Urinary Proteomics—a Tool for Biomarker Discovery

Renal Failure, 32:259–268, 2010 Copyright © Informa UK Ltd.ISSN: 0886-022X print / 1525-6049 onlineDOI: 10.3109/08860221003599759

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LRNFSTATE OF THE ART REVIEW

Urinary Proteomics—a Tool for Biomarker Discovery

Urinary Proteomics—a Tool for Biomarker DiscoveryMiljana Pejcic, Slavica Stojnev, and Vladisav StefanovicUniversity School of Medicine, Nis, Serbia

The strong need for the discovery of novel disease markerstogether with the development of high-throughput techniquesthat provide highly sensitive analysis of protein content in tissuesand bodily fluids, using proteomics, has opened the completelynew chapter in biomarker discovery. The detection of biomarkersbased on urinary proteome analysis is rapidly advancing and mayprovide new tools to improve non-invasive diagnostics, prognos-tics, and therapy enhancement. As a tool for biomarker discovery,urinary proteomics is especially fruitful in the area of early diag-nostics and differentiation of renal damage, and it possesses enor-mous potential for improving and expanding non-invasive cancerdiagnostics. An abundance of urinary proteins could provide awide variety of biomarkers for the diagnosis and follow-up ofmany systemic diseases as well. This article reviews the utility ofurinary proteomics for biomarker discovery from the perspectiveof clinical application. Despite huge potential and prompt devel-opment of urinary proteomics, many challenges are still in frontof us. Research effort and financial investment have to be orientedon providing strategies for exceeding current methodological andtechnical obstacles in a way to ensure the successful validationand implementation of newly discovered urinary biomarkers. Theresult is expected to be the development of new non-invasive testsand procedures able to guarantee higher efficiency of patient careand provide needed personalized medical approach.

Keywords urine, proteomics, biomarkers, kidney disease,diagnostics

INTRODUCTION

From the constant challenges that modern medicine isfacing emerged the desperate need for the identification of

novel disease-specific biomarkers, which may find appli-cation in preventive screening and early diagnosis, andimprove prognostic and therapeutic possibilities as well.Scientific excitement recently induced by the HumanGenome Project has been swiftly replaced by the enthusi-asm for proteomics, with the aim to find new biomarkersfor human diseases. The new era in biomarker discoveryhas started together with the revolutionary technologicalachievements for detection, quantification, and identifica-tion of proteins. Increased knowledge about molecularmechanisms hand-in-hand with the application of proteomictechniques for high-throughput analysis of proteomes isproviding identification of new specific protein biomarkerpanels for different diseases.[1]

Proteomics represents systematic study of proteomes,which refers to the whole protein content of cell, tissue, oran organism, as well as of different bodily fluids. Under theinfluence of different factors, variations in gene expression,alternative splicing, and posttranslational modificationsgenerate constant changes of proteome profiles, whichdepict the complexity of the specific proteome. Biomedicalproteome research with the aim of biomarker discovery ismainly based on expression proteomics, analyzing quan-tity of certain proteins in different conditions. Functionalproteomics interferes in interactions among proteins inorder to discover molecular functions and signaling path-ways of identified biomarkers, explain pathogenesis, anddiscover targets for novel therapeutic interventions.[2]

POTENTIAL OF URINARY PROTEOMICS IN BIOMARKER DISCOVERY

Proteinuria, a cardinal symptom of renal disease, hasalways been considered a nonspecific source for diagnosticand prognostic information. Currently, new proteomictechnologies provide greater capabilities in urinary proteininvestigation; thus, it is clear that protein content of urinecould actually be a quite accurate and specific diagnosticand prognostic tool for different diseases. Recent proteomic

Received 17 October 2009; revised 17 November 2009;accepted 13 December 2009.

