of lysosomal enzymes acting on the protein. (39] Proteases such as V-8, trypsin and Lys-C can be used to produce a peptide map of a purified protein. Other proteases can be used, preferably proteases that cause limited proteolysis ("enzyme scission”), in which a protease cleaves only one or a limited number of peptide bonds > of a target protein. The protease can be from any group of proteases, such as the serine rroteinases {~hvmetrypsin, trypsin, elastase, kallikrein, and the substilisin family), the cyste:-- proteinases (the plant proteases such as papain, actinidin or bromelain, some cathepsins, the cytosolic calpains, and parasitic proteases (e.g., from Trypanosoma, Schistosoma), the aspartic proteinases (pepsin family members such as pepsin, chymosin, some cathepsins D, and renin; certain fungal proteases (pemicillopepsin, rhizopuspepsin, endothiapepsin); and viral proteinases ‘ such as retropepsin); and the metalloproteinases (including thermolysin, neprilysin, alanyl aminopeptidase, and astacin). ) (40] In renal disease, the fragmentation process of filtered proteins is inhibited. The number of fragments 1s decreased and the size of the fragments is increased (FIG. 19). This is due to the fact that there are less points of scission by lysosmal enzymes. Therefore, in terms of the size and amino acid sequence, the fragment profile is considerably different from that obtained in normal kidneys for any particular filtered protein, such as albumin or immunoglobulin. The degree of inhibition of fragmentation will depend on the severity of the disease. As disease progresses the degree of fragmentation will become less as demonstrated in FIG A.
[41] U.S. Pat. No. 5,240,835 discloses a method of diagnosing renal diseases by detecting fragments of albumin in human urine. The '835 patent discloses that the fragments are denved from the plasma and are filtered by the kidney, unaltered, and are ultimately excreted. The method of detection of the unnary fragments in the '835 patent preferably involves the use of affinity binding to conventional albumin antibodies. In contrast to the method of present invention, there 1s an increased detection of albumin fragments in diabetes in the method of the '$35 patent. In the present invention, the diagnosis of diabetic nephropathy can occur when there is a decrease in the number of fragments. The albumin fragments examined in the present invention are not necessarily detected by albumin antibodies.
[42] In contrast to the method of the '835 patent, one embodiment of the invention is the taking urine from a patient, and separating all the fragments by HPLC (single dimensional or two dimensional or three dimensional electrophoresis and/or chromatography) and then sizing the fragments by mass spectrometry and then using amino acid sequencing to determine the peptide sequence and where peptide scission occurred.
[43] The protein fragments can be detected and separated by a vanety of methods that are well-known in the art, including, but not limited to chromatography, electrophoresis and ) sedimentation, or a combination of these, which are described in Karger BL, Hancock WS (eds.)
High Resolution Separation ad Analysis ~* hiriagical Macromolecules. Part A Fundamentals in
Methods in Enzymology, Vol. 270, 1996, Academic Press, San Diego, Cahforma, USA; Karger
N
BL. Hancock WS (eds.) High Resolution Separation and Analvsis of biological Macromolecules.
Part B Applications in Methods in Enzymology, Vol. 271, 1996. Academic Press. San Dicgo,
California, USA; or Harding SE, Rowe. AJ, Horton JC (eds.) Analvtical Ultracentrifugation in . Biochemistry and Polvmer Science. 1992, Royal Soc. Chemistry. Cambridge, UK, which references arc incorporated herein by reference in their entirety. ) [44] The electrophoresis method includes, but is not limited to, moving-boundary electrophoresis, zone electrophoresis. and isoelectric focusing.
[45] The chromatography method includes, but is not limited to, partition chromatography, adsorption chromatography, paper chromatography, thin-layer chromatography, gas-liquid chromatography, gel chromatography, ion-exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography. Preferably, the method is a sizing gel chromatography and hydrophobic interaction chromatography. More preferably, the method is hydrophobic interaction chromatography using a HPLC column.
[46] HPLC is preferred for generating a fragmentation profile. A fragmentation profile on
HPLC is charactenzed by a series of peaks representing a number of fragment species.
[47] A HPLC column for detecting modified albumin or unmodified albumin may be a hydrophobicity column, such as Zorbax 300 SB-CB (4.6mm x 150 mm). A 50ul sample loop may be used. Elution solvents suitable for HPLC in detecting albumin and its breakdown products may include standard elution solvents such as acetonitrile solvents. Preferably a buffer of water/1% tnfluoro acetic acid (TFA) followed by a buffer of 60% acetonitrile/0.09% TFA may be used. A gradient of 0 to 100% of a 60% acetonitrile/0.09% TFA has been found to be suitable.
[48] Suitable HPLC conditions for a hydrophobicity column may be as follows:
Solvent A H,0, 1% trifluoro acetic acid
Solvent B 60% acetonitrile, 0.09% TFA
Solvent A2 99.96>00.00:49.58min
Pressure 9.014Mpascalls (~1100psi)
Solvent B2 0.04>100.0:49.58min
Pressure 7.154Mpascalls ’ [49] The wavelength used in HPLC may be approximately 214 nm.
[50] For albumin, modified albumin may elute betweer J-44 minutes (o°1G. 5). Albumin fragments may elute much earlier. mainly at less than 20 minutes.
[51] "Fragmented protein or fragment albumin” includes post-glomerular breakdown products after chemical, enzymatic or physical breakdown that occurs during renal passage. These components have a reduced size and/or may have changed hydrophobicity. ’ [52] “Intact albumin, modified albumin, or modified form of albumin" as used herein means a compound having similar size and structural characteristics to native albumin, wherein the amino acid sequence is substantially the same as the native albumin. It is preferably a filtered intact protein. It elutes at or near the same position as native albumin on high-pressure liquid chromatography (HPLC) (FIG. 5). However, the structure has been moditied biochemically either by minor enzyme mediated modification or addition to its basic structure and/or physically through a change in its three dimensional structure so that it escapes detection by conventionally used anti-albumin antibodies. Biochemical modification may be made by enzymes such as endo- or exo- peptidases. The 3D structure of albumin may have been altered in some way. Ligands may have bound to the albumin, or it may be any combination of these. The modified albumin detected in the method of the invention 1s not detectable by current and conventional radioimmunoassays using available antibodies and 1s not a fragment.
