Full Review. Clinical trials in minimal change disease. Pietro Ravani 1, Enrica Bertelli 2,3, Simardeep Gill 1 and Gian Marco Ghiggeri 2,3

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1 Nephrol Dial Transplant (2017) 32: i7 i13 doi: /ndt/gfw235 Full Review Pietro Ravani 1, Enrica Bertelli 2,3, Simardeep Gill 1 and Gian Marco Ghiggeri 2,3 1 Division of Nephrology, Faculty of Medicine, Foothills Medical Centre, University of Calgary, th Street NW, Calgary, Alberta T2N 2T9, Canada, 2 Division of Nephrology, Dialysis, Transplantation, Giannina Gaslini Children s Hospital, Genoa 16148, Italy and 3 Laboratory on Pathophysiology of Uremia, Giannina Gaslini Children s Hospital, Largo Gerolamo Gaslini 5, Genoa 16148, Italy Correspondence and offprint requests to: Gian Marco Ghiggeri; gmarcoghiggeri@gaslini.org; Pietro Ravani; pravani@ucalgary.ca ABSTRACT Minimal change disease (MCD) is a pathological condition characterized by subtle glomerular lesions causing massive and reversible proteinuria that is usually steroid sensitive. Recurrence of symptoms of active disease following successful treatment (including proteinuria, oedema and oliguria) and steroid toxicity requires the use of other drugs to attain or maintain remission. Unresolved MCD is considered the initial step in the pathological pathway leading to focal and segmental glomerulosclerosis (FSGS). Historically, cyclophosphamide, chlorambucil, mycophenolate and calcineurin inhibitors have been utilized with success in MCD; however, the chronic nature of the disease and the toxicity of long-term use of these medications has pushed the development of new therapies. Synthetic corticotropin (adrenocorticotropic hormone) and anti-cd20 monoclonal antibodies, for example, are currently under investigation in clinical trials. In addition, these new interventions have dramatically impacted our understanding of the mechanisms of the disease. Phase II IV clinical trials targeting new mechanisms and/or molecules are in progress. The list is long and includes drugs blocking the adaptive immune system (abatacept and anti-cd40 antibodies), as well as retinoids and the sialic acid precursor N-acetyl-D-mannosamine (ManNAc), two agents that affect the sieving properties of the glomerular basement membrane. Other drugs are being tested against FSGS and, if successful, could also be utilized against MCD. Clinical trials currently in progress should furnish a proper solution to what appears to be a solvable problem. Keywords: calcineurin inhibitors, FSGS, minimal change disease, nephrotic syndrome, podocytes WHAT IS MINIMAL CHANGE DISEASE? Minimal change disease (MCD) is a glomerular disease resulting in nephrotic syndrome that is usually sensitive to steroid treatment. The term minimal refers to ultrastructural changes that are only detectable via electron microscopy and does not necessarily imply that the disease is benign in prognosis. There is still debate as to whether MCD is a separate entity or a morphological transition [1]. Most agree that MCD is not an evolving disease when drugs revert and stabilize proteinuria. In cases with partial drug sensitivity, MCD may instead evolve into focal and segmental glomerulosclerosis (FSGS), and it is considered to be the initial stage of a process that begins with sub-microscopic podocyte modifications that evolve into more structured lesions [2, 3]. Studies of children with persistent disease who received repeated renal biopsies support this concept [3, 4]. Although these studies were small (including a total of 50 participants), evolution from MCD to FSGS was unequivocally shown in all, strengthening the concept of transition. MCD almost always presents with severe nephrotic syndrome, often complicated by acute kidney injury. Chronicity of the process is common and, at least in children, recurrence of proteinuria occurs in almost 80% of patients following successful treatment [5]. Given the high relapse rate, drug safety and tolerability are as important as drug efficacy in the management of the disease. The clinical course following steroid treatment is key to disease classification, and patients responses to the different types of drugs used to treat relapses have provided new insights into the mechanisms of the disease. MCD is more common in patients <18 years of age, but can also occur in adults. Histological confirmation is not needed in children unless steroid treatment fails, whereas kidney biopsy is The Author Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. i7

