Banerjee, S.

Maji, B.

Sinha, R.

R. Sinha

S. Banerjee

B. Maji

Directory of Open Access Journals

Kidney International Reports

P03 RITUXIMAB IN DIFFICULT PAEDIATRIC NEPHROTIC SYNDROME: AN ACCOUNT FROM EASTERN INDIA

Elsevier - Publisher Connector 

P03 RITUXIMAB IN DIFFICULT PAEDIATRIC NEPHROTIC SYNDROME: AN ACCOUNT FROM EASTERN INDIA 

ISN NEXUS SYMPOSIUM 2016 HIGHLIGHTS: TRANSLATIONAL IMMUNOLOGY IN
KIDNEY DISEASE, BERLIN, GERMANY, APRIL 14–17, 2016P01
ANTI MICA (MAJOR
HISTOCOMPATIBILITY COMPLEX
CLASS I RELATED CHAIN A)
ANTIBODY, WHETHER TO TREAT OR
AVOID IN RENAL TRANSPLANTATION?
Godhani, U1, Balwani, M1, Kute, V1, Trivedi, HL1
1IKDRC - ITS- Ahmedabad, Nephrology, Ahmedabad, India
Introduction: Pre-transplantation anti-major histocompatibility
complex class-I related chain A (MICA) sensitization is an uncom-
mon event and its role in kidney graft evolution is not completely
deﬁned. Even when kidney allografts are well matched for HLA as
in living related transplant and anti-HLA antibodies are not
detected, graft rejection can still occur. Anti MICA antibody is
reportedly associated with poor transplant outcomes and a high
risk of acute and chronic rejection in renal transplantation.
Methods: A retrospective study of patients undergone living renal
transplantation between years 2000-2014 was performed. Recipients
were classiﬁed in two groups, pre-transplantation Anti MICA
antibody positive group (n¼17) and antibody negative comparison
group (n¼17). Patients with anti HLA antibodies were excluded
and only isolated MICA positive patients were included in the
study group. Both groups were comparable in recipient age, donor
age, donor relation, HLA and immunosuppression.
Results: Patients with pre transplant MICA antibody positivity
were associated with increased acute rejection rate as compared to
comparison group (47% vs 11.7 %, p value¼ 0.02). Renal function
in MICA positive group and comparison group were comparable
over a mean follow up of 6.5 years (mean creatinine 1.58 vs 1.53 mg/
dl). Rate of chronic rejection was same in both groups (5.8%). No
patient loss or graft loss occurred in either group over mean follow
up of 6.5 years.
Conclusions: Isolated Anti MICA antibody positivity is uncommon
event. Pre transplant anti MICA antibody positivity is associated
with increased acute rejection rates. However chronic rejection
rates and renal function are comparable in both groups. Thus our
study emphasizes that MICA antibody positive patients may
require more aggressive immunosuppression. Role of desensitiza-
tion has to be deﬁned.
P02
ENHANCED IMMUNOPATHOLOGY
EVALUATION OF HUMAN RENAL
BIOPSIES USING MULTICOLOR FLOW
CYTOMETRY AND CYTOKINE
ANALYSIS: A FOCUS ON
TRANSPLANTED KIDNEYS
Muczynski, K1, Leca, N1, Anderson, A1, Kieran, N1,
Anderson, SK1
1University of Washington, Medicine-Nephrology, Seattle, USA
Introduction: Current histologic processing of renal biopsies pro-
vides limited information about immune mechanisms causing kid-
ney injury and disease activity. To overcome this we developed a
protocol to reduce a fraction of a renal biopsy to single cells for
multicolor ﬂow cytometry and for capture and quantitation of cy-
tokines present in the biopsy. Using this technique we deﬁne newKidney International Reports (2016) 1, S1–S22criteria for evaluating rejection and renal inﬂammation that is
useful for directing therapy.
Methods: A third of a standard kidney biopsy core is reduced to a
