Rituximab treatment of idiopathic membranous nephropathy

Idiopathic membranous nephropathy is a common cause of nephrotic syndrome whose pathogenesis may involve B-cell functions. Rituximab is a monoclonal antibody that binds to the CD20 antigen on B cells thereby deleting them. We conducted an open-label pilot trial of rituximab treatment in 15 severely nephrotic patients with proteinuria refractory to angiotensin-converting enzyme inhibition and/or receptor blockade but with adequately controlled blood pressure. Rituximab was given 2 weeks apart and, at 6 months, patients who remained proteinuric but had recovered B-cell counts were given a second course of treatment. Proteinuria was significantly decreased by about half at 12 months. Of the 14 patients who completed follow-up, full remission was achieved in two and partial remission in six patients based upon the degree of proteinuria. Side effects were minor; however, we found no relationship between the response and number of B cells in the blood, CD20 cells in the kidney biopsy, degree of tubulointerstitial fibrosis, starting proteinuria or creatinine values. Rituximab appears effective in reducing proteinuria in some patients with idiopathic membranous nephropathy but prospective identification of responsive patients was not possible. Idiopathic membranous nephropathy is a common cause of nephrotic syndrome whose pathogenesis may involve B-cell functions. Rituximab is a monoclonal antibody that binds to the CD20 antigen on B cells thereby deleting them. We conducted an open-label pilot trial of rituximab treatment in 15 severely nephrotic patients with proteinuria refractory to angiotensin-converting enzyme inhibition and/or receptor blockade but with adequately controlled blood pressure. Rituximab was given 2 weeks apart and, at 6 months, patients who remained proteinuric but had recovered B-cell counts were given a second course of treatment. Proteinuria was significantly decreased by about half at 12 months. Of the 14 patients who completed follow-up, full remission was achieved in two and partial remission in six patients based upon the degree of proteinuria. Side effects were minor; however, we found no relationship between the response and number of B cells in the blood, CD20 cells in the kidney biopsy, degree of tubulointerstitial fibrosis, starting proteinuria or creatinine values. Rituximab appears effective in reducing proteinuria in some patients with idiopathic membranous nephropathy but prospective identification of responsive patients was not possible. Idiopathic membranous nephropathy (IMN) is the most common cause of nephrotic syndrome in Caucasian adults and the second or third cause of a primary glomerulopathy leading to end-stage renal disease.1.Cattran D.C. Membranous nephropathy: quo vadis? [comment].Kidney Int. 2002; 61: 349-350Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Although, in most patients, the disease progresses relatively slowly, approximately 40% of patients eventually develop end-stage renal disease.2.Ruggenenti P. Chiurchiu C. Brusegan V. et al.Rituximab in idiopathic membranous nephropathy: a one-year prospective study.J Am Soc Nephrol. 2003; 14: 1851-1857Crossref PubMed Scopus (186) Google Scholar Current treatment options include corticosteroids, alkylating agents, cyclosporin A, mycophenolate mofetil, and tacrolimus, but their use may be associated with significant adverse effects and is not effective in all patients. Experimental data in MN suggest that B-cell activation results in immunoglobulin deposition along the glomerular basement membrane causing injury to the membrane and subsequent proteinuria.3.Kerjaschki D. Neale T.J. Molecular mechanisms of glomerular injury in rat experimental membranous nephropathy (Heymann nephritis).J Am Soc Nephrol. 1996; 7: 2518-2526Crossref PubMed Google Scholar In humans, there is evidence that therapy directed against B cells, for example, cyclophosphamide, is effective in MN.4.Ponticelli C. Zucchelli P. Passerini P. et al.A 10-year follow-up of a randomized study with methylprednisolone and chlorambucil in membranous nephropathy.Kidney Int. 1995; 48: 1600-1604Abstract Full Text PDF PubMed Scopus (313) Google Scholar Cyclophosphamide has striking, but non-selective, effects on B-cell function and suppresses the secretion of immunoglobulins.5.Cupps T.R. Edgar L.C. Fauci A.S. Suppression of human B lymphocyte function by cyclophosphamide.J Immunol. 