[53] Conventional anti-albumin antibodies can be purchased from any purveyor of immunochemicals. For example, monoclonal antibody catalog numbers A6684 (clone no. HSA- 11), and A2672 (clone no. HSA-9), as well as liquid whole serum, lyophilized fractionates, liquid IgG fraction, and the monoclonal antibodies in liquid ascites fluids form, can be obtained from Sigma. St. Louis, MO, as found in the Immunochemicals section at pages 1151-1152 in the 1994 Sigma - Biochemicals Organic Compounds for Research and Diagnostic Reagents catalog.
[54] As used herein, intact/modified albumin includes albumin that is substantially full-length, fragmented, chemically modified, or physically modified. As used herein, intact/modified albumin is meant to indicate albumin that ts less than, equal to, or greater in molecular weight than the full-length albumin, and elutes at or near the native albumin position in a separation medium, such as chromatography, preferably HPLC, and most preferably hydrophobicity HPLC. ) As used herein, fragmented albumin is meant to refer to the fragment of albumin that is not detec'sd hv ~onventional anti-albumi.: antibody, and its presence is detected in diagnoesi=g an early stage of renal disease and/or renal complications of a disease. The detection of the presence of intact/modified albumin 1s an indication of a predisposition to renal disease.
[35] “Intact protein. modified protein or modified form of a protein” as used herein includes . those torms of substantially full-length protein which are undetectable by conventional radioimmunoassay. The protein includes, but is not limited to, albumin, globulin (c-globuhin(a, - : globulin, ay-globulin), -globulin,y -globulin), euglobulin, pscudoglobulin [ and II, fibrinogen. cy acid glycoprotein (orosomucoid). or; glycoprotein, a; lipoprotein. ceruloplasmin, wu, 19S ¢lycoprotein. 3, transferrin, {iy lipoprotein, immunoglobulins A. E, G. and M, horseradish peroxidase, lactate dehydroucnase. glucose oxidase, myoglobin, lysozyme. protein hormone, growth hormone, insulin, or parathyroid hormone..
[56] "Kidney disease" as used herein includes any malfunction of the kidney. Kidney disease may be identified by the presence of intact or modified albumin in the urine. Preferably, an early diagnosis of the kidney disease may be made by detecting the presence of modified protein in the urine, or an increase in the modified protein in the urine over time. [57} "Low lysosome activity” as used herein is compared against normal levels of lysosome activity and/or lysosome machinery that traffics protein to the lysosome in a normal individual.
The activity is insufficient for the lysosome to fragment proteins so that intact protein is excreted at a greater amount than at normally low levels.
[58] "Lysosome-activating compound” as used herein refers to a compound that is beneficial to reactivation of the lysosome. The compound may work directly or indirectly on the lysosome resulting in activation of lysosomal function. These compounds may be selected from the group including, but not limited to, anticancer compounds, antiproliferation compounds, paracetamol, vitamin A (retinoic acid) or derivatives of retinol, or compounds, including antibodies, to neutralize TGF beta.
[59] “Macroalbuminuria” is a condition where an individual excretes greater than 200pg albumin/min in the unne as measured by conventional radioimmunoassay (RIA).
[60] "Microalbuminuria" is a condition where an individual excretes at Jcast 20 pg albumin/min in the urine as measured by conventional radioimmunoassay (RIA). RIA measures ) down to 15.6 ng/ml and is able to measure albumin in urine of norr al subjects who have clearance of less than 6 pg/min. However. when albumin excretion exceeds 20 g/min. treatment of the kidney disease 1s hmited and full recovery 1s difficult trom this point.
[61] "Microalbuminuric" as used herein is a condition when albumin 1s detected in the urine at ‘ an excretion rate of at least 20 pg/min as measured by conventional RIA.
[62] As used herein. “native” and “unmodified” are used interchangeably to describe a protein ) that 1s naturally found in an organism, preferably a human, which has not been modified by the filtering process of the renal glomeruli. (63] "Normal individual” as used herein is an individual who does not have a disease in which intact protein found in urine is an indicator of the disease. Preferably, the disease is kidney disease.
[64] "Normal levels of lvsosome activity” are levels of lysosome activity found in undiseased kidney of a normal individual.
[65] "Normoalbuminuric” as used herein means a condition where albumin is excreted in the urine and 1s not detectable by RIA, or less than 20 g/min (as measured by RIA) is excreted.
[66] "Propensity for a disease” as used herein means that a disease may result in an individual as judged by a determination of the presence and excretion rate of a modified protein such as modified albumin. (67) “Proteinuria” as used herein is the existence of protein in the urine, usually in the form of albumin, a protein that is soluble in water and can be coagulated by heat. Related to this, "specific proteinuria” refers to the existence of a particular protein in the urine.
[68] "Radioimmunoassay" as used herein is a method for detection and measurement of substances using radioactively labeled specific antibodies or antigens.
[69] "Reactivation of the lysosome" as used herein includes an activation of lysosome activity preferably so that breakdown of proteins, particularly albumin, is increased compared with an inactivated state of the lysosome.
[70] "Restore" as used herein means to restore in full or in part so that the component being restored has an improved function compared with its previous function. {71] The "sum of intact and intact modified protein” as used herein refers to the total amount ' of intact protein, and intact modified protein present in a biological sample.
[72] “Total protein” as used herein refers to a parc. lar filtc, cu p.o.ein present in native, unmodified, modified or fragmented form that is excreted in urine. It includes protein that is not detected bv conventional radioimmunoassay or conventional methods, which are currently available 10 detect the protein. Preferably the protein 1s albunun.
