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-
mary glomerular disease in the world.1,2 Among
patients with reduced renal function and proteinuria
.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
presence of immune complexes in the kidney suggest
that immunosuppression would be beneficial in this
disease. Indeed, many small studies have suggested
Received June 13, 2016. Accepted September 11, 2016.

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

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


that corticosteroid therapy may be effective in attenuating pro-
teinuria and retarding progression, but they are attended by
risks for serious adverse events.7–9 However, a recent highly
publicized study questioned the efficacy of corticosteroids
and other immunosuppression in IgAN.10 In this study, com-
pared with the control group (without immunosuppression),
the rate of decrease of eGFR was not reduced in patients treated
with corticosteroids alone (patients with eGFR$60 ml/min) or
those who were treated with corticosteroids and cyclophospha-
mide for 3 months followed by corticosteroids and azathio-
prine thereafter (patients with eGFR between 30 and 60 ml/min
per 1.73 m2). Similarly, immunosuppressive therapy with
other approaches, such as mycophenolate mofetil, has yielded
conflicting findings with regards to efficacy in retarding pro-
gressive disease.11,12 Thus, there remains a strong need to find
better, safer therapies for this disease.
A potentially novel therapy is suggested on the basis of the
current understanding of the pathogenesis of IgAN. A key event
is the development of IgG and/or IgA autoantibodies against
polymeric galactose–deficient IgA1 (Gd-IgA1), the levels of
which are elevated in the circulation of most patients with
IgAN.13,14 Increased circulatory levels of autoantigen or auto-
antibody levels correlate with increased risk of progres-
sion.15,16 Because B cell–depleting therapies are now known
to be effective in many renal diseases mediated by the presence
of autoantibodies (for example, membranous nephropathy,
lupus nephritis, and ANCA-associated vasculitis),17–19 such
an approach is appealing in IgAN, because it would deplete
antibody–producing B cells and presumably, the autoanti-
bodies that drive progression of this CKD. Indeed, isolated
case reports support this approach, because rituximab, which
depletes CD20–bearing B cells, reportedly improved renal
function and reduced proteinuria in individuals with IgAN
or the associated systemic disease, Henoch–Schönlein pur-
pura with nephritis (HSPN).20,21
We conducted an open label, multicenter, randomized, con-
trolled study to formally assess the hypothesis that B cell de-
pletion with rituximab, added to the standard of care, would
decrease the production of IgG anti–Gd-IgA1 autoantibodies
and reduce immune complex formation and glomerular in-
flammation, leading thereby to less proteinuria and renal dys-
function in patients with IgAN at high risk of progression.
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.
J Am Soc Nephrol 28: 1306–1313, 2017
www.jasn.org CLINICAL RESEARCH
RESULTS
Thirty-four patients met inclusion criteria for this study and
were randomized, leading to 17 subjects in the rituximab group
as well as in the standard care group. Their ultimate disposition
is shown in Figure 1. Two patients had HSPN with inactive
systemic features and were randomly divided between the two
groups. Six of the rituximab and five of the standard care
patients had received prior corticosteroids. Baseline charac-
teristics are detailed in Table 1. All subjects had biopsy-proven
IgAN, with ,50% glomerular sclerosis or interstitial fibrosis.
All patients were maintained on RAAS inhibitors, with five
patients on combined therapy with angiotensin–converting
enzyme inhibitor and angiotensin receptor blocker treatment,
three in the rituximab arm and two in the conservative group.