1982; 128: 2453-2457PubMed Google Scholar Thus, a case could be made for using an agent capable of selectively depleting B cells and therefore halting the production of immunoglobulins directed against antigen(s) present in the glomeruli, and improving or even resolving the glomerular pathology and reducing proteinuria. There is evidence that this strategy is effective in the treatment of other antibody-mediated diseases such as systemic lupus erythematosus and rheumatoid arthritis (RA).6.Leandro M.J. Edwards J.C. Cambridge G. Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion [see comment].Ann Rheum Dis. 2002; 61: 883-888Crossref PubMed Scopus (315) Google Scholar, 7.Anolik J.H. Campbell D. Felgar R.E. et al.The relationship of FcgammaRIIIa genotype to degree of B cell depletion by rituximab in the treatment of systemic lupus erythematosus.Arthritis Rheum. 2003; 48: 455-459Crossref PubMed Scopus (388) Google Scholar, 8.Silverman G.J. Anti-CD20 therapy and autoimmune disease: therapeutic opportunities and evolving insights.Front Biosci. 2007; 12: 2194-2206Crossref PubMed Scopus (17) Google Scholar Rituximab, a monoclonal antibody (mAb) against the CD20 antigen present on B cells, was approved by the Food and Drug Administration (FDA) in 1997 for the treatment of relapsed or refractory non-Hodgkin's lymphoma and more recently for patients with RA. Because the CD20 antigen is not expressed on hematopoietic stem cells, normal plasma cells, or other normal tissues, the use of this mAb allows for selective depletion of B cells. Recently, Ruggenenti et al.2.Ruggenenti P. Chiurchiu C. Brusegan V. et al.Rituximab in idiopathic membranous nephropathy: a one-year prospective study.J Am Soc Nephrol. 2003; 14: 1851-1857Crossref PubMed Scopus (186) Google Scholar treated eight patients with IMN and nephrotic syndrome with four weekly doses of rituximab (375 mg m−2) and followed them for 1 year.9.Remuzzi G. Chiurchiu C. Abbate M. et al.Rituximab for idiopathic membranous nephropathy.Lancet. 2002; 360: 923-924Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar Treatment resulted in a 60% reduction in urinary protein excretion with notably modest side effects and no major adverse events. However, the patient's response to rituximab was quite variable and the reasons for that variability are not clear. Given these encouraging preliminary results, we prospectively treated 15 patients with IMN with rituximab 1 g i.v. (intravenous) on days 1 and 15. We also studied the pharmacokinetics (PK) and pharmacodynamics (PD) since previously no studies had evaluated the effect of proteinuria on these parameters despite its increasing frequency of use in autoimmune diseases such as vasculitis, systemic lupus erythematosus, and RA.6.Leandro M.J. Edwards J.C. Cambridge G. Clinical outcome in 22 patients with rheumatoid arthritis treated with B lymphocyte depletion [see comment].Ann Rheum Dis. 2002; 61: 883-888Crossref PubMed Scopus (315) Google Scholar, 10.Keogh K.A. Ytterberg S.R. Fervenza F.C. et al.Rituximab for refractory Wegener's granulomatosis: report of a prospective, open-label pilot trial.Am J Respir Crit Care Med. 2006; 173: 180-187Crossref PubMed Scopus (360) Google Scholar, 11.Looney R.J. Anolik J.H. Campbell D. et al.B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/II dose-escalation trial of rituximab.Arthritis Rheum. 2004; 50: 2580-2589Crossref PubMed Scopus (666) Google Scholar We wanted these measurements since we postulated that in patients with IMN and non-selective proteinuria, rituximab, an IgG monoclonal protein, may be lost in the urine affecting its PK and PD and perhaps its therapeutic efficacy. We also addressed the question of whether renal pathology might help in the study of either pathogenesis or patient responsiveness to this agent. We did this by examining the specific cellular markers in the renal tissue as well as the degree of tubular interstitial damage in the biopsies of the study patients. Previous studies have demonstrated B-cell infiltrates in increased number in renal biopsies from patients with IMN, and it has been hypothesized that they could play a role in the pathogenesis of the disease.12.Cohen C.D. Calvaresi N. Armelloni S. et al.CD20-positive infiltrates in human membranous glomerulonephritis.J Nephrol. 