This document discusses non-invasive methods for diagnosing acute rejection in renal transplant patients. Currently, graft biopsy is the gold standard but it is invasive and detects rejection at a late stage. The presentation evaluates various potential biomarkers being studied through genomics, proteomics, and metabolomics approaches. Several individual studies are highlighted that found biomarkers like urinary MCP-1, VEGF, cytokines, and certain metabolites that showed potential for detecting early acute rejection non-invasively. Magnetic resonance imaging is also discussed as a non-invasive imaging technique being researched. In summary, the ideal non-invasive biomarker has yet to be identified but a combination of markers may provide a more realistic approach for different clinical scenarios.

1. Non biopsy diagnosis of
acute rejection
Presenter: Dr Bakshish Singh
Preceptor: Prof SK Agarwal

2. Introduction
 Acute rejection key factor for long-term graft
function and survival in RT patients
 Timely detection and treatment of rejection an
important goal.
 The standard care with S.Creat measurements and
biopsy usually detects AR in an advanced stage
 Hence the need for Non invasive markers that can
detect AR at an earlier stage.
2

3. Current Approach
 Clinical signs
 Decreased urine output
 Graft tenderness
 Hypertension
 USG
 Serial USG with echo enhancers
 S creatinine
 Greater than 15% rise suspicious
 Biopsy
 GOLD STANDARD
3

4. Graft biopsy
 Graft biopsy currently
the GOLD STANDARD
for diagnosis of acute
rejection.
 Criteria for AR defined
by consensus (Updated on
regular intervals)
 Can pick up other causes
of graft dysfunction
 Invasive
 Subjective variability
 May be non
representative
 Timing of biopsy
 Detects injury at a late
stage
4

5. Acute Rejection
 Heterogenous
 Involved structures
 Mechanism
 Clinical presentation
 Severity
 Time related
 Immunosuppression
ALL THE ABOVE AFFECT THE BIOMARKERS
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6. Rejection: A Time-line Model
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8. 8
 Given the the heterogeneity and complexity of AR,
it is unlikely that one marker will fit all facets of
this process.
 Different markers may detect incipient, fully
established and resolving stages of rejection.
 Different rejection types (T-cell vs. antibody-
mediated, tubulo-interstitial vs. vascular) may
have distinct sets of markers.
 Further, quantifiable markers could give additional
information about the severity of rejection

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13. Approaches to biomarkers
 Two Principles
 Monitor Immune System

Allo-immune recognition/activation

Effector pathway of inflammation
 Detect injury to renal structures

Tubular epithelial cells

Interstitium

Vasculature

Glomerular structures
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14. 14

15. Pyramid of life
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16. 16

17. Genomics
 “Candidate Gene Approach”
 Gene products implicated in
 Cellular traffic
 Physical contact between lymphocytes
and targeted cells
 Target cell damage
 The counter-regulatory responses
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19. 19

20. 20

21. 21

22. Acute T Cell Rejection : Molecular events Invitation: IP10
Contact: CD103
Induced suicide:
Granzyme B/ Perforin
Collateral Protection:
PI-9
Damage control: FoxP3
22

23. Proteomics
 DEFINITION
 The systematic analysis of proteins for their identity, quantity
and function
23

24. Why study proteins
 Proteins, rather than nucleic
acids, mediate most of the
physiologic functions within the
cell.
 Analysis of body fluids such as
urine can only be accomplished
by proteomics approaches
because nucleic acids play no
direct functional role in
extracellular fluids
 Proteomics can be viewed as
being complementary to the
area of functional genomics
24

25.  The common element of proteomics studies is
“Multiplexing”
 The simultaneous study of multiple proteins rather than one
protein at a time (as in traditional biochemistry)
 Most proteomics-based investigations focus on defined
subpopulations of proteins
 Set of all proteins found in urine or blood plasma,
 Set of proteins present in endosomes from collecting duct principal
cells
 Set of all proteins that form complexes with the proximal tubule Na-H
exchanger NHE3
 Set of all Na transporters expressed along the renal tubule
 Set of all renal cortical proteins detectable in two-dimensional gels
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26. Flow chart for protein identification
26

