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  Assessment of Proteinuria Gautham Viswanathan and Ashish Upadhyay Proteinuria is a strong predictor of adverse cardiovascular and kidney events, and an accurate assessment of proteinuria isimportant for the evaluation and management of CKD. Total urinary protein can be assessed using dipstick, precipitation,and electrophoresis methods. Urinary albumin, the predominant urinary protein in most proteinuric kidney diseases, can beassessedusinganalbumin-specificdipstick,immunochemicaltechniques,andsize-exclusionhigh-performanceliquidchroma-tography. Urine albumin may be immune-reactive, immune-unreactive, fragmented, and biochemically modified, and labora-tory techniques have variable abilities to detect different types of albumin. Urine specimen for proteinuria assessment caneither be obtained from a timed-collection or a spot urine sample. Spot urine protein- or albumin-to-creatinine ratios are pre-ferredtoa24-hoururinesampleinroutinepractice.Assessmentofalbuminuriaratherthanproteinuriaismoreclinicallymean-ingful in patients with diabetic kidney disease, and proteinuria and albuminuria assessments both have a role in nondiabetickidney disease and in general population screening. As measurement and sampling procedures for proteinuria assessmenthave yet not been standardized, it is important for physicians to be aware of different types of urinary proteins, albumins, lab-oratory techniques, and urine sampling methods. Q 2011 by the National Kidney Foundation, Inc. All rights reserved. Key Words:  Proteinuria, Albuminuria, Immunoassay, High performance liquid chromatography H igh urinary protein excretion amplifies risks foradverse cardiovascular and kidney events in indi-viduals with CKD, 1-3 and the assessment of proteinuriais a cornerstone for identification, evaluation, andmanagement of CKD. 4 The optimal method for measur-ing urinary protein, however, is uncertain, and there areinconsistencies among guidelines on whether the mea-surement of total urinary protein excretion or only uri-nary albumin excretion should be recommended forrisk assessment and therapeutic decisions. 4-6 Urine primarily consists of 2 groups of proteins––plasma proteins that cross the filtration barrier and non-plasma proteins that srcinate in renal tubules or urinarytract. Albumin is the principal plasma protein and is thepredominant urinary protein in most proteinuric kidneydiseases, and uromodulin or Tamm–Horsfall protein isthe major nonplasma urinary protein. Apart from albu-min and uromodulin, urine also contains variable pro-portions of immunoglobulins, low molecular weightproteins, and light chains. Normal individuals usuallyonly excrete small amounts of protein in the urine anda persistent excretion of  . 200 mg of protein or  . 30 mgof albumin a day is considered to be a marker of kidneydamage. 4 There is a lack of standardization in the labora-tory techniques and in the collection of urine samplesused for proteinuria and albuminuria quantification.This review elaborates on our current understanding of different laboratory techniques and measurement meth-odologies for proteinuria and albuminuria assessment. Methods for Measuring Proteinuria Wide variation in the type and concentration of proteinsseen in urine during different states of health and diseasemakes it very difficult to standardize proteinuria assess-ment. Dipstick, precipitation, and electrophoresis are 3commonly used methods for measuring total urinaryprotein in clinical practice. Dipstick Method The dipstick method is the most common method usedfor proteinuria assessment, and it uses a paper or plasticstrip impregnated with a pH-sensitive dye that changescolor in the presence of negatively charged urinary pro-teins. The degree of color change corresponds with thedegree of proteinuria, with an accompanying scale pro-viding concentrations as a range from 0 to 3 or  $ 4. Fortetrabromophenol blue, a commonly used pH-sensitivedye, yellow color means low protein concentration and blue color means high protein concentration. Althoughdipsticks are convenient to use, they are not very sensi-tive for the detection of proteinuria. The test is heavilydependent on the protein concentration in urine anddoes not reflect the protein excretion rate. False negativeresults may be seen when the urine volume is high andurine is dilute, whereas false positive results may beseen when urine volume is low and it is concentrated.Similarly, as this is a pH-sensitive dye, false positive re-sults may also be seen when urine is alkaline. Eventhough different types of reagent strips may perform dif-ferently, 7 the United Kingdom National Institute of