cASe RePORT
Bortezomib as a Novel Approach to Early Recurrent
Membranous Glomerulonephritis After Kidney Transplant
Refractory to Combined Conventional Rituximab Therapy
Antoine Barbari,1 Rima Chehadi,1 Hala Kfoury Assouf,2 Gaby Kamel,1 Mahassen Jaafar,1
Ayman Abdallah,1 Sylvana Rizk,3 Marwan Masri3
Abstract
We report a case of early recurrent membranous
glomerulonephritis after kidney transplant from a
deceased donor. The patient received induction
therapy and was discharged with a serum creatinine
level of 0.78 mg/dL on triple maintenance immuno-
suppressive therapy, which included tacrolimus,
mycophenolate mofetil, and prednisone. At 7 months
after transplant, a graft biopsy for new-onset isolated
proteinuria (2.7 g/day) revealed stage 2 recurrent
membranous glomerulonephritis. In the face of
persistent proteinuria despite combined conservative
rituximab therapy over several months and the total
eradication of CD20-positive cells, bortezomib was
introduced. This resulted in a substantial decline in
proteinuria within 2 months and its subsequent
disappearance several months later. This was
paralleled by a considerable drop in plasma CD34-
positive and CD138-positive cell counts. These
preliminary observations indicate that recurrent
posttransplant membranous glomerulonephritis is
associated in part with a B-cell-mediated immunologic
process that may involve both CD20-positive
and plasma cells. Rituximab-resistant or partially
responsive recurrent posttransplant membranous
glomerulonephritis may benefit from a proteasome
inhibitor-based therapy.
Key words: Autoimmune disease, Plasma cell, Proteasome
inhibitors, Proteinuria, Recurrent posttransplant
glomerulonephritis
From the 1Rafik Hariri University Hospital, Beirut, Lebanon; the 2King Saud University,
Riyadh, Saudi Arabia; and the 3Transmedical for Life, Beirut, Lebanon
Acknowledgements: The authors declare that they have no sources of funding for this study,
and they have no conflicts of interest to declare.
Corresponding author: Antoine Barbari, Lebanese Faculty of Medical Sciences, Department
of Internal Medicine, Renal Transplant Unit, Rafik Hariri University Hospital, Bir Hassan,
Beirut, Lebanon
Phone: +961 1832040-1831550-1831551 E-mail: barbariantoine@gmail.com
Experimental and Clinical Transplantation (2017) 3: 350-354
Copyright © Başkent University 2017
Printed in Turkey. All Rights Reserved.
Introduction
A 50-year-old male patient presented with end-stage
renal disease secondary to membranous glomerulo-
nephritis (MGN). He underwent deceased-donor
kidney transplant using induction therapy with
rabbit antithymocyte globulin (ATG Fresenius,
Grafalon; Neovii, Waltham, MA, USA), followed by
a triple maintenance immunosuppressive regimen
that included tacrolimus (Prograf; Astellas Pharma,
Tokyo, Japan), mycophenolate mofetil (Cellcept;
Hoffmann-La Roche, Basel, Switzerland), and
prednisone. The patient was discharged with a
serum creatinine level of 0.72 mg/dL. Tacrolimus
dosage was adjusted according to whole blood
trough levels. Mycophenolate mofetil was
maintained at 2 g/day, and prednisone was tapered
and maintained at 5 mg/day.
Overt proteinuria was first detected at 5 months
posttransplant (2.2 g/day). Ramipril (Tritace; Sanofi-
Aventis, Paris, France) was started at a dose of
10 mg/day. In the face of persistent isolated
proteinuria with a negative cross-match and panel
reactive antibodies, a graft biopsy was performed,
which revealed characteristic features of stage
2 recurrent posttransplant MGN (PTMGN)
(Figure 1). Irbesartan (Aprovel; Sanofi-Aventis) and
subsequently spironolactone (Aldactone; Konoshima
Chemical, Osaka, Japan) were added and increased
to optimally tolerated dose levels. Proteinuria
worsened to reach 8.5 g/day despite optimal triple
renin angiotensin aldosterone system blockade,
tacrolimus at a therapeutic whole blood trough level,
and adequate mycophenolate mofetil dosage, which
allowed systolic and diastolic arterial blood pressure
levels to be maintained at 100 to 120 mm Hg and 70
to 80 mm Hg (Figure 2). Rituximab (Mabthera;
Roche, Basel, Switzerland) was introduced in four
DOI: 10.6002/ect.2016.0350
 Antoine Barbari et al/Experimental and Clinical Transplantation (2017) 3: 350-354 351