27. 27

28.  Two broad areas
 Mass spectrometry to
detect and identify
proteins and
 Approaches using
arrays or ensembles of
binding molecules to
detect and identify
proteins using
antibodies as the
binding molecules
 MS: Initial separation
technique by one of the
following followed by
identification
 2 D Electrophoresis
 Liquid chromatography
 Surface-enhanced laser
desorption/ ionization
 Capillary electrophoresis
 Protein microarrays
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29. 29

30. Mass spectrometry
 Two basic approaches using trypsin
digest
 Peptide mass finger printing usually
employing matrix-assisted laser
desorption and ionization–time of- flight
(MALDI-TOF) mass spectrometers
 Peptide sequencing using tandem mass
spectrometers
30

31. 31

32. A. Diagram of a
quadrupole/time-of-flight
(Q-TOF) tandem mass
spectrometer. Peptide
ionization uses the
electrospray method B. Peptide sequencing using tandem mass spectrometry.
Y- series of peaks is derived from sequential elimination
of amino acids from the amino terminus of the peptide
by collision-induced dissociation. Differences in masses
between successive peaks correspond to residue masses
of individual amino acids. For example, the difference
between y12 and y11 peak is 115.03 Daltons
corresponding to aspartate (D). Difference between y11
and y10 is 113.08 Daltons corresponding to leucine (L).
32

33. How protein structure is elucidated
How protein Structure is Elucidated 33

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35. 35

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37. 37

38. Metabolomics
 DEFINITION
 Dresdale coined this term in the year 2000.
 Also known as metabonomics or metabolic
profiling
 High-throughput identification and
quantification of the small molecule (1,000 Da)
metabolites in the metabolome
 Meatabolome
 Collection of all small molecule metabolites
(endogenous or exogenous) that can be found in
a cell, organ or organism
38

39. This is possible due to
 High-resolution mass spectrometry (MS) instruments for
precise mass determination
 High-resolution, high-throughput nuclear magnetic
resonance (NMR) spectrometers for accelerated
compound identification
 Capillary electrophoresis, high-pressure liquid
chromatography (HPLC), and ultra-high pressure liquid
chromatography systems for rapid compound separation
 Software programs to rapidly process spectral or
chromatographic patterns
39

40. Approaches to metabolomics
Two Approaches
 Chemometrics
 compounds are not formally identified
 spectral patterns and intensities are recorded, compared
and used to make diagnoses, identify phenotypes or draw
conclusions
 Targeted profiling
 compounds are formally identified and quantified
 Being preferred these days
 Human Metabolome Database available online
40

41. 41

42.  Most associated with generic metabolic processes
 glycolysis
 gluconeogenesis
 lipid metabolism
 Changes in relative concentrations of certain
‘universal’ metabolites such as glucose, citrate,
lactate, alpha-ketoglutarate and others can
reflect changes in
 cell viability (apoptosis),
 levels of oxygenation (anoxia, ischemia, oxidative stress)
 local pH
 general homeostasis
42

43.  Methyl-histidine, creatine, taurine and glycine
reflect the extent of tissue repair or tissue
damage
 Trimethylamine-N-oxide (TMAO) act as buffer to
stabilize serum proteins from the effects of
accumulated waste product
EACH PRODUCT TELLS A UNIQUE STORY
43

44. Transplanted, dysfunctional, rejected
kidneys show increased
 Urine and serum concentrations of TMAO
 Organic amines : trimethylamine & dimethylamine
 Amino acids : glycine & alanine
 Nephrotoxins : hippuric acid & uric acid in serum
 Nitric oxide synthase inhibitors : phenylacetic acid &
dimethylarginine in the serum
 Markers of Kreb cycle distress: lactate, acetate, succinate,
citrate and urea in the serum & urine
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45. Rat models
 Proximal straight
tubules injury (via the
toxin D-serine)
 Increased lactate,
phenylalanine,
tryptophan, tyrosine
and valine
 Straight tubule injury
 Reduced levels of
methylsuccinic, sebacic
and xanthurenic acid
 Proximal convoluted
tubule injury (via the
toxin gentamicin)
 Elevated levels of urinary
glucose
 Reduced levels of TMAO,
xanthurenic acid and
kynurenic acid
45

