Treatment of severe lupus nephritis: the new horizon

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1 Treatment of severe lupus nephritis: the new horizon Tak Mao Chan Abstract Lupus nephritis is a common and severe manifestation of systemic lupus erythematosus, and an important cause of both acute kidney injury and end-stage renal disease. Despite its aggressive course, lupus nephritis is amenable to treatment in the majority of patients. The paradigm of immunosuppressive treatment for lupus nephritis has evolved over the past few decades from corticosteroids alone to corticosteroids combined with cyclophosphamide. Sequential treatment regimens using various agents have been formulated for induction and long-term maintenance therapy, and mycophenolate mofetil has emerged as a standard of care option for both induction and maintenance immunosuppressive treatment. The current era has witnessed the emergence of multiple novel therapeutic options, such as calcineurin inhibitors and biologic agents that target key pathogenetic mechanisms of lupus nephritis. Clinical outcomes have improved in parallel with these therapeutic advances. This Review discusses the evidence in support of current standard of care immunosuppressive treatments and emerging therapies, and describes their roles and relative merits in the management of patients with lupus nephritis. Chan, T. M. Nat. Rev. Nephrol. advance online publication 25 November 2014; doi: /nrneph REVIEWS Room PB408, 4 th Floor, Professorial Block, Department of Medicine, University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong Special Administrative Region (SAR), China. dtmchan@hku.hk Introduction The natural course of systemic lupus erythematosus is characterized by periods of disease activity, termed flares, interspersed with periods of quiescence. Disease flares result in cumulative organ damage; 1,2 thus, adequate suppression of an acute flare and prevention of subsequent flares are important to achieve favourable long-term renal outcomes and optimize prognosis. Renal involvement, termed lupus nephritis affects over half of all patients with systemic lupus erythematosus. The natural course of disease in the majority of patients with lupus nephritis also comprises relatively long periods of quiescence interspersed with episodic flares. 3 5 Lupus nephritis is characterized by inflammatory damage to nephrons during the acute stage. 3 5 The relative proportions of normal nephrons, acute inflammatory lesions, and chronic damage varies between patients, and at different time points in the same patient. Acute inflammatory lesions destroy normal nephrons quickly but are potentially reversible by potent immunosuppressive treatment. By contrast, chronic lesions such as fibrosis and tubular atrophy are not amenable to immuno suppressive treatment, and lead to chronic renal impairment through a relatively slow but inexorable process (Figure 1). 6 Lupus nephritis is, therefore, a major cause of acute kidney injury and chronic kidney disease especially in young adults, and a leading cause of endstage renal disease (ESRD) in some parts of the world, such as southeast Asia. 7 Severe renal damage portends Competing interests T.M.C. declares that he has acted as a consultant for the following companies: Astellas, Teva and Vifor Pharma. inferior patient survival, 8,9 and ESRD is associated with a fold increase in the standardized mortality ratio. 10,11 However, unlike many diseases of the renal parenchyma, for which there are no effective treatments, the majority of patients with severe active lupus nephritis respond well to current standard of care immunosuppressive therapies, which highlights the importance of early recognition and t reatment of this condition. The objective of treatment for active lupus nephritis is to preserve nephrons by reversing the acute inflammatory process and achieving a state of disease quiescence. Management of patients with active lupus nephritis comprises two phases: in patients with active disease, potent immunosuppressive medications are administered with the objective of inducing disease quiescence, referred to as the induction phase. Induction treatment is followed by long-term maintenance therapy, in which immunosuppressive medications are used at reduced doses to maintain a stable state and prevent further attrition of nephron mass by renal flares, while minimizing treatment- related adverse effects. The selection of immuno suppressive treatment should, therefore, include a long-term perspective, and entail a fine balance between efficacy and risk considerations. This Review summarizes the evidence base and discusses the relative merits of currently available standard of care immunosuppressive treatments for severe lupus nephritis, focusing on class III (focal proliferative), class IV (diffuse proliferative) and class V (membranous) lupus glomerulopathy. Histological classification of lupus nephritis is based on International Society of Nephrology and Renal Pathology Society 2003 criteria 12 NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 1

2 REVIEWS Key points The objective of immunosuppressive treatment for lupus nephritis is to abate ongoing damage to nephrons by active disease, and to prevent disease flares during the long-term maintenance phase Current standard of care induction therapy for active, severe proliferative or membranous lupus nephropathy is dual immunosuppression with high-dose corticosteroids and either mycophenolate mofetil or cyclophosphamide Low-dose corticosteroids combined with either mycophenolate or azathioprine are recommended as maintenance immunosuppression; mycophenolate mofetil for a minimum of 2 years is preferred in patients with previous renal flares The selection of immunosuppressive agent and dose should take into account ethnic variation in response to different treatments Antimalarial treatment is associated with reductions in renal flares and accrual of renal damage, as well as improved patient survival Holistic management of patients with lupus nephritis should include prevention of and surveillance for complications, blood pressure control, renal preservation, vascular risk minimization and attention to overall quality of life Glomerulus Podocyte injury Mesangial cell activation Soluble mediators Endothelial cell activation Thrombosis Collecting duct Lymphocytic infiltration Endothelial activation and death