2 commonly performed in adults to rule out other causes of nephrotic syndrome. Because steroid sensitivity carries far more meaningful clinical and prognostic information than histology in children, nephrotic syndrome is classified as either steroid responsive or steroid resistant, with MCD being the prevalent cause of steroid-responsive cases. PATHOGENESIS Overview In MCD, light microscopy reveals glomeruli to be normal or minimally altered and no immune deposits can be identified by immunofluorescence or electron microscopy. Typical ultrastructural changes include podocyte foot-process fusion or effacement, which are constant and diffuse during active disease and resolve during remission. Transitory modification of the sieving properties of the slit diaphragm underlies proteinuria, although the real cause of these changes is poorly defined. Several factors may be involved, and the pathogenesis of MCD is now considered to be multifactorial. Considering the transience and/or reversibility of these lesions, and the usual correlation of proteinuria with infectious triggers, a highly regulated immune model has been proposed to explain the pathogenesis of MCD; for a review, see Bertelli et al. [6]. An early step in the process involves the innate immune system, with the activation of professional cells [antigen presenting cells (APCs) and B cells/toll-like receptors] and production of active oxygen species; adaptive immunity has a parallel role at this stage with the involvement of co-stimulatory molecules (Figure 1a). During the second step, alternative pathways of podocyte injury may be prevalent. One possibility involves inflammatory mechanisms with the activation of both T effector (Teff) and Th17 cells. Regulatory T cell (Treg) expansion may counterbalance this inflammatory pathway and reduce oxidation by metabolizing adenosine triphosphate (ATP) to adenosine. Such multipoint regulation of a system that responds to frequent and ubiquitous inflammatory triggers represents an efficient way to ensure that the immunologic cascade is turned on only in response to specific dangers. It is noteworthy that podocytes contain several of the molecules involved in adaptive immunity, implying that drugs targeting these molecules may directly interact with podocytes (Figure 1b). Poor understanding of the mechanisms of disease in MCD, including the potential transition of podocyte injury from MCD to FSGS, limits our ability to identify key pathogenetic targets. While some studies in humans support the transition of podocyte lesions to advanced degenerative lesions, many cases of MCD do not evolve and some cases with FSGS present with degenerative lesions at disease onset. Animal models have not helped to elucidate this issue. Several, if not all, animal models begin with subtle pathological lesions that evolve to glomerulosclerosis. Examples include chemical-induced proteinuria (e.g. adriamycin and puromycin) [7, 8] andconditioned models such as Buffalo rats [9]. Proteinuria induced by infectious triggers [with lipopolysaccharide (LPS) as the most classical example] is more controversial, since LPS produces only transient proteinuria and minimal lesions at the FIGURE 1: (a) Overview of blood cells participating in the immune response. Several cells are activated following an immunogenic trigger: in grey are those cells that constitute the interactive network (i.e. APCs, CD4+, CD8+, Tregs, B cells and Th17); elements shown in colours are interactive molecules that are mainly involved in the co-stimulatory pathways (i.e. CD80, CTLA4, CD28, CD40 and CD40L). (b) Podocytes contain several of the co-stimulatory molecules involved in adaptive immunity. At the same time, podocytes can interact with rituximab via SMPDL3b and be modified upon its binding. Finally, the sieving properties of podocytes can be improved by utilizing ManNAc, which increases sialylation of Angptl4, a secretory glycoprotein that is essential for maintenance of the negative charge of the GBM. start and then evolves slowly to glomerulosclerosis in the absence of proteinuria [10]. Finally, rats over-expressing Angptl4 (angiopoietin-like protein 4) represent a novel model (see below) that seems better related to MCD, since Angptl4 expression in glomeruli is glucocorticoid sensitive [11]. Overall, despite the fact that animal models can provide limited information about the transition from MCD to FSGS, they have the advantage of permitting the identification of the stage and severity of the disease and related disease mechanisms. Human studies are commonly based on samples with large variability in disease onset or treatment history. Potential molecular targets Increased production of oxidants has been demonstrated in both adriamycin/puromicin nephrosis in rats and humans [8], and antioxidant administration prior to exposure reduces the severity of proteinuria and disease progression. Apyrase, adenosine agonists (e.g. 20-chloroadenosine and 50-N-ethylcarboxamidoadenosine) and antagonists of ATP efflux (e.g. carbenoxolone) or ATP blockers can reduce oxidant generation by 30 50% [12]. However, antioxidants appear to have a limited role in humans, probably because they are active when administered before or in concomitance with the pathogenic trigger. i8 P. Ravani et al.

