CLINICAL RESEARCH www.jasn.

org

A Randomized, Controlled Trial of Rituximab in IgA

Nephropathy with Proteinuria and Renal Dysfunction
Richard A. Lafayette,* Pietro A. Canetta,† Brad H. Rovin,‡ Gerald B. Appel,† Jan Novak,§
Karl A. Nath,| Sanjeev Sethi,¶ James A. Tumlin,** Kshama Mehta,* Marie Hogan,|
Stephen Erickson,| Bruce A. Julian,§†† Nelson Leung,| Felicity T. Enders,‡‡ Rhubell Brown,§
Barbora Knoppova,§§§ Stacy Hall,§ and Fernando C. Fervenza|
*Division of Nephrology and Hypertension, Stanford University, Stanford, California; †Division of Nephrology and
Hypertension, Columbia University Medical Center, New York, New York; ‡Division of Nephrology, Ohio State University,
Columbus, Ohio; Departments of §Microbiology and ††Medicine, University of Alabama at Birmingham, Birmingham,
Alabama; |Division of Nephrology and Hypertension, ¶Department of Laboratory Medicine and Pathology, and
‡‡
Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester,
Minnesota; **Division of Nephrology, University of Tennessee, Chattanooga, Tennessee; and §§Department of
Immunology, Faculty of Medicine and Dentistry, Palacky University and University Hospital, Olomouc, Czech Republic

ABSTRACT
IgA nephropathy frequently leads to progressive CKD. Although interest surrounds use of immunosuppressive
agents added to standard therapy, several recent studies have questioned efficacy of these agents. Depleting
antibody–producing B cells potentially offers a new therapy. In this open label, multicenter study conducted over
1-year follow-up, we randomized 34 adult patients with biopsy–proven IgA nephropathy and proteinuria .1 g/d,
maintained on angiotensin–converting enzyme inhibitors or angiotensin receptor blockers with well controlled BP
and eGFR,90 ml/min per 1.73 m2, to receive standard therapy or rituximab with standard therapy. Primary outcome
measures included change in proteinuria and change in eGFR. Median baseline serum creatinine level (range) was
1.4 (0.8–2.4) mg/dl, and proteinuria was 2.1 (0.6–5.3) g/d. Treatment with rituximab depleted B cells and was well
tolerated. eGFR did not change in either group. Rituximab did not alter the level of proteinuria compared with that at
baseline or in the control group; three patients in each group had $50% reduction in level of proteinuria. Serum
levels of galactose-deficient IgA1 or antibodies against galactose-deficient IgA1 did not change. In this trial,
rituximab therapy did not significantly improve renal function or proteinuria assessed over 1 year. Although rituximab
effectively depleted B cells, it failed to reduce serum levels of galactose-deficient IgA1 and antigalactose–deficient
IgA1 antibodies. Lack of efficacy of rituximab, at least at this stage and severity of IgA nephropathy, may reflect
a failure of rituximab to reduce levels of specific antibodies assigned salient pathogenetic roles in IgA nephropathy.

J Am Soc Nephrol 28: 1306–1313, 2017. doi: 10.1681/ASN.2016060640

IgA nephropathy (IgAN) is the most common pri- presence of immune complexes in the kidney suggest
mary glomerular disease in the world.1,2 Among that immunosuppression would be beneficial in this
patients with reduced renal function and proteinuria disease. Indeed, many small studies have suggested
.1 g/24 h, outcomes remain poor. Up to 50% of such
patients will progress to ESRD over 10 years.3,4 Trials
have established the benefit of agents that antagonize Received June 13, 2016. Accepted September 11, 2016.

the renin-angiotensin-aldosterone system (RAAS) in Published online ahead of print. Publication date available at
reducing or delaying progression,5,6 but even patients www.jasn.org.
treated with RAAS blockade still face deterioration of Correspondence: Dr. Fernando C. Fervenza, Mayo Clinic, Division
their kidney function over time. of Nephrology and Hypertension, 200 First Street SW, Rochester,
MN 55905. Email: fervenza.fernando@mayo.edu
The presence of inflammation on histologic ex-
amination of the kidney in IgAN along with the Copyright © 2017 by the American Society of Nephrology

