assessment of renal tubular function

East Kent HospitalsEast Kent HospitalsNHS TrustNHS Trust

Kidney Function Testing - 2Kidney Function Testing - 2

Dr Edmund LambDr Edmund Lamb

ACB National ACB National Training Course, Training Course,

September 2007September 2007

Ucr x VPcr x T


OverviewOverview

Part onePart one• Classification of CKDClassification of CKD• GFR GFR • Creatinine and eGFRCreatinine and eGFR

Part twoPart two• Cystatin CCystatin C• Proteinuria/albuminuriaProteinuria/albuminuria

East Kent HospitalsEast Kent HospitalsNHS TrustNHS TrustCystatin CCystatin C

13 kD basic protein13 kD basic protein120 AAs, single pp. chain120 AAs, single pp. chaincysteine-protease inhibitorcysteine-protease inhibitorproduced at constant rate produced at constant rate by all nucleated cellsby all nucleated cellsfreely filtered at glomerulusfreely filtered at glomerulusreabsorbed/catabolised in reabsorbed/catabolised in proximal tubuleproximal tubuleserum concentration mainly serum concentration mainly determined by GFRdetermined by GFRproposed as improved GFR proposed as improved GFR markermarker? especially useful in ? especially useful in moderate CKDmoderate CKD


CKD: sensitivityCKD: sensitivity to nephron lossto nephron loss

<30 31-40 41-50 51-60 61-70 71-80 81-90 91-100101-110111-120 >1200

1

2

3

4

Cystatin C

Creatinine

EDTA GFR (ml/min/1.73 m2)

Pro

port

ion

al in

cre

ase in

an

aly

te

****

*

*

Newman et al KI 1995Newman et al KI 1995

206 nephrology out-patients with SCr <300 umol/L


Sensitivity in older peopleSensitivity in older people

0

1

2

3

4

5

6

0 20 40 60 80 100 12051Cr EDTA clearance (mL/min/1.73 m2)

Pro

po

rtio

na

l in

cre

as

e in

se

rum

m

ark

er

Cystatin CCreatinine

O’Riordan et al O’Riordan et al 20032003

53 patients, 53 patients,

mean age 80 ymean age 80 y


Newman, Newman,

Ann Clin Ann Clin Biochem 2002Biochem 2002

Cystatin C reflects GFR in childrenCystatin C reflects GFR in children


Other settingsOther settings

• CKD (numerous studies)CKD (numerous studies)• Paediatrics (numerous studies)Paediatrics (numerous studies)• Renal Tx monitoring (Le Bricon et al Clin Chem 1999)Renal Tx monitoring (Le Bricon et al Clin Chem 1999)• Chemotherrapy monitoring (Stabuc et al Clin Chem 2000)Chemotherrapy monitoring (Stabuc et al Clin Chem 2000)• Pre-eclampsia (Strevens et al BJOG 2003)Pre-eclampsia (Strevens et al BJOG 2003)• Type 2 diabetes (Mussap et al KI 2002)Type 2 diabetes (Mussap et al KI 2002)• Spinal cord injury (Jenkins et al Ann Clin Biochem 2003)Spinal cord injury (Jenkins et al Ann Clin Biochem 2003)• Renovascular disease (Olivieri et al Clin Chem 2002)Renovascular disease (Olivieri et al Clin Chem 2002)• Myeloma (Lamb et al 2004)Myeloma (Lamb et al 2004)• Rheumatoid arthritis on NSAIDs (Mangge et al CCA 2000)Rheumatoid arthritis on NSAIDs (Mangge et al CCA 2000)

All demonstrate benefits c.f. creatinineAll demonstrate benefits c.f. creatinine


ROC Meta-AnalysisROC Meta-Analysis

ROC curve analysis of relative ROC curve analysis of relative diagnostic accuracydiagnostic accuracy

20 studies included20 studies included

AUC Cystatin C = 0.95AUC Cystatin C = 0.95

AUC Creatinine = 0.91AUC Creatinine = 0.91

P=0.003P=0.003

Laterza et al 2002Laterza et al 2002


1/CysC 1/Creat

META-ANALYSISMETA-ANALYSISSerum Cystatin C Is Superior to Serum Cystatin C Is Superior to Serum Creatinine as a MarkerSerum Creatinine as a Markerof Kidney Functionof Kidney Function