3 CD80 acts as a co-stimulatory molecule on professional APCs. It targets CD28 or cytotoxic T-lymphocyte antigen 4 (CTLA4) on T cells with stimulatory or inhibitory effects, respectively. CD80 is also the effector of LPS nephropathy in mice, an experimental condition leading to podocyte reorganization and proteinuria. LPS nephropathy is abrogated in mice missing CD80 [10]. Urinary levels of full-length CD80 are high during relapse of proteinuria in patients with MCD, whereas CTLA4 (the negative modulator of CD80) is not modified, suggesting that the former molecule is upregulated in MCD [13]. Serum from MCD patients with relapsing MCD also has high levels of a splicing form of CD80 and stimulates full-length CD80 expression by podocyte in vitro. High podocyte expression and high urinary levels of full-length CD80 suggest that this molecule derives from the kidney during relapse of proteinuria. It is of interest that CD80 modification is transitory and does not occur in patients with FSGS [13]. This observation constitutes the basis of a two-hit hypothesis for MCD, according to which transient hyperexpression of CD80 determines proteinuria only in association with other triggers such as CTLA4, interleukin-10 (IL-0) and interleukin-13 (IL-13) etc. CD40 and CD40L are members of the TNF superfamily CD40 and CD40 ligand (CD40L) constitute the second co-stimulatory system present on both B lymphocytes and APCs and are expressed in the kidney by both podocytes and tubule epithelia. CD40 CD40L interaction activates the immune system via T cell stimulation and/or by direct interleukin production. CD40L also exists in a soluble form, produced by shedding, that is circulating and may produce remote responses from the site of production. In animal models with adriamycin nephrosis, and/or in other experimental autoimmune and degenerative conditions, blockade of CD40L has renal protecting properties [14]; CD40L may increase protein renal permeability and it is now considered to be a potential permeability factor in patients with glomerulosclerosis. Epitope mapping of CD40 showed altered immunogenicity in patients with post-transplant recurrence of FSGS, and high pretransplant serum levels of anti-cd40 antibodies are highly predictive of recurrence [15]. Investigation of potential links between anti-cd40 antibodies and recurrence of nephrotic syndrome are warranted both in FSGS and in MCD patients. TNF activation is now considered to be a mechanism for proteinuria and, in some ways, mimics some compound effects in diabetic kidneys. In animal models, high circulating levels and/or local activation of TNF, TNFR1 and TNFR2 are associated with the development of proteinuria and progression of chronic renal disease [16, 17]. It has been proposed that TNF may modulate cholesterol metabolism in podocytes and that some of the molecules involved in cholesterol metabolism may become targets of selective therapies. Experimental studies are now in progress to support this hypothesis. IL-2 is a potent stimulator of circulating Tregs that plays a regulatory role in several steps of the immune cascade (e.g. antioxidant, anti-inflammatory via IL-10 and pro-inflammatory via IL-17 effects). Initial studies conducted in several models of experimental proteinuria (e.g. adriamycin, LPS and Buffalo rats) showed a beneficial effect on proteinuria. In these cases, Tregs were infused directly with or induced by IL-2, or their effect was mimicked by adenosine [18, 19]. However, successive studies in children with steroid-resistant nephrotic syndrome failed to show any positive effects of IL-2, challenging the effects observed in mice [20]. Retinoids can be involved in proteinuria. Evidence supporting their role is entirely experimental and is specifically based on the finding that experimental nephrosis induced by puromycin is worsened in knockout mice for retinoic acid receptor-α (RAR-α) [21]. A RAR-α agonist attenuates proteinuria and glomerulosclerosis in the same model. Angptl4 is a secretory glycoprotein that is essential for maintenance of the negative charge of the glomerular basement membrane (GBM). There is evidence that Angptl4 modifies the clinical outcome of experimental nephrosis: (i) hyperexpression of podocyte Angptl4 in rats induces nephrotic proteinuria, loss of GBM charge and foot process effacement; (ii) Angptl4 / mice are less prone to develop proteinuria if injected with LPS than control mice; and (iii) Angptl4 is highly upregulated in the sera and podocytes of patients affected by MCD [11]. Sialylation of Angptl4 seems to be the crucial point involved in proteinuria, since it