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.
Call Girls Service Jaipur {9521753030} ❤️VVIP RIDDHI Call Girl in Jaipur Raja...
Non biopsy diagnosis of acute rejection of Renal allograft
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
5
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
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
25
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
28
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
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
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
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
44
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
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
48
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
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
57
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
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
80