1306 ISSN : 1046-6673/2804-1306 J Am Soc Nephrol 28: 1306–1313, 2017

 www.jasn.org CLINICAL RESEARCH

that corticosteroid therapy may be effective in attenuating pro- RESULTS

teinuria and retarding progression, but they are attended by
risks for serious adverse events.7–9 However, a recent highly Thirty-four patients met inclusion criteria for this study and
publicized study questioned the efficacy of corticosteroids were randomized, leading to 17 subjects in the rituximab group
and other immunosuppression in IgAN.10 In this study, com- as well as in the standard care group. Their ultimate disposition
pared with the control group (without immunosuppression), is shown in Figure 1. Two patients had HSPN with inactive
the rate of decrease of eGFR was not reduced in patients treated systemic features and were randomly divided between the two
with corticosteroids alone (patients with eGFR$60 ml/min) or groups. Six of the rituximab and five of the standard care
those who were treated with corticosteroids and cyclophospha- patients had received prior corticosteroids. Baseline charac-
mide for 3 months followed by corticosteroids and azathio- teristics are detailed in Table 1. All subjects had biopsy-proven
prine thereafter (patients with eGFR between 30 and 60 ml/min IgAN, with ,50% glomerular sclerosis or interstitial fibrosis.
per 1.73 m2). Similarly, immunosuppressive therapy with All patients were maintained on RAAS inhibitors, with five
other approaches, such as mycophenolate mofetil, has yielded patients on combined therapy with angiotensin–converting
conflicting findings with regards to efficacy in retarding pro- enzyme inhibitor and angiotensin receptor blocker treatment,
gressive disease.11,12 Thus, there remains a strong need to find three in the rituximab arm and two in the conservative group.
better, safer therapies for this disease. BP was well controlled, averaging 122/79610/11 mmHg. The
A potentially novel therapy is suggested on the basis of the median baseline serum creatinine concentration was 1.4 (0.8–
current understanding of the pathogenesis of IgAN. A key event 2.4) mg/dl. Randomization resulted in a higher but not statis-
is the development of IgG and/or IgA autoantibodies against tically significant baseline serum creatinine concentration in
polymeric galactose–deficient IgA1 (Gd-IgA1), the levels of the rituximab group compared with the standard care group
which are elevated in the circulation of most patients with (1.7 [0.8–2.3] mg/dl versus 1.3 [0.8–2.4] mg/dl, respectively),
IgAN.13,14 Increased circulatory levels of autoantigen or auto- but the resultant eGFR was significantly lower in the rituximab
antibody levels correlate with increased risk of progres- group (Table 1). The baseline proteinuria of all subjects was
sion.15,16 Because B cell–depleting therapies are now known 2.1 (range =0.6–5.3) g/d, and the difference between the rit-
to be effective in many renal diseases mediated by the presence uximab group and the standard care group was not statistically
of autoantibodies (for example, membranous nephropathy, significant, although it trended higher in the rituximab group
lupus nephritis, and ANCA-associated vasculitis),17–19 such (2.6 versus 1.7 g/d). There was no difference in components of
an approach is appealing in IgAN, because it would deplete the Oxford classification score22 between the two groups (rit-
antibody–producing B cells and presumably, the autoanti- uximab group versus standard care group: M1, 100% versus
bodies that drive progression of this CKD. Indeed, isolated 94%; E1, 41% versus 24%; S1, 82% versus 88%; T1–T2, 35%
case reports support this approach, because rituximab, which versus 50%, respectively; all nonsignificant) (Table 1). The
depletes CD20–bearing B cells, reportedly improved renal standard care group was significantly younger and had a
function and reduced proteinuria in individuals with IgAN greater percentage of men than women. Two subjects in the
or the associated systemic disease, Henoch–Schönlein pur- standard care group dropped out as did three in the rituximab
pura with nephritis (HSPN).20,21 group, such that 15 subjects completed the study in the stan-
We conducted an open label, multicenter, randomized, con- dard care group and 14 completed it in the rituximab group;
trolled study to formally assess the hypothesis that B cell de- all 16 rituximab-treated subjects had post-treatment data for
pletion with rituximab, added to the standard of care, would the intention-to-treat analysis (they received their first round
decrease the production of IgG anti–Gd-IgA1 autoantibodies of rituximab).
and reduce immune complex formation and glomerular in- BP was stable and well controlled throughout the study, and
flammation, leading thereby to less proteinuria and renal dys- it was consistently comparable between treatment groups. Kid-
function in patients with IgAN at high risk of progression. ney function was also stable throughout the study in both

Figure 1. Trial organization. Patients were randomly assigned, in a 1:1 ratio, to rituximab and control groups. No patient was withdrawn
from the study due to an adverse event.