Dendritic cell activation Loop of Henle Tubular atrophy Podocyte Dendritic cell Autoantibody * Complement proteins Mesangial cell Lymphocyte of immuno suppressive treatment resulted in a better outcome than a smaller reduction (15-year renal survival 71% versus 25%, respectively).16 Nevertheless, because renal reserve (both at baseline and after treatment) differs between patients with lupus nephritis, a favourable response to induction therapy does not necessarily guarantee long-term freedom from ESRD. Moreover, complete and partial renal responses are defined differently by different investigators, although definitions typically include an inactive urinary sediment and significant but variable degrees of improvement in proteinuria and renal function. Differences in the way that study outcomes are defined in clinical trials can profoundly influence their results, as illustrated by the abatacept trial results discussed below Although renal survival and patient survival are regarded as hard outcomes, in lupus nephritis these end points represent the cumulative effect of prior disease flares and responses to treatment, and are affected by induction and maintenance immunosuppression as well as by adjunctive therapies. Although disease flare is an uncontroversial indicator of the inefficacy of maintenance immuno suppression, the nature and severity of disease flares varies, and thus the effect of disease flares on the subsequent disease course and hard outcomes can differ. Also, the distinction between the induction and maintenance phases can be blurred (except in sequential regimens), since the same agents can be used for both, and remission is reached at different times in different patients. The optimal duration of induction therapy can also vary with different treatment regimens. These complexities add considerably to the challenges in d esigning clinical trials of interventions for lupus nephritis. The next sections describe key developments in the evolution of treatments for lupus nephritis, in the context of evidence from clinical trials of immunosuppressive agents. Fibrosis Cyclophosphamide Figure 1 Pathogenesis of lupus nephritis. In situ formation of immune complexes with complement activation and induction of inflammatory mediators results in chemotaxis and activation of inflammatory cells within the kidney and further release of proinflammatory signals and mediators, leading to injury and damage of cellular and structural elements within the renal parenchyma. Permission obtained from Nature Publishing Group Davidson, A. & Aranow, C. Lupus nephritis: lessons from murine models. Nat. Rev. Rheumatol. 6, (2010). unless otherwise stated. Data on emerging therapies and their potential roles in the therapeutic armamentarium are also discussed. Challenges to clinical trial design A satisfactory response to induction treatment for active lupus nephritis is associated with a favourable longterm renal prognosis.3,13,14 Data from the Collaborative Study Group showed that 10-year renal survival was 94% in patients who achieved complete remission, 45% in those who achieved partial remission and 19% in patients who did not achieve remission.15 A similar trend was observed in 10-year patient survival.15 In another study, a 50% reduction in proteinuria after 6 months Corticosteroids plus cyclophosphamide has been the standard therapy for severe lupus nephritis against which novel treatments have been compared since the 1980s (Table 1) Clinical trials indicated that cyclophosphamide and corticosteroids was more effective than corticosteroids alone in sustaining disease quiescence and improving renal outcome,21,22,24 although combined treatment was associated with an increased incidence of adverse events and significantly greater mortality (18.2%, versus 3.7% in patients treated with corticosteroids alone).21,22,25 Concerns regarding the adverse effects of cyclophosphamide (including leukopenia, alopecia, vulnerability to infections, gonadal toxicity, haemorrhagic cystitis, uroepithelial tumours and increased incidence of other malignancies) led researchers to investigate approaches to reduce cyclophosphamide exposure. Several investigators have examined the efficacy and tolerability of immunosuppressive regimens using reduced doses or durations of cyclophosphamide. In the Collaborative Study Group trial, the rate of renal remission among patients with lupus nephritis (defined as protein uria <0.33 g daily and serum creatinine <124 μmol/l) 2 ADVANCE ONLINE PUBLICATION

3 Table 1 Key trials of induction treatment for severe lupus nephritis Study Participants Treatment groups Trial design and duration Chan (2000) 46 Chan (2005) Chinese patients with lupus nephritis class IV (WHO) 64 Chinese patients with lupus nephritis class IV Yap 65 Chinese patients (2013) 65 with lupus nephritis class III or IV Houssiau (2002) 29 Houssiau (2010) 30 Ginzler (2005) patients (76 white, 6 Asian, 8 Afro-Caribbean or black) with lupus nephritis class III, IV, Vc or Vd (WHO) 84 participants from the Euro-Lupus Nephritis Trial 140 patients (79 black, 28 Hispanic, 24 white and 8 Asian) with lupus nephritis class III V (WHO) Bao 40 Chinese patients (2008) 78 with lupus nephritis class IV ± V Appel (2009) 55 Austin (2009) 85 Chen (2011) patients (147 white, 123 Asian and 100 other race) with lupus nephritis class III V 42 patients (12 white, 30 non-white) with lupus nephritis class V and proteinuria >2 g daily 81 Chinese patients with lupus nephritis class III V Prednisolone plus mycophenolate mofetil versus prednisolone plus oral cyclophosphamide followed by azathioprine Prednisolone plus mycophenolate mofetil versus prednisolone plus oral cyclophosphamide followed by azathioprine Prednisolone plus mycophenolate mofetil Intravenous methylprednisolone (3 doses of 750 mg) for all patients, then prednisolone plus high-dose cyclophosphamide versus prednisolone plus low-dose cyclophosphamide Intravenous methylprednisolone (3 doses of 750 mg) for all patients, then prednisolone plus high-dose cyclophosphamide versus prednisolone plus low-dose cyclophosphamide Prednisolone plus mycophenolate mofetil versus prednisolone plus cyclophosphamide Intravenous methylprednisolone (3 doses of 500 mg) for all patients, then prednisolone plus mycophenolate mofetil plus tacrolimus versus prednisolone plus cyclophosphamide Prednisolone plus mycophenolate mofetil versus prednisolone plus