Chronic kidney disease progression and proteinuria: role of endoplasmic reticulum

stress and lipocalin2

KHALIL EL KAROUI, AMANDINE VIAU, FRANK BIENAIME, BERTRAND KNEBELMANN, MORGAN GALLAZZINI, FABIOLA TERZI

INSERM U845 Mécanismes et Stratégies Thérapeutiques des Maladie Rénales Chroniques

Actualités Néphrologiques, 23 avril 2013

Chronic kidney disease

Therapeutic strategies & Biomarkers

> Prevalence of CKD

~7 million people in France

> Prevalence of ESRD

60000 people

1/2 dialysis

1/2 transplantation

> Incidence of ESRD

+ 6 à 8% each year

Go et al. N Engl J Med 2004

Chronic kidney disease

Kidney Function

End Stage Renal Disease

Auto-aggravation

Chronic Kidney Disease Prognosis Consortium, Lancet, 2010

Proteinuria

Proteinuria-Albuminuria

control proteinuria

The axolotl experiment

Intraperitoneal proteins infusion induce tubular cell exposition to

proteins without glomerular lesions

Tubular cell toxicity and fibrosis induction

Gross, Kidney Int, 2002

ProteinsSaline

Perico, Nat rev Drug Discov, 2008

Hypothesis

ER stress?

ER stress: mismatch between polypeptide load and ER capacity to fold it

Unfolded protein response (UPR) induction

Multiple pathophysiological conditions induce ER stress:

nutrient deprivation, Ca homeostasis, hypoxia, ROS…

Albuminuric CKD are associated with ER stress

Albumin may induce ER stress in tubular cells

… But undetermined role of ER stress in vivo !

Endoplasmic Reticulum stress hypothesis

Ohse, KI, 2006Kitamura, AJPRP, 2008

Ohse, KI, 2006

Wu, OMICS, 2010Lindenmeyer, JASN, 2008

ER stress and UPR

CHOPCHOPCHOPCHOP

JNK cJun

Zhang, Nature, 2008

Does albumin/proteinuria induce ER stress in tubular cells ?

Albuminuria experimental models

X

Nphs2: key component of podocyte slit diaphragm

Podocin (Nphs2) invalidation

Nphs2Fl/Fl Actin-CreER

Mollet, JASN, 2009

WT1: Transcription factor with key role in podocyte homeostasis

Mutant WT1 Knock-In mice

Ratelade, HMG, 2009

WT1+/mut

Genetic models

Doxorubicin injection

Zheng, PNAS, 2005

Doxorubicin: podocyte apoptosis

Doxo

Ctrl

Albumin injection

Eddy, KI, 2000

Bovine albumin: glomerular toxicity, and murine proteinuria

Albuminuria experimental modelsPharmacologic models

Proteinuria and ER stress

Does inhibition of ER stress slow down CKD progression?

PBA

ER stress inhibition by PBA (chemical chaperone)

PBA treatment in WT1+/mut mice

WT1+/mut

ER stress inhibition in vivo

PBA

Proteinuric CKD progression ?

Morphology

Function

PBA treatment in WT1+/mut mice

control WT1 +/mut Vehicle-treated WT1 +/mut PBA-treated

Perico, Nat rev Drug Discov, 2008

Hypothesis

Lcn2 ?

C:\Documents and Settings\amandine\Mes documents\Mes images\ngal structure.gif

Lipocalin 2/NGAL(Neutrophil Gelatinase Associated Lipocalin)

Negatively-charged

Traps iron with high affinity

small secreted polypeptide, member of the lipocalin family, with a hydrophobic calyx

lipocalin 2 or NGAL

a small non-peptide iron binding chemical, of bacterial or mammalian origin (catechol)

Lcn2 ligands: Siderophores

Goetz, 2002

Lcn2 is highly expressed during CKD progression

Another model of CKD: Nephron reduction

Lcn2 invalidation in vivo

Tubular lesions

Glomerular lesions

Interstitial fibrosis

Contr

ol

Nx L

cn2

+/+

Nx L

cn2

-/-

Lcn2-/-

Tubular cell proliferation

Lcn2 expression in WT1+/mut mice

IgG in mIMCD-3 cells

Lcn2 expression in albumin-treated cells

mIMCD-3 cells

Is Lcn2 induced by proteinuria-associated ER-stress ?

