Diagnostic Potential of Urine Proteome: A Broken Mirror of Renal Diseases
Michael S. Goligorsky,
Francesco Addabbo and
Edmond O'Riordan
Departments of Medicine and Pharmacology and Renal Research Institute, New York Medical College, Valhalla, New York
Correspondence: Dr. Michael S. Goligorsky, New York Medical College, 26 Research Way, Valhalla, NY 10595. Phone: 914-594-4731; Fax: 914-594-4732; E-mail: michael_goligorsky{at}nymc.edu
This brief overview of studies into the urine proteome illustratesits potential value for diagnostic, prognostic, and pathophysiologicdiscovery. Hypothesis-targeted investigations of individualproteins as well as proteome-wide searches for urinary biomarkersof various diseases and their progression are reviewed. Themajority of urine proteins appear as cleavage products thatare found not only as free solutes but also in secreted membranevesicles called exosomes. Described are several recent examplesof important diagnostic findings using urine proteomics alongwith the idea that signature profiles of injury to individualnephron segments can be measured by this technology. Sharedare some thoughts on the most challenging step: Integrationof seemingly unrelated findings of various protein fragmentsinto a rational pathogenetic pathway(s). The future chance thatthe centuries-old technique of uroscopy will reveal its secretsusing modern proteomic approaches makes gradual improvement.
Proteinuria, a cardinal symptom of renal disease, has long beenconsidered as a potential "black box" for diagnostic and evenprognostic information. Urine proteins arise from various sources,including filtration of plasma proteins; impaired reabsorptionof filtered proteins; and appearance of proteins that originatefrom injured glomeruli, tubules, infiltrating inflammatory cells,or connective tissue as well as those that enter the urine inthe urinary tract below the kidney. Not surprising, many traditionalstudies have focused on the excretion of (1) individual proteinssuch as enzymes or albumin, (2) selectivity of proteinuria,and (3) attempts at global characterization of urine proteinsusing two-dimensional electrophoresis. Insufficient resolutionof many techniques, the lack of understanding of the physiologicand pathologic underpinnings of proteinuria, and frequent appearanceof protein fragments that are undetectable using standard approachesall hampered early attempts to extract information from thisubiquitous resource. Recent progress in our understanding ofmolecular mechanisms of renal handling of proteins, especiallyalbumin,1,2 combined with the ongoing revolution in the technologicaltools for peptide detection, quantification, and identification,3–7have re-energized attempts to obtain proteomic footprints ofrenal disease. In this brief review, we recount the most importantfindings made so far in the field of urine biomarker proteins,reflect on the capabilities and limitations of these biomarkers—frequentlyprotein fragments—in diagnosing the disease, and discussthe tortuous intellectual routes that lead to the faithful reconstructionof pathogenetic mechanisms of the disease from these seeminglyunrelated protein fragments (integrating the image from piecesof the broken mirror).
Although the actual amount of protein and albumin filtered byglomeruli remains a highly controversial issue,1 a complex mapof the slit diaphragm and podocyte with their protein makeupis rapidly emerging. This map should help in the understandingof the breadth of glomerular proteins that are contributed tothe tubular fluid after glomerular injury. Since the discoveryof nephrin, a host of protein components of the filtration barrierare known.8 Nephrinuria, for example, has been recognized asa feature of diabetes and diabetic nephropathy.9,10 Among 40normoalbuminuric patients with type 1 diabetes, nephrinuria,as judged by immunorecognition of protein bands with molecularweight of 18-, 32-, 40-, 60-, 75-, and full-length 185-kD proteins,was detected in 30% of cases, whereas nephrin was undetectableby Western blot analysis in the urine of healthy individuals.Is it possible that nephrinuria may serve as an early warningof impending nephropathy, and studies to address this questionare in progress in Holthofer's laboratory.