Also, PENIA studies (r=0.846) Also, PENIA studies (r=0.846) better than PETIA studies better than PETIA studies (r=0.784)(r=0.784)

Dharnidharka et al 2002Dharnidharka et al 2002

1/CysC 1/Creat

p<0.001p<0.001

p<0.001p<0.001

p<0.001p<0.001


Measurement of cystatin CMeasurement of cystatin C

• Measured by immunoassayMeasured by immunoassay• No international standard.No international standard.• Generally free from spectral interferences (haemolysis, Generally free from spectral interferences (haemolysis,

icterus, lipaemia) ? Effects of rheumatoid factoricterus, lipaemia) ? Effects of rheumatoid factor• Precision as good as creatininePrecision as good as creatinine• Cost £2-£3Cost £2-£3


Possible caveatsPossible caveats

• Malignant progressionMalignant progression

• Thyroid diseaseThyroid disease

• Biological variationBiological variation


Malignant progressionMalignant progressionSuggested up-Suggested up-regulation of regulation of cystatin C in tumour cystatin C in tumour progressionprogression

BUT, didn’t present BUT, didn’t present renal function data renal function data (other than (other than “creatinines “creatinines equivalent”)!equivalent”)!

Kos et al 1998Kos et al 1998


Malignant progressionMalignant progression• Expression of cystatin C has been observed in human lung and colon Expression of cystatin C has been observed in human lung and colon

cancer cell linescancer cell lines• Cathepsins (which cystatin C inhibit) implicated in a variety of Cathepsins (which cystatin C inhibit) implicated in a variety of

models of malignant progressionmodels of malignant progression

But, to date:But, to date:

• Multiple myeloma - no evidence of effect (Lamb et al 2004)Multiple myeloma - no evidence of effect (Lamb et al 2004)• Multiple myeloma – no evidence of effect (Finney et al 2001)Multiple myeloma – no evidence of effect (Finney et al 2001)• Proliferative haematological disorders - no evidence of effect Proliferative haematological disorders - no evidence of effect

(Mojiminiyi et al 2002)(Mojiminiyi et al 2002)


Thyroid function and cystatin CThyroid function and cystatin C

Discrepancy between Discrepancy between GFR assessed by GFR assessed by creatinine and cystatin Ccreatinine and cystatin C

(BUT, no gold standard (BUT, no gold standard GFR used)GFR used)

Jayagopal et al 2003Jayagopal et al 2003


Thyroid functionThyroid function““Cystatin C should not be Cystatin C should not be used without knowledge used without knowledge of thyroid status”of thyroid status”

(BUT, no gold standard (BUT, no gold standard GFR used)GFR used)

den Hollander et al 2003den Hollander et al 2003


Biological variability and cystatin CBiological variability and cystatin C• Healthy volunteersHealthy volunteers• Cystatin C better as a screening test than creatinineCystatin C better as a screening test than creatinine• Creatinine better for following changes in an individual Creatinine better for following changes in an individual

patientpatient

• Children with CKDChildren with CKD• Total variability (analytical + biological)Total variability (analytical + biological)• Cystatin C 12%, creatinine 13% (p=0.0012)Cystatin C 12%, creatinine 13% (p=0.0012)

Keevil et al 1998Keevil et al 1998

Sambasivan et al 2005Sambasivan et al 2005


Monitoring function over timeMonitoring function over time

• 20 Pima Indians with type 2 diabetes20 Pima Indians with type 2 diabetes• All hyperfilteringAll hyperfiltering• Iothalamate GFR over 4 yearsIothalamate GFR over 4 years• Cystatin CCystatin C• MDRDMDRD• C&GC&G

Perkins et al 2005Perkins et al 2005


Monitoring function over timeMonitoring function over timeMeasureMeasure BaselineBaseline Annual % Annual %

changechange

Iothalamate GFRIothalamate GFR 156156 -8.1-8.1

100/cystatin C100/cystatin C 163163 -6.9-6.9

100/creatinine100/creatinine 148148 -3.8-3.8

C&GC&G 166166 -4.5-4.5

MDRDMDRD 127127 -4.4-4.4

Perkins et al 2005Perkins et al 2005


Cystatin C can predict GFRCystatin C can predict GFR

• 536 adults and children536 adults and children• Iohexol gold standardIohexol gold standard• GFR = 84.69 x cystatin CGFR = 84.69 x cystatin C-1.680-1.680 [ x 0.948 if female] [ x 0.948 if female]• RR22 = 0.868, median bias 1.9%, within 30% =82% = 0.868, median bias 1.9%, within 30% =82%• Estimation superior/equivalent to MDRDEstimation superior/equivalent to MDRD• RR22 = 0.846, median bias 0.02%, within 30% =79% = 0.846, median bias 0.02%, within 30% =79%• (Also better than Counahan-Barrat, Schwartz)(Also better than Counahan-Barrat, Schwartz)