BP was well controlled, averaging 122/79610/11 mmHg. The
median baseline serum creatinine concentration was 1.4 (0.8–
2.4) mg/dl. Randomization resulted in a higher but not statis-
tically significant baseline serum creatinine concentration in
the rituximab group compared with the standard care group
(1.7 [0.8–2.3] mg/dl versus 1.3 [0.8–2.4] mg/dl, respectively),
but the resultant eGFR was significantly lower in the rituximab
group (Table 1). The baseline proteinuria of all subjects was
2.1 (range =0.6–5.3) g/d, and the difference between the rit-
uximab group and the standard care group was not statistically
significant, although it trended higher in the rituximab group
(2.6 versus 1.7 g/d). There was no difference in components of
the Oxford classification score22 between the two groups (rit-
uximab group versus standard care group: M1, 100% versus
94%; E1, 41% versus 24%; S1, 82% versus 88%; T1–T2, 35%
versus 50%, respectively; all nonsignificant) (Table 1). The
standard care group was significantly younger and had a
greater percentage of men than women. Two subjects in the
standard care group dropped out as did three in the rituximab
group, such that 15 subjects completed the study in the stan-
dard care group and 14 completed it in the rituximab group;
all 16 rituximab-treated subjects had post-treatment data for
the intention-to-treat analysis (they received their first round
of rituximab).
BP was stable and well controlled throughout the study, and
it was consistently comparable between treatment groups. Kid-
ney function was also stable throughout the study in both
Rituximab in IgA Nephropathy 1307
 CLINICAL RESEARCH www.jasn.org

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
group. In the rituximab group, median proteinuria decreased
from 2.6 (range =0.9–5.3) g/d to 1.9 (range =0.4–8.8) g/d, but
this change was not statistically significant (P=0.30). The final
proteinuria in the rituximab group was not different from the
proteinuria in the standard care group, which was unchanged
from baseline: 1.8 (range =0.5–4.0) g/d to 1.8 (range =0.4–4.3)
g/d (Figure 3). There was no statistical difference found using
absolute level of quantitative proteinuria or the change of pro-
teinuria during the study period. Three subjects in each group
experienced a .50% reduction in proteinuria from baseline. Six
subjects in the rituximab group and four in the control group
had a .25% reduction in proteinuria.
As expected, rituximab depleted B cells as measured 6
months into the study. Peripheral blood B cells (CD19+)
decreased from a median of 84 (range =41–222) cells per mi-
croliter prerituximab to 0 (range =0–45) cells per microliter
Figure 2. eGFR trends in (A) rituximab versus (B) control groups.
The red line represents average data.
Our study enrolled 34 subjects with IgAN who were likely
to progress (on the basis of decreased eGFR and substantial
proteinuria), assigning 17 to treatment with rituximab and
resulting in 16 receiving therapy and having outcome data.
At baseline, there were no differences in 24-hour proteinuria
or serum creatinine concentration between the active treat-
ment group and control group treated with diet, RAAS block-
ade, and fish oil. Although there was a trend toward lower
proteinuria after rituximab treatment, neither group experi-
enced ;a significant reduction in proteinuria, and a significant
number of individuals did not manifest a substantial, sustained
reduction in proteinuria or improvement in renal function.
There were equal numbers of subjects achieving a 50% or
greater reduction in proteinuria (three of 16 in the rituximab
arm and three of 15 in the standard therapy arm) and similar
numbers of subjects achieving a 25% or greater reduction
(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-
spection and restraint in considering the use of this agent in
patients with IgAN at this stage of the disease who exhibit

1308 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 1306–1313, 2017
 www.jasn.org CLINICAL RESEARCH

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

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

J Am Soc Nephrol 28: 1306–1313, 2017 Rituximab in IgA Nephropathy 1309

 CLINICAL RESEARCH www.jasn.org
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
the rituximab group than in the standard care group. It is
impossible to rule out individual responses, but if present,
they were the exception rather than the rule. Our experience
is also limited in that our patients receiving rituximab had
significant proteinuria and abnormal kidney function, perhaps
making it too late in their disease to respond to any immuno-
therapy, including B cell depletion. Patients in the rituximab
group were older and had lower eGFR at baseline (by
21 ml/min per 1.73 m2), more than expected by the age dif-
ference. This finding suggests that the disease was of longer
duration in the rituximab group, which may have biased the
outcomes, despite equal Oxford MEST scores at baseline.