In parallel, a more detailed picture of the machinery for tubularprotein reabsorption is emerging. Nonspecific receptors megalinand cubilin and their internalization, lysosomal degradationof protein cargo, and recycling to the luminal plasma membranein the proximal epithelium provided important insights intothe origins of tubular proteinuria.11 In fact, it has becomeincreasingly clear that most proteins are excreted as proteolyticfragments rather than intact molecules and, therefore, are poorlydetectable using today's standard approaches.12 For instance,90% of albumin is degraded to small, <10-kD fragments. Similardegradation has been documented for other proteins, includingtransferrin, apolipoprotein, IgG, glucose oxidase, and lactatedehydrogenase.13,14
Commonality in the excretion patterns of certain proteins ortheir fragments in various diseases offers critical insightsinto the default mechanisms of pathologic processes. For instance,there are well-established markers of tubular injury (e.g.,2-microglobulin, prealbumin, lactate aminopeptidase) or infectiousprocesses, such as the release of defensins.15 Megalin itselfis normally processed via intramembrane proteolysis characterizedby the protein kinase C–regulated metalloprotease-mediatedshedding of its large ectodomain,16 and it remains to be elucidatedhow this process may be altered in kidney disease.
One of the techniques used to distinguish between generic andthe site- or disease-specific excretory proteins during proteome-wideanalyses is by stratifying overlapping occurrence of the proteinsin the former category among patients with diverse diseasesand contrasting it with unique occurrences in the latter. Thistype of analysis can be represented by a Venn diagram, as shownin Figure 1. The figure demonstrates a representative analysisperformed by Gene@work-based software of protein peaks characteristicof interstitial fibrosis, tubular injury/atrophy, and vasculopathyin patients with biopsy-graded chronic allograft nephropathy(CAN). The presentation is segregated into classes of proteinpeaks that are characteristic for the individual histologicfeature, peaks that are shared by two histologic features, andpeaks that are common to all three major histologic presentationsof CAN. The presence of peptides in the first group suggeststhat there is a theoretical possibility that each morphologicabnormality of CAN is definable, whereas the other two categoriesprovide a basis for believing that certain proteomic markersare common to all patients with CAN.
Figure 1. Segregation of protein peaks (shown as the type of protein chip and molecular mass) with the specific histologic manifestations of chronic allograft nephropathy. Ci, interstitial fibrosis; Ct, tubular atrophy; Cv, chronic vascular injury. Note that a subset of proteins is common for all three, some protein peaks segregate with only two patterns, and several protein peaks are specific for each pattern.
INTUITIVE SELECTION OF URINARY CANDIDATE BIOMARKER PROTEINS
Enzymuria
Enzymuria has long been considered as a potentially informativemarker for the detection of tubular injury on the basis of theprediction that the enzymes should leak into the urine fromdamaged tubular epithelia. Increased urinary excretion of alysosomal enzyme N-acetyl-glucosaminidase has been reportedin contrast nephropathy.17 Brush border enzymes, such as alkalinephosphatase, -glutamyltranspeptidase, and ala(leu-gly)-aminopeptidase,are elevated in acute renal injury.18 Westhuyzen et al.19 demonstratedthat alkaline phosphatase and -glutathione S-transferase markersof the brush border and distal tubular epithelia, respectively,have predictive value for development of acute renal failure(sensitivity 100%, specificity 91%) in patients who are admittedto a general intensive care unit. The major limiting factorfor these markers consists of the high sensitivity of releaseby tubular epithelia even in mild injury, which does not necessarilyportend development of acute renal failure. This is exemplifiedby a study of patients who underwent coronary bypass surgeryand almost uniformly had postoperative increases in urine excretionof N-acetyl-glucosaminidase but did not succumb to acute renalfailure.20
Devarajan's group21 screened 71 patients who were undergoingcardiopulmonary bypass (a carefully selected group of patientswithout additional confounding problems) for the urinary excretionof neutrophil gelatinase–associated lipocalin (NGAL).NGAL excretion 2 h after cardiopulmonary bypass was a powerfulindependent predictor of acute kidney injury, diagnosed in thisstudy as a 50% rise in plasma creatinine from baseline, showingthe sensitivity of 100% and specificity of 98% for a cutoffvalue of 50 µg/L (only one of 51 patients without acutekidney injury had urinary NGAL level >50 µg/L). Thissuccessful strategy capitalized on the previous unbiased experimentalfindings obtained through cDNA microarray screening and putforward a role for infiltrating inflammatory cells as importantcontributors to biomarker profiles from urine. Another inflammatorymediator, IL-18, at levels >100 pg/ml in the urine of 52patients with acute respiratory distress syndrome was associatedwith a 6.5-fold increased odds for acute kidney injury in thenext 24 h.22 Importantly, elevated IL-18 was detected 24 to48 h before development of this acute kidney injury.
