THE URINARY PROTEOMICS:A TOOL TODISCOVER NEW AND POTENT BIOMARKERS FORKIDNEY DAMAGE

1. THE URINARY PROTEOMICS:A TOOL TO
DISCOVER NEW AND POTENT
BIOMARKERS FOR
KIDNEY DAMAGE
ByBy
Prof. Moustafa Rizk
Prof. of Clinical Pathology
Faculty of Medicine, University of Alexandria
Wednesday 07/04/2010
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2. PROTEOMICS
TranscriptomicsTranscriptomics
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3. What is proteomics?
Proteomics is the systematic study of proteomes,
which describes the entire protein content of
one or all cells of an organism as well as of
bodily fluids such as blood, urine and sweat.
While the genome of an organism is considered to be mostly static,
the proteome shows dynamic properties with protein profiles
changing depending on a variety of extra- and intracellular stimuli
(i.e. cell cycle, temperature, differentiation, stress, apoptotic signals)
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4. TYPES OF PROTEOMICS
1-Expression proteomics, : This is the
qualitative and quantitative study of the
expression of total proteins under two different
conditions. For example, expression
proteomics of normal cells and diseased cells
can be compared to understand the protein that
is responsible for the diseased state or the
protein that is expressed due to disease.
3-Functional Proteomics:This is an
assembly type of proteomic method to
analyze and understand the properties of
macromolecular networks involved in the life
activities of a cell.
4-Clinical proteomics, a comparative approach of
normal and abnormal status of cells, tissues or bodily
fluids , is still a promising new analytic discipline with the
following main aims:
(i) discovery of biomarkers allowing early detection, risk
management or therapeutic monitoring of diseases for the
establishment of individualized treatment procedures.
(ii) identification of protein targets for the development of
new mechanistic intervention therapies with the promise of
an improved clinical outcome.
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2-Structural Proteomics:Structural
proteomics, as the name indicates, is about
the structural aspects, including the three-
dimensional shape and structural
complexities, of functional proteins

5. So a major goal in the field of clinical proteomics
is to identify disease biomarkers in biological
fluids that can be measured relatively
inexpensively for early diagnosis of disease.
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6. An important challenge in this process is to develop a
rational means of reducing the complexity of the
proteome of body-fluid samples to enhance the
detectability of relatively low-abundance proteins that
may have special pathophysiological significance.
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7. Most of the focus thus far has been on
proteomics of blood (serum or plasma) .
Because urine can be collected noninvasively
in large amounts, it provides an attractive
alternative to blood plasma as a potential
source of disease biomarkers .
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8. The increasing number of patients suffering from
chronic renal failure represents one of the
major challenges which the nephrologists are
facing worldwide.
For a better therapeutic outcome of this
disease, earlier detection is urgently warranted
in routine clinical practice.
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9. Origin of urinary proteins and peptides :
1- Glomerular filtration
2- Tubular secretion
3- Epithelial cells shed from the kidney and
urinary tract
4- Secreted exosomes
5- Semen.
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10. .
Glomerular filtration Tubular reabsor.
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11. Urinary exosomes containing apical membrane
and intracellular fluid are normally secreted
into the urine from all nephron segments, and
may carry protein markers of renal dysfunction
and structural injury.
Derived from all cell types that face the urinary
space including glomerular podocytes, renal
tubule cells, and the cells lining the urinary
drainage system 05:04 AM11