However, all patients had biopsies within 2 years before treat-
ment, and those with advanced glomerulosclerosis or inter-
stitial fibrosis (.50%) were excluded. Two patients with
HSPN were included, but their outcomes did not influence
the results. The rituximab group was more racially and ethnically
diverse, but an analysis, restricted to white patients only also
revealed an absence of an effect of rituximab on proteinuria
and eGFR (data not shown). Thus, because reduction in
1310 Journal of the American Society of Nephrology
proteinuria is an important determinant in outcome,30 if there
were signals of benefit in this population, we should have been
alerted by this trial. The implications of the numerical, not
statistically significant, fall in proteinuria in the rituximab-
treated group merit comment. To achieve 80% power for any
significant reduction in proteinuria to be possibly observed,
our results show that a randomized study comparing the
change in proteinuria for rituximab with control would re-
quire 272 patients. The need to treat such a large number of
patients for any statistical change in proteinuria to be evinced
thus seriously questions whether any meaningful pathoge-
netic and/or therapeutic importance can be applied to this
numerical reduction in proteinuria observed in the rituximab-
treated group.
Finally, although the presence of circulatory Gd-IgA1 and
IgG autoantibodies to this protein are thought to be central to
the pathogenesis and progression of IgAN, our data strikingly
show no favorable effect of rituximab on these Igs (either Gd-
IgA1 or autoantibodies to Gd-IgA1), supporting the lack of clin-
ical evidence of benefit observed in the study. Because the serum
levels of Gd-IgA1 and anti–Gd-IgA1 autoantibodies predict
J Am Soc Nephrol 28: 1306–1313, 2017

disease progression15,16 and disease recurrence,31 both Gd-IgA1
and the corresponding autoantibodies may be valid candidate
biomarkers for assessment of responses to treatment.27
In summary, in patients with IgAN and relatively high risk
for progressive renal dysfunction, treatment with rituximab
resulted in statistically insignificant reductions in proteinuria
or partial remissions that could not be distinguished from the
response to supportive therapy alone. These data do not sup-
port the use of rituximab for the treatment of IgAN, at least for
this stage and severity of the disease. Future studies could
consider examining earlier use of B cell depletion in IgAN or
longer follow-up periods or focus on other approaches to re-
duce the progression of renal disease. Finally, if CD19+ cells of
B cell lineage, such as plasma cells, are confirmed as the source
of autoantigen and autoantibody production in IgAN, it is
possible that rituximab may not be effective at any stage and
severity for this disease.
CONCISE METHODS
The protocol was approved by the institutional review board of each
participating center. The study was registered with clinicaltrials.gov, pro-
tocol NCT00498368. Informed consent was obtained before all study
procedures, and the study adhered to the Declaration of Helsinki.
Subjects
Adults, ages 18–70 years old, with biopsy-proven IgAN shown within 2
years of enrollment were included. Patients were excluded if their biopsy
showed .50% glomerular sclerosis or interstitial fibrosis or .10%
glomerular crescents. Baseline Oxford classification score22 was recorded
in blinded fashion by an expert renal pathologist (S.S.). eGFR (by
Modification of Diet in Renal Disease [MDRD]) or measured creatinine
clearance had to be ,90 and .30 ml/min per 1.73 m2. To establish
continued risk, baseline proteinuria needed to be .1000 mg/d while on
stable doses of angiotensin–converting enzyme inhibitor, angiotensin
receptor blocker, or renin inhibitor therapy for at least 2 months. How-
ever, patients who were on dual therapy with agents that inhibit angio-
tensin II required a lower proteinuria threshold of .500 mg/d. Baseline
BP was controlled to ,130/80 mmHg. Patients with secondary forms of
IgAN, such as cirrhosis, were excluded, although subjects with HSPN
could be included. Patients were excluded if they had previously received
rituximab, were receiving other immunosuppressive therapy, or had
ever received .6 months of prednisone or other systemic corticosteroid
therapy in the past. There was no corticosteroid exposure within 3
months of study initiation. Randomization was done centrally by a ran-
dom assignment by prefilled envelopes. The study was unblinded as to
treatment arm.
