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Title: GFR talk


1
Kidney Function Testing - 2
Dr Edmund Lamb ACB National Training Course,
September 2007
Ucr x V Pcr x T
2
Overview
  • Part one
  • Classification of CKD
  • GFR
  • Creatinine and eGFR
  • Part two
  • Cystatin C
  • Proteinuria/albuminuria

3
Cystatin C
  • 13 kD basic protein
  • 120 AAs, single pp. chain
  • cysteine-protease inhibitor
  • produced at constant rate by all nucleated cells
  • freely filtered at glomerulus
  • reabsorbed/catabolised in proximal tubule
  • serum concentration mainly determined by GFR
  • proposed as improved GFR marker
  • ? especially useful in moderate CKD

4
CKD sensitivity to nephron loss
206 nephrology out-patients with SCr lt300 umol/L
Cystatin C
Proportional increase in analyte
Creatinine




EDTA GFR (ml/min/1.73 m2)
Newman et al KI 1995
5
Sensitivity in older people
53 patients, mean age 80 y
ORiordan et al 2003
6
Cystatin C reflects GFR in children
Newman, Ann Clin Biochem 2002
7
Other settings
  • CKD (numerous studies)
  • Paediatrics (numerous studies)
  • Renal Tx monitoring (Le Bricon et al Clin Chem
    1999)
  • Chemotherrapy monitoring (Stabuc et al Clin Chem
    2000)
  • Pre-eclampsia (Strevens et al BJOG 2003)
  • Type 2 diabetes (Mussap et al KI 2002)
  • Spinal cord injury (Jenkins et al Ann Clin
    Biochem 2003)
  • Renovascular disease (Olivieri et al Clin Chem
    2002)
  • Myeloma (Lamb et al 2004)
  • Rheumatoid arthritis on NSAIDs (Mangge et al CCA
    2000)
  • All demonstrate benefits c.f. creatinine

8
ROC Meta-Analysis
  • ROC curve analysis of relative diagnostic
    accuracy
  • 20 studies included
  • AUC Cystatin C 0.95
  • AUC Creatinine 0.91
  • P0.003

Laterza et al 2002
9
plt0.001
META-ANALYSIS Serum Cystatin C Is Superior to
Serum Creatinine as a Marker of Kidney
Function Also, PENIA studies (r0.846) better
than PETIA studies (r0.784)
Dharnidharka et al 2002
1/CysC
1/Creat
plt0.001
1/CysC
1/Creat
plt0.001
10
Measurement of cystatin C
  • Measured by immunoassay
  • No international standard.
  • Generally free from spectral interferences
    (haemolysis, icterus, lipaemia) ? Effects of
    rheumatoid factor
  • Precision as good as creatinine
  • Cost 2-3

11
Possible caveats
  • Malignant progression
  • Thyroid disease
  • Biological variation

12
Malignant progression
Suggested up-regulation of cystatin C in tumour
progression BUT, didnt present renal function
data (other than creatinines equivalent)!
Kos et al 1998
13
Malignant progression
  • Expression of cystatin C has been observed in
    human lung and colon cancer cell lines
  • Cathepsins (which cystatin C inhibit) implicated
    in a variety of models of malignant progression
  • But, to date
  • Multiple myeloma - no evidence of effect (Lamb
    et al 2004)
  • Multiple myeloma no evidence of effect (Finney
    et al 2001)
  • Proliferative haematological disorders - no
    evidence of effect (Mojiminiyi et al 2002)

14
Thyroid function and cystatin C
Discrepancy between GFR assessed by creatinine
and cystatin C (BUT, no gold standard GFR
used) Jayagopal et al 2003
15
Thyroid function
Cystatin C should not be used without knowledge
of thyroid status (BUT, no gold standard GFR
used) den Hollander et al 2003
16
Biological variability and cystatin C
  • Healthy volunteers
  • Cystatin C better as a screening test than
    creatinine
  • Creatinine better for following changes in an
    individual patient
  • Children with CKD
  • Total variability (analytical biological)
  • Cystatin C 12, creatinine 13 (p0.0012)

Keevil et al 1998
Sambasivan et al 2005
17
Monitoring function over time
  • 20 Pima Indians with type 2 diabetes
  • All hyperfiltering
  • Iothalamate GFR over 4 years
  • Cystatin C
  • MDRD
  • CG