PBA treatment in mIMCD3 cells

ATF4

control WT1 +/mut Vehicle-treated WT1 +/mut PBA-treated

PBA treatment in WT1+/mut mice

Lcn2 expression

Protocol

Thapsigargin: induces ER Ca depletion

Tunicamycin: inhibits N-glycosylation

ER stress and Lcn2 expression

ThapsigarginTunicamycin

ER stress and Lcn2 expression

Thapsigargin Tunicamycin

Is Lcn2 a critical mediator during proteinuric CKD progression ?

Protocol

Lcn2-/-

XWT1+/mut

Lcn2 inactivation in WT1+/mut mice

Morphology

Mortality

Lcn2 inactivation in WT1+/mut mice tubular lesions

control WT1 +/mut X Lcn2+/+ WT1 +/mut X Lcn2-/-

What is the mechanism by which Lcn2 favors proteinuric CKD progression ?

Lcn2 and tubular cells apoptosis in vivo

control WT1 +/mut X Lcn2+/+ WT1 +/mut X Lcn2-/-control WT1 +/mut X Lcn2+/+ WT1 +/mut X Lcn2-/-

Lcn2 and tubular cells apoptosis in vitro

MEF Lcn2+/+ MEF Lcn2-/-

MEF Lcn2+/+ MEF Lcn2-/-

EGFR UPR

C:\Documents and Settings\amandine\Mes documents\Mes images\ngal structure.gif

Lcn2

Cell apoptosisCell proliferation

Conclusions

Lcn2 is a key mediator of CKD progression

CKD progression

Control Proteinuric patient

Lcn2 in proteinuric patients

Currently: renin-angiotensin system (RAS) inhibition in proteinuric nephropathies: proteinuria

However: residual proteinuria (persisting despite RAS inhibition) is a crucial problem

So: targeting tubular effects of proteinuria ?

Pilot study: PBA for a multi-target therapy in proteinuric nephropathies

Lcn2/NGAL as a true actor of CKD progression

Lcn2

Perspectives

Correlation of urinary NGAL expression and GFR degradation

Lcn2/NGAL as a biomarker of proteinuric CKD progression

Multi-target therapy in proteinuric nephropathies ?

PBA has been already tested in patients with a good tolerance profile

Urea Cycle disorders32 patients, different protocols, follow-up > 9 years

Cystic fibrosis18 patients, PBA 19 g/d, 1 week

Diabetes/insulin resistance8 patients, PBA 7,5 g/d, 2 weeks

Rubinstein, AJRCCM, 1998Zeitlin, Mol Therapy, 2002

Maestri, NEJM, 1996

Xiao, Diabetes, 2011

Pilot studyProteinuric patients

UrinePlasma

UrinePlasma

D0 D15

Lcn2 / ER stress markers expression (cells: mRNA, supernatant: ELISA) ?

Oral PBA

Multi-target therapy in proteinuric nephropathies ?

D30

Wash out period

UrinePlasma

Many thanks to

Service de Néphrologie et Transplantation rénale

Necker Hospital

Histology facilitySophie Berissi

Noémie Gadessaud

Amandine ViauMorgan Gallazzini

Ariane Ambolet-CamoitWilliam BaronFrank BienaiméMartine BurtinMélanie Broueilh

Guillaume CanaudAnne Druilhe

Bertrand KnebelmannDenise LaouariMordi Muorah

Clément Nguyen

Fabiola Terzi

Terzi Team

Corinne AntignacCristelle Arrondel

Marie-Claire GublerLaurence HeidetGéraldine Mollet

Antignac Team

L.E.A.TNecker Hospital

AnatomopathologieNecker Hospital, Dr Noel

AnatomopathologieHEGP, Dr Nochy