cyclophosphamide Prednisolone versus prednisolone plus cyclophosphamide (in alternate months) versus prednisolone plus ciclosporin for 12 months Prednisolone plus tacrolimus versus prednisolone plus cyclophosphamide Key findings 12 months High response rate (>90%) in both groups Median 63 months* Mean 91.9 months Median 41 months Comparable renal outcome in both groups; fewer adverse events with mycophenolate mofetil than cyclophosphamide Relapse rate lowest in patients receiving mycophenolate mofetil for over 24 months compared with patients who stopped mycophenolate earlier; renal survival 5 years 93%, 10 years 86% No difference between high-dose and lowdose groups: treatment failure, 20% versus 16%; renal remission 54% versus 71% Mean Death, doubling of serum creatinine 10 years levels and renal failure rates did not differ between high-dose and low-dose cyclophosphamide groups 24 weeks Prednisolone plus mycophenolate mofetil had a better safety profile and was more effective than prednisolone plus cyclophosphamide: complete remission 22.5% versus 5.8%; partial remission 29.6% versus 24.6% 9 months Triple therapy was associated with fewer adverse events and was more effective than prednisolone plus cyclophosphamide: complete remission 65% versus 15%; partial remission 30% versus 40% 24 weeks # Similar response rates: 56.2% versus 53%; similar proportions of patients experienced adverse events: 96.2% versus 95%; 40.6% more adverse events in the cyclophosphamide group Median post-treatment follow-up 60 months Prednisolone monotherapy less effective than prednisolone plus cyclophosphamide or prednisolone plus ciclosporin: remission rates 27% versus 60% versus 83% Relapse of nephrotic-range proteinuria was more common after discontinuation of ciclosporin than cyclophosphamide 6 months** Similar efficacy: response rates 90.5% versus 82.1%; complete remission rates 52.4% versus 38.5% Prednisolone plus tacrolimus associated with fewer adverse events than prednisolone plus cyclophosphamide *Extension of Chan (2000). 46 Euro-Lupus Nephritis Trial. Extension of Houssiau (2002). 29 Noninferiority trial, assuming 30% complete remission with prednisolone plus cyclophosphamide. ALMS induction phase. # Powered to demonstrate superiority of prednisolone plus mycophenolate mofetil. **Noninferiority trial, assuming 70% complete remission with prednisolone plus cyclophosphamide. receiving standard therapy comprising corticosteroids in combination with 8 weeks of oral cyclophosphamide was 41%. 26 Sequential immuno suppression with corticosteroids and oral or intravenous cyclophosphamide for 6 months followed by a switch to azathioprine maintenance therapy resulted in stable renal function in 80% of Chinese patients with severe lupus nephritis after 10 years of follow-up. 3,27,28 NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 3

4 Table 2 Key trials of maintenance treatment for lupus nephritis Study Participants Treatment groups Trial duration Key findings Contreras (2004) 34 Houssiau (2010) 64 * Dooley (2011) patients (29 Hispanic, 27 black and 3 white) with lupus nephritis class III or IV 105 patients (83 white, 13 black and 9 Asian) with lupus nephritis class III, IV, Vc and Vd (WHO) 227 patients (99 white, 76 Asian, 23 black and 29 other ethnicity) with lupus nephritis class III V who had responded to induction therapy Prednisolone plus cyclophosphamide ( 7 monthly pulses) for all patients, then prednisolone plus mycophenolate mofetil, or prednisolone plus azathioprine or prednisolone plus cyclophosphamide (quarterly) Intravenous methylprednisolone (3 doses 750 mg) then prednisolone plus low-dose cyclosphosphamide induction for all patients until week 12, then prednisolone plus mycophenolate mofetil versus prednisolone plus azathioprine Prednisolone plus mycophenolate mofetil versus prednisolone plus azathioprine Median 29, 30 and 25 months respectively Mortality and renal-failure-free survival inferior with prednisolone plus cyclophosphamide; relapse-free survival better with prednisolone plus mycophenolate mofetil than with prednisolone plus cyclophosphamide Mean Time to renal or systemic flare did 48 months not differ between groups; similar proportions of patients developed renal flares: 19% with mycophenolate mofetil versus 25% with azathioprine 36 months Mycophenolate mofetil was superior to azathioprine with regard to time to treatment failure, time to renal flare and time to rescue therapy: treatment failure rates 16.4% versus 32.4%, renal flare rates 12.9% versus 23.4% *MAINTAIN trial. Powered to demonstrate superiority of mycophenolate mofetil over azathioprine, assuming a 35% renal flare rate with azathioprine at 5 years and 10% difference between groups. ALMS maintenance phase. 55.9% of participants completed the study. Powered to demonstrate superiority of mycophenolate mofetil over azathioprine, assuming failure rates of 20.0% versus 37.5%, respectively. The Euro-Lupus Nephritis Trial compared high-dose (six monthly pulses plus two quarterly pulses, starting at 500 mg and titrated up to 1,500 mg per pulse in the absence of leukopenia) and low-dose (six 500 mg pulses every 2 weeks) intravenous cyclophosphamide treatment. Both induction treatments were combined with initial pulse methylprednisolone (750 mg daily for 3 days) followed by oral corticosteroids, and were followed by azathio prine maintenance (Table 1). 29 The results showed similar rates of treatment failure (20% and 16%) and renal flare (29% and 27%) for high-dose and low-dose cyclophosphamide, respectively, and both groups had similar improvements in serum creatinine, proteinuria, disease activity score and lupus serology. 29 The two groups also showed no significant differences in the incidence of severe infections and haematological or gonadal toxicity. 29 Extended follow-up for 115 ± 30 months revealed similar rates of progression to chronic kidney disease (11% versus 5%), death (4% versus 11%), serum creatinine doubling (11% versus 14%) and ESRD (9% versus 5%) in high-dose and reduced-dose cyclophosphamide groups, respectively. 