Clin-ical Excellence guidelines have stated that, on average,dipsticks have low sensitivity and variable specificityfor detecting total protein, and positively charged pro-teinslikeimmunoglobulinlightchainsmaynotbepicked From Division of Nephrology, Tufts Medical Center, Boston, MA;and Department of Medicine, Tufts University School of Medicine, Boston, MA. Address correspondence to Ashish Upadhyay, MD, William B. SchwartzDivision of Nephrology, Tufts Medical Center, Tufts University School of  Medicine, 800 Washington Street, #391, Boston, MA 02111. E-mail:aupadhyay@tuftsmedicalcenter.org  2011 by the National Kidney Foundation, Inc. All rights reserved.1548-5595/$36.00doi:10.1053/j.ackd.2011.03.002  Advances in Chronic Kidney Disease, Vol 18, No 4 (July), 2011: pp 243-248  243  up even when the concentrations of these proteins arehigh. 8 For detecting albumin, a negatively charged pro-tein, sensitivity in generalremains low,whereasthe spec-ificity is high and ranges from 93% to 98%. 8 However,sensitivity is higher for albumin-specific dipsticks (Clini-tek and Micral-test II). High inter-observer variation fur-ther limits the interpretation of dipstick results, withinconsistencies among technologists ranging f rom 33%to 93% based on the level of their experience. 7,9 Precipitation Method Intheprecipitationmethod,acidisaddedtotheurineandproteinuria is quantified by measuring the turbiditycaused by protein precipitation using a photometer ornephelometer and comparing it with the standard. Sulfo-salicylic acid, nitric acid, and trichloroacetic acid are thecommonly used acids. Sulfosalicyclic acid is more sensi-tive for detecting albumin and light chains as comparedwith globulins, whereas the sensitivity for globulin ishigher with trichloroacetic acid. Precipitation methodsare cumbersome, have low sensitivity for immunoglobu-lin light chains, and lackprecision, with the coeffi-cient of variation for sulfosa-licyclic acid method beingaround 20%. 10-12 Further-more, the use of iodinatedcontrast agents and, withtrichloroacetic acid method,drugs like tolbutamide, pen-icillin, nafcillin, and oxacillinmay also give rise to false-positive results. 11,13-15 Electrophoresis Method Electrophoresis method is used primarily for detectingthe presence of immunoglobulins and immunoglobulinlight chains in the urine. Urine electrophoresis showsmonoclonal peak and immunoelectrophoresis identifiesthe specific protein with very high sensitivity. 16 Methods for Measuring Albuminuria Albumin molecules in urine arenot homogenous. A typi-cal albumin molecule is heart-shaped, weighs about69,000 Da, and consists of 585 amino acids arranged as 3homologous domains, with each domain consisting of 2subdomains. 17 Binding of urinary albumin to ions, pep-tides,hormones,anddrugsmodifythisuniquemolecularconfiguration. 18 Inaddition,albuminisalsosubjecttopro-teolysisinbothplasmaandrenaltubulesresultinginalbu-min fragments of various sizes in the urine. 19 A recentstudy has revealed that albumin fragments may accountfor 25% to 30% of total urinary albumin in patients withnephrotic syndrome. 20 Sometimes, as has been shown indiabetic nephropathy, the tubular lysosomal action may just result in the conformational change of the albuminepitope resulting in the formation of immune-unreactiveintact albumin. 19 Therefore, even the intact urinary albu-min may be immune-reactive or immune-unreactive,and may exist as monomers or dimers. 21 The clinical sig-nificance of these various types of albumin is unclear, but this heterogeneity makes it challenging for accuratealbuminuria assessment because different laboratorymethodshavedifferentabilitiestodetectanddiscriminatedifferent types of albumin. 22 Laboratory techniques for albuminuria assessmentcan broadly be divided into immunochemical methodsand size-exclusion high-performance liquid chromatog-raphy (SE-HPLC) method. Table 1 summarizes proper-ties of different methods for albuminuria assessment. Immunochemical Methods The conventional immunochemical methods only mea-sureimmune-reactivealbumins,polymeralbuminaggre-gates, and albumin fragments . 12 kDa. 23 Double-AntibodyRadioimmunoassay Inthismethod,urinaryalbu-mins compete with radiola- beled albumins for fixed binding sites on anti-albumin antibodies (first an-tibody). Antibodies boundto albumins are then sepa-rated by immunoabsorptionusing anti-immunoglobulinantibody (second antibody). The radioactivity of theresultantsupernatantisinverselyproportionaltotheuri-nary albumin concentration and radioactivity counts aremeasured against standardized curves to quantify uri-nary albumin. Radioimmunoassay (RIA) is highly sensi-tive for detecting immune-reactive albumins andalbuminfragments . 