cell suspension amenable for multi-color ﬂow cytometry without
losing cells or epitopes of interest. The resulting supernatant is used
for measuring cytokines with high-sensitivity Milliplex reagents on
a Luminex platform. After analysis of hundreds of transplant bi-
opsies we now generate clinically useful reports describing
lymphocyte subsets, endothelial antibody and eculizumab binding
and IL-6, IL-8 and IL-10 levels.
Results:A ratio of CD8+ to CD4+ T lymphocytes greater than 1.2 in
transplanted allografts is associated with rejection, even before it is
apparent by microscopy. Elevated numbers of CD45 leukocytes and
higher levels of IL-6, IL-8 and IL-10 within the biopsy indicate
more severe injury.
Peripheral blood T lymphocyte subsets do not correlate with
those found within the renal allograft. In addition, lymphocytes
within the kidney express variable degree of activation based on
expression of CD69 and HLA-DR, while these markers are low or
absent on peripheral blood lymphocytes.
Antibody binding to renal microvascular endothelial cells can
be measured by cytometry and corresponds to antibody-mediated
forms of allograft rejection. Eculizumab binding to endothelial cells
suggests complement activation, which may be independent of
bound antibody, or associated with it.
Conclusions: Assessment of leukocyte subsets, renal microvascular
endothelial properties and measurement of cytokines within a renal
biopsy enhance understanding of pathogenesis, provide disease
activity markers and identify potential targets for therapy.
P03
RITUXIMAB IN DIFFICULT PAEDIATRIC
NEPHROTIC SYNDROME: AN
ACCOUNT FROM EASTERN INDIA
Sinha, R1, Banerjee, S2, Maji, B3
1Institute of Child Health, Paedaric Nephrologyt, Kolkata, India; 2CMRI,
Paediatric, Kolkata, India; 3Institute of Child Heath, Paediatric Nephrology,
KOlkata, India
Introduction: Reports on the utility of rituximab in difﬁcult
nephrotic syndrome (NS) have been varied. We retrospectively
analyzed the outcome of rituximab, used in a multi centre cohort of
difﬁcult nephrotic syndrome (NS) from Eastern India.
Methods: Data was collected for all children with NS who received
rituximab from May 2011 to Nov 2015. Steroid resistant (SRNS) and
steroid dependent / frequently relapsing (SDNS /FRNS) were
identiﬁed as per standard deﬁnition. Complete response (CR) for
SRNS was deﬁned as normalization of serum albumin and urinary
protein creatinine ratio (UPCR) whereas for partial response (PR);
50% improvements in these parameters along with albumin at last
follow up $ 2gm/dl. In cases of SDNS/FRNS; CR was deﬁned as
stoppage of steroid and absence of relapses for at least a year and PR
as discontinuation of steroid without any relapses for at least six
month or reduction in steroid threshold by at least 50%.
Results: 34 children (56 % male) were identiﬁed (SRNS ¼12, SDNS/
FRNS ¼22). Among SRNS all had failed steroid (S), mycophenola-
te(M) as well as calcineurin inhibitor (CNI) except two who were
CNI naïve prior to rituximab. Among the SDNS/ FRNS group, 11
(50%) children had failed all drugs (S, M, CNI & cyclophospha-
mide) and the rest were not exposed to CNI. Majority were minimalS1
TRANSLATIONAL IMMUNOLOGY IN KIDNEY DISEASE, ISN NEXUS SYMPOSIUM 2016change nephrotic syndrome (MCNS) (62%) followed by focal
segmental glomerulosclerosis (FSGS, 27%) and IgM nephropathy
(11%). Median age was 7.8 (Range 2.5 – 16.5) years with median
follow up post rituximab 33 (Range 1.5 to 87) months. Rituximab
