chronic kidney disease progression and proteinuria: role ... · amandine viau morgan gallazzini...
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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
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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
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Chronic kidney disease
Kidney Function
End Stage Renal Disease
Auto-aggravation
Chronic Kidney Disease Prognosis Consortium, Lancet, 2010
Proteinuria
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Proteinuria-Albuminuria
control proteinuria
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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
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Perico, Nat rev Drug Discov, 2008
Hypothesis
ER stress?
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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
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ER stress and UPR
CHOPCHOPCHOPCHOP
JNK cJun
Zhang, Nature, 2008
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Does albumin/proteinuria induce ER stress in tubular cells ?
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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
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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
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Proteinuria and ER stress
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Does inhibition of ER stress slow down CKD progression?
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PBA
ER stress inhibition by PBA (chemical chaperone)
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PBA treatment in WT1+/mut mice
WT1+/mut
ER stress inhibition in vivo
PBA
Proteinuric CKD progression ?
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Morphology
Function
PBA treatment in WT1+/mut mice
control WT1 +/mut Vehicle-treated WT1 +/mut PBA-treated
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Perico, Nat rev Drug Discov, 2008
Hypothesis
Lcn2 ?
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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
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Lcn2 invalidation in vivo
Tubular lesions
Glomerular lesions
Interstitial fibrosis
Contr
ol
Nx L
cn2
+/+
Nx L
cn2
-/-
Lcn2-/-
Tubular cell proliferation
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Lcn2 expression in WT1+/mut mice
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IgG in mIMCD-3 cells
Lcn2 expression in albumin-treated cells
mIMCD-3 cells
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Is Lcn2 induced by proteinuria-associated ER-stress ?
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PBA treatment in mIMCD3 cells
ATF4
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control WT1 +/mut Vehicle-treated WT1 +/mut PBA-treated
PBA treatment in WT1+/mut mice
Lcn2 expression
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Protocol
Thapsigargin: induces ER Ca depletion
Tunicamycin: inhibits N-glycosylation
ER stress and Lcn2 expression
ThapsigarginTunicamycin
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ER stress and Lcn2 expression
Thapsigargin Tunicamycin
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Is Lcn2 a critical mediator during proteinuric CKD progression ?
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Protocol
Lcn2-/-
XWT1+/mut
Lcn2 inactivation in WT1+/mut mice
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Morphology
Mortality
Lcn2 inactivation in WT1+/mut mice tubular lesions
control WT1 +/mut X Lcn2+/+ WT1 +/mut X Lcn2-/-
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What is the mechanism by which Lcn2 favors proteinuric CKD progression ?
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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-/-
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Lcn2 and tubular cells apoptosis in vitro
MEF Lcn2+/+ MEF Lcn2-/-
MEF Lcn2+/+ MEF Lcn2-/-
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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
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Control Proteinuric patient
Lcn2 in proteinuric patients
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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
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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
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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
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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