Address correspondence to Vladisav Stefanovic, M.D., Ph.D.,Faculty of Medicine, Zorana Djindjica 81, 18000 Nis, Serbia; Tel.:381-18-570 029; Fax: 381-18-238 990; E-mail: [email protected]

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260 M. Pejcic et al.

analysis has identified more than 1500 proteins in urine ofhealthy individuals,[3] and their quantitative and qualitativechanges might have a significant biomarker role.

Urine represents a modified ultrafiltrate of plasma,with protein concentration approximately 1000-fold lowerthan plasma.[4] However, urinary proteins/peptides havehigher thermodynamic stability and are less complex andinteractive compared with the plasma proteins,[5] so thatlow protein concentration does not make it a less promisingdiagnostic specimen than plasma. Furthermore, noninva-sive accessibility of urine and simplicity of samplingmakes it very convenient diagnostic source. As a reflectionof serum composition and kidney function, urine is a sourceof most plasma proteins, with increased proportions of low-molecular-weight protein and peptide components, and it isenriched with proteins released along the urinary tract.[4]

Changes of urinary proteome are reflecting disease-relatedchanges of tissue or cellular proteomes. This gives urineenormous potential for tasks such as early detection of dis-ease, classification of disease, choice of therapeuticagents, assessment of prognosis, and monitoring of aparticular therapeutic regimen.[6] In that way, urinary pro-teomics is especially fruitful in the area of early diagnos-tics and differentiation of renal damage, and it possessesenormous potential for improving non-invasive cancerdiagnostics.[7] Furthermore, systemic diseases associatedwith the generation of small circulating proteins and pep-tides as proteolytic fragments, which pass glomerular filter,could also manifest themselves by changes in urinary pro-teome. Thus, urine could be the source for biomarkers ofdifferent systemic diseases as well.

Soluble urinary proteins originate mainly fromplasma by glomerular filtration, or they could be pro-teolytically cleaved membrane bound proteins, secreted bythe thick ascending limb of Henle loop (Tamm-Horsfallprotein or uromodulin). Solid phase elements consist ofslough epithelial cells and casts, small membrane frag-ments from shedding of microvilli or by apoptosis, andurinary exosomes.[6,8] Because of the different origins ofthis proteins, each of this protein fraction and sub-fractioncontains potential biomarkers for relevant disorders.Urinary prefractionation by different speed centrifugationor ultrafiltration is an important step prior to proteomeinvestigation, and could be the method for enriching markersfor particular types of diseases.[6]

TECHNIQUES USED IN URINARY PROTEOMICS

There are several different techniques for proteomicstudies. Most of the approaches start with a separationstep, followed by ionization and subsequent mass spec-trometry (MS) analysis. Ionization of the compound of

interest can be achieved using matrix-assisted laser des-orption ionization (MALDI) or electro-spray ionization(ESI). Now, different MS analyzers are in use, andindividual advantages of the different mass detectors arefrequently combined. Tandem mass spectrometry (MS/MS) is used to obtain protein sequence information.[9]

For pre-MS fractionation, different techniques providevery different types and quality of data. Two-dimensionalgel electrophoresis (2-DE) is most commonly appliedmethod, which allows separation and characterization ofproteins according to their isoelectric point and molecularmass. The application of 2-DE to urine is a labor- andtime-consuming low-throughput approach that requires arelatively large amount of proteins.[10,11] Liquid chroma-tography coupled to mass spectrometry (LC-MS) offerssensitive urine proteome analysis. LC/MS seems to besuitable for urine analysis due to the small amount of pro-tein required. CE-MS couples the high-resolution proper-ties of capillary electrophoresis (CE) with the powerfulidentification ability of the electro-spray time-of-flight MSto profile and sequence urinary proteins.

Array technology, using specific antibodies immobi-lized onto specially treated surfaces in an array format,could provide rapid examination of urine samples.[7] Thesurface-enhanced laser desorption/ionization (SELDI)technique effectively couples high-end MS with arrayformats. This method has the capacity to analyze multiplesamples in a short time, and it is widely used in urinaryproteomic analysis.