2005; 18: 328-333PubMed Google Scholar In contrast, other studies have suggested that the degree of fibrosis may portend unresponsiveness to this agent.13.Ruggenenti P. Chiurchiu C. Abbate M. et al.Rituximab for idiopathic membranous nephropathy: who can benefit?.Clin J Am Soc Nephrol. 2006; 1: 738-748Crossref PubMed Scopus (76) Google Scholar Finally, the formation of human anti-chimeric antibodies (HACAs) may be induced by administering any chimeric mAb such as occur with the anti-tumor necrosis factor mAb infliximab. We tested for the development of these antibodies since their appearance may affect the efficacy of rituximab. Fifteen patients (13 men and 2 women), age 47±8 years (mean±s.d.) were enrolled. Seven patients had failed previous immunosuppressive treatment: two patients had received prednisone alone (n=2); combined prednisone and cytotoxic agent (n=2); cyclosporin A (n=2); and mycophenolate mofetil (n=1). All patients were severely nephrotic. At entry, systolic and diastolic blood pressures averaged 130±14 and 76±9 mm Hg, respectively. The mean serum creatinine was 1.4±0.5 mg per 100 ml and the mean creatinine clearance was 85.2±28 ml per min per 1.73 m2. The mean time from the diagnostic biopsy to entrance into the study was 13.1±13.6 months (range: 6–60) (Table 1).Table 1Main clinical and laboratory characteristics at study entry (baseline) of individual patients with IMNPatient number123456789101112131415Age (years)633753603353534546444642523840Gender (male/female)MFMMMMMMMFMMMMMDisease duration (months)6106135137108860286106Histology stage (I–IV)I–III–IIIII–IVIIIII–IIIIIIIII–IIIIIIIIIIIUrinary protein excretion (g per 24 h)9.08.48.520.010.823.516.68.810.16.115.87.814.723.411.8Serum creatinine1.50.91.32.61.11.81.81.01.30.72.11.21.51.81.0Creatinine clearance (ml per min per 1.73 m2)631027540109585911481116611457468113IMN, idiopathic membranous nephropathy. Open table in a new tab IMN, idiopathic membranous nephropathy. In the 14 patients who completed 12 months of follow-up, proteinuria decreased from 13.0±5.7 g per 24 h (range: 8.4–23.5) to 6.0±7.3 g per 24 h at 12 months (mean±s.d.) (P<0.05) (Figure 1 and Table 2). Ten patients who had proteinuria >3 g per 24 h and >15 CD19+ B cells per μl at 6 months were retreated with a second identical course of rituximab. Prior to the 6-month point and repeat rituximab treatment, four patients had achieved a partial remission (PR) and were not retreated. By the end of 12 months, complete remission (CR) (proteinuria <0.3 g per 24 h) was achieved in two patients and PR (<50% peak value and <3 g per 24 h) in six patients. The mean drop in proteinuria from baseline to 12 months was 6.2±4.8 g or a 48% reduction (P=0.0003, paired t-test). The reduction in proteinuria was gradual, and in patients who responded to treatment, the lowest level was not achieved until month 12. This meant that the exact role of the second course of therapy was hard to determine since in all cases but one proteinuria was already reduced from baseline value at 6 months in all patients who went to achieve a PR or CR by 12 months. In the five patients who did not respond, proteinuria at 6 months was not significantly different from baseline and was unaltered by the second course of rituximab. The reduction in proteinuria was paralleled by a progressive and significant increase in serum albumin levels from 2.3±0.6 g per 100 ml at baseline (<3 g per 100 ml in 13/15 patients; 87%) to 3.5±0.8 g per 100 ml at 12 months (<3 g per 100 ml in 4/14 patients; 28%), respectively (mean±s.d.), representing a 53% increase over basal levels (Table 3). There were significant changes in serum IgG and IgM levels with baseline IgG levels increasing and IgM levels falling at 12 months while serum IgA levels remained relatively stable (Table 3). Total serum