Figure 1. Histological Findings on the Graft Biopsy

(A) Photomicrograph showing glomerulus with minimal capillary wall thickening (silver staining, ×400).
(B) Immunofluorescence study for immunoglobulin G shows diffuse finely granular staining along the
glomerular capillary walls (3+/4+). (c) C4d immunostaining showing diffuse strong staining along the
glomerular capillary walls (×250). (D) Electron photomicrograph highlighting the numerous subepithelial
dense deposits (Churg Ehrenreich stage 2), with diffuse effacement of the epithelial cell foot processes and
microvillous degeneration of the visceral epithelial cell cytoplasm (uranyl acetate, lead citrate, ×800).

Figure 2. Posttransplant Clinical Course Represented by Progression of Proteinuria, Renal Function Defined by Serum
Creatinine, Systolic and Diastolic Arterial Blood Pressures, and Body Weight

Abbreviations: ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin type 2 receptor blocker; ATG,
antithymocyte globulin; BP, blood pressure; MMF, mycophenolate mofetil; T0, trough level
352 Antoine Barbari et al/Experimental and Clinical Transplantation (2017) 3: 350-354 Exp Clin Transplant

500-mg doses 1 month apart. Partial remission in
proteinuria was noted, followed by a rebound that
occurred after the third dose.
In the face of persistent proteinuria despite near
eradication of CD20-positive cells (Figure 3),
bortezomib (Velcade; Takeda Oncology, Cambridge,
MA, USA), a proteasome inhibitor, was started at a
dose of 1.3 mg/m for a total of 4 doses over 2 weeks.
Within 2 months, considerable drops in CD34-
positive and CD138-positive cell counts were noted
that were paralleled by significant reductions in
proteinuria, which resolved several months later
(Figure 3).
Discussion
Membranous glomerulonephritis may recur or
develop de novo after transplant. The de novo form
is twice as common as the recurrent form.1 Recurrent
PTMGN can occur in approximately 40% of patients,
usually within the first year.2 In our case, the overt
proteinuria appeared at 7 months (Figure 2).
Posttransplant MGN is characterized by a sub-
epithelial immune complex dense deposit with
isolated linear C4d deposition in the glomerular
capillary basement membrane, as in our case, leading
to predominantly epithelial cell damage without
significant endothelial cell injury or inflammatory
cell infiltration (Figure 1). The presence of
multilayering of the peritubular capillary basement
membrane with C4d deposition indicates its
association with antibody-mediated injury3 and may
represent another manifestation of chronic humoral
rejection.4
Two types of antibodies are recognized:
antibodies against the major human histo-
compatibility HLA complex and antibodies against
a non-HLA major histocompatibility complex class I-
related chain gene A (MICA) and gene B.5 Unlike
classical HLA molecules, MICA expression is limited
to dendritic cells, monocytes, activated T cells,
keratinocytes, fibroblasts, and endothelial and
epithelial cells. Products of the gene, MICA
molecules, act as a ligand for several immune cells. A

Figure 3. Progression of Proteinuria and Variations Observed in the Different Immunologic Markers (total lymphocyte count and CD20-,
CD34-, CD105-, and CD138-positive cell counts)

