ΑΞΙΟΛΟΓΗΣΗ ΤΟΥ ΡΟΛΟΥ ΤΩΝ PLA2R Ags-Abs) ΣΕ ΑΣΘΕΝΕΙΣ ΜΕ ΙΔΙΟΠΑΘΗ ΜΕΜΒΡΑΝΩΔΗ ΣΠΕΙΡΑΜΑΤΟΠΑΘΕΙΑ

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1 ΑΞΙΟΛΟΓΗΣΗ ΤΟΥ ΡΟΛΟΥ ΤΩΝ PLA2R Ags-Abs) ΣΕ ΑΣΘΕΝΕΙΣ ΜΕ ΙΔΙΟΠΑΘΗ ΜΕΜΒΡΑΝΩΔΗ ΣΠΕΙΡΑΜΑΤΟΠΑΘΕΙΑ Π.Κούκη 1,Α.Δαμιανάκη 1,Ε.Βεντούρη 2,Ε.Δαφνής 3,Κ. Στυλιανού 3,Χ.Γακιοπούλου 2,Α.Καποτά 1, Δ.Πετράς 1 1 Νεφρολογική Κλινική Γενικό Νοσοκομείο Αθηνών Ιπποκράτειο 2 Α Εργαστήριο Παθολογικής Ανατομικής Ιατρική Σχολή Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών 3 Πανεπιστημιακή Νεφρολογική Κλινική Πα.Γ.Ν.Η 20ο Πανελλήνιο Συνέδριο Νεφρολογίας 4/5/2018

2 A In situ formation of immune deposits and disease progression Endogenous antigen NEP PLA2R THSD7A A-enol AR SOD2 Cell injury ER stress Podocyte Protective cytotoxic Exogenous antigen Y Activation of complement pathways C5b-9 complex formation Insertion into membrane Cell injury Disease-induced alterations Up-regulation of cytoprotective mechanisms Prolonged ER stress Apoptosis ERT enzymes Cationic BSA HEpB B Optium therapeutic approach Eliminating environmental factors diet, toxins Reducing autoantibodies Inhibiting complement activation Protecting the podocyte Antioxidants Promotion of autophagy Inhibitors of ER stress Mechanisms of immune mediated podocyte Injury in MN and pathogenesis based Therapeutic approach A) The in situ formation of immune complexes is initiated by binding of circulating antibodies to antigens that are endogenous integral membrane proteins of the podocyte, or to exogenous Antigens planted in the glomerular Basement membrane. Complement activation and formation of the C5b 9 attack complex, which is triggered by Immune complex deposition, have major roles in sublethal podocyte injury and proteinuria. Distinct cytoprotective responses are observed over time in cells undergoing endoplasmic reticulum (ER) stress. Prolonged ER stress beyond threshold results in apoptosis B) Optimum therapeutic intervention requires that four injury mechanisms are simultaneously targeted Ronco F,Hannad,2015. Membranous nephropathy.a fairy tale for immunopathologists, nephrologists and patiensts. Molecular Immunology. 58:57-62

3 in situ IC Complement activation MAC deposits, ROS, protein leakage podocytes C3 C5b-C9 = autoag, GMB e.g. Pla2R endothelial cells Capillary lumen = anti-podocyte autoantibody (IgG4), e.g. anti-pla2r, anti-thsd7a, others Mechanism of anti-podocyte autoantibody-mediated disease in membranous nephropathy. Circulating autoantibodies can target surfaceexposed intrinsic podocyte proteins to form in-situ immune deposits. The functional impairment represented by proteinuria is the result of formation of the membrane attack complex (C5b-C9, MAC), which leads to sublethal podocyte injury resulting in the activation of transcription factors encoding for mediators of fibrosis and cytoskeletal podocyte rearrangement. It also increases production of potentially nephritogenic molecules such as reactive oxygen species (ROS), proinflammatory cytokines, proteases and vasoactive molecules Sinico R.A et al.immunology of membranous nephropathy :from animal models to humans.2015.clin and Exper Immunology 183:

4 PLA2R Collagens Integrin β1 Putative PLA2 binding domain Negative regulator Internalization Degradation CTLD Cys-R FNII N C Conformation dependent epitopes Mannose coupled protein Positive regulator Lipid mediator production MARK activation ROS production Activation of DNAdamage pathways High magnification of PLA2R an Structural and functional properties of PLA 2 R. This receptor is a type I transmembrane glycoprotein composed of a large extracellular portion that consists of an N-terminal cysteine-rich region,a fibronectin-like type II domain (FNII), a tandem repeat of 8 C-type lectin domains (CTLD), a transmembrane domain and a short intracellular C-terminal domain. Known interactions and domains involved are indicated. PLA 2 R can function as a positive regulator of PLA 2 by inducing a variety of biological responses, or as a negative regulator through rapid internalization and degradation of PLA 2. The blue loop shows that in the bent configuration, the N terminal domain folds back to interact with C-type lectin-like domains Ronco P and Debiec h. Anti phospholipase A2 receptor antibodies and the pathogenesis of membranous nephropathy 2014.Nephron Clin Pract.128:

5 3-D fitting of the Ab-PLA 2 R Best fit 3D model of the antibody-n-c8 complex. Scale bar = 50 Å

6 Σκοπός της μελέτης Σκοπός της μελέτης ήταν η συσχέτιση της καθήλωσης των PLA2Rαντιγόνων(Ags) στη βιοψία και της ύπαρξης των anti-pla2rαντισωμάτων(abs) στον ορό με τη λευκωματουρία, τη νεφρική λειτουργία (egfr)