30 However, 76 of the 90 participants in the trial were white, 29,30 and the Euro-Lupus regimen is usually not adopted in black patients, who often have severe disease that might not respond well to treatment and in whom efficacy data of this regimen is lacking In 2004, three maintenance immunosuppressive regimens were compared in 59 patients who had all received corticosteroids and intravenous cyclophosphamide induction treatment of these patients were black or Hispanic, and all but one had WHO class III or IV lupus nephritis. Mycophenolate mofetil maintenance therapy was associated with significantly improved relapse-free survival compared with quarterly intravenous cyclophosphamide. Moreover, cyclophosphamide was also associated with inferior event-free survival (a composite of death and ESRD) compared with both mycophenolate and azathioprine maintenance therapy (Table 2). 34 In view of these results, and given that uroepithelial and gonadal toxicity as well as a predisposition to malignancies are all related to cumulative cyclophosphamide exposure, the use of quarterly pulse cyclophosphamide as maintenance immunosuppression has decreased. The latest Kidney Disease: Improving Global Outcomes (KDIGO) guidelines recommend that lifetime exposure to cyclophosphamide should not exceed 36 g. 39 Mycophenolate mofetil Mycophenolate mofetil has greater efficacy than azathioprine for preventing acute rejection of kidney grafts, and is well tolerated in most patients. 40 The adverse effects of mycophenolate mofetil include gastrointestinal irritation, leukopenia, anaemia and a predisposition to infections. The antiproliferative, anti-inflammatory and anti oxidant actions of mycophenolate mofetil, and its relative selectivity for lymphocytes make it an attractive candidate for the treatment of lupus nephritis. This suggestion is corroborated by experimental findings in Fas lpr/lpr lupus-prone mice Induction therapy One randomized controlled trial compared mycophenolate mofetil for 12 months with oral cyclophosphamide for 6 months followed by azathioprine for 6 months (both groups also received prednisolone) in 42 Chinese patients with active class IV lupus nephritis. 46 After 12 months, response rates in the two groups were similar, with approximately 80% and 15% of patients achieving complete and partial remission, respectively (Table 1). 46 However, mycophenolate mofetil was associated with 4 ADVANCE ONLINE PUBLICATION

5 improved quality of life and a reduced incidence of adverse effects, including infections. 47,48 Subsequent reports confirmed that mycophenolate mofetil was at least as effective as intravenous cyclophosphamide when given as induction treatment for active lupus nephritis A response rate of 83% was reported in white patients of European ancestry treated with prednisolone and mycophenolate mofetil for 1 year. 54 Results from a trial in which over 75% of patients were of black or Hispanic ethnicity with Class III, IV and/or V lupus nephritis showed that the rate of complete or partial remission was 52.1% after treatment with corticosteroids and mycophenolate mofetil, versus 30.4% after treatment with corticosteroids and intravenous cyclophosphamide for 24 weeks (Table 1). 52 The Aspreva Lupus Management Study (ALMS) is the largest trial of treatment for lupus nephritis conducted to date (Table 1). 55,56 ALMS was an industry-sponsored, multinational prospective study with two phases. The first phase of ALMS compared the efficacy of mycophenolate mofetil and intravenous cyclophosphamide as induction therapy, 55 and the second phase compared the efficacy of mycophenolate mofetil and azathioprine as maintenance therapy. 56 For the induction phase, 370 patients with class III, IV or V lupus nephritis were randomly assigned to treatment with corticosteroids plus either monthly intravenous cyclophosphamide ( g/m 2 ) or mycophenolate mofetil ( g daily). 55 The primary outcome was the proportion of patients who responded to treatment (defined as a decrease in urinary protein:creatinine ratio to <3 in those with baseline levels 3, or a decrease of at least 50% in those with baseline levels <3, in addition to maintaining serum creatinine levels within 25% of baseline) after 24 weeks. The primary outcome was not significantly different between the two groups in either the intention-to-treat analysis (56.2% for mycophenolate mofetil versus 53.0% for cyclophosphamide) or the per-protocol analysis (63.7% versus 57.1%, respectively). Complete remission (protein uria <0.5 g daily with normal serum creati nine levels and inactive urinary sediment) rates were similar in both groups, attained by 8.6% of patients in the mycophenolate mofetil group and 8.1% in the cyclo phosphamide group after 6 months. 57 The response rates were similar in Asian and white patients, being in the range of 53 56% with myco pheno late mofetil and 54 64% with cyclophosphamide. 57 By contrast, the response rate was significantly higher with mycophenolate mofetil than cyclophosphamide in black patients and those in the other race group (60.4% versus 38.5%) or patients of Hispanic ethnicity (60.9% versus 38.8%), respectively. 57 Results from a post-hoc analysis of data on the 32 patients in ALMS with baseline estimated glomerular filtration rate <30 ml/min/1.73 m 2 showed similar response rates to induction with either mycophenolate mofetil or cyclophosphamide (20% versus 16.7%, respectively). 58 Significant improvements in renal function occurred in similar proportions of patients in each group (55% versus 42%, respectively). 58 By contrast, another post-hoc analysis of pooled data from different trials suggested that cyclophosphamide might be more effective than mycophenolate mofetil in patients with aggressive crescentic disease or who have had multiple relapses. 59 In the induction phase of ALMS, although the proportion of patients who experienced adverse events was similar in the mycophenolate mofetil and cyclophosphamide groups (96.2% versus 95.0%), cyclophosphamide treatment was associated with 40.6% more episodes. 55 Infections occurred in 12.0% of patients in the mycophenolate mofetil group and in 10.0% of patients in the cyclophosphamide group. Infections accounted for seven of the nine deaths in the mycophenolate mofetil group, and two of the five deaths in the cyclophosphamide group. 55 Of the nine Asian patients who died, seven were in the mycophenolate mofetil group. Geographical variation in the risk of infection, and the higher target mycophenolate mofetil dose used in Asian patients in this trial compared with previous trials in Chinese patients are possible contributory factors. In this regard, data from kidney transplant recipients have demonstrated an increased incidence of adverse reactions, including infections, in patients treated with myco phenolate mofetil at 3 g daily compared with those receiving 2 g daily, while increasing the daily dose of mycophenolate mofetil from 2 g to 3 g decreased the incidence of acute rejection in African-American patients but not in other ethnic groups. 