Treatment and Follow-Up
Fish oil supplements were required at a minimal dose of 3 g/d. Subjects
were randomly assigned to receive rituximab or continue standard
care. The study was an open label trial; those assigned to rituximab
received a 1-g infusion of rituximab followed by an identical dose 2
weeks later. All patients were premedicated with acetaminophen (1 g)
J Am Soc Nephrol 28: 1306–1313, 2017
www.jasn.org CLINICAL RESEARCH
and diphenhydramine HCl (50 mg) by mouth from 30 to 60 minutes
before the start of an infusion. Premedication with corticosteroids
(10 mg dexamethasone intravenously) was also given 30 minutes before
the first infusion of each series of rituximab. They received an identical
2-g course of rituximab 6 months later. Subjects were assessed at least
every 3 months or as needed for clinical events. This assessment in-
cluded physical examination, a questionnaire for adverse events, and
measurement of routine hematology, serum chemistry, timed urine
protein excretion, and, for those assigned to rituximab, B cell subsets.
eGFR was calculated by the four–component MDRD formula using
serum creatinine concentration measured in the central laboratory.
Follow-up was considered complete at 12 months.
Outcomes
The primary outcome measures were the change in proteinuria and
the change in eGFR from baseline to 12 months. The secondary out-
come was safety related, comparing overall adverse events and mon-
itoring for potential intervention–specific complications, such as
infusion-related reactions, hypogammaglobulinemia, and infections.
Determination of Serum Total IgA and Serum Levels of
Gd-IgA1
The concentration of serum total IgA was measured by ELISA.32
Briefly, 96-well plates were coated with goat F(ab9)2 anti–human
IgA (Jackson ImmunoResearch Laboratories), blocked with 1%
BSA in PBS plus 0.05% Tween 20 (PBS-T), washed, and incubated
with serially diluted samples or standard serum with known concen-
tration of IgA. Bound IgA was detected by biotinylated goat F(ab9)2
anti–human IgA (GenWay Biotech Inc.) followed by incubation
with horseradish peroxidase–conjugated streptavidin (ExtrAvidin-
Peroxidase; Sigma-Aldrich) and developed with the peroxidase chro-
mogenic substrate o-phenylenediamine-H2O2. OD was measured at
490 nm on an EL808 Microplate Reader, and the concentration of
IgA in the samples was calculated on the basis of a calibration curve
of standard serum.
Gd-IgA1 was measured by lectin ELISA.26,33 Briefly, 96-well plates
coated with goat F(ab9)2 anti–human IgA were blocked with 1%
BSA in PBS-T, washed, and incubated with serum samples added at
dilutions corresponding to 100 ng IgA per well overnight at 4°C.
Captured IgA was desialylated using neuraminidase.26 Gd-IgA1 was
detected using biotinylated lectin from Helix aspersa (HAA; Sigma-
Aldrich) specific for terminal N-acetylgalactosamine and diluted to
final concentration of 2 mg/ml in 1% BSA in PBS-T. HAA was de-
tected by ExtrAvidin-Peroxidase and developed with the peroxidase
chromogenic substrate o-phenylenediamine-H2O2. OD was mea-
sured at 490 nm using an EL808 Microplate Reader. The serum
Gd-IgA1 concentration was expressed in units defined as the ratio
of OD determined for individual samples and a standard Gd-IgA1
(Ale) myeloma protein; 100 U Gd-IgA1 was defined as the OD of
HAA lectin binding to 50 ng standard Gd-IgA1 (Ale).
Determination of Serum Levels of IgG Autoantibody
Specific for Gd-IgA1
Serum levels of IgG autoantibody specific for Gd-IgA1 were measured
by ELISA 34 ; 96-well plates were coated with 3 mg/ml Gd-IgA1
Rituximab in IgA Nephropathy 1311
 CLINICAL RESEARCH www.jasn.org
myeloma protein (Ale). Plates were washed and then blocked with 1%
BSA in PBS-T. Samples of serum were diluted 500-fold in PBS. Bound
IgG was detected with a biotin–labeled F(ab9)2 fragment of goat IgG
anti–human IgG antibody (Invitrogen). ExtrAvidin-Peroxidase was
added, and the reaction was developed with the peroxidase chromogenic
substrate o-phenylenediamine-H2O2. OD was measured at 490 nm us-
ing an EL808 Microplate Reader. Serum levels of IgG autoantibody
specific for Gd-IgA1 were expressed in units (1 U IgG autoantibody
defined as the OD of 1.0 measured at 490 nm) using a standard re-
combinant IgG autoantibody specific for Gd-IgA1.34
Statistical Analyses
Data were stored centrally within a secure database. Our preliminary
experience (one investigator) with rituximab in IgAN indicated that
approximately 80% of patients would achieve a significant reduction
in proteinuria. On the basis of the one-half width of a 95% confidence
interval, the study targeted 25 patients per arm to achieve 80% power
and allowed for an interim analysis after 15 per arm were recruited.