Perkins et al 2005
18
Monitoring function over time
Measure Baseline Annual change
Iothalamate GFR 156 -8.1
100/cystatin C 163 -6.9
100/creatinine 148 -3.8
CG 166 -4.5
MDRD 127 -4.4
Perkins et al 2005
19
Cystatin C can predict GFR
  • 536 adults and children
  • Iohexol gold standard
  • GFR 84.69 x cystatin C-1.680 x 0.948 if
    female
  • R2 0.868, median bias 1.9, within 30 82
  • Estimation superior/equivalent to MDRD
  • R2 0.846, median bias 0.02, within 30 79
  • (Also better than Counahan-Barrat, Schwartz)

Grubb et al 2005
20
Summary cystatin C
  • Cystatin C detects CKD earlier than creatinine
  • It more sensitively predicts earlier
    complications of CKD
  • We need better markers of GFR
  • If we are really serious about early detection
    (and management) of CKD, then cystatin C may find
    a place
  • Possible roles Tx monitoring, paediatric
    nephrology, pregnancy

21
Proteinuria the cardinal sign of kidney disease

22
History of proteinuria
  • Hippocrates (400 bc) noted association between
    bubbles on surface of urine and kidney disease
  • Richard Bright (1827), Guys Hospital, London
    discovered that oedema and proteinuria linked
    with renal disease Brights disease (albuminous
    nephritis).
  • Detected protein by boiling urine until white
    precipitate appeared

23
Proteinuria is the strongest predictor of
progressive disease
Progression to ERF per tertile of protein
excretion
GISEN study, KI 1998
24
Independently of hypertension
GISEN study, KI 1998
25
Clinical proteinuria
  • Normal protein excretion lt150 mg/day (of which
    albumin about 30 mg, THG predominates)
  • Proteinuria typically considered present when
    1 on disptick
  • Equivalent to approx 300 mg/L or 500 mg/day (0.5
    g/day)

26
Proteinuria can be assessed from a single urine
sample (preferably an EMU)24 h urine collections
are therefore unnecessary for this
27
Proteincreatinine ratio and 24 h
proteinexcretion are closely related
Ginsberg (NEJM 1983) proposed 24 h urine
collections could be replaced by PCR ratios
Ruggenenti et al (1998) study of 177 non-diabetic
patients with nephropathy
28
also showed that the ratio (r-0.40) is a better
predictor of progression than 24 h excretion
(r-0.27)
Ruggenenti et al 1998
29
ProteinCreatinine Ratios
  • The proteincreatinine ratio on a random urine
    specimen provides evidence to rule-out the
    presence of significant proteinuria as defined by
    a 24 h urine excretion measurement

Systematic review. Price et al, Clin Chem
September 2005
30
ProteinCreatinine Ratios
  • Assumptions normal volume 1.5 L/24 h, normal
    creatinine excretion 10 mmol/24 h
  • Normal protein excretion often considered lt150
    mg/24 h
  • 1 on a dipstick 300 mg/L or 450 mg/24 h
  • Therefore, normal becomes lt15 mg/mmol and 1
    becomes 45 mg/mmol

31
ProteinCreatinine Ratios (2)
  • correct for urinary dilution/concentration
  • easier
  • cheaper
  • more acceptable to the patient
  • closely predict 24 h excretion
  • consistent with guidelines (K-DOQI, PARADE, NSF)
  • more accurately predict progression
  • but require re-education in interpretation

32
Classification of proteinuria
  • Glomerular
  • Tubular
  • Overflow
  • Quantitatively and clinically, glomerular
    proteinuria is most significant

33
Glomerular proteinuria
  • The glomerulus is a filter, retaining proteins of
    Mr gt approximately 65 kDa (e.g. albumin)
  • Therefore the appearance of high Mr proteins in
    urine implies glomerular damage
  • May be selective (mainly albumin) or unselective
    (larger proteins e.g. IgG) classification
    rarely used
  • In most conditions, albumin is quantitatively the
    most significant protein

34
Total protein versus albumin
  • Proteinuria is predominantly albuminuria, but
    there is not a linear relationship between the
    two
  • 150 mg/L TP contains 30 mg/L albumin (20)
  • 300 mg/L TP contains 150 mg/L albumin (50)
  • 1000 mg/L TP contains 700 mg/L albumin (70)
  • Relationship more variable at low protein
    concentrations
  • TP stick tests and laboratory methods
    particularly sensitive to albumin

35
Albuminuria
36
Renal handling of albumin
  • r3.6 nm, filtration fraction lt0.01 (cf
    dextran of same r0.1)
  • pI 4.7 highly anionic repulsed by glomerular
    polyanion
  • 37,000 g/day pass through glomerular capillaries,
  • 1.3 g/day pass into urinary space (0.004)
  • Where is the barrier to filtration?