60,61 In summary, although the induction phase of ALMS failed to demonstrate the superiority of mycopheno late mofetil over cyclophosphamide, which the study was primarily designed and powered to investigate, the results confirmed earlier reports that myco phenolate mofetil was no worse than cyclophosphamide as an induc tion treatment for active lupus nephritis. 46 How ever, mycophenolate mofetil could be the preferred induction treatment choice for both black and Hispanic patients, in view of the inferior outcome of cyclophospha mide therapy in these groups. Furthermore, the ALMS results suggest that ethnicity, geographic factors, mycophenolate mofetil dose and overall immuno suppressive potency might contribute to determining the optimal balance between efficacy and risk. Subgroup analysis of data from 84 patients with Class V lupus nephritis from a US study and ALMS showed similar reductions in proteinuria (50 70%) after treatment with corticosteroids and mycophenolate mofetil or corticosteroids and intravenous cyclophosphamide for 24 weeks. 62 In patients with nephrotic syndrome, complete and partial remission rates were 5.9% and 58.8%, respectively, after mycophenolate mofetil and 0% and 60.9%, respectively, after cyclophosphamide. A study in 16 Chinese patients with Class V lupus nephritis and nephrotic syndrome showed similar response rates (~60%) to either corticosteroids and m ycophenolate mofetil or corticosteroids and tacrolimus. 63 Maintenance therapy The Mycophenolate Mofetil Versus Azathioprine for Maintenance Therapy of Lupus Nephritis (MAINTAIN) trial involved 105 patients, mostly of white European ancestry, who were recruited during from NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 5

6 European centres. 64 All participants received the Euro- Lupus reduced-dose cyclophosphamide induction regimen. From week 12, all patients were randomly assigned to treatment with either mycophenolate mofetil 2 g daily or azathioprine 2 mg/kg daily, plus low-dose corticosteroids. 32 The primary end point of the trial was time to renal flare. At follow-up (48 ± 14 months), renal flares had occurred in 19% of patients treated with mycophenolate mofetil and in 25% of patients treated with azathioprine, but the difference was not statistically significant. 64 The results also showed no statistically significant differences between mycophenolate mofetil or azathioprine maintenance therapy in terms of preventing non-renal flares (despite numerically fewer episodes in the mycophenolate mofetil group), rate of doubling of serum creatinine level or infectious complications. By contrast, in ALMS only the 227 patients classified as responders after the first 24 weeks of treatment were included in the maintenance phase (Table 2). 56 These patients were randomly assigned to receive low-dose cortico steroids and either mycophenolate mofetil 2 g daily or azathioprine 2 mg/kg daily for 3 years. The primary efficacy end point in the maintenance phase was the time to treatment failure (a composite of renal flare, use of rescue immunosuppressive therapy, doubling of baseline serum creatinine levels, ESRD or death). 127 (55.9%) patients completed 3 years of treatment, and renal flares or adverse events were the major reasons for withdrawal. The actual prescribed daily doses of mycopheno late mofetil and azathioprine were 1.87 ± 0.43 g and ± mg, respectively. Treatment failure occur red in significantly fewer patients receiving myco phenolate mofetil than azathioprine (16.4% versus 32.4%, HR 0.44, 95% CI ). The cumulative rate of renal flares was fairly low (~10%) after 36 months of continuous mycophenolate mofetil therapy. 56 Renal flares occurred in significantly fewer patients in the mycophenolate mofetil group than in the azathioprine group (12.0% versus 23.4%, HR 0.50, 95% CI ). However, the two groups showed similar rates of serum creatinine doubling (0.9% versus 0%), renal failure (4.5% versus 2.7%), extrarenal flares (6.9% versus 6.3%) and serious infections (9.6% versus 11.7%) for mycophenolate mofetil versus azathioprine, respectively. Withdrawal owing to adverse events was significantly more frequent in patients treated with azathioprine than in those receiving mycophenolate mofetil (39.6% versus 25.2% respectively). Notably, the superiority of mycophenolate mofetil over azathioprine was irrespective of i nduction regimen, ethnicity and geographic region. 56 Although only 23 black patients were included in the ALMS maintenance phase, this group showed a 7.0% treatment failure rate with mycophenolate mofetil versus 34.3% with azathioprine. In patients of other ethnicities, the treatment failure rate in the azathioprine group was approximately double that in the mycopheno late mofetil group. In summary, the maintenance phase of the ALMS demonstrated the superiority of mycophenolate mofetil over azathioprine when given for 3 years to patients who responded to induction treatment by 24 weeks. The most important difference detected in the maintenance phase of ALMS was the prevention of renal flares. 56 Induction maintenance (continuous) therapy Our research group conducted an extended study involving 33 patients who were treated with prednisolone and mycophenolate mofetil as both induction and maintenance therapy and 31 patients who were treated with sequential prednisolone and oral cyclophosphamide induction treatment followed by azathioprine maintenance therapy. 47 At follow-up (median 63 months), both groups showed notably high and comparable response rates of >90%. 47 Relapse-free survival and flare rates were similar in the two groups (11 patients in the mycophenolate mofetil group and nine in the sequential immunosuppression group developed flares). The latest analysis of long-term follow-up data showed a satisfactory longterm safety profile and good efficacy for p rednisolone and mycophenolate mofetil given continuously as both induction and maintenance immunosuppression: relapse-free survival was 76% after 5 years and 69% after 10 years (Table 1). 