Cystatin c 2014

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Cystatin c 2014

  1. 1. MAHARISHI MARKANDESHWAR INSTITUTE OF MEDICAL SCIENCES & RESEARCH (M.M.I.M.S.R), MULLANA, AMBALA, HARYANA
  2. 2. CYSTATIN C
  3. 3. HISTORY • It was first described as “gamma trace in 1961 as a trace protein together with other ones (such as beta trace) in CSF and in urine of patient with renal failure. • Grubb and Lofberg first reported its amino acid sequence. • They noticed it was increased in patient of CRF. • It was first proposed as a measure of GFR by Grubb and co-workers in 1985
  4. 4. CYSTATIN C • Cystatin C is a small 13 kDa protein (most potent inhibitor of cysteine proteases). • Constant production rate, regulated via house-keeping gene • Not influenced by muscle mass , or inflammation • Free renal filtration • No tubular secretion • Tubular reabsorption followed by degradation.
  5. 5. • The family 3 Cystatins, high and low molecular weight kininogen, contain three Cystatin domains and are mainly intravascular proteins, which in addition to being inhibitors of cysteine proteases also are involved in the coagulation process and in the production of vasoactive peptides. • Cystatin C is abundant in various tissues and bodily fluids, the highest levels having been determined in cerebrospinal fluid, seminal fluid, plasma, and synovial fluid. CYSTATIN C
  6. 6. PROTEIN TURNOVER • Half life (N-END RULE). • Protein with N-Terminal Met,Ser,Thy,Val, or Gly. Have half life more than 20 hours. • Protein with N-Terminal Phe,Leu,Asp,Lys, or Arg. Half life of 3 minutes or less. • PEST: Proteins with Pro(P),Glu (E),Serine (S) Thr(T) are more rapidly degraded.
  7. 7. CYSTEINE PROTEASES • Papain is well studied plant (CP). • Cathepsins are large family of lysosomal cysteine proteases. • Caspase are involved in activation & implementation of Apoptosis.. • Calpains are Ca++ activated cysteine proteases that cleave intracellular proteins. • They regulate processes such as cell migration and wound healing.
  8. 8. PROTEASES • Exoproteases. • Endoproteases. • Sereine proteases. • Cysteine proteases. • Aspartyl proteases. • Metal ion proteases (Zn++) • Threonine proteases. • *Best substrate is unfolded proteins.
  9. 9. CYSTATIN C • The human Cystatin family presently comprises 11 identified proteins. • Two of these, Cystatin A and B, form the family 1 Cystatin and are mainly, or exclusively, intracellular proteins, while Cystatin C, D, E, F, S, SA and SN are mainly extracellular and/or transcellular proteins and constitute the family 2 Cystatins. • The family 3 cystatins are mainly intravascular proteins, produces vasoactive peptide & involved in coagulation.
  10. 10. FATE OF CYSTATIN-C • Produced by all nucleated cells and its rate of production is constant. • In human, all cells with nucleus produce cystatin C as a chain of 120 amino acids • It is freely filtered at the Glomerulus. • Practically completely reabsorbed by proximal renal tubules. • It is totally catabolized in the proximal renal tubule. • No re-entry into circulation. FATE OF CYSTATIN C
  11. 11. MOLECULAR BIOLOGY • Cystatin superfamily encompasses proteins that contain multiple cystatin like sequences • Some members are active cysteine proteinase inhibitors • There are 3 inhibitory families in the superfamily: 1) Type 1 cystatins (stefins) 2) Type 2 cystatins 3) Kininogens • Type 2 are a class of cysteine proteinase inhibitors found in human fluids and are protective in function
  12. 12. SOME FACTS ABOUT CYSTATIN-C • Cystatin-C is a non glycosylated basic protein (isoelectric pH 9.3) • It has a crystal structure characterized by short alpha helix and a long alpha helix running across a large anti-parallel 5 stranded beta sheet • It has 2 disulfide bonds • 50% of the molecule carry a hydroxylated proline • It forms two dimers • It is a potent inhibitor of lysosomal proteinases • It is also an important inhibitor of extracellular cysteine proteases • It has a low molecular weight of 13.3 kilodaltons
  13. 13. Types of cystatin • The human cystatin family presently comprises of 11 identified proteins. • FAMILY 1: Two cystatin A and B, and are mainly, or exclusively, intracellular proteins, • FAMILY 2: C, D, E, F, S, SA and SN are mainly extracellular and/or transcellular proteins and constitute the family 2 Cystatins. • Family 3 cystatins: high and low molecular weight kininogen, contain three cystatin domain & are mainly intravascular proteins. • In addition to inhibitors of cysteine protease also involved in coagulation.