Abbreviations: ACEI, angiotensin-converting enzyme inhibitor; ARB, angiotensin type 2 receptor blocker; ATG, antithymocyte globulin;
MMF, mycophenolate mofetil
Cell counts are expressed as percentage of total lymphocyte counts. Progression is in relation to different sequential therapeutic
interventions from the time of transplant until the introduction of bortezomib and the subsequent decline and later disappearance of
proteinuria.
 Antoine Barbari et al/Experimental and Clinical Transplantation (2017) 3: 350-354
sizeable proportion of kidney transplant recipients
may have anti-MICA antibodies present in their sera
at the time of transplant that may be directed against
their own MICA antigens, and many develop de
novo MICA antibodies after transplant that may be
either donor or nondonor specific.6 Antibodies
directed against MICA may occur as a consequence
of allogeneic donor sensitization and from some
novel autoreactive mechanism yet to be identified,
such as autoimmune disease.6
Analysis of MICA is neither easily nor routinely
evaluated in most transplant centers. In our case,
MICA antibodies were detected at the time of graft
biopsy. Their donor specificity could not be identified
and their de novo occurrence could not be confirmed.
Given that MICA expression occurs on epithelial cells
and that the predominant epithelial cell injury
(podocyte) observed in our patient was in the
absence of any alloimmune response, one can
postulate a possible role of anti-MICA antibodies in
the pathogenesis of PTMGN, irrespective of the
timing of their occurrence. In the case of recurrence,
they may represent preformed anti-MICA antibodies
that might have been potentially responsible for the
native kidney disease and may cause early
recurrence posttransplant.
Phospholipase A2 receptor (PLA2R), a podocyte
structure, has been recognized as an important
specific marker for idiopathic MGN and PTMGN.1,7
Persistence or reappearance of posttransplant anti-
PLA2R is associated with increasing proteinuria and
resistant disease.8,9 Recent reports have described the
presence of antibodies against MICA in kidney
allografts localized in podocytes within the glomeruli
of renal transplant recipients without or with acute
rejection, together with infiltrating mononuclear
cells, B cells, CD8-positive T cells, and natural killer
cells.10 Whether PLA2R is a product of the MICA gene
or the MICA molecule family remains to be
determined. Unfortunately, staining and testing for
PLA2R and the specific antibodies were not available
at our center.
Induction therapy and standard maintenance
immunosuppression in some cases are sufficient to
induce immunologic remission; however, they do not
preclude the possibility of recurrence of PTMGN,7 as
was the case in our patient. Interestingly, in our
patient, the increasing proteinuria was paralleled by
a progressive rise in total lymphocyte count (TLC)
while on the same maintenance immunosuppressive
353
regimen (Figure 3). Surprisingly, the peak TLC
coincided with the highest level of proteinuria in the
absence of any rejection process, which was followed
by partial simultaneous reduction in both TLC and
proteinuria after induction therapy with rituximab
(Figure 3). The considerable and persistent increase
in TLC despite rituximab treatment and in the
absence of any histologic or serologic evidence of any
type of rejection may be explained, although on a
purely speculative basis, by the proliferation of
yet to be identified immune cell, such as memory B
cells that may be involved in the pathogenesis of
PTMGN. Alternatively, the significant increase in
TLC may be related to a process of immuno-
tolerance.11 Unfortunately, none of the markers
associated with immunotolerance or micro-
chimerism have thus far been identified. The partial
response to rituximab in our patient indicates, at least
in part, a pathogenic role of CD20-positive B cells in
PTMGN, as also reported previously.12 Rituximab
binds to the CD20 receptor of B cells and depletes
them through cellular-dependent cytotoxicity and
stimulation of B-cell apoptosis.13 It may also target
short-lived plasma cells still expressing CD20.14
CD20 is expressed later in the B-cell lineage than
CD19 and is not found on pro-B cells of mature
plasma cells known to produce 90% of circulating
immunoglobulin G.14
In our patient, CD20-positive cell counts
decreased after the first dose of rituximab and then
increased again. Rituximab eliminates peripheral B
cells without preventing the regeneration of B cells
from precursor cells and does not directly affect
immunoglobulin G levels.14 This may explain at least
in part the reappearance of proteinuria during
treatment with rituximab. The partial remission
observed with anti-CD20 therapy and the relapse
in proteinuria despite near elimination of CD20-
positive cells suggests a possible additional CD20-
independent mechanism of antibody production.
The possible role of memory plasma cell in the
persistent production of alloantibodies or auto-
antibodies has also been suggested but so far remains
unproven.11 Classically, memory B cells can become
activated plasmablasts, which then convert to
plasmocytes within hours of exposure to an antigen.
Plasma cells produce large quantities of protein
immunoglobulin G. Inhibition of proteasome activity
results in accumulation of misfolded proteins and
consequent apoptosis of plasma cells.14
354 Antoine Barbari et al/Experimental and Clinical Transplantation (2017) 3: 350-354
In vitro, bortezomib induces apoptosis of all
antibody-producing plasma cells.15 In the absence of
rituximab-induced complete remission despite near
eradication of a CD20-positive cell count, bortezomib
was introduced. Within 2 months, considerable
drops in CD34-positive and CD138-positive cell
counts were noted in our patient, which was
paralleled by a significant reduction in proteinuria
that disappeared several months later (Figure 3).
Both CD34 and CD138 are established plasmocyte
markers.
Our clinical observation suggests a possible role
of plasma cells in the pathogenesis of recurrent
PTMGN, mainly in those patients who are resistant to
CD20-positive targeted therapy. To our knowledge,
this is the first case report describing the successful
use of a proteasome inhibitor (bortezomib) in
recurrent PTMGN with persistent proteinuria that
was refractory to combined conventional rituximab
therapy.
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