All of these high-throughput techniques have limita-tions in detecting proteins, as well as in protein identificationand quantification, but they can be used as screening toolsfor biomarker discovery.

STRATEGY FOR APPLICATION OF URINARY BIOMARKERS

Biomarker discovery based on proteomic approachesis comparative analysis and requires quantification of pro-teins. High-throughput proteomic techniques facilitatewhole-expression proteomics, and make possible to deter-mine protein profiles between normal and disease states.However, targeted analysis, oriented on predefined subsetof proteins, is useful whenever is possible.[12] Once apresumptive biomarker or a set of biomarkers has beenidentified, its predictive capabilities have to be determinedthrough validation studies on large patient cohorts in orderto define sensitivity and specificity. The final step is theimplementation of specific biomarker in clinical settingsby development of a clinical assay incorporated on a singleantibody array chip to allow personalized medicine to becarried out at costs that will allow everyone to benefit.[6]

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Urinary Proteomics—a Tool for Biomarker Discovery 261

The future will probably bring interesting opportunities forintroducing proteomics in routine laboratory for urinediagnostics.

URINARY BIOMARKERS WITH POTENTIAL FOR DIAGNOSTIC APPLICATION

The discovery of new urinary biomarkers with thepotential for early diagnostics is particularly useful formany kidney diseases that could lead to chronic renalfailure, especially because current diagnostics is based onnon-specific parameters and invasive procedures. There-fore, research interest has been aiming to discover specificurinary proteome patterns for many nephrourologicaldiseases and cancers.

Glomerular Disease

The most common cause of glomerulonephritisworldwide is IgA nephropathy (IgAN). Although thisimmune complex-mediated disease has the potential fordevelopment of chronic renal failure, diagnosis still relieson kidney biopsy. Comparison of urinary polypeptide pro-file of IgAN with the profiles of normal controls and otherglomerular diseases, using CE-MS, has brought a specificIgAN urinary polypeptide pattern. This biomarker panelalso showed high sensitivity and specificity for predictionof IgAN.[13] Additional research has led to establishmentof 2-D urinary proteomic map for IgAN, with the total of84 differentially expressed spots, representing 59 differentproteins comparing to urine of healthy controls.[14] Thesefindings could be crucial for discovering a reliable non-invasive early diagnostic test for IgAN.

Membranous nephropathy, an idiopathic antibody-mediated autoimmune disease, is one of the most commoncauses of nephrotic syndrome in adults. In order to definebiomarker candidates for human membranous nephropa-thy, serial analysis of urinary proteome profile of a ratmodel of passive Heymann nephritis, which resembleshuman membranous nephropathy, revealed a group of sig-nificantly differed proteins in the disease course.[15] Earlystage of the disease was characterized by markedlyincreased level of urine haptoglobin, which could be abiomarker for early membranous nephropathy.[16] Similarserial urinary proteome profiling in murine models ofinduced focal segmental glomerulosclerosis (FSGS) identi-fied a group of urinary proteins that showed characteristicpatterns of dynamic changes along the disease course.Urinary proteins that are identified to appear before glom-erular sclerotic changes could serve as early diagnosticbiomarkers of FSGS.[17]

Allograft Rejection and Dysfunction

Rejection constitutes the major impediment to thesuccess of renal transplantation, and currently availablemethods often fail to detect rejection until late stages ofprogression. Discovery of biomarkers using urinary pro-teomic could improve early non-invasive diagnosis ofallograft rejection and prognosis of renal transplants.Although substantial differences in concentration of severalproteins have been found in the urine of patients whoreceived a transplant comparing to healthy individuals,these differences are mostly due to immunosuppressant.[18]