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Table 1. Baseline characteristics postrituximab (only one patient exhibited

Baseline Characteristics All Patients, n=34 Control, n=17 Rituximab, n=17 P Value partial depletion of CD19+ B cells to 45 and
Age, yr 40 (21–63) 33 (21–59) 43 (29–63) 0.04 68 cells per microliter at 6 and 12 months,
Sex, women, men 9, 25 2, 15 7, 10 0.12 respectively). CD19+ B cells depletion was
Weight, kg 89.7 (57–120) 92.3 (57–119) 85.1 (61–120) 0.46 sustained at 12 months. There was no signif-
Race 0.07 icant effect on serum Ig levels, including total
White 24 15 9 0.07 IgA levels (Table 2). There were no differ-
Black 1 0 1 0.07 ences in serum levels of Gd-IgA1 or anti–
Asian/Pacific Islanders 6 2 4 0.07 Gd-IgA1 autoantibodies in the rituximab
Hispanic/Latino 3 0 3 0.07 compared with control group at baseline or
Systolic BP 121 (102–147) 118 (102–147) 124 (107–144) 0.17
during the study. The serum levels did not
Diastolic BP 78 (56–106) 78 (56–106) 81 (59–94) 0.24
change during observation. There was no
Creatinine, mg/dl 1.4 (0.8–2.4) 1.3 (0.8–2.4) 1.7 (0.8–2.3) 0.07
eGFR 49 (30–122) 61 (32–122) 40 (30–78) 0.02
correlation between changes in serum levels
24-h Proteinuria, g 2.1 (0.6–5.3) 1.7 (0.6–4.0) 2.6 (0.9–5.3) 0.09 of Gd-IgA1 or anti–Gd-IgA1 autoantibody
Oxford pathology score, % and proteinuria during the study (data not
M0/M1 3/97 6/94 0/100 0.30 shown). There was no serious adverse event
E0/E1 68/32 76/24 59/41 0.30 in either group during the study, although
S0/S1 15/85 12/88 18/82 0.60 adverse events per subject were more com-
T0/T1/T2 56/41/3 53/47/0 65/29/6 0.30 mon in the rituximab group, including mild
Data presented as median and range. infections (Figure 4).

groups, whether assessed by eGFR or serum creatinine concen- DISCUSSION

tration (Figure 2, Table 1). The other primary outcome, pro-
teinuria at 12 months, was not changed from baseline in either Our study enrolled 34 subjects with IgAN who were likely
group. In the rituximab group, median proteinuria decreased to progress (on the basis of decreased eGFR and substantial
from 2.6 (range =0.9–5.3) g/d to 1.9 (range =0.4–8.8) g/d, but proteinuria), assigning 17 to treatment with rituximab and
this change was not statistically significant (P=0.30). The final resulting in 16 receiving therapy and having outcome data.
proteinuria in the rituximab group was not different from the At baseline, there were no differences in 24-hour proteinuria
proteinuria in the standard care group, which was unchanged or serum creatinine concentration between the active treat-
from baseline: 1.8 (range =0.5–4.0) g/d to 1.8 (range =0.4–4.3) ment group and control group treated with diet, RAAS block-
g/d (Figure 3). There was no statistical difference found using ade, and fish oil. Although there was a trend toward lower
absolute level of quantitative proteinuria or the change of pro- proteinuria after rituximab treatment, neither group experi-
teinuria during the study period. Three subjects in each group enced ;a significant reduction in proteinuria, and a significant
experienced a .50% reduction in proteinuria from baseline. Six number of individuals did not manifest a substantial, sustained
subjects in the rituximab group and four in the control group reduction in proteinuria or improvement in renal function.
had a .25% reduction in proteinuria. There were equal numbers of subjects achieving a 50% or
As expected, rituximab depleted B cells as measured 6 greater reduction in proteinuria (three of 16 in the rituximab
months into the study. Peripheral blood B cells (CD19+) arm and three of 15 in the standard therapy arm) and similar
decreased from a median of 84 (range =41–222) cells per mi- numbers of subjects achieving a 25% or greater reduction
croliter prerituximab to 0 (range =0–45) cells per microliter (six of 16 rituximab and four of 15 standard care). There
was no significant difference in kidney function over the 1
year of follow-up. Two rituximab subjects and one standard
care subject had a 25% or greater increase in eGFR, and one
rituximab subject and no standard care subjects had a .25%
reduction in eGFR. Thus, over the time course studied and for
this stage and severity of IgAN, treatment with rituximab failed
to significantly reduce proteinuria or benefit renal function.
We believe that these negative findings with regards to the
efficacy of rituximab are important for several reasons. First,
these data add another type of immunosuppression to the
current list of such agents wherein there is no clear evidence
of therapeutic efficacy. Second, our findings call for circum-
Figure 2. eGFR trends in (A) rituximab versus (B) control groups. spection and restraint in considering the use of this agent in
The red line represents average data. patients with IgAN at this stage of the disease who exhibit