12kDa. 29 Theassay,however,isex-pensive,hasshortshelflife,andcanonlybeperformedinlaboratories that are able to handle radioactive sub-stances. 30 Immunonephelometry In this method, specialized anti-albumin antibodies areadded to the urine sample resulting in the precipitationof antibody-albumin complexes. These complexes scatterlight passing through the sample and the scatter signal ismeasured by a laser nephelometer. Urinary albumin con-centration is directly proportional to the scatter sig-nal. 31,32 Immunonephelometry technique is as sensitiveas RIA for early detection of microalbuminuria. 33 CLINICALSUMMARY   Laboratory techniques for proteinuria assessment are notstandardized, and have variable abilities to measuredifferent types of proteins and albumins.   Spot urine protein- or albumin-to-creatinine ratio can beused in place of 24-hour urine protein or albumin in mostclinical situations.   Decisiononwhethertomeasureproteinuriaoralbuminuriais determined by the underlying kidney disease. Viswanathan and Upadhyay 244  Immunoturbidimetry Immunoturbidimetry method measures the absorbanceof light by the urine sample containing antibody–albumin complexes. 34 The absorbency is measured usingaspectrophotometerandisproportionaltotheurinaryal- bumin concentration. In general, immunoturbidimetry isless sensitive than immunonephelometry. The sensitivitycan be enhanced by using the latex-enhanced method,whichenlargesthesizeoftheimmunecomplexesandam-plifies the reaction. 35 Other Immunochemical Methods Enzyme-linked immunosorbent assay, enzyme im-munoassay, fluoroimmunoassay, chemiluminescence,and electrochemiluminescence are other methods thathave also been used to measure urinary albumin. Size-Exclusion-High-Performance LiquidChromatography SE-HPLC uses chromatographic techniques to measure both immune-reactive and immune-unreactive albu-mins. 36 Intact albumin molecules are separated fromother proteins when the urine sample is passed througha column that contains size-selective gel, as the flowrate through the column is determined by the proteinsize. 37 SE-HPLC is much more sensitive than immuno-chemical methods, especially when albumin excretionrate is  , 100  m g/min (Fig 1). 21 SE-HPLC also detectsmicroalbuminuria in patients with diabetes about 2 to 4years earlier than RIA and the data from the Heart Out-comes Prevention Study show that the prevalence of microalbuminuria is 2 to 3 times higher when SE-HPLCis used instead of RIA. 38 Even though the clinical signif-icance of immune-unreactive albumins that SE-HPLCdetects is unclear, it has been shown that the use of SE-HPLC to detect albuminuria identifies more peopleat risk for vascular diseases and death than the use of conventional immunochemical methods. 39,40 The maindrawback of SE-HPLC is its inability to discriminate Figure 1.  Comparison of urinary albumin excretion ratesdeterminedbySE-HPLCascomparedwithRIAforfreshurinesamples obtained from patients with diabetes. Grid linesdenote value of 20  m g/min. Reprinted with permission. 21 Table 1. Properties of Immunochemical Methods and Size-Exclusion-High Performance Liquid Chromatography (SE-HPLC)for Assessing Albuminuria PropertiesImmunochemical MethodsSE-HPLCRadioimmunoassay Immunonephelometry ImmunoturbidimetryType of intact albumindetectedImmune-reactive Immune-reactive Immune-reactive Immune-reactive, andImmune-unreactiveMolecular weight of albuminfragments detected . 12 kDa  . 12 kDa  . 12 kDa  . 12 kDaDetection limit (for immune-reactive albumin inimmunochemical methodsand for total albumin inSE-HPLC)0.1-0.3 mg/L 0.5-1 mg/L 5-8 mg/L 2 mg/LFalse negatives vs SE-HPLCfor detection of microalbuminuria23% No data 36% ReferenceFalse positives vs SE-HPLCfor detection of microalbuminuria0% No date 0% ReferenceInter-assay coefficients of variation9.2% at 12.2 mg/L4.8% at 33 mg/L4.2% at 12.1 mg/L5.3% at 45 mg/L8.5% at 8 mg/L3.4% at 35 mg/L5.6% at 44.7 mg/L6.0% at 141 mg/LPercentage of patients withnormoalbuminuira byimmunochemical methodsreclassified as havingmicroalbuminuria bySE-HPLC16% 17% No data ReferenceAbility to predict vascularoutcomes and death 1 1 1 11 Data obtained from. 23-28  Assessment of Proteinuria: Current Methods and Techniques  245  albumins from coeluting globulins of similar molecularsize resulting in the overestimation of albuminuria. 24 The amount of albumin-sized globulins coeluting withalbumin may in some instances be as high as 20% to30%. 