46.  Papillary and medullary
injury (via
bromoethaneamide)
 Increased urinary
concentrations of glutaric
acid, creatine and adipic
acid
 Reduced levels of citrate,
succinate, oxoglutarate
and TMAO
 Cortical damage
(induced via mercuric
chloride)
 Increased urinary
glucose, alanine, valine,
lactate, hippurate
 Decreased citrate,
succinate and
oxoglutarate
46

47. Summary of significant metabolomics
47

48.  Metabolomics may well be the most useful
biomarkers for monitoring kidney function and
detecting adverse renal events.
 This is because the kidney is specifically designed to
concentrate or filter small molecule metabolites and
small molecule toxins.
 As a result, one would expect changes in metabolite
levels in blood or urine to be more detectable and
reflective of kidney function than subtle changes to
the kidney proteome or transcriptome
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50. 50

51. 51

52. Important Individual Studies
52

53.  Initial Study : Rapid multi component analysis of
low molecular weight compounds in urine
 The patterns of metabolic changes associated
with early renal allograft dysfunction
Increased urinary levels of trimethylamine-N-
oxide (TMAO), dimethylamine (DMA), lactate,
acetate, succinate, glycine and alanine during
episodes of graft dysfunction
53

54.  100 pts: AR (n=35) Stable graft fxn (n=65)
 Urinary sediments by cytospin
 Stained with anti-CD3, anti-CD64, anti-HLA-DR labeled monoclonal
antibodies.
 Urinary expression of MCP-1 was assayed by ELISA
 AR :
 MCP-1 level was ten-fold higher
 The number of CD3+ cells was over 5 times higher
 The number of HLA-DR+ cells was 6 times higher
 CD3+, HLA-DR+ and CD64+ cell counts strongly correlated with urine
excretion of MCP-1.
54

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56. 56

57.  Urinary concentration of VEGF was determined by an ELISA
in 215 renal allograft recipients and 80 healthy controls
 AR pts higher urinary excretion of VEGF
 Urinary VEGF/creatinine ratio of 3.64 pg/mmol cut-off
point: Sensitivity and specificity for diagnosing acute
rejection were 85.1 and 74.8%, respectively
 SRAR had significantly greater urinary VEGF concentration
than patients with SSAR
 Urinary VEGF/creatinine ratio of 22.48 pg/mmol cut-off
point: sensitivity and specificity of the prediction to graft
loss after AR were 85.7% and 78.3%, respectively
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59. 59

60. 60
 61 patients studied, 13 had no rejection
episodes, 8 had a proven acute
rejection, and 40 were excluded for
graft dysfunction causes
 Mitogen induced peripheral blood
mononuclear cells tested for IL-2, IL-
4, IL-5, IL-6, IL-10, IL-15, IFN-g,
Perforin, Granzyme B, and Fas L using
(RT-PCR)
 IL-4, IL-5, IL-6, IFN-g, Perforin, and
Granzyme B mRNA levels were
associated with AR
 Using 2 markers 75% pt with AR
identified

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IDO = indoleamine 2,3-dioxygenase

76. Role of MRI
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77. 77
Blue represents the lowest R2* value (lowest deoxy hemoglobin concentration),
and green, yellow, and red show increasing R2* values

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79. 79

80. Summary
 Non invasive diagnosis of
acute rejection DESIRABLE
GOAL
 Biomarker Development in
early stages
 Ideal Biomarker still to be
found
 Possibly combination markers
for different scenario more
realistic
 Genomics, proteomics &
metabolomics complimentary
approach appears promising
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Transplantation •
Volume 88, Number
3S, August 15, 2009

82. THANKS