Membranous nephropathy pathogenesis and treatment

1. MEMBRANOUS
NEPHROPATHY

2. INTRODUCTION
• The term membranous refers to thickening of the glomerular capillary
wall on light microscopy of a renal biopsy
• Membranous nephropathy is defined by immunofluorescence and
electron microscopy (EM).
• Immune deposits of immunoglobulin G (IgG) and complement
components beneath podocytes on the subepithelial surface of the
glomerular capillary wall
• Most common cause of primary nephrotic syndrome in older (>60
years)

3. PRIMARY MN
• Anti-PLA2R associated (70%-80%)
• Idiopathic (20%-30%)
• Anti-THSD7A (up to 5%)

4. SECONDARY MN
Infections
HBV, HCV, HIV, parasites (filariasis,
schistosomiasis, malaria), leprosy,
syphilis, hydatid disease, sarcoid
Malignancy
Solid tumors (lung 26%, prostate
15%, hematologic [plasma cell
dyscrasias, non-Hodgkin
lymphoma, CLL] 14%, colon 11%),
mesothelioma, melanoma,
pheochromocytoma; some benign
tumors

5. Autoimmune diseases
SLE (class V), thyroiditis, diabetes, rheumatoid arthritis,
Sjogren syndrome, dermatomyositis, mixed connective
tissue disease, ankylosing spondylitis, retroperitoneal
fibrosis, renal allografts
Anti-GBM disease, IgAN, ANCA-associated vasculitis
IgG4 disease
Membranous-like glomerulopathy with masked
IgG κ deposits

6. Alloimmune
diseases
Graft versus host disease, autologous stem cell transplants,
de novo MN in transplants/transplant glomerulopathy
Fetometernal alloimmunization to neutral endopeptidase
Drugs/toxins
NSAIDs and cyclooxygenase-2 inhibitors, gold, d-penicillamine,
bucillamine, captopril, probenecid, sulindac, anti-TNFα, thiola,
trimetadione, tiopronin
Mercury, lithium, hydrocarbons, formaldehyde, environmental air
pollution (China)
Miscellaneous
Cationic Bovine Serum Albumin (infants)
Sarcoidosis

7. Experimental Membranous Nephropathy
• Heymann nephritis model of MN in rats - late 1970s
• Subepithelial immune deposits form in situ when circulating
antibodies bind to an intrinsic antigen in the glomerular capillary wall
• Antigen was subsequently identified as megalin,(internsic)
• A large (~600 kD) transmembrane receptor of the low-density lipoprotein
receptor family expressed on the basal surface of rat podocytes
• Capping and shedding of the antigen-antibody complexes, where they
bind to the underlying GBM, resist degradation, and persist for weeks
or months as immune deposits characteristic of MN
• Sublethal podocyte injury induced by the complement membrane
attack complex C5b-9

8. • Podocyte foot process effacement is likely the result of the collapse of
the actin cytoskeleton and loss of cell-GBM adhesion complexes, and
the loss and displacement of slit diaphragms are associated with the
onset of severe, nonselective proteinuria
• ECM proteins that are laid down between and around the immune
deposits, giving rise to the characteristic “spikes” and GBM thickening
that are hallmarks of MN
• Planted antigens - ainimal models immunized with cationized bovine
serum albumin (cBSA). The cBSA binds to negatively charged residues
in the GBM where it serves as a target for circulating anti-BSA
antibodies

11. Human Membranous Nephropathy
• First demonstration of an intrinsic podocyte antigen -by an unusual
case of antenatal MN induced by the transplacental passage of
alloantibodies to neutral endopeptidase (NEP), a known podocyte
protein
• The mother of the affected child was found to be deficient in NEP and
had been immunized during a previous pregnancy
• Same mechanism seen in
• de novo MN after renal transplantation and
• MN in the setting of chronic graft-versus-host disease after allogeneic
hematopoietic stem cell transplantation

13. •AUTOANTIBODIES
•PLA2R
• autoantibodies directed at the M-type phospholipase
A2 receptor (PLA2R) on podocytes
• 75% to 80% of patients with primary MN
• IgG4
•THSD7A
• thrombospondin type 1 domain–containing 7A
• 5% of cases of primary MN

14. • Planted antigen mechanism in MN
• children with MN who have been exposed to cBSA,
presumably in bottled milk
• class V (membranous) lupus nephritis and
• hepatitis B virus (HBV)–associated MN

15. Serum Antibody (±) Glomerular Antigen (±)
Percent of Patients Who
Underwent Biopsy, %
Diagnosis
Anti-PLA2R (+) PLA2R (+) 70
PLA2R-mediated PMN
(active)
Anti-PLA2R (−) PLA2R (+) 15
PLA2R-mediated PMN
(inactive)
Anti-THSD7A (+) THSD7A (+) 3–5
THSD7A-mediated PMN
(active)
Anti-THSD7A (−) THSD7A (+) Unknown
THSD7A-mediated PMN
(inactive)
Anti-PLA2R/THSD7A (−) PLA2R/THSD7A (−) 10
Non-PLA2R/THSD7A–
mediated (pathogenesis
unknown)a