Grubb et al 2005Grubb et al 2005


Summary – cystatin CSummary – cystatin C

• Cystatin C detects CKD earlier than creatinineCystatin C detects CKD earlier than creatinine• It more sensitively predicts earlier complications of CKDIt more sensitively predicts earlier complications of CKD• We need better markers of GFRWe need better markers of GFR• If we are really serious about early detection (and If we are really serious about early detection (and

management) of CKD, then cystatin C may find a placemanagement) of CKD, then cystatin C may find a place• Possible roles – Tx monitoring, paediatric nephrology, Possible roles – Tx monitoring, paediatric nephrology,

pregnancypregnancy


Proteinuria – the Proteinuria – the cardinal sign of cardinal sign of kidney diseasekidney disease

East Kent HospitalsEast Kent HospitalsNHS TrustNHS TrustHistory of proteinuriaHistory of proteinuria

• Hippocrates (400 bc) noted Hippocrates (400 bc) noted association between bubbles on association between bubbles on surface of urine and kidney surface of urine and kidney diseasedisease

• Richard Bright (1827), Guy’s Richard Bright (1827), Guy’s Hospital, London discovered Hospital, London discovered that oedema and proteinuria that oedema and proteinuria linked with renal disease – linked with renal disease – Bright’s disease (albuminous Bright’s disease (albuminous nephritis). nephritis).

• Detected protein by boiling Detected protein by boiling urine until white precipitate urine until white precipitate appearedappeared


Proteinuria is the strongest Proteinuria is the strongest predictor of progressive diseasepredictor of progressive disease

GISEN study, KI 1998GISEN study, KI 1998

Progression to Progression to ERF per ERF per tertile of tertile of protein protein excretionexcretion


……Independently of hypertensionIndependently of hypertension

GISEN study, KI 1998GISEN study, KI 1998


‘‘Clinical’ proteinuriaClinical’ proteinuria

• Normal protein excretion <150 mg/day (of Normal protein excretion <150 mg/day (of which albumin about 30 mg, THG which albumin about 30 mg, THG predominates)predominates)

• Proteinuria typically considered present when Proteinuria typically considered present when ‘1+’ on disptick‘1+’ on disptick

• Equivalent to approx 300 mg/L or 500 mg/day Equivalent to approx 300 mg/L or 500 mg/day (0.5 g/day)(0.5 g/day)


““Proteinuria can be Proteinuria can be assessed from a single assessed from a single urine sample urine sample (preferably an EMU)(preferably an EMU)…24 h urine …24 h urine collections are collections are therefore unnecessary therefore unnecessary for this”for this”

East Kent HospitalsEast Kent HospitalsNHS TrustNHS TrustProtein:creatinine ratio and 24 h proteinProtein:creatinine ratio and 24 h protein

excretion are closely relatedexcretion are closely related

Ruggenenti et al (1998) study Ruggenenti et al (1998) study of 177 non-diabetic patients of 177 non-diabetic patients with nephropathywith nephropathy

Ginsberg (NEJM 1983) Ginsberg (NEJM 1983) – proposed 24 h urine – proposed 24 h urine collections could be collections could be replaced by PCR ratiosreplaced by PCR ratios


Ruggenenti et al 1998Ruggenenti et al 1998

……also showed also showed that the ratio that the ratio (r=-0.40) is a (r=-0.40) is a better better predictor of predictor of progression progression than 24 h than 24 h excretion excretion (r=-0.27)(r=-0.27)


Protein:Creatinine RatiosProtein:Creatinine Ratios

““The protein:creatinine ratio on a random urine The protein:creatinine ratio on a random urine specimen provides evidence to rule-out the presence specimen provides evidence to rule-out the presence of significant proteinuria as defined by a 24 h urine of significant proteinuria as defined by a 24 h urine excretion measurement”excretion measurement”

Systematic review.Systematic review. Price et al, Clin Chem September 2005Price et al, Clin Chem September 2005