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levels of the anti–Gd-IgA1 autoantibodies may thus account

for the absence of a beneficial effect of rituximab on the course
of IgAN at this stage. Another consideration is that increased
circulating levels of anti–Gd-IgA1 IgG autoantibodies may
originate from plasma cells in bone marrow. Plasma cells are
derived from B cells but in the process, have lost CD19 ex-
pression. It is thus possible that our failure to observe efficacy
of rituximab may reflect that, although CD19+ B cells are
effectively depleted by rituximab, plasma cells are not, and it
is these latter cells, rather than the CD19+ expressing B cells,
that account for production of antiglycan antibodies. This
Figure 3. Proteinuria trends in (A) rituximab versus (B) control consideration may also be germane to other antibody–driven
groups. The red line represents median data. nephropathies in which rituximab use is considered: if CD19+
plasma cells are the source of such antibodies, rituximab may
increasing proteinuria and/or decline in eGFR, despite conser- not be effective. There is a similarity of our results with those
vative therapy and/or other immunosuppressive regimens. showing an ineffectiveness of rituximab to induce remission
Third, because rituximab is not without significant adverse in patients with moderately active ulcerative colitis who had
effects and risks, the current failure to observe a beneficial not responded to oral corticosteroids. 28 Both IgAN and
effect at this stage of the disease argues against its use in this ulcerative colitis display a prominent involvement of
setting, thereby sparing patients from unjustified risks. the mucosal immune system, and B cell depletion therapy
In our study, we confirmed that rituximab achieved its does not seem to provide long-lasting effects. In contrast,
stated objective of effectively depleting CD19+ B cells, the latter patients with rheumatoid arthritis or primary membranous
considered, in earlier studies, a primary producer of IgA and a nephropathy benefit from B cell depletion therapy.18,19 These
contributor to the pathogenesis of IgAN.23 We also assessed the disease-specific and divergent effects thus raise the following
effect of rituximab on serum levels of Gd-IgA1 and anti–Gd- questions. Is it the significant role of mucosal immunity in
IgA1 IgG autoantibodies and found, much to our surprise, IgAN and ulcerative colitis that renders B cell depletion ther-
that levels of neither were reduced. Serum Gd-IgA1 levels apy less effective? Are the autoantibodies in patients with
are known to be genetically codetermined,24,25 although the primary membranous nephropathy of systemic origin, and
levels can be further enhanced by some cytokines).26 Thus, it is thus, are the antigen-specific cells in these subjects an easier
not clear whether a B cell depletion protocol that would sig- target of rituximab? As a recent review concluded, we have
nificantly reduce serum levels of Igs, including Gd-IgA1, much more to learn about B cell biology to be able to design
would have a long-lasting effect on serum Gd-IgA1 levels. optimal approaches to test for therapeutic response to B cell–
We regard the absence of reduction in the autoantibody pro- targeted agents.29
duction as notable for at least two reasons that center on path- This trial is limited by the relatively small numbers of pa-
ogenetic and therapeutic considerations. The pathogenesis of tients studied. However, we wish to emphasize that this is the
IgAN conforms to a four-hit process: (1) circulating levels of first randomized, controlled study that prospectively evaluated
Gd-IgA1 are increased, (2) anti–Gd-IgA1 autoantibodies are the use of B cell depletion added to standard care in patients
produced, (3) complexes containing the two are formed in the with IgAN. Moreover, given the nature of IgAN, this study
systemic circulation, and (4) deposition of these complexes in serves as a relatively large series of patients treated with biopsy-
the glomerulus ensues, leading to glomerular inflammation defined disease who were given full doses of rituximab,
and injury.13,14,27 The failure of rituximab to reduce the serum resulting in effective B cell depletion. The presence of a control