Screening of subtraction libraries in an animal model of acuterenal failure revealed an early upregulation of a secreted cysteine-richprotein 61 (CYR61), a growth factor–inducible immediateearly gene, and was found to be enriched in the urine of rats4 to 8 h after renal ischemia.23 Similarly, kidney injury molecule-1,found to be highly upregulated in postischemic rat kidney,24has been detected in the urine of patients with ischemic acuterenal failure confirmed by renal biopsy,25 as was the elevatedurinary excretion of liver-type fatty acid–binding protein.26
Aquaporinuria in the Diagnosis of Hypo-osmolar and Other Syndromes
Aquaporin-2 (AQP2) is one of the most studied proteins excretedin urine; it represents a reliable example of a well-establishedurinary biomarker that is critical for diagnosis of severaldisorders of renal water balance. Kanno et al.27 studied theurinary excretion of AQP2 in diabetes insipidus using a RIA.Elliot et al.28 demonstrated urinary AQP2 as a marker of collectingduct responsiveness to vasopressin. Valenti et al.29 showedchanges in AQP2 expression and excretion in conditions suchas nocturnal enuresis characterized by hypercalciuria or duringfollow-up in patients who underwent surgical relief of congenitalureteral obstruction.30 Martin et al.31 also provided evidencethat the excretion of AQP2 in the urine of patients who hadcongestive heart failure (NYHA class II to III) and receiveda V2-vasopressin receptor antagonist was decreased in proportionwith the resulting increase in free water clearance. These findingsintroduce the idea that AQP2 may serve as a marker of therapeuticefficacy.
Recently it was demonstrated that AQP2 is also excreted in theurine as part of secreted exosomes.32 Exosomes are small vesiclesthat are derived indirectly from the apical endosomal system.Among 295 proteins identified, AQP2 (potential marker of nephrogenicdiabetes insipidus), polycystin-1 (autosomal dominant polycystickidney disease type 1), podocin (autosomal recessive steroid-resistantnephrotic syndrome), nonmuscle myosin II (potential marker ofFechner syndrome and Epstein syndrome), angiotensin-convertingenzyme (hypertension), Na+K+/2Cl– co-transporter (Barttersyndrome type 1), thiazide-sensitive Na+/Cl– co-transporter(Gitelman syndrome), and epithelial sodium channel (Liddle syndromeand autosomal recessive pseudohypoaldosteronism type 1) areexcreted on exosomes. The studied exosomes were obtained byultracentrifugation of a large volume of urine (400 ml), andproteins were initially separated by electrophoresis followedby in-gel trypsin digestion. The tryptic peptides were analyzedby nanospray liquid chromatography–tandem mass spectrometry(MS). A modification of this technique was recently used todetect more than a 50-fold increase in fetuin-A excretion inexperimental animals and patients with acute kidney injury.33
Technological platforms that are used in the discovery phaseof proteome-wide research have been reviewed elsewhere,34 andwe only recount them here.
MS
There are several types of mass spectrometers35 that use theassortment of electrical, radio frequency, and magnetic fields:Time-of-flight, quadrupole, ion trap, and Fourier transformion cyclotron resonance.34 Mass spectrometers lack the abilityto quantify the protein detected, the drawback that can be circumventedusing isotope-coded affinity tagging. Because of the competitionbetween proteins and peptides for capture of charges, the detectionof low-abundance species may be compromised. Therefore, additionalseparation strategies are required.