12. Process of exosome formation and release into the urine
Hoorn, E. J., Pisitkun, T and Knepper, M. A. (2005) Prospects for urinary proteomics:
exosomes as a source of urinary biomarkers. Nephrology (Carlton) 10, 283–290
1- Apical membrane proteins
undergo endocytosis followed
by targeting to the
MVB.
2- The membrane proteins are
segregated initially in the MVB
outer membrane and then are
internalized by membrane
invagination, encapsulating
cytosolic proteins in the process.
3- After accumulation of
numerous internal vesicles, the
outer membrane of the MVB
fuses with the apical plasma
membrane releasing its internal
vesicles, called exosomes, into
the urinary space.
4- Exosomes contain both membrane and cytosolic proteins.
Multivesicular bodies
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13. Urine sample collection, storage, and processing
procedures for isolation of urinary exosomes
Sample collection (protease inhibitor) solution (volume per 50 ml urine)
1.67 ml of 100 mM sodium azide (NaN3)
2.5 ml PMSF (2 mg/ml in isopropyl alcohol, stable 4°C for several months)
50 µl Leupeptin (1 mg/ml in ddH2O, stable 1 week at 4°C, 6 months at -20°C)
Collection
If possible, determine the collection time and volume.
Collect the first or second morning urine, 10~100 ml .
First morning urine: first urine after waking (no fluid or fruits after 9 PM the prior evening).
Second morning urine: Discard the first urine. Collect the next voided urine. May have breakfast and undergo regular
activity between first and second morning urine.
Can also use random urine samples
Storage
Add sample collection (protease inhibitor) solution
If possible, process samples immediately (refrigerate at 4 °C).
If the urine samples cannot be processed immediately, the samples should be frozen at -80 °C (NOT -20 °C).
50 ml of urine samples are aliquoted in 50 ml tubes and stored at -80°C as soon as possible after collection.
Defrosting and processing
Frozen urine samples should be thawed at room temperature (requires 3 hrs for a 50 ml urine sample). Avoid prolonged
thawing on ice (4 °C).
While urine is defrosting (i.e., is still a mixture of ice and water), extensively and vigorously vortex for one minute.
After sample completely thaws, vigorously vortex for an additional 30 sec, then proceed to differential centrifugation for
urinary exosome isolation.
Insufficient vortexing will result in major loss of urinary exosomes.
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14. Classifications of exosomal identified proteins
from renal epithelia
1 - Glomerular podocytes (podocin and podocalyxin)
2 - Proximal tubule (e.g., megalin, cubilin, APN, AQP1)
3 - Ascending limb of Henle (e.g., THP, CD9)
4 - Distal convoluted tubule (e.g., NCC),
5 - Collecting duct (e.g., AQP2, mucin-1, and the Rh type C )
6 - Transitional epithelium of the urinary bladder ( uroplakin-1 and -2)
Thus, proteomic analysis of urine can potentially provide insight into the
physiological or pathophysiological processes in every epithelial cell type
facing the urinary space.
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15. Naturally occurring human urinary peptides and proteins
Establishment of a high resolution proteome database ranging
from 800–17,000 Da from over 3,600 individual samples
using capillary electrophoresis coupled to mass
spectrometry, yielding an average of 1,500 peptides per
sample.
All processed data were deposited in an SQL database,
currently containing 5,010 relevant unique urinary peptides
that serve as classifiers for diagnosis and monitoring of
diseases, including kidney and vascular diseases.
Of these, 352 have been sequenced to date.
Nature Precedings : hdl:10101/npre.2007.1219.1 : Posted 11 Oct 2007 05:04 AM15