65 Calcineurin inhibitors Calcineurin inhibitors are potent immunosuppressive agents and have been the standard of care for prophyl actic immunosuppression after kidney transplantation since the 1980s. 66 This class of drugs also has the unique ability to stabilize the podocyte actin cyto skeleton by inhibiting dephosphorylation and degradation of synaptopodin, an actin-associated protein that regulates cell shape and motility and the organization of podocyte foot processes. Synaptopodin downregulation and increased calcineurin activity are both associated with proteinuric glomerular diseases In view of their dual actions on the immune system and podocytes, calci neurin inhibitors are frequently used in the treatment of glomer ular diseases associated with heavy proteinuria. 70,71 The efficacy of calcineurin inhibitors in the treatment of non renal (skin and haematological) manifestations of systemic lupus erythematosus has also been reported. 72,73 For clinical use, tacrolimus is usually preferred, owing to the adverse effects (facial hair and coarsening, dyslipidaemia and hypertension) associated with ciclosporin. 74 However, tacrolimus has a higher diabetogenic potential than ciclosporin, especially when given with high-dose corticosteroids. 74 Preliminary data indicate that combined calcineurin inhibitor and corticosteroid therapy is effective in the treatment of class III, IV or V lupus nephritis, although the data on tacrolimus is largely from patients of Asian ethnicity. 63,75 84 Ciclosporin given continuously as both initial and maintenance therapy was as effective as cyclophosphamide (both in combination with corticosteroids) in 19 patients with lupus nephritis. 75 In Chinese and Japanese patients, various small observational studies 77,79 and randomized controlled trials 81,82 have investigated the efficacy of ciclosporin and corticosteroids in induction treatment of lupus nephritis. Tacrolimus and cortico - steroids gave satisfactory response rates, 77,79 and was a 6 ADVANCE ONLINE PUBLICATION

7 potential alternative to cyclophosphamide and corticosteroids 81,82 as induction therapy for proliferative lupus nephritis. Triple immunosuppression with cortic o- steroids, tacrolimus and mycophenolate mofetil, which is similar to the standard prophylactic immunosuppression used after kidney transplantation, has also been investigated in a small prospective study. 78 Triple immunosuppression was associated with a higher short-term complete remission rate (65%) than that associated with corticosteroids and intravenous cyclophosphamide (15%) in Chinese patients with combined class IV and class V lupus nephritis (Table 1). 78 In another randomized controlled trial, treatment of 42 patients with membranous lupus nephropathy with ciclosporin for 1 year resulted in a higher response rate than was achieved with cyclophosphamide (83% versus 60%), although discontinuation of ciclosporin precipitated disease flares in the majority of patients (2.0 versus 0.2 relapses of nephrotic syndrome occurred per 100 patient-months in the ciclosporin versus cyclophosphamide groups, respectively). 85 Anecdotal reports suggested tacrolimus might be an effective treatment of class V lupus nephritis, although data on long-term outcomes is unclear. 86,87 A pilot study that compared tacrolimus with mycophenolate mofetil (both in combination with corticosteroids) in 16 patients with membranous lupus nephritis presenting as nephrotic syndrome showed comparable response rates (approximately 60%) to either regimen, with reduction of proteinuria from approximately 4 g daily to below 1 g daily. 63 Acute and chronic nephrotoxicity are concerns with calcineurin inhibitor therapy. Acute nephrotoxicity is generally associated with excessive blood concentrations of calcineurin inhibitors, whereas chronic nephrotoxicity is related to both cumulative drug exposure and organ susceptibility. Thus, some patients can develop chronic calcineurin inhibitor nephrotoxicity despite apparently acceptable blood levels of the drug. 88 Chronic calcineurin inhibitor nephrotoxicity is often subclinical in the early stages, but histopathological changes can occur despite apparently stable serum creatinine levels. Whether therapeutic drug monitoring can be safely omitted when calcineurin inhibitors are used at low doses remains to be investigated. 75 Treatment with calcineurin inhibitors needs to be long term in view of the high incidence of protein uria rebound after discontinuation. 89 Long-term clinical data (especially on renal function and calcineurin inhibitor nephrotoxicity) is, therefore, important. A prospective study involved 19 Japanese patients treated with corticosteroids and tacrolimus, with or without mizoribine, to achieve trough blood tacrolimus levels of 3.9 ± 1.5 ng/ml. This treatment achieved satisfactory control of disease activity, indicated by stable serum creatinine levels, at the end of follow-up (mean 42 months), in all but two patients. 84 A retrospective study investigated the clinical outcomes of 29 Chinese patients with lupus nephritis who were treated with tacrolimus for 46.0 ± 37.9 months. 90 The target 12 h trough blood tacrolimus level was 4 6 μg/l, which achieved satisfactory suppression of proteinuria. Tacrolimus was used as add-on therapy in 17 patients with class III V lupus nephritis who had persistent proteinuria >2 g daily despite receiving standard immunosuppression; the response rate (complete or partial) in this group was 66.7% after 1 year and 80.0% after 2 years of tacrolimus treatment. 90 Renal function remained stable except in patients with pre-existing progressive renal impairment, and one patient developed biopsy-proven chronic calcineurin inhibitor nephrotoxicity. 90 Azathioprine Azathioprine is commonly used in combination with low-dose corticosteroids as long-term maintenance immunosuppression, owing to its good tolerability and safety during pregnancy. A study by the Dutch Working Party on Systemic Lupus Erythematosus compared azathioprine plus intravenous methylprednisolone versus intravenous cyclophosphamide plus oral prednisone as induction treatment in 87 patients with proliferative lupus nephritis. 91 Although response rates in the first 2 years were comparable between the two regimens, repeat renal biopsy samples from 39 patients showed a greater increase in chronicity index (reflecting accrual of renal damage) in the azathioprine group. 