was given as infusion at 375 mg / m2 . Each cycle constituted of
2 injections at an interval of 1-2 weeks followed by conﬁrmation of
B cell depletion. Single cycle achieved total B-cell depletion in all.
5 cases (all SRNS) received 2. Median duration for normalisation of
CD 19 was 4.65 (3 – 7) months. Among the SRNS, serum albumin
rose from mean 1.87 (SD  0.53) to 2.63 (SD  1.1) g/dl, (p¼0.06)
and UPCR fell from mean 19.3 (SD  12.04) to 9.8 (SD  10.96)
p¼ 0.15. 20% of SRNS (n¼2) achieved CR another 20% PR. Steroid
threshold among SDNS/FRNS fell from mean 0.47 (SD  0.19) to
0.17 (SD  0.23) mg/kg, p ¼0.0003 and dose of steroid at last follow
up fell men 0.88 (SD  0.56) to 0.22 (SD  0.43) mg/kg, p ¼0.0009.
50% of SDNS/FRNS (n¼11) did not have any relapse during the
follow up period and median time to ﬁrst relapse was 6.6 (Range 0.3
– 25) months.
Conclusions: Rituximab was demonstrated to be useful with sig-
niﬁcant beneﬁt particularly in the SDNS group.
P04
A NOVEL MOUSE MODEL OF
MEMBRANOUS NEPHROPATHY
INDUCED WITH HETEROLOGOUS
RABBIT ANTI-THSD7A ANTIBODIES
Tomas, N1, Meyer-Schwesinger, C1, von Spiegel, H1,
Zahner, G1, Hoxha, E1, Helmchen, U2, Koch-Nolte, F3,
Stahl, RAK1
1Universitätsklinikum Hamburg-Eppendorf, III. Medizinische Klinik-Neph-
rologie, Hamburg, Germany; 2Universitätsklinikum Hamburg-Eppendorf,
Nierenregister, Hamburg, Germany; 3Universitätsklinikum Hamburg-
Eppendorf, Institut für Immunologie, Hamburg, Germany
Introduction: Phospholipase A2 receptor 1 (PLA2R1) and throm-
bospondin type-1 domain-containing 7A (THSD7A) are podocyte
membrane proteins that have been identiﬁed as target antigens for
autoimmunity in membranous nephropathy (MN). The investiga-
tion of the pathogenicity of the involved autoantibodies has been
hampered by the fact that PLA2R1 is not expressed on rodent
podocytes. On the other hand, THSD7A is expressed on mouse
podocytes and shares over 90% of sequence homology with the
human protein. We have recently demonstrated that anti-THSD7A
antibodies isolated from a patient with THSD7A- associated MN can
cause morphological and clinical MN in mice, allowing further
investigations of disease pathogenesis. However, THSD7A-associ-
ated MN is a rare entity, incapacitating patient antibodies for larger
experimental procedures.
Methods: We generated polyclonal antibodies against human and
mouse THSD7A in rabbits using cDNA immunization. IgG from the
immunized rabbits and from preimmune control rabbits was af-
ﬁnity-puriﬁed and intravenously injected into mice. Urine was
collected daily and investigated for the development of proteinuria
by measurement of albumin-to-creatinine ratios. Mice were sacri-
ﬁced ﬁve, nine, and fourteen days after injection of rabbit IgG and
kidneys were analyzed using confocal and light microscopy.
Results: Puriﬁed IgG from immunized rabbits recognize, like pa-
tient autoantibodies, (a) conformation-dependent epitope(s) present
in both native human and mouse THSD7A in vitro. Moreover, two
hours after intravenous injection into mice, rabbit IgG is bound
along the glomerular ﬁltration barrier. Two days later, mice developS2proteinuria that rapidly increases and reaches around 10 g/g after
5 days and 200-300 g/g albumin-to-creatinine after 14 days with
some mice developing a severe nephrotic syndrome with ascites and
hyperlipidemia. In immunoﬂuorescent analysis, granular rabbit IgG
is found subepithelially along the glomerular ﬁltration barrier after