Outcome variables were measured as median and range. Primary
outcomes of the rituximab and standard care groups were compared
using the Wilcoxon rank sum test, rejecting the null hypothesis
at a P value of ,0.05. Within-group comparisons were done with a
Wilcoxon signed rank test, again with a P value ,0.05 denoting a
significant variance. Fisher exact test and chi-squared analysis were
used to measure categorical variables. Between-group analyses were
performed according to the principle of intention to treat using all
patients who received any dose of rituximab as part of the treatment
group.
ACKNOWLEDGMENTS
This study was an investigator-initiated study sponsored by Gen-
entech/Roche, Inc. and the Fulk Family Foundation.
The abstract was previously published at the American Society of
Nephrology Meeting, November 5–8, 2015, San Diego, CA, abstract
number 6192.
The sponsors had no role in study design, protocol development,
data analysis, or preparation of the manuscript.
DISCLOSURES
J.N. and B.A.J. are founders of Reliant Glycosciences, LLC (Birmingham,
AL). F.C.F. has received unrestricted grant support from Genentech/Roche,
Inc (South San Francisco, CA).
REFERENCES
1. Li LS, Liu ZH: Epidemiologic data of renal diseases from a single unit in
China: Analysis based on 13,519 renal biopsies. Kidney Int 66: 920–
923, 2004
2. Zaza G, Bernich P, Lupo A; “Triveneto” Register of Renal Biopsies
(TVRRB): Incidence of primary glomerulonephritis in a large North-
Eastern Italian area: A 13-year renal biopsy study. Nephrol Dial Trans-
plant 28: 367–372, 2013
1312 Journal of the American Society of Nephrology
3. Barratt J, Feehally J: IgA nephropathy. J Am Soc Nephrol 16: 2088–
2097, 2005
4. Barbour SJ, Reich HN: Risk stratification of patients with IgA nephrop-
athy. Am J Kidney Dis 59: 865–873, 2012
5. Boyd JK, Cheung CK, Molyneux K, Feehally J, Barratt J: An update on
the pathogenesis and treatment of IgA nephropathy. Kidney Int 81:
833–843, 2012
6. D’Amico G: Natural history of idiopathic IgA nephropathy: Role of clinical
and histological prognostic factors. Am J Kidney Dis 36: 227–237, 2000
7. Wang W, Chen N: Treatment of progressive IgA nephropathy: An up-
date. Contrib Nephrol 181: 75–83, 2013
8. Pozzi C, Andrulli S, Del Vecchio L, Melis P, Fogazzi GB, Altieri P,
Ponticelli C, Locatelli F: Corticosteroid effectiveness in IgA nephropa-
thy: Long-term results of a randomized, controlled trial. J Am Soc
Nephrol 15: 157–163, 2004
9. Lv J, Zhang H, Chen Y, Li G, Jiang L, Singh AK, Wang H: Combination
therapy of prednisone and ACE inhibitor versus ACE-inhibitor therapy
alone in patients with IgA nephropathy: A randomized controlled trial.
Am J Kidney Dis 53: 26–32, 2009
10. Rauen T, Eitner F, Fitzner C, Sommerer C, Zeier M, Otte B, Panzer U,
Peters H, Benck U, Mertens PR, Kuhlmann U, Witzke O, Gross O,
Vielhauer V, Mann JF, Hilgers RD, Floege J; STOP-IgAN Investigators:
Intensive supportive care plus immunosuppression in IgA nephropathy.
