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