cholesterol and triglyceride levels also decreased progressively by 12 months (P<0.05, paired t-test) (Table 3 and Figure 2). The reduction in proteinuria was paralleled by a progressive and significant decline in body weight (95.5±12 kg at baseline versus 92.1±11 kg at month 12; P<0.05, Table 3) likely reflecting decreasing peripheral edema. Systolic and diastolic blood pressures were stable during the study. Taking the group as a whole, renal function did not change significantly during the 12-month period of the study. The serum creatinine concentrations at baseline and at 12 months were similar (1.4±0.5 versus 1.6±1.0 mg per 100 ml; P=not significant (NS); Table 3). Similarly, there were no significant changes in 24 h creatinine clearances obtained at baseline versus at 12 months (85.2±28 versus 85.6±37 ml per min per 1.73 m2, respectively; P=NS). However, in the two patients who had the lowest creatinine clearance at entry (40 and 59 ml per min per 1.73 m2), proteinuria remained unchanged, renal disease was progressive, and both patients reached end-stage renal disease at 6 and 7 months after completion of the study.Table 2Time course of urinary protein excretion (g per 24 h) in individual patients with IMN from entry into the study (baseline) to end of study (month 12)Patient no.Month -6BaselineMonth 1Month 3Month 6Month 9Month 12Baseline to month 12aPaired t-test P=0.0003 for 12-month change.1bPatients who underwent retreatment with rituximab.10.69.17.45.918.87.47.71.42bPatients who underwent retreatment with rituximab.12.98.48.96.33.92.20.67.837.98.56.11.20.60.40.28.34bPatients who underwent retreatment with rituximab.16.420.116.521.817.915.420.2-0.1458.610.85.51.71.10.80.210.5613.323.531.35.0————7bPatients who underwent retreatment with rituximab.10.416.67.514.817.217.319.9-3.384.68.83.62.20.80.71.07.89bPatients who underwent retreatment with rituximab.7.710.18.97.54.88.10.79.410bPatients who underwent retreatment with rituximab.4.86.13.95.76.00.60.35.8117.915.816.53.82.13.80.914.912bPatients who underwent retreatment with rituximab.12.47.83.81.84.93.52.65.213bPatients who underwent retreatment with rituximab.8.914.714.022.416.912.56.08.714bPatients who underwent retreatment with rituximab.12.323.416.422.026.517.414.19.415bPatients who underwent retreatment with rituximab.8.811.89.215.28.911.19.82.0Mean9.813.010.69.89.37.26.06.2s.d.3.15.77.37.98.46.57.34.8Median8.910.88.95.95.55.61.87.8IMN, idiopathic membranous nephropathy.a Paired t-test P=0.0003 for 12-month change.b Patients who underwent retreatment with rituximab. Open table in a new tab Table 3Main clinical and laboratory parameters in 14 patients with IMN from rituximab administration (baseline) to study end (month 12)BaselineDay 283 months6 months9 months12 monthsSystolic BP (mm Hg)131±14129±19124±14127±16122±12131±20Diastolic BP (mm Hg)76±977±1376±875±1372±879±12Body weight (kg)95.5±1295±1091±11aP≤0.05 versus month 0.93±1093±13aP≤0.05 versus month 0.92±11aP≤0.05 versus month 0.Serum creatinine (mg per 100 ml)1.4±0.51.5±0.51.5±0.61.5±0.81.4±0.71.6±1.0Serum albumin (g per 100 ml)2.3±0.62.7±0.5aP≤0.05 versus month 0.2.9±0.7aP≤0.05 versus month 0.3.0±0.8aP≤0.05 versus month 0.3.2±0.9aP≤0.05 versus month 0.3.5±0.8aP≤0.05 versus month 0.Serum IgG (g per 100 ml)468±345493±289587±265aP≤0.05 versus month 0.680±283aP≤0.05 versus month 0.768±377aP≤0.05 versus month 0.745±301aP≤0.05 versus month 0.Serum IgM (g per 100 ml)105±48105±4783.5±42aP≤0.05 versus month 0.78±44aP≤0.05 versus month 0.68±30aP≤0.05 versus month 0.61±28aP≤0.05 versus month 0.Serum IgA (g per 100 ml)193±80208±93179±0.64194±58187±68176±74Serum cholesterol (mg per 100 ml)332±82297±91aP≤0.05 versus month 0.271±77aP≤0.05 versus month 0.259±85aP≤0.05 versus month 0.250±101aP≤0.05 versus month 0.215±69aP≤0.05 versus month 0.Serum triglycerides (mg per 100 ml)339±218381±217283±217aP≤0.05 versus month 0.285±171240±184aP≤0.05 versus month 0.241±175aP≤0.05 versus month 0.BP, blood pressure; Ig, immunoglobulin; IMN, idiopathic membranous nephropathy.Mean±s.d.a P≤0.05 versus month 0. Open table in a new tab Figure 2Changes in serum IgG, IgA, and IgM levels following treatment with rituximab. *P<0.05.View Large