Table 2. Disease markers and outcomes in control versus rituximab treatment groups
Baseline End of Study
Markers and Outcomes
Control, n=11 Rituximab, n=11 P Value Control, n=10 Rituximab, n=10 P Value
Biochemical markersa
IgA, mg/ml 4.762.0 5.061.8 0.68 4.661.9 4.461.4 0.77
Gd-IgA1, per 100 ng IgA, U 56.866.9 54.8610.0 0.58 58.965.6 60.5613.0 0.73
IgG autoantibodies, U/ml 858.46647.3 1492.561672.9 0.25 10756908.7 1751.362469.4 0.42
Outcomes, n
Patients with .50% reduction in proteinuria — — — 3 3 .0.99
Patients with .50% increase in proteinuria — — — 2 1 0.54
Patients with ,500 mg/d protein — — — 2 2 .0.99
—, not applicable.
a
Data presented as mean 6SD.

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Figure 4. Adverse events in the control group and the rituximab group. Type of event and number of each of these events are depicted,
with the control group to the left of the red line and the rituximab group to the right of the red line.

group allows us to conclude that results were not different in proteinuria is an important determinant in outcome,30 if there
the rituximab group than in the standard care group. It is were signals of benefit in this population, we should have been
impossible to rule out individual responses, but if present, alerted by this trial. The implications of the numerical, not
they were the exception rather than the rule. Our experience statistically significant, fall in proteinuria in the rituximab-
is also limited in that our patients receiving rituximab had treated group merit comment. To achieve 80% power for any
significant proteinuria and abnormal kidney function, perhaps significant reduction in proteinuria to be possibly observed,
making it too late in their disease to respond to any immuno- our results show that a randomized study comparing the
therapy, including B cell depletion. Patients in the rituximab change in proteinuria for rituximab with control would re-
group were older and had lower eGFR at baseline (by quire 272 patients. The need to treat such a large number of
21 ml/min per 1.73 m2), more than expected by the age dif- patients for any statistical change in proteinuria to be evinced
ference. This finding suggests that the disease was of longer thus seriously questions whether any meaningful pathoge-
duration in the rituximab group, which may have biased the netic and/or therapeutic importance can be applied to this
outcomes, despite equal Oxford MEST scores at baseline. numerical reduction in proteinuria observed in the rituximab-
However, all patients had biopsies within 2 years before treat- treated group.
ment, and those with advanced glomerulosclerosis or inter- Finally, although the presence of circulatory Gd-IgA1 and
stitial fibrosis (.50%) were excluded. Two patients with IgG autoantibodies to this protein are thought to be central to
HSPN were included, but their outcomes did not influence the pathogenesis and progression of IgAN, our data strikingly
the results. The rituximab group was more racially and ethnically show no favorable effect of rituximab on these Igs (either Gd-
diverse, but an analysis, restricted to white patients only also IgA1 or autoantibodies to Gd-IgA1), supporting the lack of clin-
revealed an absence of an effect of rituximab on proteinuria ical evidence of benefit observed in the study. Because the serum
and eGFR (data not shown). Thus, because reduction in levels of Gd-IgA1 and anti–Gd-IgA1 autoantibodies predict