Two different works succeeded to detect potential urinarybiomarkers for allograft rejection in kidney-transplantedpatients using SELDI-TOF MS. However, they pointed tocompletely different biomarkers for the same disor-der.[19,20] Other researchers discovered that in patientswith rejection, there is a reduction in b-Defensin-1, whilea-1-antichymotrypsin increased compared with clinicallystable transplants.[21] The differences in detected peaks forallograft rejection are probably a result of the complexity ofurinary proteome and variation in the performance ofapplied techniques. Urinary polypeptide analysis in differ-ent types of chronic allograft dysfunction established a pat-tern for two different lesions associated with distinct graftoutcomes (viz., pure interstitial fibrosis and tubular atrophyand chronic active antibody-mediated rejection), which con-stitutes a first step toward the design of a specific, noninva-sive diagnostic tool for chronic allograft dysfunction.[22]

Acute Kidney Injury

Acute kidney injury (AKI) is a common clinical problemassociated with high morbidity and mortality. Diagnosis ofAKI is based on unreliable and nonspecific indicators,such as serum creatinine, detection of casts, and fractionalexcretion of sodium. Nevertheless, early identification ofAKI is an imperative, because it demands rapid andaggressive treatment. A number of candidate urinary pro-teins, mainly renal tubular proteins, have been evaluatedas biomarkers of renal injury. It has been suggested that a“urine panel” consisting of interleukin-18, kidney injurymolecule-1 (KIM), and neutrophil gelatinase-associatedlipocalin may provide more reliable information.[23]

Recently, exosomal fetuin-A has been identified as apotential urinary biomarker of AKI.[24]

Diagnostic Potential of Urinary Exosomes

Urinary exosomes are derived from all cell types thatface the urinary space.[25] Exosomes are small vesicles that

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originate as internal vesicles of multivesicular bodies andare excreted from the cell by fusion of the multivesicularbody and plasma membrane. They contain membrane pro-teins and cytosolic proteins as well. Many cell types havebeen shown to secrete exosomes.[6]

The proteomics analysis of urinary exosomes fromnormal human subjects using LC-MS identified a numberof proteins involved in exosome biogenesis and also proteinproducts known to be responsible for renal and systemicdiseases, such as autosomal dominant polycystic kidneydisease, Gitelman syndrome, Bartter syndrome, autosomalrecessive syndrome of osteopetrosis with renal tubularacidosis, familial renal hypomagnesemia, and hyperten-sion.[25] The isolation of exosomes can provide very largeenrichment of urinary proteins that are derived from renaltubule epithelial cells, and they may be the best source ofbiomarkers for renal tubulopathies. In addition, they couldbe useful in cancer proteomics studies, because erythro-leukemia and other tumor cells also secrete exosomes.[6]

Urinary Biomarkers of Renal and Urological Cancers

Renal cell cancer (RCC) is often detected inciden-tally, and at the time of presentation, is frequently inadvanced stage. Currently, there are no routinely usedtumor markers for RCC. The examination of potentialclinical utility of SELDI profiling of urine samples todiagnose clear cell RCC has proved diagnostic potential ofdefined protein pattern, with sensitivity and specificity ofabout 82%. However, the model could not maintainrequired sensitivity and specificity due to factors thataffect methodology standardization.[26] Kininogen levelswere found to be elevated in the urine of RCC patients, andthe concentration of this protein fell after nephrectomy.[27]

This link between RCC and kininogen levels could help indefining RCC biomarkers. In analyzing the expression ofhuman kidney injury molecule 1 (hKIM) in patients withRCC, its presence in tissue samples and urine is shown.Urinary levels of hKIM were significantly higher inpatients with RCC compared to patients with prostatecancer and normal subjects, with significant decreasing ordisappearance after nephrectomy.[28] This suggests hKIMas a new possible biomarker for RCC. A very interestingfield in cancer biomarker discovery is tissue proteomicswith definition of RCC proteomic map[29] and analysis ofproteins from conditioned media of RCC cell lines.[30]

However, the potential of identified proteins as biomarkersin clinical settings has to be further explored.