16. Nature Precedings : hdl:10101/npre.2007.1219.1 : Posted 11 Oct 2007
CE-MS data from 28 different pre-selected pathophysiological conditions
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17. Distribution of native peptides identified with respect to their protein precursor (described
by SwissProt protein name and gene symbol). Comparison of peptides to other references)
A database of naturally occurring human urinary peptides and proteins for use in clinical applications
Nature Precedings : hdl:10101/npre.2007.1219.1 : Posted 11 Oct 2007
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18. HC=healthy control;
CRD= chronic renal disease;
MNGN=membranous
glomerulonephritis;
FSGS=focal segmental
glomerulosclerosis;
MCD=minimal change disease;
SLE=systemic lupus
erythematosus;
IgAN=IgA nephropathy,
DN=diabetic nephropathy;
CAD=coronary artery disease;
NTx=renal transplantation;
LTx=liver transplantation;
HSCT=hematopoietic stem cell
transplantation;
PTLD=post-transplant
lymphoproliferative disorders;
Fanconi=Fanconi’s syndrome;
AD=Alzheimer’s disease;
DM=diabetes mellitus.
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19. HC=healthy control;
CRD= chronic renal disease;
MNGN=membranous
glomerulonephritis;
FSGS=focal segmental
glomerulosclerosis;
MCD=minimal change
disease;
SLE=systemic lupus
erythematosus;
IgAN=IgA nephropathy,
DN=diabetic nephropathy;
CAD=coronary artery disease;
NTx=renal transplantation;
LTx=liver transplantation;
HSCT=hematopoietic stem cell
transplantation; PTLD=post-
transplant
lymphoproliferative disorders;
Fanconi=Fanconi’s syndrome;
AD=Alzheimer’s disease;
DM=diabetes mellitus.
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20. Proteomics is increasingly used in the discovery of
renal biomarkers mostly in:
1 - Cancer screening
2 - Glomerular diseases in native kidneys
3 - Acute rejection
4 - BK virus nephropathy as a complication in kidney
transplants
Early detection of graft rejection
Specific new biomarker candidates for other renal disease
detected by proteomic analysis
5- Predict the need for operation in newborns presenting
with unilateral uretero-pelvic junction obstruction05:04 AM20

21. Cancer screening
1- Renal cell carcinoma (RCC) is the sixth leading
cause of cancer death and is responsible for 11,000
deaths per year in the US.
2- In this study, a comprehensive proteomic analysis
to identify biological processes involved in clear cell
RCC (ccRCC).
3- Urinary markers of RCC were investigated
which could be applied to high-risk patients, or to
those being followed for recurrence, for early
diagnosis and treatment, thereby substantially
reducing mortality of this disease.
Pathway analysis of kidney cancer using proteomics and metabolic profiling.Bertrand Perroud, et
al Mol Cancer. 2006; 5: 64. 05:04 AM21

22. Using 2-dimensional electrophoresis and mass
spectrometric analysis, 31 proteins were identified which
were differentially expressed with a high degree of
significance in ccRCC as compared to adjacent non-
malignant tissue, and some of these were confirmed by
immunoblotting, immunohistochemistry, and comparison
to published transcriptomic data.
Pathway analysis of kidney cancer using proteomics and metabolic profiling.Bertrand Perroud, et al Mol
Cancer. 2006; 5: 64.
BlueBlue : Underexpression
BlackBlack: Equal expression
RedRed : Overexpression
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23. A highthroughput proteomic approach was applied to the evaluation of
urine samples from type 2 diabetic patients and identified a protein
profile that accurately predicted nephropathy in advance of an
increase in albuminuria. These results warrant further studies to
determine the applicability of this approach within other populations,
and, more specifically, within other larger cohorts of diabetic patients
with prospectively collected samples.
Finally,further characterization and evaluation of the proteins in the
biomarker profile are needed to demonstrate whether they are
biologically important in the development of nephropathy.
Prediction of Diabetic Nephropathy Using Urine Proteomic Profiling 10 Years Prior to
Development of Nephropathy.Diabetes Care 30:638–643, 2007
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24. 05:04 AM24