  14. 14. CALCULATION OF GFR • CKD-EPI cystatin equation adjusted for age, sex and race: • Formula for calculation of eGFR: • eGFR = 127.7 X (Cys C)-1.17 X (age)-0.13 X 0.91 (if female) X 1.06 (if African American) CALCULATION OF GFR
  15. 15. ROLE IN MEDICINE • KIDNEY FUNCTION – It is removed from bloodstream by glomerular filtration by kidneys – If the function of kidneys decrease and GFR falls, level of cystatin-C in blood increases – So it has been suggested that cystatin-C might predict the development of CRF
  16. 16. ROLE IN MEDICINE (CONT.) • Levels of cystatin-C are altered in following conditions 1) Cancer patient 2) Thyroid dysfunction 3) Glucocorticoid therapy 4) Cigarette smoking 5) HIV infection 6) Increased levels in MI,stroke,heart failure,peripheral arterial syndrome 7) Increased in metabolic syndrome 8) Increased in Alzheimers disease 9) Levels decreased in atherosclerosis and aneurysmal(saccular bulging) lesions of aorta
  17. 17. • A reliable marker of GFR in patients with mild-to-moderate kidney dysfunction (stages 2–3 of CKD) in both type 1 and type 2 diabetes. • Elevated serum cystatin C levels identified as a significant prognostic indicator for the development of cardiovascular disease in people with diabetes. • Cystatin C is not only a better indicator of GFR in diabetes, it has the best correlation with changes in GFR over two years, making it a useful measure for follow-up of patients with diabetes. DIABETES MELLITUS
  18. 18. HUMAN IMMUNODEFICIENCY VIRUS (HIV) INFECTION • Studies have reported increased cystatin C levels in HIV. • Because of an increase of cystatin C levels with active HIV infection, an overestimation of kidney impairment may occur, particularly in treatment-naive patients with renal disease.
  19. 19. • Like creatinine concentrations, cystatin C levels are also lower in the hypothyroid and higher in the hyperthyroid state as compared with the euthyroid state. Thyroid Function
  20. 20. CARDIOVASCULAR DISEASE • Cystatin C has been reported to be a potent predictor of cardiovascular mortality beyond classical risk factors in patients with CAD and normal or mildly reduced kidney function. • Serum cystatin C may have a stronger association with mortality and cardiovascular disease than serum creatinine in patients without CKD, as reported in a large study of older adults.
  21. 21. OBESITY • Serum cystatin C concentrations are increased in human obesity in relation to over-production by the adipose tissue. So, cystatin C levels are higher in obese subjects as compared to lean. • Adipose tissue is a source of cystatin C in a way that is not related to eGFR but to the status of adipose tissue itself, including enlarged adipocytes, hypoxia, pro-inflammatory cytokines production, increased number of macrophages, and probably other cellular and molecular alterations known to occur in obesity
  22. 22. METHODS FOR GFR ESTIMATION GFR measurement Creatinine Cystatin C Direct measurement ( reference method) • Clearance determination by exogenous substances - inulin - iohexol - 51Cr-EDTA - 125 I-iothalamate not routinely USED • costly • time & labor intensive - invasive, stress-full for patient •Small molecule, 113 D ( breakdown product of creatine as part of muscle metabolism) • Routine clinical chemistry method - Jaffe method -enzymatic method •different formulas for eGFR, e.g. MDRD • Small protein, 13 kD •Cysteine protease Inhibitor • Fully automated immuno-assays Available •Method of standardization program in progress. •Different formulas for
  23. 23. CYSTATIN C • HIGHLY SENSITIVE • SPECIFIC MARKER • CHRONIC KIDNEY DISEASE • ACUTE RENAL FAILURE • CARDIOVASCULAR EVENTS • ALL CAUSE MORTALITY prognostic marker • HEART FAILURE • STROKE • DIABETES • UNSUCCESSFUL AGING
  24. 24. CKD – a silent threat Kidneys do not hurt! • Laboratory testing = key to early diagnosis • Prevention as more efficient as earlier started • Sensitive detection method required • Continuous strong increase in the prevalence of chronic kidney disease CKD – a silent threat
  25. 25. CKD – a silent threat • Outcomes of CKD: - progression of renal disease to end-stage renal disease (ESRD) • Complications of CKD: - hypertension - anemia - bone and mineral disease - increased risk of cardiovascular disease (CVD)
  26. 26. IN Seniors • GFR declines with age and cystatin C may better reflect true kidney function in older people because muscle mass does not influence it. • After age 50, reference values of serum cystatin C concentration are higher.
  27. 27. IN OBSTETRICS • Serum cystatin C concentration varies in pregnancy, because it is not consistently produced. • In preeclampsia, however, altered kidney function is more likely to be detected by CysC– GFR than by creatinine-based formulas.