Two-Dimensional Electrophoresis.
The cartesian position of a protein is a product of its pI andmolecular mass. This technique has the ability to be quantitativeeither through comparison of spot size between two gels or bya differential in-gel electrophoresis. The shortcomings of two-dimensionalelectrophoresis include that more than one protein per spotcan exist and that multiple spots can contain the same posttranslationallymodified protein, at times making abundance profiling problematic.Furthermore, very small or large proteins and very acidic orbasic proteins are not visualized.
HPLC.
HPLC is applied to the urine and includes size exclusion, reversephase, strong and weak cation binding and affinity binding (i.e.,Ig adsorbing to protein A). One of the disadvantages of thissystem is that a buffer dilutes the proteins, thus requiringrepeated re-concentration of the sample and making quantificationproblematic.
Capillary Electrophoresis.
Capillary electrophoresis is a powerful separation technology,but it lacks the ability to quantify absolutely the proteome.36
Protein Array Technologies
This concept is similar to gene microarray with antibodies ortissue sections robotically placed on a glass slide. Techniquesfor protein–protein (including antigen–antibody),protein–DNA, protein–lipid, and protein–druginteractions exist.37 Limitations include accurate quantificationand dependence on the availability of antibodies. This technologyhas yet to be applied to renal proteomics. A variation of thistechnique is Luminex multiplex analysis, which is based on thearray of polystyrene microspheres with two spectrally distinctfluorochromes. Using the precise ratio of these fluorochromes,100 bead sets have been created, each with its unique color-codedsignature. Each signature bead is conjugated to an analyte-specificantibody and combined in a single assay to measure up to 100analytes in up to 96 samples simultaneously. The assays arebased on the conventional two-site sandwich method. After conjugationreaction, a mixture of beads is analyzed using a dual wavelengthlaser flow cytometer–like apparatus. One laser beam detectscolor-coded beads, and another quantifies the reporter signalon each bead. These techniques may be better suited for a moretargeted analysis of isolated proteins.
Bioinformatics
Several databases are used to identify protein fragments bypeptide mass fingerprinting after trypsin digestion or afterMS/MS sequencing. Databases compare the size of the fragmentsrecorded by the mass spectrometer with the translated DNA sequence;search for trypsin digestion sites, comparing the theoreticalwith the measured values; and calculate the probability of acorrect match. Logistical analysis of the findings is conductedusing the following:
Tree-based technologies such as RandomForest:As the samplesare classified according to the proteins thatmost accuratelyclassify the whole population, an inverted treeis formed.
Adaboost: A method for combining weak classifiersto createa summary and stronger classifier: The basic principleis thatafter selection of the variable that is most likelyto predictcorrectly the class of a sample, the samples arereweightedwith increasing weight applied to the misclassifiedsamples;the next best classifier variable is then selected.This processis then repeated with summation of the classifiersto createa robust and accurate classifier.
Genetic algorithms,neural networks, and unified maximum separabilityanalysis:These are alternative approaches that have also provedto beuseful in classification that is based on spectral data.
In summary, it is critical to appreciate that there is no singleproteomic or informatics technique to fit the diverse requirementsof analyses; therefore, combination of several approaches offersthe optimal solution to the problem.
Using the arsenal of technological and bioinformatics toolsalready discussed, the following findings have been reported.
Nephropathies
In a study of 57 control urine samples compared with samplesfrom patients with minimal-change disease (n = 16), membranousnephropathy (n = 18), and FSGS (n = 10), a group of 690 polypeptideswere present in >50% of all normal samples. Plots of >500polypeptides typical of each disease were compiled. The ratesof correct classification were 71.4% for minimal-change disease/FSGSand 92.9% for membranous nephropathy.36
Woroniecki et al.38 studied steroid-resistant nephrotic syndromein a pediatric population with idiopathic nephritic syndrome.A protein of mass 4144 Da was identified as the single mostimportant marker for distinguishing steroid-sensitive and steroid-resistantpatients with a high level of confidence.