25. Detection of Acute Tubulointerstitial Rejection by Proteomic
Analysis of Urinary Samples in Renal Transplant Recipients
A distinct urinary polypeptide pattern identified 16 out
of 17 cases of acute tubolointerstitial rejection, but
was absent in two cases of vascular rejection. Urinary
tract infection resulted in a different polypeptide
pattern that allowed to differentiate between infection
and acute rejection in all cases..
Detection of acute rejection by CE-MS offers a
promising non-invasive tool for the surveillance of
renal allograft recipients..
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26. Copyright restrictions may apply.
Mosley, K. et al. Rheumatology 2006 45:1497-1504; doi:10.1093/rheumatology/kel351
Candidate biomarker discovery
Urine samples from inactive (n= 49) and active (n= 26) lupus nephritis patients
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27. Gel based and gel free proteomics methods in urinary
proteome analyses:
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28. Urine collection methods advantages and
disadvantages for urine proteome analysis
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29. Several methods have been used to search for biomarkers
in urine samples :
1- Two-dimensional electrophoresis
2- Surface-enhanced laser desorption/ionization
time- of-flight (SELDI-TOF)
3- Capillary electrophoresis.
4- Matrix-assisted laser desorption/ionization
time-of-flight (MALDI-TOF) to identify -β2
microglobulin (B2M) in the urine as a
potential biomarker in acute rejection for
kidney transplant recipients.
5- Another method recently introduced is iTRAQ.
(Multiplexed Isobaric Tagging Technology)
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30. 05:04 AM30

31. Ionization Source
Electrospray Ionisation (ESI)
Matrix Assisted Laser Desorption Ionisation
(MALDI)
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Mass Spectrometer.htm

32. Electrospray Ionization 05:04 AM32

33. Electrospray Ionisation (ESI)
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34. Electrospray needle
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35. ESI characteristics:
1. Soft ionization method
2. Provides molecular weight information.
3. Suitable for analyzing large bio- or synthetic polymers.
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36. Matrix Assisted Laser Desorption Ionization 05:04 AM36

37. MALDI characteristics
1. Soft ionization method, so results predominantly in the
generation of singly charged ions hence the spectra are relatively
easy to interpret.
2. Provides molecular weight information.
3. MALDI deals well with thermolabile, non-volatile organic
compounds especially those of high molecular mass
4. MALDI is used successfully for the analysis of proteins,
peptides, glycoproteins, oligosaccharides, and oligonucleotides..
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38. Types of Mass Analyzers
• Sector Mass Analyzers
• Time Of Flight (TOF)
• Quadrupole
• Quadrupole Ion Trap
• Ion Cyclotron Resonance
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39. 05:04 AM39

40. Time Of Flight Mass AnalyzerTime Of Flight Mass Analyzer
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41. The firing of a laser at the sample / matrix
mixture.
Protein Chips SELDI-
TOFhow_proteins_are_ionised_SELDI.pps
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42. Quadrupole Mass AnalyzerQuadrupole Mass Analyzer
acts as a mass selective filter. 05:04 AM42

43. Quadrupole Ion Trap Mass AnalyzerQuadrupole Ion Trap Mass Analyzer 05:04 AM43

44. Ion Cyclotron Resonance
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45. FT
Time domain
signal
Ion Cyclotron Resonance Mass AnalyzerIon Cyclotron Resonance Mass Analyzer 05:04 AM45

46. Detectors
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47. Mass SpectrumMass Spectrum
M/Z05:04 AM47

48. What is a mass spectrum?
Mass spectrum: graphic
representation of ions
separated according to their
m/z ratio.
It is a plot of relative
abundance versus mass-to-
charge ratio (mz).
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49. Mass SpectrumMass Spectrum
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50. Summary of the proteomic platforms used for urine
analysis, their advantages and disadvantages.
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51. Conclusion
1- The adequate diagnosis of complex diseases e.g., renal
disease with a single biomarker seems to be an illusion.
2- A Multiple biomarker assay could deliver a better and a
more individualized diagnosis and allow therapeutic
strategies that delay or prevent the progression of the
disease.
3- Due to many limitations and uncertainties,
urinary proteomics at present cannot replace invasive
standardized diagnostic procedures such as the renal
biopsy, but holds great promise and potential for future
highly improved diagnosis and care of the patient in
nephrology.
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52. www3.niddk.nih.gov
THANK YOU
Prof./ Moustafa rizk
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53. Copyright restrictions may apply.
Thongboonkerd, V. Nephrol. Dial. Transplant. 2010 25:11-16; doi:10.1093/ndt/gfp476
Schematic summary of methodologies and applications of renal and urinary proteomics
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