Methods ot Detection
[73] Unnary protein profiles can be created and examined using the methods of Hampel DJ et : al. J. Am. Soc. Nephrol 12(3): 1020-35 (2001), who have developed a sensitive, high- throughput technique, namely surface-enhanced laser desorption/ionization (SELDI)
ProtenChip ® array-time of flight mass spectrometry. Hampel er af. tested the applicability of the technique for protein profiling of urine and to exemplify its use for patients rcceiving radiocontrast medium. Assessment of the accuracy, sensitivity, and reproducibility of SELDI in test urinary protein profiling was performed in rats before and after intravenous administration of either 1oxilan or hypertonic saline solution as a control. Administration of 1oxilan to rats resulted in changes in the abundance of proteins of varying weights. Then, urine samples from patients undergoing cardiac catheterization were obtained. For patients, even in uncomphcated cases of radiocontrast medium infusion during cardiac cathetenzation, perturbations in the protein composition occurred but returned to baseline values after 6 to 12 hours. Proteins with certain defined molecular masses changed in abundance. For patients with impaired renal function, these changes were not reversible within 6 to 12 hours. As a proof of principle, one ot the proteins was identified as ;-microglobulin. Even for patients without renal complications, proteins with a broad range of molecular masses either appear in or disappear from the urine. (74) Unnary protein profiles can also be created and examined using the commercially available ProteinChip® System (Ciphergen Biosystems, Fremont, CA, USA), which uses SELDI (Surface-Enhanced Laser Desorption/Ionization) technology to rapidly perform the separation, detection and analysis of proteins at the femtomole level directly from biological samples. Each aluminum chip contains eight individual, chemically treated spots for sample application; this set-up facilitates simultaneous analysis of multiple samples. A colored, hydrophobic coating retains samples on the spots and simultaneously allows for quick identification of chip type.
Typically, a few microliters of sample applied on the ProteinChip® Array yield sufficient protein ‘ for analysis with the ProteinChip® Reader. r78Y Tor more dilute samples, a ProteinChip® Bioprocessor can be used to api: up to S77
The mass determination of protein samples 1s accomplished by sample crystallization, sample ionization, flight through a vacuum tube, and detection of the ionized proteins. After washing off non-specifically bound proteins and other contaminants from the ProteinChip® Array. a chemical Energy Absorbing Molecule (EAM) solution is applied and allowed to dry, during ‘ which time minute crystals form on the chip. These crystals contain the EAM and the protein(s) of interest. After inserting the ProteinChip Array into the ProteinChip Reader. a laser beam 1s focused upon the sample, which causes the proteins embedded in the EAM crystals to desorb and ionize. Released ions then experience an accelerating electrical field that causes them to "fly" through a vacuum tube. towards the ion detector. Finally, the ionized proteins are detected and an accurate mass is determined based on the time of flight (TOF).
[76] Proteases such as V-$, trypsin and Lys-C can be used to produce a peptide map ofa purified protein bound to the ProteinChip® Array by on-chip protease digestion as shown in the figure to the right. The molecular weights of the resulting fragments can be compared to a peptide database for identification. The process takes less than an hour.
[77] Additionally, twelve ProteinChip Arrays aligned side-by-side create a 96-well plate footprint. A typical experiment using ProteinChip Array technology requires one to three hours of work at the bench followed by automated sample analysis with the ProteinChip Reader. The entire process thus can be completed in a single afternoon.
Other Methods
[78] According to the present invention, the diseases to be treated include, but are not limited to renal disease (glomerulonephritis, bacterial and viral glomerulonephritides, [gA nephropathy and Henoch-Schénlein Purpura, membranoproliferative glomerulonephritis, membranous nephropathy, Sjégren's syndrome, diabetic nephropathy, nephrotic syndrome (minimal change disease, focal glomerulosclerosis, and related disorders), acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, GU tract inflammatory disease, Pre-clampsia, renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis), genetic renal disease (medullary cystic, medullar sponge. polycystic kidney disease (autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tuborous sclerosis), von Hippel-Lindau disease, familial thin-glomerular basement membrane disease, collagen 11 glomerulopathy, fibronectin glom:r-lopathy *'rar'; syndrome, Fabry's disease, Nail-Patella Syndrome, congenital urologic anomalies). )
[79] In one aspect of the invention, there is provided a method for determining a propensity for or early diagnosis of renal discase and/or renal complications of a discasc. The method includes determining a change tn the albumin content in a urine sample. The discase may be a kidney disease, although not necessarily limited to o kidney disease. (80] Inthe method of the invention. albumin is used herein only as an example of a protein to ’ be detected 1n urine. When the albumin in a patient is analyzed by conventional RIA, it is expected that a normoalbuminuric patient or normal individual would have albumin in the urine in the range of 3-10 pg/min in young people and greater in older people. However, normoalbuminuric patients also show levels of albumin in the urine if measured by HPLC.
Applicant has found that these levels may be in the order of 5 pg/min. As kidney disease progresses, the level of intact/modified albumin will increase to microalbuminuria levels in the order of 20 10 200 ug/min as determined by RIA. This will be much higher when determined by
HPLC or a method that determines the sum of intact albumin and intact modified albumin. By monitoring the increase in intactmodified albumin, early signs of kidney disease may be detected. However, these levels are not detectable by the methods currently available such as radioimmunoassay using antibodies currently commercially in use. possibly for the reason that antibodies detect certain epitopes. If the albumin is modified in any way as described above, the epitope may be destroyed thereby leaving the modified albumin undetectable.
[81] A patient suspected of having diabetic kidney disease will not show signs of kidney degeneration until well after 10 to 15 years when albumin is detected by currently available methods such as RIA methods. Urinary excretion rates of at least 20 png/min may be detected by
RIA when an individual enters a microalbuminuric state. Again, by observing the excretion of modified albumin, a change in the kidney and possibly onset of a kidney disease may be detected.
[82] A normoalbuminuric subject, or normoalbuminurnic diabetic patient may continue to have a low albumin excretion rate of less than 20 pg/min as determined by RIA, for many years. The presence of albumin in the urine is a sign that functions of the kidney may be impaired. Once this level begins to change. treatment may be initiated. (83] In anormal individual a small amount of albumin is detectable in the urine. Total “tered albumin appears mainly as fragmented albumir. ©.. unne. Juice w.oumin may be detected in normoalbuminuric individuals. However, the excretion rate of albumin in urine in a lS normoalbuminuric individual may be as low as 5 pg/min. This level is generally detectable by
RIA.
