Sei sulla pagina 1di 8
CLINICAL RESEARCH www.jasn.org A Randomized, Controlled Trial of Rituximab in IgA Nephropathy with Proteinuria and

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 efcacy of these agents. Depleting antibodyproducing 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 biopsyproven IgA nephropathy and proteinuria .1 g/d, maintained on angiotensinconverting enzyme inhibitors or angiotensin receptor blockers with well controlled BP and eGFR,90ml/minper 1.73m 2 , to receive standard therapy or rituximabwith standard therapy. Primary outcome measures included change in proteinuria and change in eGFR. Median baseline serum creatinine level (range) was 1.4 (0.82.4) mg/dl, and proteinuria was 2.1 (0.65.3) 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 comparedwith that at baseline or in the control group; three patients in each group had $50% reduction in level of proteinuria. Serum levels of galactose-decient IgA1 or antibodies against galactose-decient IgA1 did not change. In this trial, rituximab therapydid not signicantly improve renal functionorproteinuria assessedover 1 year.Although rituximab effectively depleted B cells, it failed to reduce serum levels of galactose-decient IgA1 and antigalactosedecient IgA1 antibodies. Lack of efcacy of rituximab, at least at this stage and severity of IgA nephropathy, may reect a failure of rituximab to reduce levels of specic 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 benet of agents that antagonize 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 in ammation on histologic ex- amination of the kidney in IgAN along with the

1306 ISSN : 1046-6673/2804-1306

presence of immune complexes in the kidney suggest that immunosuppression would be bene cial in this disease. Indeed, many small studies have suggested

Received June 13, 2016. Accepted September 11, 2016.

Published online ahead of print. P ublication 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

J Am Soc Nephrol 28: 1306 1313, 2017

www.jasn.org CLINICAL RESEARCH

www.jasn.org CLINICAL RESEARCH

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 ef cacy 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 m 2 ). Similarly, immunosuppressive therapy with other approaches, such as mycophenolate mofetil, has yielded con icting ndings with regards to ef cacy in retarding pro- gressive disease. 11,12 Thus, there remains a strong need to nd 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 de cient 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- ammation, leading thereby to less proteinuria and renal dys- function in patients with IgAN at high risk of progression.

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 ve 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 brosis. All patients were maintained on RAAS inhibitors, with ve 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 122/79 6 10/11 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 signi cant 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 signi cantly 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 signi cant, although it trended higher in the rituximab group (2.6 versus 1.7 g/d). There was no difference in components of the Oxford classi cation score 22 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 nonsigni cant) (Table 1). The standard care group was signi cantly 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 rst 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

ney function was also stable throughout the study in both Figure 1. Trial organization. Patients were
ney function was also stable throughout the study in both Figure 1. Trial organization. Patients were
ney function was also stable throughout the study in both Figure 1. Trial organization. Patients were
ney function was also stable throughout the study in both Figure 1. Trial organization. Patients were
ney function was also stable throughout the study in both Figure 1. Trial organization. Patients were
ney function was also stable throughout the study in both Figure 1. Trial organization. Patients were

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

Rituximab in IgA Nephropathy

1307

CLINICAL RESEARCH www.jasn.org

CLINICAL RESEARCH www.jasn.org

Table 1. Baseline characteristics

Baseline Characteristics All Patients, n =34 Control, n =17 Rituximab, n =17 P Value

Age, yr

40 (21 63)

33 (21 59)

43 (29 63)

0.04

Sex, women, men

9, 25

2, 15

7, 10

0.12

Weight, kg

89.7 (57 120)

92.3 (57 119)

85.1 (61 120)

0.46

Race

0.07

White

24

15

9

0.07

Black

1

0

1

0.07

Asian/Paci c Islanders

6

2

4

0.07

Hispanic/Latino

3

0

3

0.07

Systolic BP

121 (102 147)

118 (102 147) 124 (107 144)

0.17

Diastolic BP

78 (56 106)

78 (56 106)

81 (59 94)

0.24

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

24-h Proteinuria, g Oxford pathology score, %

2.1 (0.6 5.3)

1.7 (0.6 4.0)

2.6 (0.9 5.3)

0.09

M0/M1

3/97

6/94

0/100

0.30

E0/E1

68/32

76/24

59/41

0.30

S0/S1

15/85

12/88

18/82

0.60

T0/T1/T2

56/41/3

53/47/0

65/29/6

0.30

Data presented as median and range.

postrituximab (only one patient exhibited partial depletion of CD19+ B cells to 45 and 68 cells per microliter at 6 and 12 months, respectively). CD19+ B cells depletion was sustained at 12 months. There was no signif- icant effect on serum Ig levels, including total IgA levels (Table 2). There were no differ- ences in serum levels of Gd-IgA1 or antiGd-IgA1 autoantibodies in the rituximab compared with control group at baseline or during the study. The serum levels did not change during observation. There was no correlation between changes in serum levels of Gd-IgA1 or antiGd-IgA1 autoantibody and proteinuria during the study (data not shown). There was no serious adverse event in either group during the study, although adverse events per subject were more com- mon in the rituximab group, including mild infections (Figure 4).

groups, whether assessed by eGFR or serum creatinine concen- 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 =0.95.3) g/d to 1.9 (range =0.48.8) g/d, but this change was not statistically signicant (P=0.30). The nal proteinuria in the rituximab group was not different from the proteinuria in the standard care group, which was unchanged from baseline: 1.8 (range =0.54.0) g/d to 1.8 (range =0.44.3) 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

prerituximab to 0 (range =0 – 45) cells per microliter Figure 2. eGFR trends in (A)

Figure 2. eGFR trends in (A) rituximab versus (B) control groups. The red line represents average data.