14 days and immunohistochemistry for rabbit IgG shows the classic
picture of human MN. Mice injected with puriﬁed IgG from rabbit
serum that was taken before THSD7A-immunization fail to develop
any of these changes.
Conclusions: Our study introduces a heterologous mouse model
that allows further mechanistic investigations of the molecular
events leading to membranous nephropathy.
P05
IDENTIFICATION OF
GLYCOSAMINOGLYCANS THAT
SPECIFICALLY INHIBIT THE LECTIN
PATHWAY OF COMPLEMENT
Talsma, D1, Poppelaars, F1, Vives, RR2, Lortat-Jacob, H2,
Naggi, A3, Torri, G3, Seelen, M1, Daha, M1, Stegeman, CA1,
van den Born, J1
1University Medical Centre Groningen, Nephrology, GRONINGEN, The
Netherlands; 2Univ of Grenoble, Inst. for Structural Biology, Grenoble,
France; 3Ronzoni Institute, Carbohydrate Science Group, Milano, Italy
Introduction: The complement system can be activated via three
pathways, the classical (CP), alternative (AP) and lectin pathway
(LP). Studies have shown a role for complement in renal diseases
such as hemolytic uremic syndrome, C3 glomerulopathy and renal
transplantation. It is well known that heparin and some other
glycosaminoglycans (GAGs) inhibit complement activation. We
tested the pathway speciﬁc complement inhibiting potential of >70
GAGs. The results showed that some GAGs had a speciﬁc inhibitory
effect on the LP only. In this work we unravel the inhibitory
mechanism of GAGs on the LP.
Methods: GAGs were tested in the Wieslab complement screening
assay for a dose-dependent inhibition assay of the complement
pathways. Inhibition of ﬁcolin-3 mediated LP activation by GAGs
was tested in the Wieslab ﬁcolin-3 assay. To assess whether GAGs
inhibit the MBL – mannan interaction, GAGs were added to diluted
serum and incubated on mannan coated plates where after depo-
sition of MBL was measured. To test the inhibition of GAGs on
MASP activity, the GAGs were added to puriﬁed C4 and incubated
on MBL-MASP coated plates, C4 activation was measured by
detection of C4b. To assess whether the MBL-MASP complex binds
to heparin, serum was incubated on heparin-albumin coated plates
and binding of MBL, MASP-1 and MASP-2 was measured.
Results: In the Wieslab complement screening assay, (LMW-)hep-
arin dose-dependently inhibited all three pathways of complement.
Depolymerization of heparin followed by size-fractionation of the
resultant oligosaccharides revealed hexasaccharides and tetra-
saccharides to exclusively inhibit the LP, without any inhibition
potential for the CP and AP. Heparin (oligo’s) also show a dose-
dependent inhibition of the ﬁcolin-3 mediated LP activation,
however failed to inhibit the binding of MBL to mannan, indicating
inhibition by GAGs of MASP1/2 enzymes. This was proven in the
MASP-1/2 inhibition assays. Heparin (oligo’s) inhibited the C4
cleavage by MASP-1/2 in a dose-dependent way. Comparison of
deﬁned heparin-derived tetrasaccharides with various sulfation
revealed the fully N-, 2-O, 6-O-sulfated fragment the most potent
MASP-1/2 inhibitor. Vice versa the MBL-MASP complex showsKidney International Reports (2016) 1, S1–S22


file:///data/core-remote/dit/data/elsevier/pdf/970/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMjQ2ODAyNDkxNjMwMDg4Mg==.pdf

P03 RITUXIMAB IN DIFFICULT PAEDIATRIC NEPHROTIC SYNDROME: AN ACCOUNT FROM EASTERN INDIA

Abstract

Similar works

Full text

Available Versions

Directory of Open Access Journals

Elsevier - Publisher Connector

Elsevier - Publisher Connector