[84] The modified protein of the invention can be detected by a variety of methods that are ‘ well-known in the art, including, but not hmited to chromatography, clectrophori:sis and sedimentation, or a combination of these, which are described in Karger BL, Hancock WS (eds.)
High Resolution Separation and Analysis of biological Macromolecules. Part A Fundamentals in
Methods in Enzymology, Vol. 270. 1996, Academic Press. San Diego, California, USA; Karger
BL, Hancock WS (eds.) High Resolution Separation and Analvsis of biological Macromolecules.
Part B Applications in Methods in Enzvmology, Vol. 271, 1996, Academic Press, San Diego,
California, USA; or Harding SE, Rowe, AJ, Horton JC (eds.) Analytical Ultracentrifugation in
Biochemistry and Polvmer Science. 1992, Royal Soc. Chemistry, Cambridge, UK, which references are incorporated herein by reference in their entirety.
[85] The clectrophoresis method includes, but is not limited to, moving-boundary electrophoresis, zone electrophoresis, and isoelectric focusing.
[86] The chromatography method includes, but is not limited to, partition chromatography, adsorption chromatography, paper chromatography, thin-layer chromatography, gas-liquid chromatography, gel chromatography, ion-exchange chromatography, affinity chromatography, and hydrophobic interaction chromatography. Preferably, the method is a sizing gel chromatography and hydrophobic interaction chromatography. More preferably, the method is hydrophobic interaction chromatography using a HPLC column.
[87] The modified protein can also be detected by the use of specific albumin dyes. Such methods are described by Pegoraro er al., American Journal of Kidney Diseases 35(4): 739-744 (April 2000), the entire disclosure of which is hereby incorporated by reference. The modified albumin, as well as the whole albumin, 1s detectable by this dye method to provide the sum of modified albumin and whole or intact albumin. This detection method may be used with or without an initial separation of the albumin components from unne. Such dyes normally do not detect fragments <10,000 in molecular weight, but will detect the modified albumin.
[88] In this dye method of detection, a dye such as Albumin Blue S80 is used. Such dyes are ’ naturally non-fluorescent, but fluoresce on binding to intact albumin as well as the modified ~'-amin, but do not bind to globulins. Therefore, globulins do not interfere ‘vit the ase; ~~ at measurements can be made in unfractionated urine. .
[89] Applicant has found that among diabetics. a normoalbuminuric diabetic patient has almost undetectable levels of modified or fragments of albumin when analvzed by conventional
RIA. They appear to be normal. However, when the urine is tested by HPLC. the levels of : modified albumin are much greater than found in a normal individual. This difference in albumin may be attributed to the inability of conventional RIA's to adequately detect all albumin } (total albumin) in intact or modified forms. Thus, HPLC is preferred for generating a fragmentation profile. A fragmentation profile on HPLC is characterized bv a serics of peaks representing a number of species of albumin as fragments or in intact or modified forms.
[90] Ina preferred aspect of the present invention, the method of determining a propensity for or early diagnosis of a kidney disease in a subject is determined before the subject becomes microalbuminuric.
[91] Measuring albumin content in a sample by an HPLC method of the present invention may provide different results from its measurement by conventional RIA. In the HPLC technique, a low level of albumin is observed in normal individuals. When the level of modified albumin begins to be detected and its level increases, and progresses toward microalbuminuria then a patient can be determined to have a propensity for kidney disease.
[92] In anormal individual, the HPLC generated fragmentation profile is characterized by the absence of a peak in a region where full-length native albumin elutes. Instead, multiple fragmented albumin is detectable. A pure protein product (unmodified) produces essentially a single peak. For example, using a hydrophobicity HPLC, albumin was observed to elute in the range of 39-44 minutes (FIG. 5). Thus, a normal individual would provide a distinct fragmentation profile indicative of an absence of kidney disease or no propensity for a kidney disease. However, as kidney disease progresses, an increasing amount of modified albumin first, and then native form later are detectable. The fragmentation profile begins to change and more products in the region of full-length albumin manifests as additional spikes or an enlarged peak indicative of more intact/modified albumin in the urine.
[93] Ina HPLC generated fragmentation profile of a urine sample, the modified albumin may appear in a region where native albumin elutes but may be manifest as multiple peaks indicating ’ the presence of multiple forms of modified albumin.
[94] Ina farher pref~—e4 ~rabodiment, the propensity for kidney disease may be measured by ) determining the presence of or identifying at least one species of modified albumin. This may be determined or identified bv the presence of a specific peak on a HPLC profile, preferably the peak is within the range of position that corresponds to the clution position of the native albumin.
[95] The method for determining the propensity for kidney disease is applicable to any ‘ individual. Kidney disease may be caused by a number of factors including bactenal infection, allergic, congenital defects, stones, tumors, chemicals or from diabetes. Preferably, the method ) is applicable for determining a propensity for kidney diseasc in diabetic patients that may progress to 4 kidney disease. Preferably, the individual is a normoalbuminunic diabetic.
However, normal individuals may be monitored for propensity for the discase by determining increased levels of intact or modified albumin in the urine.
[96] The method of the invention can be carried out using non-antibody separation procedures as described above. However, antibody specific for modified protein may also be used to detect the presence of the modificd protein.
[97] The anubody 10 the modificd protein may be obtained using the following method. The procedure is described specifically for albumin by way of example only, and can be readily applied to antibody production against any other protein in the urine. The method seeks to determine which modified albumin molecule is the most sensitive marker to identify diabetic patients, for example, who will progress to kidney complications.
