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Natàlia Puig's predoctoral presentation at the 6th VHIR Scientific Session. Watch the video of the presentation after the last slide.
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A form of Apolipoprotein A-I is a potential
biomarker of focal segmental
glomerulosclerosis relapse following
transplantation
Natàlia Puig Gay Fisipatologia Renal- CIBBIM-Nanomedicine
VI VHIR Scientific Session November, 29th 2012
Introduction: Glomerular diseases
Glomeruli are the parts of the nephron that act as molecular filters,
preventing the pass of the molecules over 70KDa from blood to
urine.
Glomerular diseases are usually associated to proteinuria.
Protein Loss
Hypoalbuminemia
Hypoproteinemia
Oedema
Dyslipidemia
Nephrotic Syndrome (NS)
Treated with Corticoids
Protein Loss Do not respond Steroid-Resistant Nephrotic Syndrome
(SRNS)
Focal Segmental Glomerulosclerosis (FSGS)
SRNS is associated to Focal Segmental Glomerulosclerosis (FSGS)
FSGS
Genetic Idiopathic
Relapsing (R) Non-Relapsing (NR)
60%
New transplantation
80%
End-Stage Renal Disease (ESRD)
Transplantation
90% 10%
End-Stage Renal Disease (ESRD)
Transplantation
scar found in some glomeruli
40%
Unknown putative plasmatic
permeabilising factor Mutations of podocyte proteins
Recovery
Remission
NPHS1
NPHS2
CD2AP
ACTN4
TRPC6
WT1
PLCE1
Healthy kidney FSGS Patient
Frequent
relapse
of FSGS
Transplantation Plasma aphaeresis
(change of plasma
proteins) can
diminish proteinuria
Serum form FSGS patients
can induce proteinuria in
rats and permeabilise
isolated glomeruli
Relapsing FSGS Project
- Identification of proteins useful as early biomarkers that will enable prompt diagnosis and prognosis of
relapsing FSGS.
- Identification of proteins that provide new insights into the pathogenic mechanism of idiopathic FSGS.
Aims
Experimental design
Proteomic comparison of relapsing (R) and non-relapsing (NR) patients. Individual validation and specificity
in respect to non-FSGS proteinuria (P) and familiar FSGS (FAM) by WB. [Samples from GREAT group]
HJC: Hospital Juan Canalejo (A Coruña)
HCA Hospital Central de Asturias (Oviedo)
HRC Hospital Ramón y Cajal (Madrid)
HUC Hospital universitario de Canarias (Tenerife)
HPM Hospital Puerta del Mar (Cadiz)
HCH Hospital Carlos Haya (Malaga)
HRS Hospital Reina Sofia (Cordoba)
HVN Hospital Virgen de las Nieves (Granada)
HGA Hospital General de Alicante
HVA Hospital Virgen de la Arrixaca (Murcia)
HVH Hospital Vall d’Hebron
FP Fundació Puigvert (Barcelona)
HULP Hospital Universitario La Paz (Madrid)
HMV Hospital Marqués de Valdecilla (Santander)
LF Hospital La Fe (Valencia)
HVR Hospital Virgen del Rocío (Sevilla)
HC Hospital de Cruces (Barakaldo)
HMS Hospital Miguel Servet (Zaragoza)
HCM Hospital Clínico de Madrid
HCP Hospital Clínico de Barcelona
HCR Hospital Germans Trias i Pujol (Badalona)
Proteomic Comparation (urine samples)
Relapsing Non-Relapsing
No significant differences were found in plasma samples, but significant differences
were found in urine samples, even after rejecting the proteins from blood.
A
-- 13.8
-- 16.9
-- 38.5
-- 23.5
-- 20.0
|
5.52
|
5.2 3
|
4.86 |
5.02 |
5.70 pI
KDa
B
-- 13.8
-- 16.9
-- 38.5
-- 23.5
-- 20.0
|
5.52
|
5.23
|
4.86 |
5.02 |
5.70
KDa
pI
Proteomic analysis (urine samples)
ApoA-Ib ApoA-Ib ApoA-I
ApoA-I
“standard” ApoA-I
“standard”
NON-RELAPSING RELAPSING
We found, among several proteins previously known altered in nephrotic syndrome, 6 spots
corresponding to a differential protein not previously associated to this disease: ApoA-I
ApoA-Ib is a modified, higher molecular weight form of ApoA-I
This spot is not present in the proteomic maps of non-relapsing patients.
Apolipoprotein A-I
The apolipoprotein AI (apoA-I) is a medium-sized protein (30 kDa) that form part of the HDL lipoproteins,
with ApoA-II, ApoL-I and ApoE and others proteins.
Apo A-I forms a "belt" around HDL lipids, enabling his transport in blood.
ApoA-I is absent in the urine of healthy subjects and most patients with glomerular proteinuria. The
presence of ApoA-I in urine is associated to postrenal proteinuria or hematuria.
HDL
ApoA-I
Validation: ApoA-Ib in different groups Western blot was used to determine ApoA-Ib in concentrated urine. A fixed amount of 60 ug of protein was
used for each patient.
R: relapsing
NR: non-relapsing
FAM: familiar FSGS (genetic)
P: FSGS-unrelated proteinuria
R NR R NR R R FAM FAM FAM P P P R R R NR R
ApoA-Ib
In plasma is detected the standard form of ApoA-I, but not ApoA-Ib.