1308 Journal of the American Society of Nephrology

DISCUSSION

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 sh oil. Although there was a trend toward lower proteinuria after rituximab treatment, neither group experi- enced ;a signi cant reduction in proteinuria, and a signi cant 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 signi cant 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 signicantly reduce proteinuria or benet renal function. We believe that these negative ndings with regards to the ef cacy 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 ef cacy. Second, our ndings 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

J Am Soc Nephrol 28: 1306 1313, 2017

Figure 3. Proteinuria trends in (A) rituximab versus (B) control groups. The red line represents

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 signi cant adverse effects and risks, the current failure to observe a bene cial effect at this stage of the disease argues against its use in this setting, thereby sparing patients from unjusti ed risks. In our study, we con rmed 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- ni cantly 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 in ammation and injury. 13,14,27 The failure of rituximab to reduce the serum

www.jasn.org CLINICAL RESEARCH

www.jasn.org CLINICAL RESEARCH

levels of the anti Gd-IgA1 autoantibodies may thus account for the absence of a bene cial 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 ef cacy of rituximab may re ect 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 bene t from B cell depletion therapy. 18,19 These disease-speci c and divergent effects thus raise the following questions. Is it the signi cant 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-speci c 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 rst 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- de ned 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

Markers and Outcomes

Baseline

End of Study

Control, n =11 Rituximab, n =11 P Value Control, n =10 Rituximab, n =10 P Value

Biochemical markers a IgA, mg/ml

4.7

62.0

5.0

6

1.8

0.68

4.6

6

1.9

4.4

6

1.4

0.77

Gd-IgA1, per 100 ng IgA, U

56.8

66.9

54.8

6

10.0

0.58

58.9

6

5.6

60.5

6

13.0

0.73

IgG autoantibodies, U/ml

858.4

6647.3

1492.5 6 1672.9

0.25

1075 6

908.7

1751.3 6 2469.4

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 6 SD.

J Am Soc Nephrol 28: 1306 1313, 2017

Rituximab in IgA Nephropathy

1309

CLINICAL RESEARCH www.jasn.org

CLINICAL RESEARCH www.jasn.org

CLINICAL RESEARCH www.jasn.org Figure 4. Adverse events in the control group and the rituximab group. Type

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 signicant 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 m 2 ), more than expected by the age dif- ference. This nding 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 brosis ( . 50%) were excluded. Two patients with HSPN were included, but their outcomes did not in uence 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 bene t in this population, we should have been alerted by this trial. The implications of the numerical, not statistically signi cant, fall in proteinuria in the rituximab- treated group merit comment. To achieve 80% power for any signi cant 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 benet 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 progression 15,16 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 insigni cant 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 con rmed 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 registeredwith 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 1870 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 brosis or .10% glomerular crescents. Baseline Oxford classication score 22 was recorded in blinded fashion by an expert renal pathologist (S.S.). eGFR (by Modication of Diet in Renal Disease [MDRD]) or measured creatinine clearance had to be ,90 and .30 ml/min per 1.73 m 2 . To establish continued risk, baseline proteinuria needed to be .1000 mg/d while on stable doses of angiotensinconverting 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 prelled 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

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 rst 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 fourcomponent 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 speci c 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 Brie y, 96-well plates were coated with goat F(ab 9 ) 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(ab 9 ) 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-H 2 O 2 . 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 Brie y, 96-well plates coated with goat F(ab 9 ) 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 IgAwas desialylated using neuraminidase. 26 Gd-IgA1 was detected using biotinylated lectin from Helix aspersa (HAA; Sigma- Aldrich) speci c for terminal N -acetylgalactosamine and diluted to nal concentration of 2 m g/ml in 1% BSA in PBS-T. HAA was de- tected by ExtrAvidin-Peroxidase and developed with the peroxidase chromogenic substrate o -phenylenediamine-H 2 O 2 . OD was mea- sured at 490 nm using an EL808 Microplate Reader. The serum Gd-IgA1 concentration was expressed in units de ned as the ratio of OD determined for individual samples and a standard Gd-IgA1 (Ale) myeloma protein; 100 U Gd-IgA1 was de ned as the OD of HAA lectin binding to 50 ng standard Gd-IgA1 (Ale).

Determination of Serum Levels of IgG Autoantibody Specic for Gd-IgA1

Serum levels of IgG autoantibody speci c 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

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 biotinlabeled F(ab9 ) 2 fragment of goat IgG antihuman IgG antibody (Invitrogen). ExtrAvidin-Peroxidase was added, and the reactionwas developed with the peroxidase chromogenic substrate o-phenylenediamine-H 2 O 2 . OD was measured at 490 nm us- ing an EL808 Microplate Reader. Serum levels of IgG autoantibody specic for Gd-IgA1 were expressed in units (1 U IgG autoantibody dened as the OD of 1.0 measured at 490 nm) using a standard re- combinant IgG autoantibody specic 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 signi cant reduction in proteinuria. On the basis of the one-half width of a 95% con dence 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 signicant 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 strati cation 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. DAmico G: Natural history of idiopathic IgA nephropathy: Role of clinical and histological prognostic factors. Am J Kidney Dis 36: 227237, 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- de cient 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-de cient 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 Grou p: 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: Ef cacy 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, Taou k 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: 136139, 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 classi cation of IgA nephropathy:

Rationale, clinicopathological correlations, and classi cation. 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

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- de cient 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