Urothelial cancer is the second most frequent type ofcancer in urogenital tract, with the highest recurrence rateof any cancer. Bladder cancers are mostly transitionalcell carcinomas (TCC); squamous carcinomas and

adenocarcinomas rarely occur. With the use of SELDI-TOF,multiple protein changes in urine of patients with TCCcompared to healthy individuals were found. Among fivepotential biomarkers and seven protein clusters that werefound to be specific for TCC patients, one that was identi-fied belongs to the defensin family, a group of endogenousantibiotic peptides that are locally secreted in response toinflammation. The combination of these protein biomarkersand clusters provided sensitivity of 87% and specificity of66% for detecting TCC, with much higher sensitivityfor detecting low-grade TCC comparing to voided urine orbladder-washing cytology.[31] An additional set of polypep-tides associated with bladder cancer is demonstrated usingprotein-chip technology, and had 80% sensitivity and90–97% specificity in the training set, as well as about55% and 60% in the test set, with the possibility to segre-gate different stages of TCC.[32] However, urinary biomar-ker panel of TCC, that was validated in the prospectivestudy, has been discovered using CE-MS. Discoveredpanel was further refined using additional urine samplesfrom healthy volunteers and patients with malignant andnon-malignant genitourinary disease. The final patternconsisted of 22 polypeptide masses, and in maskedassessment, it showed 100% sensitivity and specificityfor TCC. One of the identified protein was fibrinopeptideA, known as biomarker of ovarian and gastric cancer.[33]

Performing 2-DE with subsequent MS, other researchpointed to orosomucoid and zinc-a2-glycoprotein aspotential tumor markers of TCC. Furthermore, it wasnoticed that the abundance of these markers in urine wasincreasing according to the stage of the tumors, and thatboth of proteins identified might be related to the devel-opment of bladder cancer.[34] 2-DE analysis of tissue pro-teome identified calreticulin, g-synuclein, and a solubleisoform of catechol O-methyltransferase as other poten-tial TCC biomarkers.[35,36] These proteins were subse-quently identified in urine samples immunochemically,and later analysis of urine specimens supported the useof urinary calreticulin as a biomarker for bladdercancer.[35] Recent research proved that biomarkers forbladder cancer could be in glycosylated protein fractionof urine, by detecting a-1B-glycoprotein, specific forTCC.[37] Squamous cell carcinoma is a much less com-mon type of bladder cancer, and potential biomarker forthis type of cancer is identified as the calcium-bindingprotein psoriasin.[38]

Research interests have made urinary proteomics anespecially fruitful source for biomarkers of cancers.Despite these efforts, most of these studies are based onsmall patient cohorts, and the reported sensitivity andspecificity of urine biomarkers are wide ranging. Largervalidation studies are underway to assess the use ofdetected biomarkers in clinical settings.

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Diagnostic Biomarkers for Diseases of Other Systems

Biomarkers for graft-versus-host disease after bonemarrow transplantation have been defined using CE-MS-based urine proteomics[39] and validated in a prospectivemulticenter study. Recent investigations identified urinarybiomarkers predictive of acute pancreatitis,[40] obstructivesleep apnea,[41] pancreatic ductal adenocarcinoma,[42] andnon-small-cell lung cancer.[43] It is likely that urine testingwill be used in the future to screen for a wide range ofsystemic disorders, the majority of which will have no orlimited renal involvement.