ApoAIb of the relapsing patients has a higher molecular weight (arrow) than the ApoAI of control sera; this
is the type of patients that we consider positives for ApoAI.
ApoA-Ib
standard ApoA-I
Relapsing patients Plasma
R NR R Ct R
Plasma ApoAI rec
0
10
20
30
40
50
60
70
R NR FAM PTx PnTx
Num
ber
of
patients
ApoA1b positive
ApoA1b negative
(*): ApoA-Ib positive
proportion significantly higher
than in other groups (Chi-
Squared test p<0.0001)
*
Post Plasma apheresis sample
Results of the Validation
Sensitivity 92.8%
Specificity 98.1%
PPV 86.6%
NPV 99%
After analyzing the WB and considering positive the patients with the ApoAIb band, we observe
that this form of ApoA-I discriminates correctly the relapsing patients from other groups, so it
might be a good marker of recurrence.
119 patients
R: Relapsing patients
NR: Non- Relapsing patients
FAM: Familiar FSGS
PTx: FSGS-unrelated proteinuria,
kidney transplanted
PnTx: FSGS-unrelated proteinuria,
not transplanted
Is Apo A-Ib involved in the pathogenesis of FSGS?
Other apolipoproteins from HDL have been implicated in FSGS pathogenesis:
a) Polymorphisms of Apo L1 are associated to FSGS in Afro-American population
b) Polymorphisms of Apo E and paraoxonase are risk factors for FSGS and glomerular diseases
Search of Genetic variants/Single nucleotide polymorphisms (SNPs) in ApoA1 gene
Groups of patients:
• 13 Relapsing Patients
• 13 Non-relapsing patients
Control Group:
• 1 Familiar FSGS
• 3 Controls without FSGS
SNPs Distribution in Groups
0 5 10 15 20 25 30 35 40 45 50
R
NR
CT
FAM
IVS1-75G>A
IVS1+ 68G>A
IVS1+ 67C>T
IVS3+33T>C
IVS3+134T>C
IVS4-211T>C
IVS4-274C>T
IVS4-63C>T
All of this SNPs are previously described an
validated in the NCBI dbSNP page
As shown in the chart, no genetic variation allows us
to separate the relapsing patients from the other
groups.
Yaacov Frishberg, Helen Toledano, Rachel Becker-Cohen, Elad
Feigin
Genetic polymorphism in paraoxonase is a risk factor for
childhood focal segmental glomerulosclerosis
Search of Post Traductional Modifications (PTM)
ApoA-Ib is a modified form of standard plasma Apo A-I. We checked for different PTMs.
Without
PNGase
With
PNGase
Digestion with PNGase (eliminates the glycations) decreases the molecular
weight of the ApoA-Ib band, suggesting that ApoA-Ib is glycated.
A glycated form of Apo A-I could destabilise the HDL or / and produce a
podocyte injury.
Activation of advanced glycated products receptors
induces podocyte injury
Altered HDL subpopulations have been related to FSGS
Glycated products can induce podocyte injury.
Is Apo A-Ib involved in the pathogenesis of FSGS?
Altered HDL may be involved in the pathogenesis of
FSGS
LC-MS/MS
25
With PNGase Without PNGase
Is Apo A-Ib involved in the pathogenesis of FSGS?
Previous results of our group showed an alteration in Apo A-II in plasma of idiopathic FSGS patients when compared
to genetic ones.
Spot appearing in idiopathic and absent in genetic:
candidate to permeabilising factor
Monomeric form of
ApoA-II
Genetic FSGS (pool of 11 patients) Idiopathic FSGS (pool of 15 patients)
Lopez-Hellin J, Chocron S, Madrid A, Vazquez A , Vilalta R, Lara E , Nieto J.
Proteomic differential analysis of sporadic and genetic focal segmental
glomerulosclerosis. Pediatric Nephrology, 24(9): 1790-1791. Sep 2009
HDL
Apo A-I
Apo A-II
Conclusions 1) Proteomic analysis detect a modified ApoA-I form (ApoA-Ib) consistently present in urine from
relapsing FSGS patients but absent in all the other patients, and not found in plasma.
2) ApoA-Ib discriminates correctly the relapsing patients from other groups, so it might be a good
biomarker of focal segmental glomerulosclerosis relapse following transplantation.
3) Our findings provide new data supporting the relationship between HDL particles and idiopathic
FSGS.
The findings of this study might be clinically relevant and could have therapeutic implications:
• FSGS differentiation from other types of proteinuria.
• Early detection FSGS patients with risk of relapse.
• Design of therapeutic strategies based on the results.
J.Lopez-Hellin, C.Cantarell, L.Jimeno, A.S.Fructuoso, N.Puig-Gay, et al.
Accepted for publication 13 October 2012.
European Patent
European Patent Application No: EP11382076.5-
2404.
Diagnostic marker for relapsing primary
idiopathic focal segmental glomerulosclerosis.
Servei de Nefrologia
Joan Josep Bech
Proteòmica VHIO
Grup GREAT
Servei de Nefrologia Pediàtrica
Grup de Fisiopatologia Renal - CIBBIM Nanomedicina