92 At follow-up (median 5.7 years), patients in the azathioprine group showed more disease flares (relative risk 8.8, 95% CI ) and a higher incidence of infections (37 versus 18 events per 100 patient-years, relative risk 1.4, 95% CI ) than did patients in the cyclophosphamide group. 91 In spite of these findings, serum creatinine levels and proteinuria were similar in the two groups, and at long-term follow-up (median 9.6 years), no statistically significant differences were evident between the two groups with regard to the rate of doubling of serum crea tinine, ESRD or mortality. 93 However, rates of serum creatinin e d oubling (16% versus 8%) and death (16% versus 10%) were numerically higher in the azathioprine group than in the cyclophosphamide group. 93 These results suggest that azathioprine, even when combined with methylprednisolone pulses, might not be sufficient to ensure sustained remission and prevent renal scarring in patients with aggressive disease, but might have a role in patients with mild to moderate lupus nephritis. Biologic agents Biologic therapies for lupus erythematosus include monoclonal antibodies and fusion proteins that target cell surface molecules or signalling pathways critical to the pathogenesis of lupus nephritis (Table 3). In theory, biologic agents offer the advantages of increased specificity and reduced interference to physiological processes such as defense responses to infectious agents. Some examples include rituximab and ocrelizumab, both of which target B lymphocyte antigen CD20, abatacept, which interferes with T cell co-stimulation by binding to T lymphocyte activation antigens CD80 and CD86, and belimumab, which interferes with B lymphocyte survival and development by binding to TNF ligand super family member 13B (also known as BAFF and BLyS). These NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 7

8 Table 3 Key trials of biologic therapies for severe lupus nephritis Study Participants Treatment groups Trial duration Key findings Rovin (2012) 17 * Mysler (2013) 23 Furie (2014) patients (45 white, 40 black, 52 Hispanic, 7 Asian or Pacific Islander) with lupus nephritis class III or IV 381 patients, of whom 214 were non-hispanic and 167 were Hispanic (180 white, 101 Asian, 49 American Indian or Alaskan, 19 black, 32 other race) with lupus nephritis class III or IV 298 patients (164 Asian, 111 white, 14 black, 9 other race) with lupus nephritis class III or IV Intravenous methylprednisolone (2 doses of 1 g) for all patients, then prednisolone plus mycophenolate mofetil and rituximab versus prednisolone plus mycophenolate mofetil and placebo Prednisolone plus mycophenolate mofetil or Euro-Lupus Nephritis Trial cyclophosphamide regimen plus ocrelizumab (1 g or 400 mg) or placebo Prednisolone and mycophenolate mofetil plus abatacept (30 mg/kg on days 1, 15, 29 and 57, followed by standard 10 mg/kg weight-tiered dose on days 85, 113, 141, 169, 197, 225, 253, 281, 309 and 337) versus prednisolone and mycophenolate mofetil plus abatacept (standard weight-tiered dose) versus prednisolone and mycophenolate mofetil plus placebo 52 weeks Renal response rates were similar in the rituximab and placebo groups: 56.9% versus 45.8%; cyclophosphamide rescue therapy only required in the placebo group (8 patients); greater improvement in lupus serology with rituximab than placebo; rituximab treatment associated with leukopenia 48 weeks Similar renal response rates in all groups # : 67.1% (ocrelizumab 1 g), 66.7% (ocrelizumab 400 mg), 54.7% (placebo) Increased rates of serious infection with ocrelizumab: 25.1 (1 g) versus 28.8 (400 mg) versus 18.7 (placebo) per 100 patient-years, which was associated with concomitant mycophenolate mofetil treatment 52 weeks** Similar time to complete response and proportion of patients who achieved complete response in the three groups; 52-week complete response rates: 9.1% versus 11.1% versus 8.0%; greater improvement in lupus serology with abatacept than placebo; abatacept treatment associated with gastroenteritis and herpes zoster infection *LUNAR trial. Powered to demonstrate a 25% increase in renal response rate with rituximab. BELONG trial. Prednisolone plus either mycophenolate mofetil or low-dose cyclophosphamide, according to local preference; initial pulse steroids were allowed. Terminated early by sponsor. # Data from the 223 patients who received at least 32 weeks of treatment. **Powered to demonstrate a 20% increase in the complete response rate with abatacept. and other agents (Figure 2) 94 are being investigated as novel treatments for systemic lupus erythematosus and/ or lupus nephritis. The overall experience to date suggests that biologic therapies are mostly well tolerated. However, their clinical role remains inconclusive. The initial positive findings from small-scale, open-label case series of anti- CD20 therapy 95,96 were contradicted by negative results from large-scale, multicentre, placebo-controlled trials, such as the LUNAR study and the BELONG study of ocrelizumab (Table 3). 17,23 Both trials focused on shortterm remission rates, and were unable to demonstrate significantly superior results when biologic agents were used as add-on therapy to standard of care immunosuppressive regimens. In the case of ocrelizumab, the excess of serious infections in patients receiving concomitant mycophenolate mofetil contributed (along with publication of the negative LUNAR results) to the trial being halted before completion. 23 Another trial also failed to demonstrate greater efficacy of abatacept (in terms of the time to complete response, or the complete response rate) over mycophenolate mofetil and glucocorticoids alone in patients with class III or IV lupus nephritis. 18 The prespecified primary end point in this trial was the time to complete response, defined as urine protein:creatinine ratio <30mg/mmol and renal function parameters not below 90% of baseline values. After 1 year, the rate of complete response to treatment was 8.0% in the placebo group (treated with cortico steroids and mycophenolate mofetil) versus 9.1% and 11.1% in the two abatacept add-on groups, but this difference was not statistically significant (Table 3). 18 However, a reanalysis of data from this trial adopting the treatment response definitions as used in other trials, with a small change in the proteinuria criterion, resulted in the finding of a significant treatment effect of abatacept. 