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disease progression15,16 and disease recurrence,31 both Gd-IgA1 and diphenhydramine HCl (50 mg) by mouth from 30 to 60 minutes
and the corresponding autoantibodies may be valid candidate before the start of an infusion. Premedication with corticosteroids
biomarkers for assessment of responses to treatment.27 (10 mg dexamethasone intravenously) was also given 30 minutes before
In summary, in patients with IgAN and relatively high risk the first infusion of each series of rituximab. They received an identical
for progressive renal dysfunction, treatment with rituximab 2-g course of rituximab 6 months later. Subjects were assessed at least
resulted in statistically insignificant reductions in proteinuria every 3 months or as needed for clinical events. This assessment in-
or partial remissions that could not be distinguished from the cluded physical examination, a questionnaire for adverse events, and
response to supportive therapy alone. These data do not sup- measurement of routine hematology, serum chemistry, timed urine
port the use of rituximab for the treatment of IgAN, at least for protein excretion, and, for those assigned to rituximab, B cell subsets.
this stage and severity of the disease. Future studies could eGFR was calculated by the four–component MDRD formula using
consider examining earlier use of B cell depletion in IgAN or serum creatinine concentration measured in the central laboratory.
longer follow-up periods or focus on other approaches to re- Follow-up was considered complete at 12 months.
duce the progression of renal disease. Finally, if CD19+ cells of
B cell lineage, such as plasma cells, are confirmed as the source Outcomes
of autoantigen and autoantibody production in IgAN, it is The primary outcome measures were the change in proteinuria and
possible that rituximab may not be effective at any stage and the change in eGFR from baseline to 12 months. The secondary out-
severity for this disease. come was safety related, comparing overall adverse events and mon-
itoring for potential intervention–specific complications, such as
infusion-related reactions, hypogammaglobulinemia, and infections.
CONCISE METHODS
Determination of Serum Total IgA and Serum Levels of
The protocol was approved by the institutional review board of each Gd-IgA1
participating center. The study was registered with clinicaltrials.gov, pro- The concentration of serum total IgA was measured by ELISA.32
tocol NCT00498368. Informed consent was obtained before all study Briefly, 96-well plates were coated with goat F(ab9)2 anti–human
procedures, and the study adhered to the Declaration of Helsinki. IgA (Jackson ImmunoResearch Laboratories), blocked with 1%
BSA in PBS plus 0.05% Tween 20 (PBS-T), washed, and incubated
Subjects with serially diluted samples or standard serum with known concen-
Adults, ages 18–70 years old, with biopsy-proven IgAN shown within 2 tration of IgA. Bound IgA was detected by biotinylated goat F(ab9)2
years of enrollment were included. Patients were excluded if their biopsy anti–human IgA (GenWay Biotech Inc.) followed by incubation
showed .50% glomerular sclerosis or interstitial fibrosis or .10% with horseradish peroxidase–conjugated streptavidin (ExtrAvidin-
glomerular crescents. Baseline Oxford classification score22 was recorded Peroxidase; Sigma-Aldrich) and developed with the peroxidase chro-
in blinded fashion by an expert renal pathologist (S.S.). eGFR (by mogenic substrate o-phenylenediamine-H2O2. OD was measured at
Modification of Diet in Renal Disease [MDRD]) or measured creatinine 490 nm on an EL808 Microplate Reader, and the concentration of
clearance had to be ,90 and .30 ml/min per 1.73 m2. To establish IgA in the samples was calculated on the basis of a calibration curve
continued risk, baseline proteinuria needed to be .1000 mg/d while on of standard serum.
stable doses of angiotensin–converting enzyme inhibitor, angiotensin Gd-IgA1 was measured by lectin ELISA.26,33 Briefly, 96-well plates
receptor blocker, or renin inhibitor therapy for at least 2 months. How- coated with goat F(ab9)2 anti–human IgA were blocked with 1%
ever, patients who were on dual therapy with agents that inhibit angio- BSA in PBS-T, washed, and incubated with serum samples added at
tensin II required a lower proteinuria threshold of .500 mg/d. Baseline dilutions corresponding to 100 ng IgA per well overnight at 4°C.
BP was controlled to ,130/80 mmHg. Patients with secondary forms of Captured IgA was desialylated using neuraminidase.26 Gd-IgA1 was
IgAN, such as cirrhosis, were excluded, although subjects with HSPN detected using biotinylated lectin from Helix aspersa (HAA; Sigma-
could be included. Patients were excluded if they had previously received Aldrich) specific for terminal N-acetylgalactosamine and diluted to
rituximab, were receiving other immunosuppressive therapy, or had final concentration of 2 mg/ml in 1% BSA in PBS-T. HAA was de-
ever received .6 months of prednisone or other systemic corticosteroid tected by ExtrAvidin-Peroxidase and developed with the peroxidase
therapy in the past. There was no corticosteroid exposure within 3 chromogenic substrate o-phenylenediamine-H2O2. OD was mea-
months of study initiation. Randomization was done centrally by a ran- sured at 490 nm using an EL808 Microplate Reader. The serum
dom assignment by prefilled envelopes. The study was unblinded as to Gd-IgA1 concentration was expressed in units defined as the ratio
treatment arm. of OD determined for individual samples and a standard Gd-IgA1
(Ale) myeloma protein; 100 U Gd-IgA1 was defined as the OD of
Treatment and Follow-Up HAA lectin binding to 50 ng standard Gd-IgA1 (Ale).
Fish oil supplements were required at a minimal dose of 3 g/d. Subjects
were randomly assigned to receive rituximab or continue standard Determination of Serum Levels of IgG Autoantibody
care. The study was an open label trial; those assigned to rituximab Specific for Gd-IgA1
received a 1-g infusion of rituximab followed by an identical dose 2 Serum levels of IgG autoantibody specific for Gd-IgA1 were measured
weeks later. All patients were premedicated with acetaminophen (1 g) by ELISA 34 ; 96-well plates were coated with 3 mg/ml Gd-IgA1