Image Figure ViewerDownload (PPT) IMN, idiopathic membranous nephropathy. BP, blood pressure; Ig, immunoglobulin; IMN, idiopathic membranous nephropathy. Mean±s.d. Initial CD19+ B-cell depletion was seen in all patients (mean 307.8±203 at baseline versus 5.6 cells per μl at day 28). At 3 months, CD19+ B cells were starting to recover (mean 35.8±46 cells per μl, range: 1–152), with five patients having >35 cells per μl. By 6 months, the majority of patients had counts in the normal range (mean 110±97 cells per μl, range: 28–317). However, no association was seen between baseline proteinuria and changes in CD19+ B-cell depletion at 6 months (r=0.244; P=NS) and 12 months (r=0.231; P=NS). There were no significant changes in hemoglobin levels, total white blood cells, or platelet counts (Table 4).Table 4Main hematology parameters in 14 patients with IMN from rituximab administration (baseline) to study end (month 12)ParameterBaseline1 month3 months6 months9 months12 monthsHemoglobin (g per 100 ml)13.6±1.213.0±1.413.0±1.513.2±1.813.4±1.713.3±1.9White blood cell count (3.5–10.5 × 103 μl−1)6.6±1.35.4±1.35.7±1.26.5±1.46.5±1.26.0±1.2Lymphocytes (0.66–4.6 × 103 μl−1)1.9±0.51.4±0.41.4±0.31.4±0.31.4±0.41.4±0.3Lymphocyte sub-populations CD19 (71–567 μl−1)308±2035.6±6.7aP<0.01 and36±46aP<0.01 and110±97bP≤0.05 versus month 0.88±77bP≤0.05 versus month 0.103±108bP≤0.05 versus month 0. CD19 (5–25%)15.1±7.80.2±0.5aP<0.01 and2.8±4.5aP<0.01 and8.1±7.4bP≤0.05 versus month 0.6.8±7.3bP≤0.05 versus month 0.8.4±10.7IMN, idiopathic membranous nephropathy.cNormal ranges in parentheses.a P<0.01 andb P≤0.05 versus month 0. Open table in a new tab IMN, idiopathic membranous nephropathy. cNormal ranges in parentheses. Data from our PK studies in the 15 treated patients show rituximab levels of 218.6±67 μg ml−1 (mean±s.d.) 1 day after the first dose, 17±11 μg ml−1 on day 15 pre-dose, and 205.2±111 μg ml−1 on day 15 post-dose. There were no differences in serum rituximab levels at any point between patients who responded to treatment versus non-responders. Immunodepletable HACAs were detected in six patients at various time points during the study. In one patient with the lowest antibody level, HACA became negative with follow-up. In the other five patients, HACA remained positive to the end of the study. The presence of HACA was not associated with responders versus non-responders. We found no relation between the total number of CD20+ or CD3+ cells, the ratio of CD20+/CD3+ cells, or the number of CD20+ cells per mm2 present in the diagnostic renal biopsies and the response to rituximab treatment. CD20+ cells were 0.88±0.7 mm−2 in the responders versus 5.6±6.2 mm−2 in the non-responders (P=NS). At the time of diagnosis, interstitial fibrosis occupied 6.0±2.2% (range: 3.2–11.2%) of the renal biopsy area. The adverse events observed were mainly infusion-related reactions and none were serious. Three patients developed itching, rigors, and a skin rash during infusion. Three patients complained of sore or scratchy throat during infusion. One patient developed muscle pain after infusion that resolved with the use of non-steroidal medication. One patient developed a serum sickness-like syndrome. Small patches of hair loss and thinning were observed in two patients. One patient was diagnosed with community-acquired pneumonia 3 months after the first infusion that resolved with oral antibiotic treatment. This patient was retreated without complications. One patient experienced fatigue and voice loss soon after the infusion ended, but recovered spontaneously. One patient with a history of herpes zoster prior to enrollment in the study developed viral reactivation. This patient was treated with oral anti-viral drugs and made a full recovery. One patient was diagnosed with adenocarcinoma of the lung 3 months after the first infusion. This patient had a normal chest X-ray 1 year prior to enrollment. Lung cancer was diagnosed from a computed tomography scan performed to assess coronary artery calcification. Participation in the study was discontinued. The patient subsequently died. This is the largest study to date to prospectively evaluate the effect of rituximab in patients