N Engl J Med 373: 2225–2236, 2015
11. Tang SC, Tang AW, Wong SS, Leung JC, Ho YW, Lai KN: Long-term
study of mycophenolate mofetil treatment in IgA nephropathy. Kidney
Int 77: 543–549, 2010
12. Hogg RJ, Bay RC, Jennette JC, Sibley R, Kumar S, Fervenza FC, Appel
G, Cattran D, Fischer D, Hurley RM, Cerda J, Carter B, Jung B,
Hernandez G, Gipson D, Wyatt RJ: Randomized controlled trial of
mycophenolate mofetil in children, adolescents, and adults with IgA
nephropathy. Am J Kidney Dis 66: 783–791, 2015
13. Suzuki H, Kiryluk K, Novak J, Moldoveanu Z, Herr AB, Renfrow MB,
Wyatt RJ, Scolari F, Mestecky J, Gharavi AG, Julian BA: The patho-
physiology of IgA nephropathy. J Am Soc Nephrol 22: 1795–1803,
2011
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
15. Berthoux F, Suzuki H, Thibaudin L, Yanagawa H, Maillard N, Mariat C,
Tomino Y, Julian BA, Novak J: Autoantibodies targeting galactose-
deficient IgA1 associate with progression of IgA nephropathy. J Am
Soc Nephrol 23: 1579–1587, 2012
16. Zhao N, Hou P, Lv J, Moldoveanu Z, Li Y, Kiryluk K, Gharavi AG, Novak J,
Zhang H: The level of galactose-deficient IgA1 in the sera of patients
with IgA nephropathy is associated with disease progression. Kidney
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
V, Mueller M, Brunetta P, Allen NB, Fervenza FC, Geetha D, Keogh
KA, Kissin EY, Monach PA, Peikert T, Stegeman C, Ytterberg SR,
Specks U; RAVE-ITN Research Group: Rituximab versus cyclophos-
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
therapy with rituximab in patients with rheumatoid arthritis. N Engl J
Med 350: 2572–2581, 2004
19. Fervenza FC, Abraham RS, Erickson SB, Irazabal MV, Eirin A, Specks U,
Nachman PH, Bergstralh EJ, Leung N, Cosio FG, Hogan MC, Dillon JJ,
Hickson LJ, Li X, Cattran DC; Mayo Nephrology Collaborative Group:
Rituximab therapy in idiopathic membranous nephropathy: A 2-year
study. Clin J Am Soc Nephrol 5: 2188–2198, 2010
20. Pindi Sala T, Michot JM, Snanoudj R, Dollat M, Estève E, Marie B,
Taoufik Y, Delfraissy JF, Lazure T, Lambotte O: Successful outcome of a
J Am Soc Nephrol 28: 1306–1313, 2017

corticodependent Henoch-Schönlein purpura adult with rituximab.
Case Rep Med 2014: 619218, 2014
21. Donnithorne KJ, Atkinson TP, Hinze CH, Nogueira JB, Saeed SA, Askenazi
DJ, Beukelman T, Cron RQ: Rituximab therapy for severe refractory
chronic Henoch-Schönlein purpura. J Pediatr 155: 136–139, 2009
22. Cattran DC, Coppo R, Cook HT, Feehally J, Roberts IS, Troyanov S,
Alpers CE, Amore A, Barratt J, Berthoux F, Bonsib S, Bruijn JA, D’Agati
V, D’Amico G, Emancipator S, Emma F, Ferrario F, Fervenza FC,
Florquin S, Fogo A, Geddes CC, Groene HJ, Haas M, Herzenberg AM,
Hill PA, Hogg RJ, Hsu SI, Jennette JC, Joh K, Julian BA, Kawamura T, Lai
FM, Leung CB, Li LS, Li PK, Liu ZH, Mackinnon B, Mezzano S, Schena FP,
Tomino Y, Walker PD, Wang H, Weening JJ, Yoshikawa N, Zhang H;
Working Group of the International IgA Nephropathy Network and the
Renal Pathology Society: The Oxford classification of IgA nephropathy:
