cystatin c 2014
TRANSCRIPT
MAHARISHI MARKANDESHWAR INSTITUTE OF MEDICAL SCIENCES & RESEARCH (M.M.I.M.S.R),
MULLANA, AMBALA, HARYANA
• 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
• 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.
• 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.
• 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.
• 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.
• Exoproteases.
• Endoproteases.
• Sereine proteases.
• Cysteine proteases.
• Aspartyl proteases.
• Metal ion proteases (Zn++)
• Threonine proteases.
• *Best substrate is unfolded proteins.
• 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.
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
• 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
• 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
• 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.
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
• 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
• 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
• 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
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.
• 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
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.
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
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 ofmuscle metabolism)
• Routine clinical chemistry method
- Jaffe method-enzymatic method
•different formulas foreGFR, e.g. MDRD
• Small protein, 13 kD•Cysteine proteaseInhibitor
• Fully automatedimmuno-assaysAvailable
•Method of standardizationprogram in progress.
•Different formulas foreGFR e.g. CKDEPI
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
• Increasing economic burden
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)
• 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.
• 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.
• 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.
PREVALANCE
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.
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.
PROGRESSION OF C.R.D.
• 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 Method
ADVANTAGE COMMENTVirtually 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
• 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
• 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)
• 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
• The calculator gives instant conversion of cystatin-C measurement to standard GFR units
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