16. Pathology
• STAGE I
• Characteristic changes in MGN are in the glomerular capillary
walls.
• The initial phase of the glomerulopathy is marked by subepithelial
granular deposits
• Seen with light microscopy
• Trichrome stain-fuchsinophilic
• Methenamine-silver stain, -mottled aspect or very small orifices (“holes”)
• Can be missed if we do not have immunofluorescence (IF) or
electron microscopy (EM); these deposits are immune and will be
positive for IgG and, in most of cases, for C3, in addition, they are
electron-dense

18. • STAGE II
• Glomerular architecture is preserved and the capillary
walls appear thickened with routine stains
• Cellularity usually is not increased (if present it suggests a
secondary MGN)
• Projections perpendicularly to this GBM: “spikes” are seen
• Spikes are originated in reaction to the deposits and go
progressively surrounding them (type IV collagen)

21. • STAGE III
• Forming thus new layers of GBM leaving the deposits
immersed
• Deposits are seen intramembranous and with silver stain
capillary walls can take an aspect in “chain” or “rosary”
• Positive with the immunostaining (IF), although
progressively they are less electron-dense

23. •STAGE IV
•The GBM is irregularly thickened
•Without the presence of electron-dense deposits or
holes.
•In this phase it is considered that the deposits have
been resorpted leaving this irregular aspect.

25. IMMUNOFLUORESCENCE
• Granular parietal IgG positivity
• C3 deposits in approximately 75% of cases.
• The IgG immunostaining usually is more intense than C3.
• IgG4 sub-class is the most frequent

27. Primary Secondary
Immunofluorescence Microscopy
IgG4 > IgG1, IgG3 IgG1, IgG3 > IgG4
IgA, IgM absent IgA, IgM may be present
Mesangial Ig staining absent Mesangial Ig staining may be present
C1q negative or weak C1q positive
PLA2R positive and co-localizes with IgG PLA2R negative
Electron Microscopy
Subepithelial deposits only ± mesangial
rarely
Subepithelial deposits ± mesangial and
subendothelial deposits

28. ELECTRON MICROSCOPY
• Electron-dense deposits in the epithelial aspect (external) of the
GBM, between this one and the epithelial cell: subepithelials or
epimembranous
• Spikes are demonstrated as irregular projections of the GBM

31. CLINICAL FEATURES
• Rare in children: Less than 5% of total cases of nephrotic syndrome
• Common in adults: 15% to 50% of total cases of nephrotic syndrome,
depending on age; increasing frequency after 40
• Males > females in all adult groups
• Nephrotic syndrome in 60% to 70%
• Normal or mildly elevated blood pressure at presentation
• Benign urinary sediment
• Nonselective proteinuria
• Tendency to thromboembolic disease
• Other features of secondary causes: Infection, drugs, neoplasia,
systemic lupus erythematosus

32. Clinical features-correlating to Anti PLA2R
• 70%–80% of patients with PMN have anti-PLA2R/THSD7A antibody
• Anti-PLA2R antibody is about 80% sensitive and 100% specific for
PMN
• Anti-PLA2R antibody can be present for many months before
proteinuria appears
• In non-nephrotic patients, low, or declining, anti-PLA2R levels predict
spontaneous remission and high levels predict progression to
nephrotic syndrome
• Anti-PLA2R–negative patients can become positive later
• High antibody levels (before and after treatment) correlate with
proteinuria, response to therapy, and (after therapy) long-term
outcomes

33. • Patients with higher antibody levels require more prolonged
immunosuppression to achieve remission rates comparable to those
with lower levels
• Expansion of the specificity of anti-PLA2R antibody to include
additional epitopes (epitope spreading) correlates with a worse
prognosis
• Anti-PLA2R levels go down in remission and return with relapse
• Elevated anti-PLA2R levels after treatment predict relapse
• Elevated anti-PLA2R levels at the time of transplantation predict
recurrence
• Disappearance of anti-PLA2R antibodies (immunologic remission)
precedes renal remission (disappearance of proteinuria) by weeks to
months

34. • ELISA assay for PLA2R
• Cell-based ALBIA assay
(Mitogen Advanced
Diagnostics Laboratory,
Calgary, Canada)
ELISA assay, levels
>20 RU/ml are
considered positive

35. Predictors of Poor Outcome
Factors Predictor PPV (%)
Clinical Features
Age Older > younger 43
Gender Male > female 30
HLA type HLA/B18/DR 3/Bffl present 71
Hypertension Present 39
Serum Levels
Albumin <1.5 g/dL 56
Creatinine Above normal 61

36. Urine Protein
Nephrotic syndrome Present 32
Proteinuria >8 g for >6 months 66
IgG excretion >250 mg/day 80
β2-Microglobulin excretion >54 µg/mmol creatinine <54 79
C5b-9 excretion >7 mg/mg creatinine 67
Biopsy Changes
Glomerular focal sclerosis Present 34
Tubulointerstitial disease Present 48