19 More patients in the abatacept groups than in the placebo group achieved a renal response when response was defined as 50% reduction in proteinuria and renal function not below 75% of baseline value: 45.5% and 39.4% in the abatacept groups, versus 33.0% in the placebo group. Also, changing the criteria for complete response to match the definition in the LUNAR trial (proteinuria <55 mg/mmol and renal function not below 85% of baseline value, respectively), 17 resulted in complete response rates of over 20% in both abatacept groups, compared with 6% in the placebo group. 19 By contrast, the positive results reported for rituxi mab are mostly confined to highly selected groups of patients, many of whom had not responded to prior treatment or had repeated relapses Analysis of pooled data from 164 patients with lupus nephritis (158 of whom had biopsy-proven class III V or mixed class lupus nephritis), comprising white, black and Asian patients (56%, 28% and 13%, respectively), showed a renal response rate of 67% after 1 year. 101 Differences in patient selection criteria and concomitant treatment also contributed to the wide variations in efficacy observed in the published 8 ADVANCE ONLINE PUBLICATION

9 Rituximab CD40 CD40L Ocrelizumab CD20 MHCII TCR B cell Ofatumumab CD86 CD28 BCR CTLA-4 Plasma cell Epratuzumab Kidney Bortezomib CD22 CD19 BCMA BAFF-R TACI Belimumab Endothelium Basement membrane Epithelial cell TGF-β Anti-CD19 Abetimus Glomerular capillary IL-4 Abatacept Immune complexes Complement IFN-γ Anti-CD40L Autoantibodies Macrophage Neutrophil Eculizumab T cell MIF inhibition Tissue fibrosis Figure 2 Biologic therapies for lupus nephritis. Activation of, and interactions between antigen presenting cells, B cells and T cells are the basis for the aberrant immune reactivity observed in patients with lupus nephritis. Multiple potential targets for interruption of these interactions exist at various steps of the inflammatory and signalling cascades. Abbreviations: BAFF R, B cell activating factor receptor (TNF receptor superfamily member 13C); BCMA, B cell maturation protein (TNF receptor superfamily member 17); BCR, B cell receptor; CTLA 4, cytotoxic T lymphocyte antigen 4; MIF, macrophage migration inhibitory factor; TACI, transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TNF receptor superfamily member 13B); TCR, T cell receptor; TGF β, transforming growth factor β; TNF, tumour necrosis factor. Permission obtained from Nature Publishing Group Smith, R. M. Biological therapy for lupus nephritis tribulations and trials. Nat. Rev. Rheumatol. 6, (2010). IL-6 Tocilizumab TNF Infliximab series. For example, the BELONG trial results suggested that ocrelizumab was more effective than placebo when administered in combination with the Euro-Lupus regimen (cyclophosphamide followed by azathioprine), but not when combined with mycophenolate mofetil. 23 Clinical trial outcomes might also vary according to patient characteristics; black patients seem to respond especially well to rituximab in some studies, whereas serious infections were more common among Asian patients in the BELONG trial. 17,23,102 In this regard, the unsatisfactory response rates (approximately 40% after 24 weeks) to induction treatment with corticosteroids and cyclophosphamide observed in black and Hispanic patients in ALMS are pertinent. A small observational study compared patients treated with g of intravenous methylprednisolone daily for 3 days plus rituximab (17 patients), mycophenolate mofetil (17 patients) or the Euro-Lupus reduced-dose cyclophospha mide regimen (20 patients) as induction immunosuppression, 103 followed by azathioprine 1 2 mg/kg daily, mycophenolate mofetil 1 2 g daily or ciclosporin 1 2 mg/kg daily as maintenance immunosuppression. The three groups showed complete remission rates of 70.6%, 52.9% and 65%, respectively, after 1 year. 103 When added to standard of care therapy, belimumab was associated with an improved response rate in two phase III trials in patients with systemic lupus erythematosus. 104,105 Although these trials excluded patients with severe active lupus nephritis, a post hoc analysis of pooled data from the 267 participants in these trials who had renal involvement at baseline suggested a beneficial effect of belimumab on lupus nephritis. 106 Treatment with belimumab also prevented renal flares in patients treated with mycophenolate mofetil or who showed active serological findings (increased antibodies to double-stranded DNA and low complement C3 and C4) at baseline. 106 Although the body of data suggests a treatment effect of biologics, these agents should not be used as add-on therapy routinely. Investigating whether biologic agents could benefit patients who do not respond adequately to conventional treatments might be more fruitful, and clinically more relevant. Whether their inclusion in treatment regimens for lupus nephritis can help to minimize patients exposure to conventional medications and reduce treatment-associated adverse effects should also be considered. 107 Mizoribine Mizoribine is an imidazole nucleoside that blocks inosine monophosphate dehydrogenase, thereby inhibiting de-novo purine synthesis, similar to mycophenolate mofetil. 108 Mizoribine inhibits both humoral and cellular immune responses through selective action on lymphocytes. Unlike azathioprine, mizoribine is not incorporated into nucleic acids inside the cell and, therefore, has low oncogenic potential. Although mizoribine is well tolerated, the experience to date, mostly reported from studies in Japan, suggests that this agent has a relatively low immunosuppressive potency compared with current standard of care agents. 108 Thus, mizoribine is often used in combination with corticosteroids for maintenance immunosuppression in patients with lupus nephritis. 108 A randomized controlled trial of corticosteroids and mizoribine 4 5 mg/kg daily versus corticosteroids alone in 28 children with lupus nephritis did not demonstrate superiority for the combination treatment (assessed by the reduction of proteinuria and improvement in renal function) after 1 year. 109 However, the efficacy of mizoribine is dose-dependent and drug bioavailability shows substantial interindividual variability, suggesting that further investigations are necessary to define an optimal dose. 110 Other anecdotal reports suggest that mizori bine has increased efficacy when given at high doses with NATURE REVIEWS NEPHROLOGY ADVANCE ONLINE PUBLICATION 9