URINARY BIOMARKERS FOR PROGNOSTIC ASSESSMENT

Diabetic Nephropathy

In patients with diabetes mellitus, diabetic nephropathy(DN) is the most important cause of end-stage renaldisease and a major health problem. Diagnosis and assess-ment of DN is based on the presence of microalbuminuria,which is neither a sensitive nor specific early marker of thedisease. By using CE-MS, it was found that urinarypolypeptide pattern of patients with Type 1[44] and Type2[45] diabetes significantly differ from the normal controls,and there was a specific polypeptide pattern of “diabeticrenal damage” in patients with overt proteinuria. Evaluationof the urinary proteome profile of the patients with DN andovert proteinuria, applying 2-D DIGE, identified one spotthat was increased in the diabetic urine as a1-antitrypsin.This finding was also confirmed by an independent groupusing ELISA.[46] Recently, SELDI proteome profiling ofurine samples from DN patients discovered the SELDIpeak that was exclusively present in the urine of patientswith DN and was subsequently identified as UbA52, anubiquitin ribosomal fusion protein. Additionally, it wasfound that the processed form of ubiquitin was selectivelymissing in urine of these patients. These findings suggestthat UbA52 could serve as a potential diagnostic biomarkerof DN, and a lack of processed form of ubiquitin may haveprognostic implications.[47] Additional research estab-lished several proteins that were found to have progres-sively increased expression related to the degree ofproteinuria, whereas the other group of proteins had pro-gressively declined levels in association with the degree ofproteinuria.[48] More recently, SELDI-TOF MS was per-formed to compare the baseline urine samples of PimaIndian patients with Type 2 diabetes who developed DN10 years later to those who remained normoalbuminuriaafter 10 years of the sample collection. This study identi-fied a molecular signature that could differentiate patients

who subsequently developed DN from those whoremained normoalbuminuria and provided 71% sensitivityand 76% specificity for prediction of diabetic nephropathyin an independent validation set.[49] Obtained data empha-size the potential of urinary proteomics to predict DN in along-term period.

Lupus Nephritis

In systemic lupus erythematosus, active glomerulone-phritis is the major cause of renal failure and an importantcause of morbidity and mortality. Disease activity in lupusnephritis (LN) may require multiple biopsies for guiding atreatment. It is expected that new urinary protein biomar-kers should be able to determine class of LN, activity,and progression of the disease, besides providing non-invasiveness. Serial analysis of the urinary SELDI profilesrevealed dynamic changes in levels of low-molecularweight proteins during the course of the LN flare.[50] Otherresearch compared the SELDI proteome profiles of theurine samples from pediatric patients with SLE and thecontrols that had juvenile idiopathic arthritis. It was possi-ble to define the molecular signature of lupus nephritis,and intensities of these protein peaks were greater in urinesamples of patients with lupus nephritis, compared tothose without nephritis and no lupus controls. Some ofthese proteins could be used for the prediction of the dis-ease activity.[51] Urinary proteomic research in patientswith LN revealed two proteins that distinguished activefrom inactive LN, with 92% sensitivity and specificityeach, and multiple regression scores could predict bothrelapse and remission even earlier than traditional clinicalmarkers.[52] Identification of these proteins will allow oneto devise specific assays to routinely monitor disease pro-gression and alter immunosuppressive drug regimensaccordingly. These proteins may also play a critical role inthe pathogenesis of LN and could therefore provide targetsfor therapeutic intervention.

Obstructive Nephropathy

Congenital unilateral ureteropelvic junction (UPJ)obstruction is a frequently encountered pathology in new-borns. Urinary proteomics has been applied to discoverspecific biomarker panel for the prognosis of the disease,which would be helpful in the assessment of the need forsurgical intervention. Urinary biomarkers that were able todistinguish different levels of UPJ obstruction have beendefined applying CE-MS. These protein biomarkers couldpredict with 97% accuracy and several months in advancethe clinical outcome of UPJ obstruction in newborns.

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Moreover, a newly identified marker, proSAAS (proproteinconvertase subtilisin/kexin type 1 inhibitor), generated anew hypothesis in the pathogenesis of UPJ obstruction.[53]

Diagnostic Biomarkers for Diseases of Other Systems

Coronary Artery Disease

Proteomics analysis of urine could yield a panel ofbiomarker peptides that would be useful as additional toolsfor the diagnosis and monitoring of coronary artery dis-ease (CAD). Urinary proteome analysis using CE-MSdefined a characteristic CAD signature panel with highsensitivity and specificity. This peptide pattern signifi-cantly changed toward the healthy signature correlatingwith the therapeutic physical activity level. Identifiedpolypeptides that showed to be upregulated were collagentype I or III fragments. These collagens are increasinglyproduced in thickened intima of atherosclerotic lesionsand fractionated by increased level of collagenases, whichproved to be an independent predictor of rapid lumendiameter reduction and fatal cardiovascular events andprobably play a crucial role in plaque rupture.[54] Therefore,non-invasive urinary proteomics can become a valuableaddition to other biomarkers used in cardiovascular riskassessment in personalized medical approach.