37
The filtration barrier
The glomerular basement membrane is a size- and
charge-selective filter
Foot processes are the final barrier to filtration
38
(A) Healthy (B) MCN
MCN associated with flattening (effacement) of
the foot processes in scanning EM
Mathieson, Clin Sci 2004107533-8
39
(No Transcript)
40
Renal handling (2) post glomerular
  • 1.3 g pass into urinary space
  • (0.004 of handled)
  • Approx 10-30 mg/day passed in urine
  • (lt1 of filtered)
  • What happens to the remainder?

41
TUBULAR LUMEN
Cub
1.3 g/day
Amn
Cub
Albumin 66,000 Da
Meg
Meg
Meg
Endosome
Returns to circulation
10 mg/day
Lysosome 500-15,000 Da fragments
Lost in urine (only intact albumin measured)
35
65
42
Urinary albumin/microalbuminuria
  • 1963 - Keen Chlouverakis _at_ Guys developed
    immunoassay for low concentrations of urine
    albumin
  • Such immunoassays can detect increased albumin in
    urine before clinical proteinuria is detectable
  • 1982 - Viberti et al _at_ Guys coined term
    microalbuminuria
  • An increase in the urinary excretion of albumin
    above the reference range for healthy
    non-diabetic subjects but at a level not
    detectable by crude clinical tests (protein stix
    tests)
  • Microalbuminuria is common in diabetes mellitus
    and predicts progression to ESRD

43
NOTE!!!
  • microalbuminuria is not about a small form of
    albumnin
  • microalbuminuria is about increased, not
    decreased, amounts of albumin in the urine

44
Microalbuminuria and progression
  • mid-1980's - studies showed that microalbuminuria
    predicted development of clinical nephropathy in
    diabetic patients and that good control slowed
    progression (e.g. Kroc study) but studies were
    small/too short
  • 1990s
  • - good glycaemic control prevents progression
    to microalbuminuria (DCCT, ngt1400) and effect
    persists (metabolic memory EDIC)
  • - antihypertensive medication in patients with
    micro- ( macro-) albuminuria may delay
    progressive loss of glomerular filtration
  • - association with other disease (e.g. CVD)
    appreciated, both in diabetics and non-diabetics
    (e.g. PREVEND)
  • In diabetes, microalbuminuria has become
    established as a marker of potentially treatable
    disease

45
Confounding factors
  • Biological variation (day-to-day CV 45)
  • Metabolic control
  • Intercurrent illness (e..g. sepsis,
    post-myocardial infarction, surgery, SIRS)
  • Haematuria/menstrual contamination
  • Non-diabetic renal disease
  • Uncontrolled hypertension
  • Strenuous exercise
  • Urinary tract infection
  • Microalbuminuria should be present in at least
    two out of three urine samples preferably
    collected within a 6(1) month period in the
    absence of ketonuria or infection

46
Microalbuminuric ranges
  • Overnight AER 20-200 ug/min seen as gold
    standard method
  • equivalent to
  • 30-300 mg/24 h or 20-200 mg/L (NICE)
  • which if you excrete 10 mmol creatinine/day is
    equivalent to
  • 3.0 30 mg/mmol creatinine or 30-300 ug/mg in
    US
  • or
  • Males ? 2.5 mg/mmol, Females ? 3.5 mg/mmol

47
Natural history of diabetic renal disease
10-15 y
10-20 y
48
PREVEND (1)
  • 40,000/85,000 residents of Groningen recruited in
    1997
  • Urine albumin measured
  • Followed for 3 y
  • 516 deaths
  • Mortality and cause of mortality recorded

Hillege et al, JIM 2001, Circulation 2002
49
PREVEND (2)
  • Microalbuminuria present in 7.2 of population
  • Independently associated with hypertension,
    diabetes, CV disease
  • After excluding diabetics and hypertensives,
    microalbuminuria still present in 6.6 of
    population.