Protein:Creatinine RatiosProtein:Creatinine Ratios

• Assumptions: normal volume 1.5 L/24 h, normal creatinine Assumptions: normal volume 1.5 L/24 h, normal creatinine excretion 10 mmol/24 hexcretion 10 mmol/24 h

• ‘‘Normal’ protein excretion often considered <150 mg/24 hNormal’ protein excretion often considered <150 mg/24 h• ‘‘1+’ on a dipstick = 300 mg/L or 450 mg/24 h1+’ on a dipstick = 300 mg/L or 450 mg/24 h

• Therefore, ‘normal’ becomes <15 mg/mmol and ‘1+’ Therefore, ‘normal’ becomes <15 mg/mmol and ‘1+’ becomes 45 mg/mmolbecomes 45 mg/mmol


Protein:Creatinine Ratios (2)Protein:Creatinine Ratios (2)

• correct for urinary dilution/concentrationcorrect for urinary dilution/concentration• easiereasier• cheapercheaper• more acceptable to the patientmore acceptable to the patient• closely predict 24 h excretionclosely predict 24 h excretion• consistent with guidelines (K-DOQI, PARADE, NSF)consistent with guidelines (K-DOQI, PARADE, NSF)• more accurately predict progressionmore accurately predict progression

……but require re-education in interpretationbut require re-education in interpretation


Classification of proteinuriaClassification of proteinuria

• GlomerularGlomerular

• TubularTubular

• OverflowOverflow

Quantitatively and clinically, glomerular proteinuria Quantitatively and clinically, glomerular proteinuria is most significantis most significant


Glomerular proteinuriaGlomerular proteinuria

• The glomerulus is a filter, retaining proteins of Mr > The glomerulus is a filter, retaining proteins of Mr > approximately 65 kDa (e.g. albumin)approximately 65 kDa (e.g. albumin)

• Therefore the appearance of high Mr proteins in urine Therefore the appearance of high Mr proteins in urine implies glomerular damageimplies glomerular damage

• May be selective (mainly albumin) or unselective (larger May be selective (mainly albumin) or unselective (larger proteins e.g. IgG) – classification rarely usedproteins e.g. IgG) – classification rarely used

• In most conditions, albumin is quantitatively the most In most conditions, albumin is quantitatively the most significant proteinsignificant protein


Total protein versus albuminTotal protein versus albumin• Proteinuria is predominantly albuminuria, but there is not a Proteinuria is predominantly albuminuria, but there is not a

linear relationship between the two:linear relationship between the two:

• 150 mg/L TP contains 30 mg/L albumin (20%)150 mg/L TP contains 30 mg/L albumin (20%)• 300 mg/L TP contains 150 mg/L albumin (50%)300 mg/L TP contains 150 mg/L albumin (50%)• 1000 mg/L TP contains 700 mg/L albumin (70%)1000 mg/L TP contains 700 mg/L albumin (70%)

• Relationship more variable at low protein concentrationsRelationship more variable at low protein concentrations

• TP stick tests and laboratory methods particularly sensitive TP stick tests and laboratory methods particularly sensitive to albuminto albumin


AlbuminuriaAlbuminuria


Renal handling of albuminRenal handling of albumin

• r=3.6 nm, filtration fraction <0.01 r=3.6 nm, filtration fraction <0.01 (cf dextran of same r=0.1)(cf dextran of same r=0.1)

• pI 4.7 – highly anionic – repulsed by glomerular pI 4.7 – highly anionic – repulsed by glomerular polyanionpolyanion

• 37,000 g/day pass through glomerular capillaries, 37,000 g/day pass through glomerular capillaries,

• 1.3 g/day pass into urinary space (0.004%)1.3 g/day pass into urinary space (0.004%)

• Where is the barrier to filtration?Where is the barrier to filtration?


The filtration barrierThe filtration barrier

The glomerular The glomerular basement membrane basement membrane is a size- and charge-is a size- and charge-selective filterselective filter

Foot processes are the final barrier to Foot processes are the final barrier to filtrationfiltration

East Kent HospitalsEast Kent HospitalsNHS TrustNHS Trust(A) Healthy (B) MCN(A) Healthy (B) MCN

MCN associated with flattening (effacement) of the foot MCN associated with flattening (effacement) of the foot processes in scanning EMprocesses in scanning EM

Mathieson, Clin Sci 2004;107:533-8Mathieson, Clin Sci 2004;107:533-8


Renal handling (2) – post glomerularRenal handling (2) – post glomerular

• 1.3 g pass into urinary space 1.3 g pass into urinary space

(0.004% of handled)(0.004% of handled)

• Approx 10-30 mg/day passed in urine Approx 10-30 mg/day passed in urine

(<1% of filtered)(<1% of filtered)

• What happens to the remainder?What happens to the remainder?


