Natàlia Puig's predoctoral presentation at the 6th VHIR Scientific Session. Watch the video of the presentation after the last slide.

1. 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

2. 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.
Hypoalbuminemia
Protein Loss Hypoproteinemia
Oedema
Dyslipidemia
Nephrotic Syndrome (NS)
Treated with Corticoids
Steroid-Resistant Nephrotic Syndrome Do not respond Protein Loss
(SRNS)

3. Focal Segmental Glomerulosclerosis (FSGS)
SRNS is associated to Focal Segmental Glomerulosclerosis (FSGS)
scar found in some glomeruli
NPHS1
NPHS2
CD2AP
ACTN4
TRPC6
WT1
Unknown putative plasmatic PLCE1
permeabilising factor Mutations of podocyte proteins
FSGS
90% 10%
Idiopathic Genetic
End-Stage Renal Disease (ESRD) End-Stage Renal Disease (ESRD)
Serum form FSGS patients Frequent
Transplantation
Transplantation Plasma aphaeresis relapse
Transplantation
can induce proteinuria in
60% 40%
(change of plasma of FSGS
rats and permeabiliseRelapsing (R) FSGS Patientcan Recovery
Healthy kidney
Non-Relapsing (NR) proteins)
80%
isolated glomeruli diminish proteinuria
New transplantation
Remission

4. Relapsing FSGS Project
Aims
- 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.
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]
HVH Hospital Vall d’Hebron HJC: Hospital Juan Canalejo (A Coruña)
FP Fundació Puigvert (Barcelona) HCA Hospital Central de Asturias (Oviedo)
HULP Hospital Universitario La Paz (Madrid) HRC Hospital Ramón y Cajal (Madrid)
HMV Hospital Marqués de Valdecilla (Santander) HUC Hospital universitario de Canarias (Tenerife)
LF Hospital La Fe (Valencia) HPM Hospital Puerta del Mar (Cadiz)
HVR Hospital Virgen del Rocío (Sevilla) HCH Hospital Carlos Haya (Malaga)
HC Hospital de Cruces (Barakaldo) HRS Hospital Reina Sofia (Cordoba)
HMS Hospital Miguel Servet (Zaragoza) HVN Hospital Virgen de las Nieves (Granada)
HCM Hospital Clínico de Madrid HGA Hospital General de Alicante
HCP Hospital Clínico de Barcelona HVA Hospital Virgen de la Arrixaca (Murcia)
HCR Hospital Germans Trias i Pujol (Badalona)

5. Proteomic Comparation (urine samples)
Non-Relapsing Relapsing
No significant differences were found in plasma samples, but significant differences
were found in urine samples, even after rejecting the proteins from blood.

6. Proteomic analysis (urine samples)
NON-RELAPSING KDa RELAPSING KDa
-- 38.5 -- 38.5
A B
ApoA-Ib ApoA-Ib
ApoA-I
-- 23.5 -- 23.5
ApoA-I -- 20.0 ApoA-I -- 20.0
“standard” “standard”
-- 16.9 -- 16.9
-- 13.8 -- 13.8
| | | | | | | |
pI pI | |
4.86 5.02 5.2 3 5.52 5.70 4.86 5.02 5.23 5.52 5.70
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.

7. 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.
ApoA-I
HDL

8. 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 NR R NR R R FAM FAM FAM P P P R R R NR R
R: relapsing
NR: non-relapsing
FAM: familiar FSGS (genetic)
P: FSGS-unrelated proteinuria
ApoA-Ib
Plasma ApoAI rec Relapsing patients Plasma
R NR R Ct R
ApoA-Ib
standard ApoA-I
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.

9. Results of the Validation
70 119 patients
ApoA1b positive R: Relapsing patients
ApoA1b negative
60
NR: Non- Relapsing patients
Sensitivity 92.8%
FAM: Familiar FSGS
Specificity 98.1%
50 PTx: FSGS-unrelated proteinuria,
PPV 86.6%
Number of patients
kidney transplanted
NPV 99%
40
PnTx: FSGS-unrelated proteinuria,
not transplanted
30
Post Plasma apheresis sample
20
* (*): ApoA-Ib positive
10 proportion significantly higher
than in other groups (Chi-
Squared test p<0.0001)
0
R NR FAM PTx PnTx
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.

10. 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
Yaacov Frishberg, Helen Toledano, Rachel Becker-Cohen, Elad
Feigin
Genetic polymorphism in paraoxonase is a risk factor for
childhood focal segmental glomerulosclerosis
Search of Genetic variants/Single nucleotide polymorphisms (SNPs) in ApoA1 gene
Groups of patients: Control Group:
• 13 Relapsing Patients • 1 Familiar FSGS SNPs Distribution in Groups IVS1-75G>A
• 13 Non-relapsing patients • 3 Controls without FSGS IVS1+ 68G>A
IVS1+ 67C>T
FAM
IVS3+33T>C
IVS3+134T>C
CT IVS4-211T>C
IVS4-274C>T
IVS4-63C>T
NR
As shown in the chart, no genetic variation allows us
to separate the relapsing patients from the other
R
groups.
0 5 10 15 20 25 30 35 40 45 50
All of this SNPs are previously described an
validated in the NCBI dbSNP page

11. Is Apo A-Ib involved in the pathogenesis of FSGS?
Altered HDL subpopulations have been related to FSGS
Glycated products can induce podocyte injury.
Altered HDL may be involved in the pathogenesis of Activation of advanced glycated products receptors
FSGS induces podocyte injury
Without With
PNGase PNGase
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.
25 A glycated form of Apo A-I could destabilise the HDL or / and produce a
podocyte injury.
LC-MS/MS

12. 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.
Genetic FSGS (pool of 11 patients) Idiopathic FSGS (pool of 15 patients)
Monomeric form of
ApoA-II
Spot appearing in idiopathic and absent in genetic:
candidate to permeabilising factor Apo A-I
HDL
Apo A-II
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

13. 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.
European Patent
European Patent Application No: EP11382076.5-
2404.
Diagnostic marker for relapsing primary
J.Lopez-Hellin, C.Cantarell, L.Jimeno, A.S.Fructuoso, N.Puig-Gay, et al. idiopathic focal segmental glomerulosclerosis.
Accepted for publication 13 October 2012.

14. Grup de Fisiopatologia Renal - CIBBIM Nanomedicina
Servei de Nefrologia
Servei de Nefrologia Pediàtrica
Grup GREAT
Joan Josep Bech
Proteòmica VHIO