with IMN. In this study, rituximab appears to be effective in reducing proteinuria in a significant number of patients with IMN with severe nephrotic syndrome and relatively well-preserved renal function. Reduction in proteinuria was accompanied by significant increases in serum albumin levels, improvements in hyperlipidemia, and amelioration of edema. CR of proteinuria was achieved in two patients while PR was seen in six patients. When taken together, a CR or PR occurred in 8 of 14 patients (nearly 60%) who completed 1 year of follow-up. Renal function remained stable in the majority of patients. We consider this outcome to be very good at least in the short term, since, based on evidence garnered from the older literature in which patients did not have the benefit of anti-proteinuric and kidney-protective therapies that we currently use, and were used in our patients, persistent heavy proteinuria is almost invariably associated with progressive loss of renal function in patients with IMN.14.Cattran D.C. Idiopathic membranous glomerulonephritis.Kidney Int. 2001; 59: 1983-1994Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar In both, diabetic and non-diabetic nephropathy patients, angiotensin (Ang)-converting enzyme inhibitor (ACEi) and/or Ang II receptor blockers (ARBs) reduce proteinuria and slow progression of renal disease. The degree of protection, however, is related to the degree of proteinuria reduction and if proteinuria is not lowered, the benefit is substantially attenuated.15.Rossing P. Hommel E. Smidt U.M. Parving H.H. Reduction in albuminuria predicts diminished progression in diabetic nephropathy.Kidney Int Suppl. 1994; 45: S145-S149PubMed Google Scholar,16.Apperloo A.J. de Zeeuw D. de Jong P.E. Short-term antiproteinuric response to antihypertensive treatment predicts long-term GFR decline in patients with non-diabetic renal disease.Kidney Int Suppl. 1994; 45: S174-S178PubMed Google Scholar The most recent data from RENAAL study confirm these observations in that the renal protective effect of Ang II blockade was nearly fully explained by its anti-proteinuric effect.17.Keane W.F. Brenner B.M. de Zeeuw D. Investigators RS et al.The risk of developing end-stage renal disease in patients with type 2 diabetes and nephropathy: the RENAAL study.Kidney Int. 2003; 63: 1499-1507Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar It is also important to acknowledge the marked dichotomy in the results with five patients having no significant improvement in proteinuria despite two full courses of the rituximab therapy. In addition, the two patients with the lowest creatinine clearances at baseline developed progressive renal failure and reached end-stage renal disease soon after completing the study. These overall results are in agreement with previous work by Ruggenenti et al. who prospectively treated eight patients with lower degrees of proteinuria (8.6±4.2 g per 24 h at baseline) with four weekly doses of rituximab (375 mg m−2) and found a significant decrease in proteinuria to an average of 3.0±2.5 g per 24 h (-66%, P<0.005) at 12 months. Two patient's proteinuria had decreased to <0.5 g per 24 h and in three others to <3.5 g per 24 h. Renal function remained stable in all patients. Adverse effects were reported as mild.2.Ruggenenti P. Chiurchiu C. Brusegan V. et al.Rituximab in idiopathic membranous nephropathy: a one-year prospective study.J Am Soc Nephrol. 2003; 14: 1851-1857Crossref PubMed Scopus (186) Google Scholar,9.Remuzzi G. Chiurchiu C. Abbate M. et al.Rituximab for idiopathic membranous nephropathy.Lancet. 2002; 360: 923-924Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar In patients with lymphoma and in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis and subnephrotic range proteinuria, treatment with rituximab had minor effects on total serum immunoglobulin levels.10.Keogh K.A. Ytterberg S.R. Fervenza F.C. et al.Rituximab for refractory Wegener's granulomatosis: report of a prospective, open-label pilot trial.Am J Respir Crit Care Med. 2006; 173: 180-187Crossref PubMed Scopus (360) Google Scholar,18.McLaughlin P. Grillo-Lopez A.J. Link B.K. et al.Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program.J Clin Oncol. 1998; 16: 2825-2833Crossref PubMed Scopus (2474) Google Scholar We postulate that the increase in serum IgG levels seen in our study may reflect a reduction in urinary losses of IgG resulting from improvement in the filtration barrier, while the decrease in IgM levels may reflect the depletion of IgM-producing B cells by rituximab and/or normalization of hepatic production with decreasing proteinuria. The increase in serum IgG levels may have accounted for the reduced infectious complications seen in our study and may be a significant and important difference from higher infection rates seen with the use of broader immunosuppressive regimens. It is possible, given the heterogeneous course of the disease and the absence of a control group, that the beneficial effect observed was due to spontaneous remission rather than a therapeutic effect of rituximab. We find this explanation unlikely because although the traditional concept is that IMN has a rate of spontaneous remission close to 30%, this outcome is very rare in a cohort such as we selected with prolonged high-grade proteinuria. Previous data have indicated that the majority of spontaneous remissions occur within the first 2 years of initial presentation and is more common in females and in patients with subnephrotic range proteinuria.19.Cattran D. Management of membranous nephropathy: when and what for treatment.J Am Soc Nephrol. 2005; 16: 1188-1194Crossref PubMed Scopus (160) Google Scholar Untreated patients with heavy proteinuria (>8 g per 24 h) rarely remit spontaneously and their clinical course is characterized by progressive renal failure.20.Pei Y. Cattran D. Greenwood C. Predicting chronic renal insufficiency in idiopathic membranous glomerulonephritis.Kidney Int. 1992; 42: 960-966Abstract Full Text PDF PubMed Scopus (152) Google Scholar All patients in our study had high-grade proteinuria that was increasing despite maximum tolerated renin–angiotensin aldosterone system blockade, including dual Ang II blockade with an ACEi and ARB treatment in eight patients. Other potential confounding factors, such as a low-protein diet, the degree of blood pressure control, statin use, and ACEi or ARB use, had all been introduced >4 months prior to rituximab treatment and were maintained unchanged during the study. We performed PK and PD analyses on our patients to determine whether this might provide further information about the basic mechanism of the disease or help in the determination of some prognostic factor that might allow more appropriate targeting of this therapy in the IMN patient population. Data from our PK studies in the 15 IMN treated patients show serum rituximab levels to be significantly lower than levels found in 38 non-proteinuric patients with RA who were given the same dosing regimen and measured by the same method and according to the same schedule as our patients, in whom levels of 341±212, 65±23, and 415±94 μg ml−1 were found at the same comparable time points as our cohort (P<0.001; RA versus IMN at day 1 post-infusion, day 15 pre-infusion, and day 15 post-infusion, respectively; Genentech Inc., unpublished observations). The half-life of rituximab is 3 weeks, with rituximab PK showing biphasic disposition. We could not calculate the area under the curve because there were no PK data between days 1 and 15 (distribution phase). Usually, however, area under the curve is highly correlated with peak and trough concentrations. Treatment of patients with non-Hodgkin's lymphoma (NHL), with either a 4- or 8-week course of rituximab, results in predictable and profound depletion of normal B cells from the circulation (to ≤1% of baseline CD19+ cell counts).18.McLaughlin P. Grillo-Lopez A.J. Link B.K. et al.Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program.J Clin Oncol. 1998; 16: 2825-2833Crossref PubMed Scopus (2474) Google Scholar,21.Piro L.D. White C.A. Grillo-Lopez A.J. et al.Extended Rituximab (anti-CD20 monoclonal antibody) therapy for relapsed or refractory low-grade or follicular non-Hodgkin's lymphoma [see comment].Ann Oncol. 1999; 10: 655-661Crossref PubMed Scopus (353) Google Scholar In these patient