Rationale, clinicopathological correlations, and classification. Kidney
Int 76: 534–545, 2009
23. Yuling H, Ruijing X, Xiang J, Yanping J, Lang C, Li L, Dingping Y, Xinti T,
Jingyi L, Zhiqing T, Yongyi B, Bing X, Xinxing W, Youxin J, Fox DA,
Lundy SK, Guohua D, Jinquan T: CD19+CD5+ B cells in primary IgA
nephropathy. J Am Soc Nephrol 19: 2130–2139, 2008
24. Lomax-Browne HJ, Visconti A, Pusey CD, Cook HT, Spector TD,
Pickering MC, Falchi M: IgA1 glycosylation is heritable in healthy twins
[published online ahead of print June 16, 2016]. J Am Soc Nephrol
25. Gharavi AG, Moldoveanu Z, Wyatt RJ, Barker CV, Woodford SY, Lifton
RP, Mestecky J, Novak J, Julian BA: Aberrant IgA1 glycosylation is in-
herited in familial and sporadic IgA nephropathy. J Am Soc Nephrol 19:
1008–1014, 2008
26. Suzuki H, Raska M, Yamada K, Moldoveanu Z, Julian BA, Wyatt RJ,
Tomino Y, Gharavi AG, Novak J: Cytokines alter IgA1 O-glycosylation
by dysregulating C1GalT1 and ST6GalNAc-II enzymes. J Biol Chem
289: 5330–5339, 2014
J Am Soc Nephrol 28: 1306–1313, 2017
www.jasn.org CLINICAL RESEARCH
27. Novak J, Rizk D, Takahashi K, Zhang X, Bian Q, Ueda H, Ueda Y, Reily C,
Lai LY, Hao C, Novak L, Huang ZQ, Renfrow MB, Suzuki H, Julian BA:
New insights into the pathogenesis of IgA nephropathy. Kidney Dis
(Basel) 1: 8–18, 2015
28. Leiper K, Martin K, Ellis A, Subramanian S, Watson AJ, Christmas SE,
Howarth D, Campbell F, Rhodes JM: Randomised placebo-controlled
trial of rituximab (anti-CD20) in active ulcerative colitis. Gut 60: 1520–
1526, 2011
29. Hoffman W, Lakkis FG, Chalasani G: B cells, antibodies, and more. Clin
J Am Soc Nephrol 11: 137–154, 2016
30. Reich HN, Troyanov S, Scholey JW, Cattran DC; Toronto Glomerulo-
nephritis Registry: Remission of proteinuria improves prognosis in IgA
nephropathy. J Am Soc Nephrol 18: 3177–3183, 2007
31. Berthelot L, Robert T, Vuiblet V, Tabary T, Braconnier A, Dramé M,
Toupance O, Rieu P, Monteiro RC, Touré F: Recurrent IgA nephropathy
is predicted by altered glycosylated IgA, autoantibodies and soluble
CD89 complexes. Kidney Int 88: 815–822, 2015
32. Moldoveanu Z, Wyatt RJ, Lee JY, Tomana M, Julian BA, Mestecky J,
Huang WQ, Anreddy SR, Hall S, Hastings MC, Lau KK, Cook WJ, Novak
J: Patients with IgA nephropathy have increased serum galactose-
deficient IgA1 levels. Kidney Int 71: 1148–1154, 2007
33. Suzuki H, Moldoveanu Z, Hall S, Brown R, Vu HL, Novak L, Julian BA,
Tomana M, Wyatt RJ, Edberg JC, Alarcón GS, Kimberly RP, Tomino Y,
Mestecky J, Novak J: IgA1-secreting cell lines from patients with IgA
nephropathy produce aberrantly glycosylated IgA1. J Clin Invest 118:
629–639, 2008
34. Suzuki H, Fan R, Zhang Z, Brown R, Hall S, Julian BA, Chatham WW,
Suzuki Y, Wyatt RJ, Moldoveanu Z, Lee JY, Robinson J, Tomana M,
Tomino Y, Mestecky J, Novak J: Aberrantly glycosylated IgA1 in IgA
nephropathy patients is recognized by IgG antibodies with restricted
heterogeneity. J Clin Invest 119: 1668–1677, 2009
Rituximab in IgA Nephropathy 1313