[98] The modified albumin is characterized by carrying out a quantitative separation of the modified albumin molecules, such as by preparative HPLC. The modified proteins are analyzed for ligand binding, such as glycation. Subsequently, amino acid sequence of the individual modified protein is determined, preferably by mass spectrometry using methods described in
Karger BL, Hancock WS (eds.) High Resolution Separation and Analysis of biological
Macromolecules. Part A Fundamentals in Methods in Enzymology, Vol. 270, 1996, Academic
Press, San Diego, California, USA; or Karger BL, Hancock WS (eds.) High Resolution
Separation and Analysis of biological Macromolecules. Part B Applications in Methods in
Enzymology, Vol. 271, 1996, Academic Press, San Diego, California, USA, for example, which references are incorporated herein by reference in their entirety. In a preferred embodiment, there may be about 3 to 4 modified albumin species. ’ [99] The method of generating antibody against the modified albumin seeks to develop a diagnostic immunoassay for the modifie! »'bumin thot nredicts those diabetic patieats, for example, that progress to kidney complications. To accomplish this, sufficient quantities of modified albumin is prepared by HPLC. Antibodies are made by sequential injection of the modified albumin in an animal such as a rabbit. 10 generate good titer. and the antibodies are isolated using conventional techniques using methods described in Goding JW. Monoclonal
Anubodies: Principles and Practice. Production and Application of Monoclonal Antibodies in
Cell Biology. Biochemistry and Immunology. 2nd Edition 1986. Academic Press. London, UK; or Johnstone A. Thorpe R. immunochemistrv in Practice. 3rd edition 1996, Blackwell Science
Ltd. Oxford, UK, for example. which references are incorporated herein by reference in their entirety. The obtained antibodies may be polyclonal antibodies or monoclonal antibodies.
[100] Preferably, at least one species of a modified albumin is isolated and identified for use in determining a propensity for kidney disease. The isolated species may be used to generate antibodies for use in immunoassays. The antibodies may be tagged with an enzymatic, radioactive, fluorescent or chemiluminescent label. The detection method may include, but is not limited to radioimmuoassay, immunoradiometric assay, fluorescent immunoassay, enzyme linked immunoassay, and protein A immunoassay. The assays may be carried out in the manner described in Goding JW, Monoclonal Antibodies: Principles and Practice. Production and
Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology. 2nd
Edition 1986, Academic Press. London, UK; Johnstone A, Thorpe R, Immunochemistry in
Practice, 3rd edition 1996, Blackwell Science Ltd, Oxtord, UK: or Price CP, Newman DJ (eds.)
Principles und Practice of Immunoassay, 2nd Edition, 1997 Stockton Press, New York, NY,
USA, for example, which references are incorporated herein by reference in their entirety.
[101] It is an object of this invention to provide an article of matter or a kit for rapidly and accurately determining the presence or absence of modified protein such as modified albumin, in a sample quantitatively or non-quantitatively as desired. Each component of the kit(s) may be individually packaged in its own suitable container. The individual container may also be labeled in a manner, which identifies the contents. Moreover, the individually packaged components may be placed in a larger container capable of holding all desired components.
Associated with the kit mav be instructions, which explain how to use the kit. These instructions may be written on or attached to the kit.
[102] The invention is also directed to a method of determining a treatment agent for renal disease and/or renal complications of a disease. comprising: (a) administering to a person an agent that is suspected of being able to treat the discasc; (b) obtaining a urine sample from the person: and i (c) assaying for the modified form of the protemn in the sample, wherein either the presence of or Jack of presence of a modified form of the protein in the urine or decreasing amount of the modified form of the protein over time indicates that the agent is a treatment agent for the disease. The treatment agent may be a lysosome activating agent that may act directly or indirectly to activate lysosome, and thereby cause the lysosome to digest post-glomerular filtered proteins, which is a sign of a healthy kidney.
[103] The process of trafficking of proteins to the lysosomes plays a role in the mechanism of albuminuna in diabetes. An intracellular molecule that is involved in trafficking 1s protein kinase
C (PKC). ltis contemplated that a drug or agent can be formulated that will activate lysosomal trafficking or inhibit PKC.
[104] Accordingly, in onc aspect of the present invention, there is provided a lysosome- activating compound for use in reactivating lysosomes or processes that direct substrates to the lysosome or products away from the lysosome.
[105] In another aspect of the present invention, there is provided a composition comprising a lysosome-activating compound and a carrier.
[106] In yet another aspect of the invention there is provided a method of preventing or treating kidney disease, said method including administering an effective amount of a lysosome- activating compound to a subject.
[107] In yet another aspect of the present invention, there is provided a method of screening a muluplhicity of compounds to identify a compound capable of activating lvsosomes or processes that direct substrates to the lysosome or products away from the lysosome. said method including the steps of: (a) exposing said compound to a lysosome and assaymyg said compound for the i ability 10 activate a lysosome wherein said lysosome when activated has a changed activity; (b) assaying for the ability to restore a cellular process to substantially normal levels in kidney tissue, wherein said kidney tissue has a low lysosome activity; and/or (c) assaying for the ability to restore tissue turnover to substantially normal levels in
Kidney ussue, wherein said kidney tissue has low lysosome activity.
[108] Lysosomes may be associated with the breakdown of proteins, particularly albumin, in the kidney. In cases of microalbuminuria, substantial amounts of albumin escape lysosomal breakdown possibly duc to a deacuivated lysosome. Restoration of lysosomal breakdown may restore the balance in the kidney of cellular processes and tissue turnover.
[109] A lysosome-activating compound may be a compound that acts directly or indirectly on the lysosome. By acting indirectly, the compound may act on a component, which influences the activity of the lysosome. Nevertheless, the outcome results in an activation of the lysosome, thereby providing enhanced protein breakdown.
[110] In another aspect of the present invention, there is provided a composition comprising a lysosome-activating compound and a carrier.
[111] The composition may be a physiologically acceptable or pharmaceutically acceptable composition. However, it will be a composition which allows for stable storage of the lysosome activating compound. Where the composition ts a pharmaceutically acceptable composition, it may be suitable for use in a method of preventing or treating kidney disease.
[112] In yet another aspect of the invention there 1s provided a method of preventing or treating kidney disease, said method including administering an effective amount of a lysosome- activating compound to a subject.
[113] As described above, the lysosome-activating compound may act by reactivating the lysosome so that cellular processes and tissue turnover are restored fully or in part, thereby resulting in the kidney being restored partially ~= 1%: In any case, administering a lysosome activating compound to an animal having kidney disease may restore lysosome activity fully or
In part.