Albumin

66,000 Da

Cub

Meg

TUBULAR LUMEN

Cub

MegMeg

Endosome

Lysosome

500-15,000 Da

fragments

35% 65%Lost in urine

(only intact albumin measured)

Returns to circulation

1.3 g/day

10 mg/day

Amn


Urinary albumin/‘microalbuminuria’Urinary albumin/‘microalbuminuria’

• 1963 - Keen & Chlouverakis @ Guy’s 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 @ Guy’s 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


NOTE!!!NOTE!!!

• ‘‘microalbuminuria’ is not about a small form of microalbuminuria’ is not about a small form of albumninalbumnin

• ‘‘microalbuminuria’ is about increased, not microalbuminuria’ is about increased, not decreased, amounts of albumin in the urinedecreased, amounts of albumin in the urine


Microalbuminuria and progressionMicroalbuminuria 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

• 1990’s

- good glycaemic control prevents progression to microalbuminuria (DCCT, n>1400) 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 In diabetes, microalbuminuria has become established as a marker of potentially treatable diseasepotentially treatable disease


Confounding factorsConfounding factors

• Biological variation (day-to-day CV 45%)Biological variation (day-to-day CV 45%)• Metabolic controlMetabolic control• Intercurrent illness (e..g. sepsis, post-myocardial infarction, surgery, Intercurrent illness (e..g. sepsis, post-myocardial infarction, surgery,

SIRS)SIRS)• Haematuria/menstrual contaminationHaematuria/menstrual contamination• Non-diabetic renal diseaseNon-diabetic renal disease• Uncontrolled hypertension Uncontrolled hypertension • Strenuous exerciseStrenuous exercise• Urinary tract infectionUrinary tract infection

Microalbuminuria should be present in at least two out of three Microalbuminuria should be present in at least two out of three urine samples preferably collected within a 6(1) month period in urine samples preferably collected within a 6(1) month period in the absence of ketonuria or infectionthe absence of ketonuria or infection

East Kent HospitalsEast Kent HospitalsNHS TrustNHS TrustMicroalbuminuric rangesMicroalbuminuric ranges

Overnight AER 20-200 ug/min Overnight AER 20-200 ug/min [seen as ‘gold standard’ method][seen as ‘gold standard’ method]

equivalent to:equivalent to:

30-300 mg/24 h 30-300 mg/24 h [or 20-200 mg/L (NICE)][or 20-200 mg/L (NICE)]

which if you excrete 10 mmol creatinine/day is equivalent to:which if you excrete 10 mmol creatinine/day is equivalent to:

3.0 – 30 mg/mmol creatinine 3.0 – 30 mg/mmol creatinine [or 30-300 ug/mg in US][or 30-300 ug/mg in US]

or:or:

Males Males 2.5 mg/mmol, Females 2.5 mg/mmol, Females 3.5 mg/mmol 3.5 mg/mmol


Natural history of diabetic renal diseaseNatural history of diabetic renal diseaseSTAGE AER

(ug/min)BP serum

creatinineproteinstix test

1 (normoalbuminuria) <10 normal normal negative

2 (microalbuminuria) 20-200 +/-increased

normal negative

3 (proteinuria) >200 increased +/- increased positive

4 (progression) >200 furtherincrease

progressiveincrease

positive

5 (ESRF) >200 positive

10-15 y

10-20 y


PREVEND (1)PREVEND (1)

• 40,000/85,000 residents of Groningen recruited in 40,000/85,000 residents of Groningen recruited in 19971997

• Urine albumin measuredUrine albumin measured

• Followed for 3 yFollowed for 3 y

• 516 deaths516 deaths

• Mortality and cause of mortality recordedMortality and cause of mortality recorded

Hillege et al, JIM 2001, Circulation 2002Hillege et al, JIM 2001, Circulation 2002



• Microalbuminuria present in Microalbuminuria present in 7.2% of population7.2% of population

• Independently associated Independently associated with hypertension, diabetes, with hypertension, diabetes, CV diseaseCV disease

• After excluding diabetics After excluding diabetics and hypertensives, and hypertensives, microalbuminuria still microalbuminuria still present in 6.6% of present in 6.6% of population.population.