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myeloma protein (Ale). Plates were washed and then blocked with 1% 3. Barratt J, Feehally J: IgA nephropathy. J Am Soc Nephrol 16: 2088–
BSA in PBS-T. Samples of serum were diluted 500-fold in PBS. Bound 2097, 2005
4. Barbour SJ, Reich HN: Risk stratification of patients with IgA nephrop-
IgG was detected with a biotin–labeled F(ab9)2 fragment of goat IgG
athy. Am J Kidney Dis 59: 865–873, 2012
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added, and the reaction was developed with the peroxidase chromogenic the pathogenesis and treatment of IgA nephropathy. Kidney Int 81:
substrate o-phenylenediamine-H2O2. OD was measured at 490 nm us- 833–843, 2012
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9. Lv J, Zhang H, Chen Y, Li G, Jiang L, Singh AK, Wang H: Combination
experience (one investigator) with rituximab in IgAN indicated that therapy of prednisone and ACE inhibitor versus ACE-inhibitor therapy
approximately 80% of patients would achieve a significant reduction alone in patients with IgA nephropathy: A randomized controlled trial.
in proteinuria. On the basis of the one-half width of a 95% confidence Am J Kidney Dis 53: 26–32, 2009
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outcomes of the rituximab and standard care groups were compared N Engl J Med 373: 2225–2236, 2015
using the Wilcoxon rank sum test, rejecting the null hypothesis 11. Tang SC, Tang AW, Wong SS, Leung JC, Ho YW, Lai KN: Long-term
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patients who received any dose of rituximab as part of the treatment nephropathy. Am J Kidney Dis 66: 783–791, 2015
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ACKNOWLEDGMENTS 14. Lai KN, Tang SCW, Schena FP, Novak J, Tomino Y, Foggo AB, Glassock
RJ: IgA nephropathy. Nat Rev Dis Primers 2016 Feb 11: 16001. doi:
10.1038/nrdp.2016.1
This study was an investigator-initiated study sponsored by Gen- 15. Berthoux F, Suzuki H, Thibaudin L, Yanagawa H, Maillard N, Mariat C,
entech/Roche, Inc. and the Fulk Family Foundation. Tomino Y, Julian BA, Novak J: Autoantibodies targeting galactose-
The abstract was previously published at the American Society of deficient IgA1 associate with progression of IgA nephropathy. J Am
Nephrology Meeting, November 5–8, 2015, San Diego, CA, abstract Soc Nephrol 23: 1579–1587, 2012
16. Zhao N, Hou P, Lv J, Moldoveanu Z, Li Y, Kiryluk K, Gharavi AG, Novak J,
number 6192.
Zhang H: The level of galactose-deficient IgA1 in the sera of patients
The sponsors had no role in study design, protocol development, with IgA nephropathy is associated with disease progression. Kidney
data analysis, or preparation of the manuscript. Int 82: 790–796, 2012
17. Stone JH, Merkel PA, Spiera R, Seo P, Langford CA, Hoffman GS,
Kallenberg CG, St Clair EW, Turkiewicz A, Tchao NK, Webber L, Ding
L, Sejismundo LP, Mieras K, Weitzenkamp D, Ikle D, Seyfert-Margolis
DISCLOSURES
V, Mueller M, Brunetta P, Allen NB, Fervenza FC, Geetha D, Keogh
J.N. and B.A.J. are founders of Reliant Glycosciences, LLC (Birmingham, KA, Kissin EY, Monach PA, Peikert T, Stegeman C, Ytterberg SR,
AL). F.C.F. has received unrestricted grant support from Genentech/Roche, Specks U; RAVE-ITN Research Group: Rituximab versus cyclophos-
Inc (South San Francisco, CA). phamide for ANCA-associated vasculitis. N Engl J Med 363: 221–
232, 2010
18. Edwards JC, Szczepanski L, Szechinski J, Filipowicz-Sosnowska A,
Emery P, Close DR, Stevens RM, Shaw T: Efficacy of B-cell-targeted
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