[114] Methods of administering may be oral or parenteral. Oral may include administering with tablets, capsules. powders, syrups, etc. Parenteral administration may include intravenous, intramuscular, subcutaneous or intraperitoneal routes. i; [115] The changed activity of the lysosome is preferably a change which enhances the activity of the lvsosome so that albumin breakdown is improved. The ability to not only activate lysosome but also improve cellular processes and/or tissue turnover is a characteristic of the most desirable lysosome activating compound. Preferably, it is desired to use the lysosome activating compound to restore kidney function.
[116] In another aspect of the present invention there is provided a method for preventing kidney disease in a subject, said method including: (a) measuring the amount of intact and modified intact albumin content in a urine sample; (b) determining a change in the amount of intact albumin in the urine that has been modified so as to be not detectable by conventional RIA methods wherein the change is indicative of a propensity for kidney disease; and (c) treating the animal for a kidney disease when a change is determined.
[117] The following examples are offered by way of illustration of the present invention, and not by way of imitation.
Example 1: Size Exclusion Chromatography of Human Serum Albumin (HSA)
[118] Normal, healthy volunteers were used to provide urine for analyzing the distribution of albumin in their urine. {119} *H[HSA] (Human Serum Albumin) was injected into healthy volunteers and urine and plasma were collected and analyzed by size exclusion chromatography using a G-100 column.
The column was eluted with PBS (pH=7.4) at 20 mi/hr at 4°C. The void volume (V,) of the column was dtermined with blue dextran T2000 and the total volume with tritiated water.
[120] Tritium radioactivity was determined in I ml aqueous suinples win 5 nu scintillant and measured on a Wallac 1410 liquid scinullation counter (Wallac Turki, Finland).
[121] FIG. 2 illustrates the distribution of albumin in urine and in plasma.
Example 2: Albumin Excretion in a Normal, Healthy Volunteer and Diabetic Patient
[122] 'H[HSA] as used in Example | was injected into a normal, healthy volunteer and a diabetic patient. Samples of urine were collected and SH[HSA] was determined as in Example 1.
[123] The normal, healthy volunteer (FIG. 3) shows the excretion of fragments of albumin on a size exclusion chromatography as performed in Example |.
[124] The diabetic patient (FIG. 4) shows the presence of substantially full-length and fragmented albumin on size exclusion chromatography. However, excretion rates of albumin detectable by these methods were in the order of 5 pg/min (control) and 1457 py/min (diabetic).
Example 3: Determination of Intact Albumin, and Intact/Modified Albumin on HPLC.
[125] Urine samples were collected from normal, healthy volunteer, normoalbuminuric diabetic patients and from macroalbuminunc patients. Urine was collected midstream in 30 mi urine specimen containers. The urine was frozen until further use. Prior to HPLC analysis the urine was centrifuged at S000 g.
[126] Samples were analyzed on HPLC using a hydrophobicity column Zorbax 300 SB-CB (4.0 mmx 150 mm). A 50 ul sample loop was used.
[127] Samples were eluted from the columns using the following conditions.
Solvent A H,O, 1% trifluoro acetic acid
Solvent B 60% acetonitrile, 0.09% TFA
Solvent A2 99.96>00.00:49.58min
Pressure 9.014Mpascalls (~1100psi)
Solvent B2 0.04>100.0:49.58min
Pressure 7.154Mpascalls
[128] A wavelength of 214 nm was used.
Example 4: Purification ot Modified Albumin for Antibody Production by Standard Techniques ‘ [129] Urine trom microalbuminunc patient which had an intact albumin concentration of 43.5 mg/L as determined by turbitimer (involving :onventional immunochemical assay) was initiatly filtered through a 30 kDa membrane to separate the modified albumin from low molecular weight (<30,000) protein fragments in urine. The material that was retained by the filter gave a vield of intact albumin of 27.4 mg/L as determined by turbitimer assay. This retained material was then subjected to size exclusion chromotography on Sephadex G100. The material collected was the peak fraction that coelutes with intact albumin. This material gave a yield of 13.2 ml/L of albumin as determined by the turbitimer method. This material was then subjected to affinity ’ chromatography on an intact albumin antibody column. This column will only bind albumin that has conventional epitopes. The yield of material that eluted from the column was <6mg/L (lowest sensitivity of the turbitimer). This is expected as the immunoreactive albumin would have bound to the affinity column. The eluate was then subject to reverse phase HPLC chromatography(as described above) to determine the amount of immuno-unreactive albumin in the sample. A 1452 unit area corresponding to 30.91 mg/L of purified modified albumin was noted as shown in FIG. 5. This purified modified albumin can then be used for antibody production by standard mcans.
Results
[130] FIG. 5 illustrates a HPLC profile of albumin alone. Essentially a single peak which elutes at approximately 39-44 minutes retention time was obtained. (131] FIG. 6 illustrates a HPLC profile of plasma showing a distinct albumin peak at approximately 39-44 minutes as well as other peaks corresponding to other plasma proteins.
[132] FIG. 7 illustrates a HPLC profile of a normal, healthy volunteer showing no albumin peak in the unne sample. This individual breaks down the albumin excreted into the urine possibly via an active lysosome. Substantial fragmented products were evident showing prominence of some species, particularly of a species at approximately less than 14.5 minutes retention time. (133] When urine from a normoalbuminunic diabetic patient (with an albumin excretion rate of 8.07 ug/min, as measured by RIA) is analyzed (FIG. 8), small amounts of modified albumin eluting at approximately 39-44 minutes retention time is evident. Whereas conventional test indicates the presence of <6 mg/l of albumin in the urine sample, the method of the invention ’ showed that the true albumin content in the urine sample was 26.7 mg/l. Treatment for the disease should have begun on this individ») AYumin by-products or fragmented albumin is present as in the normal, healthy volunteer. .