50
PREVEND (3)
  • Increasing albuminuria associated with increasing
    CV and, to a lesser extent, non-CV mortality
    (esp. cancer)
  • Albuminuria is a strong predictor of all cause
    mortality in general popn.
  • Risk begins at levels not considered
    microalbuminuric
  • (Similar data from Framingham Offspring Study on
    non-diabetic, non-HT subjects Arnlov et al,
    Circulation 2005)

51
PREVEND (4)
  • Microalbuminuria common and associated with CV
    risk factors and death
  • Advocates population screening approach to
    microalbuminuria detection, but especially
    hypertensives
  • Non-diabetics with microalbuminuria should be on
    ACEI/ARBs
  • Threshold for pathological albuminuria should be
    revised

52
Measurement issues
53
Stick tests Colorimetric, approx 0.10 Semi-quantitative, inconsistency between manufacturers, mainly measure albumin
Lab total protein Mainly colorimetric (e.g. pyrogallol red, coomassie blue, benzethonium chloride), approx 0.10 Results differ between dyes, often standardised against albumin, mainly sensitive to albumin
Albumin Immunoassay, approx 0.40 Standardised against albumin
54
Urinary Total Protein UKNEQAS
Bayer stix tests 8 negative, 30 trace, 56 1,
6 2
55
Urinary albumin UKNEQAS
56
Non-immunoreactive albumin (1)
  • HPLC (total albumin) assays suggest large amounts
    of albumin present in diabetic urine not measured
    by immunoassays termed non-immunoreactive
    albumin
  • No difference between non-diabetic urines

Comper et al AJKD 2003, Osick and Comper, Clin
Chem 2005
57
Non-immunoreactive albumin (2)
  • Albumin has similar Mr to normal albumin
  • May represent
  • (a) albumin which has undergone minimal tubular
    processing (scissions with held together by
    S-S bonds) or
  • (b) filtered forms of albumin not recognised by
    immunoassay (e.g. FA binding induces
    conformational change)
  • These processes may be affected by diabetes

1. Diabetic urine
2. Purified NIA from urine
3. Purified NIA under reducing conditions
58
Non-immunoreactive albumin (3)
  • Retrospective analysis of 15 y of stored urine
    samples from diabetics with progressive (n41) or
    non-progressive (n50) kidney disease
  • HPLC predicted onset of diabetic nephropathy 2-4
    y earlier than immunoassay
  • But
  • Only this group publishing in this area
  • Assay being marketed by the authors
  • When is albumin not albumin?

Comper et al KI 2004
59
ACR to replace PCR?
Advantages Disadvantages
More sensitive essential to identify all CKD stage 1 and 2 patients KDIGO define kidney damage ACR gt30 mg/g (approx 3.5 mg/mmol) More sensitive than nephrologists want?
Improved precision
Consistency with diabetic nephropathy literature More expensive?
Single protein (know what you are measuring and how it is calibrated) No IRP
Consistent with International practice (KDIGO/KDOQI) Nephrology literature based on PCR

60
Functional tubular proteinuria
  • Small Mr proteins filtered at glomerulus and
    reabsorbed in proximal tubule
  • Appearance of proteins in urine implies tubular
    damage
  • E.g. urinary alpha-1 microglobulin, beta-2
    microglobulin, retinol binding protein

61
Tubular proteinuria due to cell damage
  • Certain proteins present at high concentration in
    tubular cells
  • E.g. Tamm Horsfall glycoprotein and
    N-acetyl-B-D-glucosaminidase (NAG)
  • Appearance of these proteins in urine implies
    tubular damage
  • Useful in drug toxicity studies or occupational
    monitoring (e.g. heavy metal workers)

62
Overflow proteinuria
  • Excess formation of a low molecular weight
    protein that is freely filtered
  • E.g. BJP, myoglobin
  • Such proteins may be directly toxic to the
    tubular cells

63
Summary
  • Kidney disease is common
  • Perhaps more than any other disease state, its
    assessment relies on quantitative laboratory
    tests
  • and renal units rely on the laboratory
  • There are a range of tests available to assess
    the functions of the kidney - the most important
    of these are GFR and proteinuria
  • No current tests are perfect for assessing GFR
    and there is inconsistency in approach to
    proteinuria

64
END
65
Major filtration barrier now widely believed to
be the filtration slits () between
inter-digitating foot processes (?)
Mutations of nephrin and podocin, which
co-localise at the slit diaphragm (and several
other protein mutations) associated with
congenital types of nephrotic syndrome (e.g.
Finnish-type congenital NS)
Mathieson, Clin Sci 2004107533-8
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