Distinct polypeptide signatures also seem to be associated withIgA nephropathy. In a study of 45 patients—including thosewhose total urinary protein levels were within normal ranges39—theurinary peptide patterns had a sensitivity of 100% and a specificityof 90%. Three of the most promising polypeptides were sequencedand shown to be albumin fragments. IgA nephropathy could bedifferentiated from membranous nephropathy with a sensitivityof 77% and a specificity of 100% and from minimal-change disease,FSGS, and diabetic nephropathy with a sensitivity and a specificityof 100%.
Cutillas et al.40 applied three different techniques to examinethe Dent disease proteome both qualitatively and quantitatively.They found that carrier proteins, complement components, andbioactive peptides were excreted at higher concentrations inpatients with Dent disease.
Proteome of the Transplanted Kidney
Three investigative groups have reported proteomic diagnosisof acute renal allograft rejection. Clarke et al.41 found thatproteins of 6.5, 6.6, 6.7, 7.1, and 13.4 kD performed well asbiomarkers of acute rejection. Proteins in the mass ranges 5270to 5550 and 10,530 to 11,000 Da were reported to be good biomarkersby Schaub et al.,42,43 and a subsequent report identified theseproteins as -2 microglobulin and its fragments. O'Riordan etal.44 identified urine proteins with masses of 4756.3, 25,665.7,and 19,018.8 Da as candidate markers of acute kidney transplantrejection compared with recipients with stable transplants.Multiple protein peaks provided a more accurate assessment thanrelying on only single biomarkers. In a more recent study, O'Riordanet al.45 chemically identified 1-defensin and anti-chymotrypsinas valuable candidate biomarkers of acute rejection. We alsoobtained preliminary data suggesting that chronic allograftnephropathy is associated with the increased degradation ofperlecan and urinary excretion of its fragment endorepellin(unpublished observations, M.S.G. and E.O.).
Diabetic Nephropathy
When 29 healthy individuals were compared with 112 patientswith type 2 diabetes, a distinct "no albuminuria/diabetic pattern"was detected. Another distinct pattern was observed in patientswith albuminuria >100 mg/L. Found in 35% of patients withelevated urinary albumin excretion rates and only 4% of healthyvolunteers, this pattern identified individuals who were morelikely to have retinopathy. The characteristic polypeptideswere insulin-like peptide 3, uromodulin, and an albumin fragment.46The putative role of nephrinuria as a potential biomarker ofdiabetic nephropathy has been mentioned.
Renal Cancer
Rogers et al.47 analyzed urinary proteome in patients with clearcell renal carcinoma and compared it with that of healthy volunteersand patients with other urogenital diseases. In another study,kininogen levels were found to be elevated in the urine of apatient with renal cancer; the concentration fell after nephrectomy.7Urinary proteomic analysis has identified several biomarkersof bladder cancer: -Synuclein, a soluble isoform of catechol-O-methyltransferase,and calreticulin, which, when tested prospectively, were foundto have a combined sensitivity of 76.8% and a specificity of77.4%.48
It would be unrealistic to expect that each disease processis identifiable by unique signature proteins in the urine, whichhave been obtained during a previous validating snapshot analysis.Different stages of disease, variations in mechanisms, and othercomorbidities are likely to modify urine proteome. An exampleof the dynamics among signature proteins in the urine is presentedin Figure 2, where the results of proteomic analyses are plottedagainst the Banff-defined stages of CAN. Knowledge of proteindynamics in the course of disease processes may be helpful intheir staging and in monitoring response to therapy.
Figure 2. Exemplary proteins identified by mass:charge ratio show the dynamic patterns of urinary excretion in patients with differing Banff grades of chronic vascular injury in chronic allograft nephropathy. Note that individual peptides show a relative grade dependence in the level of urinary expression.