Preeclampsia

Preeclampsia has been implicated in 20% of pregnancy-related maternal deaths and is the leading cause of man-dated preterm delivery. It was reported that women withpreeclampsia requiring mandated delivery exhibit a uri-nary proteomic signature characterized by nonrandomfragments of SERPINA1 (abundant serum protease inhibi-tor) and albumin. Proteomic fingerprint in urine precedesthe onset of clinical symptoms, predicts the severity ofpreeclampsia and need for delivery better than other com-mon methods, and better differentiates preeclampsia fromother uncontrolled hypertensive disorders. Aside fromthe practicality of producing a noninvasive diagnosticand prognostic test, identifying biomarkers of preec-lampsia could lead to an explanation of pathogenesis ofthis condition.[55]

Biomarkers for Therapeutic Response Assessment

Efficacy of the treatment is determined by therapeuticresponse. Moreover, knowing the drug that the patient isnot responding to could provide early initiation of individ-ualized treatment. The aim of the clinical proteomics is to

provide personal medical approach, tailored individuallyfor every patient.

In order to prevent the progression of the disease,standard treatment for patients with IgAN is the use ofangiotensin-converting enzyme inhibitors (ACEI) orangiotensin II receptor blockers. However, some patientsdo not respond to these drugs. Therapeutic response toACEI in IgAN was evaluated by comparative urinary pro-tein analysis. Markers with significantly different urinarylevels between responders and non-responders werekininogen, inter-a-trypsin inhibitor heavy chain 4, andtransthyretin. It is confirmed that very low levels of urinekininogen could serve as a marker for the prediction of thepoor response to the ACEI therapy.[56]

Minimal change disease is the most frequent cause ofnephrotic syndrome in children. The prognosis of pediatricnephrotic syndrome, as well as the need for renal biopsy,correlates with the responsiveness to glucocorticoid therapy.Thus, several studies analyzed urinary proteome with thepurpose to identify urinary biomarkers of steroid resistance.SELDI urinary proteome profiling in pediatric patients withsteroid-sensitive (SSNS) and steroid-resistant nephroticsyndrome (SRNS) could reliably differentiate control fromthe nephrotic urine. What is more, it could distinguishSSNS from SRNS with 100% sensitivity and specific-ity.[57] In another report, a similar approach was used, butdisease stage or activity was also considered. Analysisrevealed a group of SELDI peaks that could distinguishSRNS from the other groups, and one peak was subse-quently identified as b2-microglobulin, thus proving to bea biomarker associated with steroid resistance.[58]

POTENTIAL OF URINARY BIOMARKERS FOR SCREENING

Early diagnosis and treatment are essential for betterclinical outcome in malignant diseases. Several urinarybiomarkers that show high sensitivity and specificity mayprovide the basis for reliable screening tests in the nearfuture. Prostate cancer (PC) ranks third among cancers inmen, and there is an objective need for a more sensitivetumor marker than PSA. There is a report about the identi-fication and validation of a panel of 12 novel biomarkersfor prostate cancer that was discovered in first void urineusing CE-MS. This urinary biomarker panel in combina-tion with PSA could provide a more predictive diagnostictool for prostate cancer screening.[59]

Among gynecologic malignancies, ovarian cancer isassociated with the highest death rate, largely because ofits tendency to present at an advanced stage associatedwith poorer survival. Urinary proteomic analysis in ovariancancer patients resulted in identification of glycosylated