PREVEND (3)PREVEND (3)• Increasing albuminuria associated Increasing albuminuria associated

with increasing CV and, to a lesser with increasing CV and, to a lesser extent, non-CV mortality (esp. extent, non-CV mortality (esp. cancer)cancer)

• Albuminuria is a strong predictor Albuminuria is a strong predictor of all cause mortality in general of all cause mortality in general popn.popn.

• Risk begins at levels not Risk begins at levels not considered microalbuminuricconsidered microalbuminuric

• (Similar data from Framingham (Similar data from Framingham Offspring Study on non-diabetic, Offspring Study on non-diabetic, non-HT subjects [Arnlov et al, non-HT subjects [Arnlov et al, Circulation 2005])Circulation 2005])



• Microalbuminuria common and associated with CV risk Microalbuminuria common and associated with CV risk factors and deathfactors and death

• Advocates population screening approach to Advocates population screening approach to microalbuminuria detection, but especially hypertensivesmicroalbuminuria detection, but especially hypertensives

• Non-diabetics with microalbuminuria should be on Non-diabetics with microalbuminuria should be on ACEI/ARBsACEI/ARBs

• Threshold for pathological albuminuria should be revisedThreshold for pathological albuminuria should be revised


Measurement issuesMeasurement issues


Stick testsStick tests Colorimetric, approx £0.10Colorimetric, approx £0.10 Semi-quantitative, Semi-quantitative, inconsistency between inconsistency between manufacturers, mainly manufacturers, mainly measure albuminmeasure albumin

Lab total Lab total proteinprotein

Mainly colorimetric (e.g. Mainly colorimetric (e.g. pyrogallol red, coomassie pyrogallol red, coomassie blue, benzethonium blue, benzethonium chloride), approx £0.10 chloride), approx £0.10

Results differ between Results differ between dyes, often standardised dyes, often standardised against albumin, mainly against albumin, mainly sensitive to albuminsensitive to albumin

AlbuminAlbumin Immunoassay, approx Immunoassay, approx £0.40£0.40

Standardised against Standardised against albuminalbumin


Urinary Total Protein UKNEQASUrinary Total Protein UKNEQAS

Bayer stix tests: 8% negative, 30% trace, 56% 1+, 6% 2+Bayer stix tests: 8% negative, 30% trace, 56% 1+, 6% 2+


Urinary albumin UKNEQASUrinary albumin UKNEQAS


Non-immunoreactive albumin (1)Non-immunoreactive albumin (1)

• HPLC (total albumin) HPLC (total albumin) assays suggest large assays suggest large amounts of albumin amounts of albumin present in diabetic urine present in diabetic urine not measured by not measured by immunoassays – termed immunoassays – termed ‘non-immunoreactive’ ‘non-immunoreactive’ albumin albumin

• No difference between No difference between non-diabetic urinesnon-diabetic urines

Comper et al AJKD 2003, Comper et al AJKD 2003, Osick and Comper, Clin Chem 2005Osick and Comper, Clin Chem 2005



• Albumin has similar Mr to Albumin has similar Mr to normal albuminnormal albumin

• May represent May represent (a) albumin which has (a) albumin which has undergone minimal tubular undergone minimal tubular processing (‘scissions’ with # processing (‘scissions’ with # held together by S-S bonds) or held together by S-S bonds) or (b) filtered forms of albumin (b) filtered forms of albumin not recognised by immunoassay not recognised by immunoassay (e.g. FA binding induces (e.g. FA binding induces conformational change)conformational change)

• These processes may be These processes may be affected by diabetesaffected by diabetes

2. Purified NIA from urine3. Purified NIA under reducing conditions

1. Diabetic urine



• Retrospective analysis of 15 y of stored urine Retrospective analysis of 15 y of stored urine samples from diabetics with progressive (n=41) samples from diabetics with progressive (n=41) or non-progressive (n=50) kidney diseaseor non-progressive (n=50) kidney disease

• HPLC predicted onset of diabetic nephropathy 2-HPLC predicted onset of diabetic nephropathy 2-4 y earlier than immunoassay4 y earlier than immunoassay

• But:But:Only this group publishing in this areaOnly this group publishing in this areaAssay being marketed by the authorsAssay being marketed by the authorsWhen is albumin not albumin?When is albumin not albumin?