[134] Another urine sampie from normoalbuminuric diabetic patient (with albumin excretion rate of 17.04 ug min) was analyzed (FIG. 9). RIA tests show albumin excreted in the urine for this patient. However, on HPLC (FIG. 9) an albumin or modified albumin peak is evident at : approximately 39-44 minutes retention time. Whereas conventional test indicates the presence of <6 mg/l of albumin in the urine sample, the method of the invention showed that the true albumin content in the urine sample was 81.3 mg/l. Treatment for the disease should have begun on this individual. This peak begins to show a multiple peaked appearance. A smaller peak corresponding to intact albumin shows that modified albumin may represent the peak at 39-44 minutes. The presence of this albumin peak compared with the profile of a normal, healthy volunteer having no albumin peak shows a change in the detectable levels of the amount of intactmodified albumin. This may signal a propensity for a kidney disease.
[135] A further urine sample from a normoalbuminuric diabetic patient (with an albumin excretion rate of 4.37 pg/min) was analyzed, and the HPLC profile is illustrated in FIG. 10.
Again, modified albumin was detected at approximately 39-44 minutes retention time showing multiple peaks. This patient again did register normal albumin by RIA. Whereas conventional test indicates the presence of <6 mg/l of albumin in the urine sample, the method of the invention showed that the true albumm content in the urine sample was 491 mg/l. Treatment for the disease should have begun on this individual. [tis clear that modified albumin assessment is necessary to identify these changes. This patient would be determined to have a propensity for kidney disease. As kidney disease progresses, the modified albumin peak will continue to increase.
[136] This is shown in FIG. 11 where a urine sample of a macroalbuminuric patient was analyzed. A quite significant albumin peak at approximately 39-44 minutes retention time showing multiple peaks was evident. The patient’s albumin content was 1796 mg/l. Treatment for this individual is in progress.
[137] The method of the invention results in early detection of a propensity for a renal disease as illustrated by the longitudinal studies in Figures 12-14. Figures 12-14 show situations in which the ACE inhibitor treatment for diabetes was begun later than it should have had the modified albumin detection method of the invention been used. Detecting modified protein using ’ the method according to the invention is a more effective method for predicting the onset of a renal disease than using conventional RIA.
Example 5
[138] FIG. 161s a schematic diagram illustrating the manner in which an intact filtered protein : may be degraded by nonmal functioning kidneys and diseased kidneys.
[139] FIG. 17 illustrates the HPLC profile of a trypsin digested sample of albumin that has been filtered through a 30.000 molecular weight cut-off membrane. The filtrate yields many peaks eluting between 2 to 30 minutes. (140] FIG. 18 illustrates the HPLC profile of a control, normal subject showing many fragments in the eluting range of 10 to 30 minutes. The HPLC profile of a diabetic patient with macroalbuminuria (1457 microgram per minute) shows a significantly different fragment profile in the range of 10 -30 minutes.
[141] FIG. 19 illustrates the HPLC profile of a subject with renal disease. As compared with
FIG. 18, the fragmentation process of filtered proteins is inhibited. The number of fragments is decreased and the size of the fragments 1s increased.
[142] All of the references cited herein are incorporated by reference in their entirety.
[143] Finally. it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.
Claims (16)
- CLAIMS What is claimed is: . I. A method for diagnosing a renal disease and/or renal complications of a disease in a subject, comprising: (a) obtaining a urine sample from the subject; (b) loading the urine sample on an apparatus to crate a fragmentation profile of the urine proteins; (c) treating the urine sample from the patient with a protease under conditions to proteolytically cut the proteins in the urine sample; (d) loading the proteolytically treated urine sample on an apparatus to crate a fragmentation profile of the proteolytically treated urine proteins; (c) comparing the fragmentation profiles of step (c) and step (d), wherein the protein profiles are indicative of the diseased state of the subject's kidney.
- 2. The method of claim 1, wherein the protein fragmentation profile is compared for the size and sequence of particular fragments derived from intact filtered proteins.
- 3. The method of claim 1, wherein the protein fragmentation profile is compared for the position of the protein where enzymes scission occurs.
- 4. The method of claim I, wherein the protein fragmentation profile is compared to the protein fragmentation profile of control samples.
- 5. The method of claim 1, wherein the apparatus is a chromatography, electrophoresis or sedimentation apparatus.
- 6. The method of claim 1, wherein the apparatus is a chromatography, electrophoresis or ’ sedimentation apparatus.6. The method as claimed in any one of the preceding claims, wherein the apparatus is a single dimensional or two-dimensional or three dimensional electrophoresis apparatus, or an HPLC apparatus, or a mass spectrometry apparatus.
- 7. The method as claimed in any one of the preceding claims, further comprising the step of amino acid sequencing to determine the peptide sequence.
- 8. The method as claimed in any one of the preceding claims, wherein the steps are repeated over a period of time.
- 9. The method as claimed in any one of the preceding claims, wherein the disease comprises a disease selected from the group consisting of nephropathy, diabetes insipidus, diabetes type I, diabetes II, renal disease, glomerulonephritis, bacterial and viral glomerulonephiritides, IgA nephropathy and Henoch-Schonlein Purpura, membranoproliferative glomerulonephritis, membranous nephropathy, Sjégren’s syndrome, nephrotic syndrome, minimal change disease, focal glomerulosclerosis and related disorders, acute renal failure, acute tubulointerstitial nephritis, pyelonephritis, GU tract inflammatory disease, Pre-clampsia, renal graft rejection, leprosy, reflux nephropathy, nephrolithiasis, genetic renal disease, medullary cystic, medullar sponge, polycystic kidney disease, autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tuberous sclerosis, von Hippel-Linday disease, familial thin-glomerular basement membrane disease, collagen III glomerulopathy, fibronectin glomerulopathy, Alport’s syndrome, Fabry’s disease, Nail-Patella Syndrome, congenital urologic anomalies, monoclonal gammopathies, multiple myeloma, amyloidosis and related disorders, febrile illness, familial Mediterranean fever, HIV infection-AIDS, inflammatory disease, systemic vasculitides, polyarteritis nodosa, Wegener’s granulomatosis, polyarteritis, necrotizing and crescentic glomerulonephritis, polymyositis-dermatomyositis, pancreatitis, rheumatoid arthiritis, systemic lupus erythematosus, gout, blood disorders, sickle cell disease, thrombotic thrombocytopenia purpura, hemolytic-uremic syndrome, acute cortical necrosis, renal thromboembolism, trauma and surgery, extensive injury, burns, abdominal and vascular surgery, induction of anesthesia, drugs, penicillamine, steroids, and drug abuse, malignant disease, epithelial, lung and breast, adenocarcinoma, renal, melanoma, 28 Amended sheet 14/12/2004 lymphoreticular, multiple myeloma, circulatory disease, myocardial infarction, cardiac failure, peripheral vascular disease, hypertension, coronary heart disease, non- atherosclerotic cardiovascular disease, atherosclerotic cardiovascular disease, skin disease, psoriasis, systemic sclerosis, respiratory disease, COPD, obstructive sleep apnoea, hypoia at high altitude and endocrine disease, acromegaly, diabetes mellitus, diabetes insipidus.