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form of eosinophil-derived neurotoxin (EDN) and a clusterof COOH-terminal osteopontin that were elevated. Theircombination resulted in 93% specificity and 72% sensitivityfor early-stage ovarian cancer. These findings could beused for the development of potential noninvasive screeningtests for early diagnosis of ovarian cancer.[60]

SPECIFIC PROBLEMS AND LIMITATIONS OF URINARY PROTEOMICS

Regardless of the impressive potential of urinary pro-teomics, specific methodological problems impede thetranslation of new biomarkers to clinical settings. Theshortcomings of current proteomic studies, such as modestnumber of subjects, absence of disease controls, smallnumber of defined biomarkers, and diversity of analyticalplatforms, are making it impossible to merge all biomarkersinto human urinary proteome database.[61] Moreover, thereis a lack of standardization in many proteomic techniquesfor future clinical application.

In order to obtain consistent and reliable data thatcould be comparable, protocols for urine sampling andhandling, storage, shipment, enrichment, and quantificationhave to be standardized.[62] As it is not possible to obviatesignificant changes in the urinary proteome due to age,gender, and diet, these factors should be matched betweencases and controls in the study design. To achieve greaterspecificity, controls should include not only healthy sub-jects, but also patients with similar diseases, as it is possiblethat similar pathological processes are represented withundistinguished urinary proteomic profiles.[63]

The application of a new biomarker depends on its pre-dictive capabilities to determine specific clinical state. Inmany proteomic studies, the validation process of newbiomarkers is lacking, or it has been conducted on smallnumber of patients and did not show satisfying predictivepower. For the precise determination of applicability of newbiomarkers, there is an objective need for prospective vali-dation studies on large patient cohorts, which will involvescreening test in a relevant population to determine specific-ity and sensitivity of discovered urinary biomarkers.[64]

The Human Kidney and Urine Proteome Project(HKUPP) has been designed with the goal of finding waysfor analyzing urine specimens and standardizing methodsand data so that large-scale studies can be properly com-pared. Further enhancement of networking activities hasbeen initiated as EuroKUP (European Kidney and Urine Pro-teomics) by bringing together scientists and clinicians work-ing in this field to promote expertise and collaborations.[65]

With united efforts of science and industry, it will be possibleto surpass existing limitations, and we could expect the clini-cal application of some urinary biomarkers in the near future.

CONCLUSIONS

Over the past few years, proteomics has proved itself asan extremely promising method regarding protein analysis.Current research effort and financial investment in thisfield undoubtedly confirm the great expectations ofanswers that urinary proteome analysis might offer. How-ever, the discovery of biomarkers in urine is still a hugechallenge, even with the analytical tools that have beendeveloped and steps made toward method standardization.Only a few of the potential candidates that are identified inthese studies will turn out to be clinically useful biomarkers.Nevertheless, it is crucial for discovered biomarkers tofind their way into proper clinical trials so that theirefficacy as disease specific markers can be validated. Ifthe necessary effort is not directed toward putting suchdiscoveries into clinical practice, then proteomics maylanguish as a technology that creates large databases ofminimal use.

Contemporary proteomics studies of urine areexpected to provide the possibility of the development ofnew non-invasive tests and procedures, along with thepotential advantage of lower costs and higher efficiency ofpatient care. Future clinical application of novel urinarybiomarkers is expected to improve early detection andobviate the need for invasive diagnostics, particularly inkidney diseases and cancers. Moreover, urine biomarkertesting could provide tools for prognostic assessment andfacilitate monitoring the course of the disease and responseto therapy, with the possibility to tailor the therapy accord-ingly. Finally, some of the discovered biomarkers could bethe targets for novel therapeutic interventions. Adjustmentof proteomic analytical methods to current clinical settingswill enable the application of proteomics at the bedside ofthe patient, which would direct medical practice towardpersonalized medicine.

ACKNOWLEDGMENTS

This work was supported by grant number 145004, fromthe Ministry of Science and Technological Development ofSerbia.

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Urinary Proteomics—a Tool for Biomarker Discovery