Comper et al KI 2004Comper et al KI 2004

East Kent HospitalsEast Kent HospitalsNHS TrustNHS TrustACR to replace PCR?ACR to replace PCR?

AdvantagesAdvantages DisadvantagesDisadvantages

More sensitive – essential to identify More sensitive – essential to identify all CKD stage 1 and 2 patients all CKD stage 1 and 2 patients KDIGO define kidney damage = KDIGO define kidney damage = ACR >30 mg/g (approx 3.5 ACR >30 mg/g (approx 3.5 mg/mmol)mg/mmol)

More sensitive than More sensitive than nephrologists want?nephrologists want?

Improved precisionImproved precision

Consistency with diabetic Consistency with diabetic nephropathy literaturenephropathy literature

More expensive?More expensive?

Single protein (know what you are Single protein (know what you are measuring and how it is calibrated)measuring and how it is calibrated)

No IRPNo IRP

Consistent with International Consistent with International practice (KDIGO/KDOQI)practice (KDIGO/KDOQI)

Nephrology literature based Nephrology literature based on PCRon PCR


Functional ‘tubular’ proteinuriaFunctional ‘tubular’ proteinuria

• Small Mr proteins filtered at glomerulus and Small Mr proteins filtered at glomerulus and reabsorbed in proximal tubulereabsorbed in proximal tubule

• Appearance of proteins in urine implies tubular Appearance of proteins in urine implies tubular damagedamage

• E.g. urinary alpha-1 microglobulin, beta-2 E.g. urinary alpha-1 microglobulin, beta-2 microglobulin, retinol binding proteinmicroglobulin, retinol binding protein


Tubular proteinuria due to cell Tubular proteinuria due to cell damagedamage

• Certain proteins present at high concentration in Certain proteins present at high concentration in tubular cells tubular cells

• E.g. Tamm Horsfall glycoprotein and N-acetyl-B-E.g. Tamm Horsfall glycoprotein and N-acetyl-B-D-glucosaminidase (NAG)D-glucosaminidase (NAG)

• Appearance of these proteins in urine implies Appearance of these proteins in urine implies tubular damagetubular damage

• Useful in drug toxicity studies or occupational Useful in drug toxicity studies or occupational monitoring (e.g. heavy metal workers)monitoring (e.g. heavy metal workers)


Overflow proteinuriaOverflow proteinuria

• Excess formation of a low molecular weight Excess formation of a low molecular weight protein that is freely filteredprotein that is freely filtered

• E.g. BJP, myoglobinE.g. BJP, myoglobin

• Such proteins may be directly toxic to the tubular Such proteins may be directly toxic to the tubular cellscells


SummarySummary

• Kidney disease is commonKidney disease is common• Perhaps more than any other disease state, its assessment Perhaps more than any other disease state, its assessment

relies on quantitative laboratory testsrelies on quantitative laboratory tests• ……and renal units rely on the laboratoryand renal units rely on the laboratory• There are a range of tests available to assess the functions There are a range of tests available to assess the functions

of the kidney - the most important of these are GFR and of the kidney - the most important of these are GFR and proteinuriaproteinuria

• No current tests are perfect for assessing GFR and there No current tests are perfect for assessing GFR and there is inconsistency in approach to proteinuriais inconsistency in approach to proteinuria


ENDEND


Major filtration barrier now widely believed to be the filtration Major filtration barrier now widely believed to be the filtration slits (*) between inter-digitating foot processes (slits (*) between inter-digitating foot processes (↓)↓)

Mutations of nephrin and podocin, which co-localise at the slit Mutations of nephrin and podocin, which co-localise at the slit diaphragm (and several other protein mutations) associated with diaphragm (and several other protein mutations) associated with congenital types of nephrotic syndrome (e.g. Finnish-type congenital congenital types of nephrotic syndrome (e.g. Finnish-type congenital NS)NS)

Mathieson, Clin Sci 2004;107:533-8Mathieson, Clin Sci 2004;107:533-8

assessment of renal tubular function

Documents

improved gfr marker

moderate ckd

u crx v p crx t

kd basic protein

constant rate

kidney function testing

nucleated cells

proportional increase