24 Thus, SE-HPLC can be considered as a highly sen-sitive test for albuminuria detection whose specificity isloweredbythepresenceofcoelutingglobulinsofamolec-ular size similar to albumin. Methods for Sampling Urine Quantification of urinary protein or albumin can be done by either using timed urine or a spot urine collection. Timed Urine Collection Timed urine collection is usually a 24-hour urine collec-tion or an overnight urine collection. Although thesemethods for proteinuria and albuminuria quantificationare well established in clinical practice, they are cumber-some, time-consuming, and prone to errors. 4 Given theselimitations, majorguidelines now recommend using spoturine samples instead of timed urine samples for routinecare. 4,41 Timed urine collection, however, still hasa major role for proteinuria assessment in acute kidneyinjury when creatinine excretion is not constant, inhospitalized patients when urine collection may be easyand controlled, in patients with severe proteinuria, andin people at the extremes of age and weight. A recentstudy has shown that creatinine excretion rate may beestimated with little bias and moderate precision usingcommonly available clinical variables. 42 This method of estimating creatinine excretion rate may be helpful inevaluating the accuracy of timed urine specimens. Spot Urine Collection Proteinoralbuminconcentrationinasingle-voidedurinesample is highly influenced by urinary tonicity andvaries markedly depending on the person’s state of hydration and diuresis. 43 As simultaneous measurementof creatinine adjusts for variability in protein concentra-tion, a spot urine protein-to-creatinine ratio (UPCR) orurine albumin-to-creatinine ratio (UACR) are now therecommended methods for proteinuria and albuminuriaassessment. 4 The results are also easy to interpret as thedaily creatinine excretion is approximately 1 g andUPCR or UACR in a random urine sample is approxi-mately similar to the daily protein or albumin excretionin grams. 44 Ginsberg and colleagues first established the excellentcorrelation between UPCR and 24-hour urine protein intheir landmark study published in 1983. 44 Since then,the correlation between UPCR and 24-hour urine proteinhas also been verified by other studies in patients withdiabetickidneydisease,patientswithnondiabetickidneydisease, kidney transplant recipients, and pregnantwomen. 45-48 Similarly, studies mainly performed inpatients with diabetes have also shown that there isa high degree of agreement between UACR and 24-hoururine albumin excretion (Fig 2). 50-53 More recently,a study using a receiver operator characteristic curveanalysis showed that UPCR accurately predictsproteinuria  $ 150 mg/24-hours (area under curve[AUC]: 0.90-0.92) and  . 300 mg/24 hours (AUC: 0.98-0.99), and UACR accurately predicts albuminuria  $ 30mg/24 hours (AUC: 0.98-0.99) and  $ 300 mg/24 hours(AUC: 0.99-1.00) in ambulatory clinic patients. 49 There isalso a possibility that ratios may have a better prognosticvalue because urinary creatinine has also been shown topredict adverse outcomes and ratios capture the effect of  both urinary albumin or protein and urinary creatinine. 54 This growing volume of evidence gives credence to theuse of UPCR and UACR as valid surrogates for the24-hour urine protein and albumin measurements.Although UPCR and UACR are convenient for pro-teinuria quantification, their use has several limitations.A random UPCR or UACR may not always reflect the24-hour excretion rates because protein or albuminexcretion varies with the time of the day, stress level,exertion, and other factors. 55 It is believed that the first-morning sample correlates better with the 24-hour urineand has a higher sensitivity and specificity at picking upan abnormal excretion rate than the random sample. 56 However, the collection of first-morning samples is bur-densome for patients and creates challenges for accuratelaboratory measurement as the storage time and temper-ature may alter albumin levels in the urine. 57 Given thesepotential problems, the Kidney Disease Outcomes Qual-ityInitiativeguidelineconsiderstherandomsampletobeacceptable if the first-morning sample is not available. 4 The other limitation with the use of ratios is that the cre-atinine excretion is affected by race, diet, and musclemass, and the ratios will give an erroneous estimates of the 24-hour excretion rates if the creatinine excretion isnot close to a gram per day. 22,58 Similarly, ratios are alsonot useful in estimating the 24-hour urinary excretionrate if the creatinine excretion is not constant, like in the Figure 2.  Albumin-to-creatinine ratio measured in random,early morning urine as compared with 24-hour albuminuria.Spearman rank correlation coefficient 0.97;  P   ,  .0001.Reprinted with permission. 49 Copyright 2009 Royal Societyof Medicine Press, UK. Viswanathan and Upadhyay 246
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