  28. 28. IN PEDIATRICS • After age 1, serum cystatin C concentration is constant, but higher values are found in the newborn period. In full-term newborns, cystatin C progressively declines over the first week of life. • CysC–GFR has been reported to be more accurate in children with cancer and in patients with spina bifida.
  29. 29. PREVALANCE
  30. 30. Stage (Renal dysfunction) GFR (ml/min/1.73 m2) Metabolic Consequences Normal renal function – Persons at increased risk or with early renal damage >90 Mild renal insufficiency 60-89* PTH levels start to rise (GFR ~ 60-80) Moderate renal insufficiency 30-59 Decrease in Calcium absorption (GFR <50) Lipoprotein activity falls. Malnutrition. Onset of LVH. METABOLIC EFFECTS IN CRD
  31. 31. Stage (Renal dysfunction) GFR (ml/min/1.73 m2) Metabolic Consequences Moderate renal insufficiency 30-59 Decrease in Calcium absorption (GFR <50) Lipoprotein activity falls. Malnutrition. Onset of LVH. Severe renal insufficiency (Pre- ESRD) 15-29 Triglyceride levels start to rise. Onset of Anemia (Erythropoietin deficiency). Hyperphosphatemia. Metabolic acidosis. Hyperkalemia tendency. ESRD (Uremia) <15 Azotemia develops.
  32. 32. PROGRESSION OF C.R.D.
  33. 33. CRITERIA FOR C.R.D.
  34. 34. LIMITATIONS OF CREATININE AS A MARKER OF GFR • NON RENAL FACTORS- 1) Gender 2) Ethinicity 3) Diet 4) Muscle mass 5) Drugs affecting tubular secretion of creatinine • CLINICAL FACTOR- – Poor sensitivity for CKD- Creatinine blind range – Creatinine remains normal until 50% renal function is lost – Insensitive to loss of GFR in Stage-2 and Stage-3 in CKD • ANALYTICAL FACTOR- • Non specific bias frequently reported with Jaffe Assay
  35. 35. ADVANTAGE OF CYSTATIN-C AS GFR MARKER ADVANTAGE COMMENT Virtually unaffected by non renal factors Muscle mass/weight/height,age(>1 year)-cystatin-c parallels age related decrease in GFR and can be used in children Sensitive to so called creatinine blind range Enables early detection and treatment of CKD Can be used to detect and monitor kidney diseases in patient with hepatic diseases Creatinine for GFR in liver disease not recommended Correlates to appearance of microalbuminuria Clinical studies suggest that very early renal failure may be the first clinical indication of progressive renal damage associated with diabetes
  36. 36. CONTRAINDICATION OF CYSTATIN-C ESTIMATION • THYROID FUNCTION • Levels of cystatin-C are sensitive to change in thyroid function and should not be performed without knowledge of patients thyroid status • CORTICOSTEROIDS • Cystatin-C concentrations are affected in patients of impaired renal function receiving corticosteroids
  37. 37. LABORATORY MEASUREMENT • ASSAY PRINCIPLE- – Cystatin-c in the sample binds to the specific anticystatin-c antibody which is coated on latex particles and causes agglutination – The degree of turbidity caused by agglutination is measured optically and is proportional to the amount of cystatin-c in the sample by a method called TURBIDIMETRY. • REFERENCE VALUE- – Males-0.52-0.98 mg/dl – Female-0.52-0.90mg/dl • Normal value decreases until first year of life,then remains stable before increasing after age of 50 years • NOTE- – Cystatin-c can be measured from a random sample of blood from which RBC and clotting factors have been removed(i.e. serum)
  38. 38. TURBIDIMETRY • Some analytical methods give an insoluble product in finely divided form so that the particles remain in suspension • If a beam of light passes through, some of it is scattered- TYNDALL EFFECT • Turbidimetry measures the reduction of intensity of the incident beam and is similar to the study of light absorption in spectrophotometry • Turbidimetric measurements are done with usual types of photometers
  39. 39. TURBIDIMETRY COMPONENTS
  40. 40. % INCREASE IN CYSTATIN-C LEVELS AFTER 50 YEARS
  41. 41. • The calculator gives instant conversion of cystatin-C measurement to standard GFR units
  42. 42. CYSTATIN-C(mg/dl) GFR(ml/min) 0.5 217 0.6 167 0.7 133 0.8 110 0.9 93 1.0 80 1.1 70 1.2 61 1.3 55 1.4 49 1.5 45 1.6 41 1.7 37 1.8 34
  43. 43. THANK YOU kingkul@yahoo.co.in

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