37. TORANTO RISK SCORE
Low Risk Medium Risk High Risk
Normal serum
and creatinine
plus proteinuria
over 6 mo of
Normal or near-
creatinine clearance
persistent proteinuria
8 g/day over 6 mo
maximum
treatment
Deteriorating renal
function (>30%decline
GFR)and/or persistent
proteinuria >8 g/day
(up to 6) months of
observation

40. OUTCOME
Clinical Response Definition
Complete remission Proteinuria <0.3 g/d
Partial remission
>50% reduction from baseline and between 0.3
and 3.5 g/d
With stable GFR
No remission <50% reduction or >3.5 g/d
Relapse Recurrence of >3.5 g/d after remission
ESRD
GFR<15 ml/min or requirement for
dialysis/transplant

41. • Relapse from a complete remission occurs in approximately 25% to
40%
• Relapse rate is as high as 50% in those achieving only a partial
remission
• Review of 348 nephrotic patients with MN documented a 10-year
renal survival
• complete remission of 100%
• partial remission, 90%
• no remission, only 45%.
• A recent update suggested durability of remission, whether complete
or partial, drug-induced or spontaneous, is closely related to the long-
term outcome.
• Thus goal of therapy is Complete and partial remission

42. ST Regimen Drug, Dose Comments
Cytotoxic drugs KDIGO first choice
Modified Ponticelli
Months 1, 3, 5: 1 g
methylprednisolone iv on days 1, 2,
and 3 followed by oral prednisone,
0.5 mg/kg daily for 27 d
Monitor Uprotein and WBC weekly
×8, then every 2 mo; daily oral
prednisone and cyclophosphamide
may have similar efficacy. Increased
risk of malignancy above 36 g
Months 2, 4, 6: 2.0–2.5 mg/kg oral
cyclophosphamide daily
Relapse rate 20%–30%
Dutch protocol
Months 1, 3, 5: 1 g MP days 1–3
followed by oral prednisone, 0.5–1.0
mg/kg for 6 mo, then taper Same as above
Oral cyclophosphamide, 1.5–2.0
mg/kg daily for 12 mo

43. CNIs KDIGO second choice
Cyclosporin
3.5–5.0 mg/kg daily in divided doses adjusted
to level of 120–200 μg/L for 12–18 mo and
tapered
Used in patients resistant to cytotoxic drugs
but can be used as initial therapy. Taper slowly
Prednisone 5–10 mg daily or alt days
Discontinue at 6 mo if no response
Relapse rate 40%–50%
Tacrolimus
0.05–0.075 mg/kg daily in two divided doses
adjusted to level of 3–5 μg/L for 12–18 mo
and taper slowly
Same as above
Prednisone 5–10 mg/kg per day daily or alt
days
Preferable in young women
B cell depletion
Used for patients resistant to cytotoxic drugs
or CNIs
Utility as initial therapy not yet established by
RCTs
Rituximab
375 mg/M2 weekly times 4 Follow CD20 counts and repeat dose if counts
rise before remission in proteinuria or relapse
occurs
375 mg/M2 once and follow CD20/19 counts
1000 mg on days 1 and 15
ACTH
Tetracosactrin (Synacthen) (synthetic) 1 mg IM every 2 wk for 6–12 mo
Corticotropin (ACTHAR) (purified) 80 U IM every 2 wk for 6–12 mo

44. • MULTIDRUG THERAPY
• Rituximab with low-dose cyclophosphamide and an
accelerated taper of steroids reported a 100% remission
rate over a mean follow-up of 37 months
• Rituximab and CSA, achieved remissions in 92% and
antibody depletion in 100% in 9 months
• A combination of rituximab and plasma exchange showed
promise in a third small study

45. Transplantation in PMN
• In anti-PLA2R–positive patients, subepithelial deposits can appear in
the allograft within 6 days
• Proteinuria is seen within 13–15 months in about 40%–50% of
allografts and can diminish allograft survival
• Recurrence rate of subepithelial deposits in patients positive for anti-
PLA2R antibodies at the time of transplantation may approach 90%
• Anti-PLA2R–negative de novo MN is also a common cause of
transplant nephrotic syndrome, affecting about 2% of all renal
transplant recipients whose original disease was not MN

46. • Because patients with recurrent subepithelial deposits do not all go
on to manifest clinical recurrence, treatment for recurrent MN is
usually considered only when protein excretion reproducibly exceeds
1 g/d in patients with PMN or 4 g/d in patients with de novo MN
• Rituximab is usually added to regular immunosuppressive protocols,
which often already include CNIs -one dose of 200 mg

47. NEW THERAPY
• Belimumab, an inhibitor of B cell activation, in 11 anti-PLA2R–positive
patients reported a 90% reduction in anti-PLA2R levels and a
(delayed) 70% reduction in proteinuria in patients receiving monthly
iv doses of the drug over a period of 28 weeks

48. THANK U…