7 Υλικό και μέθοδοι- 20 ασθενείς με ιδιοπαθή ΜΣ Καταγράφηκαν το egfr, η λευκωματουρία στους μήνες 0 και 6 Καταγράφηκε επίσης η ένταση καθήλωσης των PLA2R-Ags (ήπια, μέτρια, ισχυρή) με ανοσοϊστοχημεία καθώς και ανοσοφθορισμό και τα anti -PLA2R-Abs στον ορό (ανιχνεύσιμα/μη ανιχνεύσιμα) με έμμεσο ανοσοφθορισμό

8 Literature review Positive rates of Serum anti-pla2r antibody 52%-82% Positive rates of Glomerular PLA2R deposition 69%-92% Serum anti PLA2R antibody and glomerular PLA2R deposition in Chinese patients with membranous nephropathy, Lu Pang, et al.2017

9 Stage 1 Stage 2 Stage 3 Stage 4 Stage 5 Proteinuria Serum anti -PLA2R antibody Glomerular PLA2R antigen Disease process Proteinuria Serum antibody _ + + Glomerular antigen _ The temporal sequence of serum anti-pla2r antibody, glomerular PLA2R deposition, and proteinuria in the process of PMN progression. -, negative; +, positive. PLA2R=M-type phospholipase A2 receptor, PMN= primary membranous nephropathy

10 Serum PLA2R antibody measurement Biopsy proven imn (n=572) (Clinically rule out secondary causes) + - Qin Hua-Zhang et al. Combined assessment of Phospholipase A2 receptor autoantibodies and glomerular deposits in membranous nephropathy JASN 27: Serum PLA2R antibody positive (SAB +) (n=392, 68.5%) Serum PLA2R antibody positive (SAB -) (n=180, 31.5%) Glomerular PLA2R antigen detection antigen detection Glomerular PLA2R antigen detection Glomerular PLA2R antigen positive (Gag+) (n=387, 98.7%) Glomerular PLA2R antigen negative (Gag-) (n=5, 1.3%) Glomerular PLA2R antigen positive (Gag+) (n=127, 70.6%) Glomerular PLA2R antigen negative (Gag-) (n=53, 29.4%) Distribution of PLA2R SAb and GAg in 572 IMN patients and the distribution of THSD7A antigen in 53 patients with out both PLA2R SAb and GAg. SAb and GAg of PLA2R were measured in 572 IMN patients. The glomerular THSD7A antigen was detected in 53 patients who were SAb2/GAg2 Glomerular THSDFA antigen positive (Gag+) (n=4, 7.5%) Glomerular THSDFA antigen detection + - Glomerular THSDFA antigen negative (Gag-) (n=49, 92.5%)

11 Positive rate % Positive rates of SAb and GAg in patients with IMN and positive rate when patients with SAb or GAg were counted together. Fisher exact test was used to compare the SAb+ and GAg+ rates (P,0.001) Sab+ Gag+ Sab + or Gag+

12 Percent of patients with no relapse (%) Patients with sustained Gag+ Others Time (Month) Patients with sustained Gag Others Kaplan-Meier analysis for proportion of relapse in patients with sustained GAg+ and patients without Gag at the second biopsy. Patients with sustained GAg+ showed higher risk of relapse than patients without GAg (P=0.03). Log rank method was used to evaluate the significance of difference.

13 Αποτελέσματα 0m 6m μ.ο egfr 73.1ml/min /1.73m ml/m in/1.73m 2 μ.ο Λευκωμα τουρίας Καθήλωση PLA2R-Ag Ανίχνευση Anti- PLA2R- Abs 9.1gr /24h 81.25% ήπια μέτρια ισχυρή 62.5% 1.8 gr/24h

14 PLA2R (-) Immunohistochemistry (x400) PLA2R (+) Immunohistochemistry (x400) PLA2R (++) Immunohistochemistry (x400) PLA2R (+++) Immunohistochemistry (x400)

15 PLA2R (-) Immunofluorescence (x200) PLA2R (+) Immunofluorescence (x200) PLA2R (++) Immunofluorescence (x200) PLA2R (+++) Immunofluorescence (x200)

16 Αποτελέσματα 0m 6m μ.ο egfr 73.1ml/min p< ml/m /1.73m 2 in/1.73m 2 μ.ο Λευκωμα τουρίας Καθήλωση PLA2R-Ag Ανίχνευση Anti- PLA2R- Abs 9.1gr /24h 81.25% ήπια μέτρια ισχυρή 62.5% 1.8 gr/24h

17 Αποτελέσματα 0m 6m μ.ο egfr 73.1ml/min /1.73m ml/m in/1.73m 2 μ.ο Λευκωμα τουρίας 9.1gr /24h p< gr/24h Καθήλωση PLA2R-Ag Ανίχνευση Anti- PLA2R- Abs 81.25% ήπια μέτρια ισχυρή 62.5%

18 Αποτελέσματα 0m 6m μ.ο egfr 73.1ml/min /1.73m ml/m in/1.73m 2 μ.ο Λευκωμα τουρίας Καθήλωση PLA2R-Ag Ανίχνευση Anti- PLA2R- Abs 9.1gr /24h 81.25% ήπια μέτρια ισχυρή 62.5% 1.8 gr/24h

19 Συμπεράσματα: Η παρουσία PLA2R-Ags και όχι των anti-pla2r-αbs φαίνεται να σχετίζεται με τη νεφρική λειτουργία και με τη λευκωματουρία στους ασθενείς με ιδιοπαθή ΜΣ. Χρειάζονται περισσότερες μελέτες για να αποδειχθεί αυτός ο προγνωστικός τους ρόλος.