- 10. The method as claimed in any one of the preceding claims, wherein the fragmented protein comprises a protein of the group consisting of albumin, globulin, [alpha]- globulin, [alpha]1-globulin, [alpha]2-globulin, [beta]-globulin, [gamma]-globulin, euglobulin, pseudoglobulin I and II, fibrinogen, [alpha]l acid glycoprotein, orosomucoid, [alpha]l glycoprotein, [alpha]l lipoprotein, ceruloplasmin, [alpha]2 19S glycoprotein, [beta]l transferrin, [beta]l lipoprotein, immunoglobulins A, E, G, and M, horseradish peroxidase, lactate dehydrogenase, glucose oxidase, myoglobin, lysozyme, protein hormone, growth hormone, insulin, or parathyroid hormone.
- 11. A method for in virto diagnosing a non-renal disease in a subject, comprising:a. obtaining a urine sample from the subject;b. creating a fragmentation profile of peptides contained in the urine sample;C. determining whether the part or whole of the fragmentation profile of the peptides is inhibited; and d. comparing the content and profile of part or whole fragmentation profile of the peptides with the content and profile of a control sample of peptides, wherein compared contents and profiles would be indicative of the disease state of the subject.
- 12. A method for in vitro diagnosing a renal disease and/or renal complications of a disease in a subject, substantially as herein described with reference to any one of the illustrative examples.
- 13. A method for in vitro diagnosing a non-renal disease in a subject, substantially as herein described with reference to any one of the illustrative examples. 29 Amended sheet 14/12/2004
- 14. A method for diagnosing a non-renal disease in a subject. comprising: (a) obtaining a urine sample from the subject, (b) loading the urine sample on an apparatus to crate a fragmentation profile of the . urine proteins; (c) treating the urine sample from the patient with a protease under conditions to ' proteolytically cut the proteins in the urine sample; (d) loading the proteolytically treated urine sample on an apparatus to crate a fragmentation profile of the proteolytically treated urine proteins; (c) comparing the fragmentation profiles of step (c) and step (d), wherein the protein profiles are indicative of the increased passage of proteins through the subject's kidney.
- 15. The method of claim 14, wherein the disease causes increased levels of proteins into the circulation.
- 16. The method of claim 14, wherein the disease comprises a cancer. €
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US30125101P | 2001-06-28 | 2001-06-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
ZA200309593B true ZA200309593B (en) | 2004-12-10 |
Family
ID=34589896
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ZA200309593A ZA200309593B (en) | 2001-06-28 | 2003-12-10 | Method for kidney disease detection by protein profiling. |
Country Status (1)
Country | Link |
---|---|
ZA (1) | ZA200309593B (en) |
-
2003
- 2003-12-10 ZA ZA200309593A patent/ZA200309593B/en unknown
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20030003588A1 (en) | Method for kidney disease detection by protein profiling | |
US6589748B2 (en) | Method for kidney disease detection and treatment | |
US6447989B1 (en) | Kidney disease detection and treatment | |
US20050191664A1 (en) | Method for kidney disease detection | |
US8741662B2 (en) | Albumin-bound protein/peptide complex as a biomarker for disease | |
CA2733357A1 (en) | Healthy kidney biomarkers | |
JP2005510575A (en) | IG heavy chain, IG kappa, and IG lambda biopolymer markers that predict Alzheimer's disease | |
WO2006044523A2 (en) | Biomarkers for use in vessel disorders | |
Wai-Hoe et al. | Proteomics and detection of uromodulin in first-time renal calculi patients and recurrent renal calculi patients | |
ZA200309593B (en) | Method for kidney disease detection by protein profiling. | |
JP2005510721A (en) | IG lambda biopolymer marker predicts Alzheimer's disease | |
Silberring et al. | Characterization of immunoreactive dynorphin B and β-endorphin in human plasma | |
JP2005510731A (en) | Fibrinogen biopolymer marker and its use for screening for type II diabetes | |
JP2005510718A (en) | Complement C3 precursor biopolymer marker to predict type II diabetes | |
Beinfeld | Peptides in rat brain immunoreactive for the carboxyl terminal extension of cholecystokinin: distribution and chromatography | |
JP2005525535A (en) | Macroglobulin biopolymer marker showing insulin resistance | |
JP2005523419A (en) | Fibronectin and fibrinogen biopolymer markers exhibiting insulin resistance | |
JP2005510725A (en) | Fibronectin precursor biopolymer marker predicts Alzheimer's disease | |
JP2005510726A (en) | Glycoprotein and apolipoprotein biopolymer markers to predict Alzheimer's disease | |
JP2005510720A (en) | Protein biopolymer markers that predict Alzheimer's disease | |
JP2005526955A (en) | Protein biopolymer markers that predict type II diabetes | |
JP2005510730A (en) | Biopolymer marker indicating normal human | |
JP2005510716A (en) | A globin biopolymer marker showing insulin resistance | |
JP2005525791A (en) | Plasma protease C1 biopolymer marker for predicting Alzheimer's disease | |
JP2005510729A (en) | Complement C3 precursor biopolymer marker showing insulin resistance |