FROM DEDUCING PROTEIN IDENTITY TO INTEGRATING THE FINDINGS INTO THE PATHOGENETIC PATHWAY: A LONG WAY TO GO
Let's consider an optimistic scenario: With multiple databasesdeveloped for assistance in chemical identification of proteins,the investigator, after completing cross-sectional and prospectiveanalyses, eventually confirms the diagnostic value of an ensembleof biomarker peptides/proteins. As important as it is by itself,the actual understanding of the defined markers can be attainedonly when the mechanisms of their appearance/disappearance inthe urine become elucidated. Reconstruction of molecular pathwaysthat are involved in these processes and their integration intosystems biology are aided in silico by the growing collectionof publications dealing with components of individual pathways,as well as by the emergence of several software suits for pathwayanalysis, yet the connectivity of diverse pathways through multiplecomponents of an individual pathway results in a complex arborizedstructure that requires actual analysis of multiple componentsto profile the correct one, as illustrated in Figure 3. Takinginto account that urinary proteins reflect not only the pathway(s)but also the mechanism(s) of the appearance or disappearanceof a certain peptide/protein component of a pathway in the excretorycompartment, this analysis becomes even more complicated. Forinstance, we recently identified a group of matricellular proteins—allproducts of the enzymatic cleavage of larger molecules—thatappear in the urine of patients with CAN; importantly, the sameenzyme is responsible for the activation of the latent TGF-(unpublished observation, M.S.G. and E.O.), thus potentiallylinking several pathways leading to fibrosis. Integration ofproteomic findings into pathogenetically rational pathways requiressubstantial investiture. Integration can be assisted by theanalysis of cDNA microarrays (e.g., detection of increased expressionof defensin and anti-chymotrypsin mRNA corroborated the findingsmade in acute renal allograft rejection and highlighted theimportance of pro- and anti-inflammatory pathways).45 Tissueanalysis of proteins in question could be best met using immunohistochemicalanalyses of multiple intermediates and/or through the recentlydescribed MS analysis of tissue sections. The latter permitsthe acquisition of protein profiles from individual nephronsegments under microscopic guidance,53 an excellent exampleof fusion between histology and proteomics.
Figure 3. Blueprints for integration of protein/peptide findings into metabolic pathways: Systems biology approach. Proteins/peptides that belong to diverse pathways (depicted as separate planes) may be linked via cross-talk between the pathways (blue lines) or promoter elements (entries into each pathway), common enzymes (brown semicircles) acting on individual elements from different pathways, or comprising feedbacks between pathways (red lines). Proof of such links may require additional targeted studies to reconstitute connectivity, enzymes, or regulatory elements (blue). In addition, tissue analysis could unveil whether changes in the urinary proteome are a result of parallel or reciprocal changes in the abundance of biomarker proteins.
This brief overview was intended to proselytize for proteomicsapproaches to renal disease and intentionally overlook the aboundingproblems—these are addressed in several recent reviews.49–52In short, many technical and bioinformatics issues await resolution,yet initial important findings are emerging, and the whole fieldof inquiry is undergoing exponential growth. Through accumulationof candidate biomarkers and their validation in larger patientpopulations, various signature combinations of proteins andtheir fragments should eventually become available for manydiseases and their stages. Studies of the urinary proteome areinseparable from in-depth morphologic analysis of the kidney,proteomic mapping of different nephron segments (as has beenpioneered by Fogo and Caprioli's laboratories53), and intracellularorganelles (e.g., mitochondrial proteome, lysosomal proteomein health in various diseases54). With further technologicaldevelopments, more precise mapping of the urinary proteome inhealth and disease, with in-depth analysis of the proteome ofdifferent nephron segments, individual cells and intracellularcompartments, and accumulation of knowledge of posttranslationalmodifications, the ancient art of uroscopy hopefully will becomea modern tool for physiologic, pathologic, and diagnostic inquiries.
These studies were supported in part by National Institutesof Health grants DK45462, DK54602, and DK52783 (M.S.G.) andthe Westchester Artificial Kidney Foundation. E.O.R. was a recipientof the Kevin J. and Gloria B. Kiely National Kidney FoundationFellowship.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
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