modifies the pi of the protein from a cationic to a neutral charge. In fact, infusion of N-acetyl-D-mannosamine (ManNAc) reverses proteinuria induced in NPHS2-Angptl4 transgenic mice, suggesting that hyposialynation is a potential mechanism by which Angptl4 over-expression contributes to proteinuria. A final important point is that glomerular expression of Angptl4 is glucocorticoid sensitive, suggesting a direct correlation of this model with MCD. The urokinase-type plasminogen activator receptor/soluble urokinase-type plasminogen activator receptor (upar/supar) axis has been intensively studied, based on promising data in animal models [22, 23]. However, their participation in the development of nephrotic syndrome is now being questioned [24, 25]. Studies of specific components of the upar connection, including urinary supar, urokinase and plasminogen activator inhibitor-1, are needed to clarify the nature of this association. PRESENT APPROACHES The therapeutic approach to MCD has evolved over the years and has completely modified disease prognosis. In 2012, the Kidney Disease: Improving Global Outcomes (KDIGO) Guidelines for Glomerulonephritis included recommendations for therapies for MCD. However, some evolution occurred in the years after KDIGO s publication, since data concerning the use of anti-cd20 antibodies were not available at that time. They have modified the approach to both steroid and multidrug dependence, and new humanized antibodies could further improve their efficacy. KDIGO is still the cornerstone of therapy for MCD and represents the benchmark of any new drug. KDIGO recommendations for the management of nephrotic syndrome in children differ from those in adults [26]. The disease entity is named MCD in adults and steroid-sensitive and/ or -dependent nephrotic syndrome (SSNS/SDNS) in children, in whom a kidney biopsy is not required at onset. Steroids are the cornerstone of treatment. i9

4 In children, oral prednisone is recommended at a daily dose of 60 mg/m 2 for6weeksfollowedby40mg/m 2 on alternate days. The steroid tapering schedule varies over the subsequent 2 3 months. A recent clinical trial showed that the best outcomes are observed following receipt of a cumulative prednisone dose of 3750 mg/kg over 12 weeks [27]. For relapses of nephrotic syndrome, a single daily dose of prednisone of 60 mg/m 2 is recommended until complete remission is achieved for at least 3 days, followed by prednisone (40 mg/m 2 ) on alternate days for 4 weeks. In children with frequent relapses and/or steroid-dependent nephrotic syndrome, daily prednisone is recommended at the same dose until remission for at least 3 days, followed by alternate-day prednisone for at least 3 months. Prednisone can be given daily or on alternate days and at the lowest possible dose to maintain remission in children with persistent dependence. According to a recent systematic review of randomized controlled trials, prednisone therapy for 2 3 months has similar benefits compared to longer courses. The same review found that prednisone given for 5 7 days at the onset of a viral infection reduces the risk of relapse [28]. Corticosteroid-sparing agents can be started in children with steroid dependence who develop steroid-related adverse effects. A single course of cyclophosphamide (2 2.5 mg/kg/day) for 8 12 weeks or chlorambucil ( mg/kg/day) for 8 12 weeks can be administered after steroids have achieved remission [29, 30]. Levamisole (2.5 mg/kg on alternate days) for 12 months is an alternative [31]. Mycophenolate ( mg twice daily) [32] or calcineurin inhibitors (CNIs) (i.e. cyclosporine 3 5 mg/kg/day or tacrolimus mg/kg/day) [33, 34] can be utilized for 12 months. According to the KDIGO, rituximab can be considered only in children who have continuing frequent relapses despite optimal combinations of prednisone and corticosteroid-sparing agents and/or who have serious adverse effects of therapy. The use of both mizoribine and azathioprine is not recommended in children. For adults, oral prednisone can be given at a daily single dose of 1 mg/kg (up to 80 mg/day) or an alternate-day single dose of 2 mg/kg (up to 120 mg every other day); the initial high dose of corticosteroids should be maintained for a minimum of 4 weeks if complete remission is achieved or for a maximum period of 16 weeks if complete remission is not achieved. Following remission, corticosteroids should be tapered off slowly over up to 6 months. Cyclophosphamide (2 2.5 mg/kg/day) for 8 12 weeks is the drug of choice for patients with steroid dependence; cyclosporine (3 5 mg/kg/day) or tacrolimus ( mg/kg/day) for 1 2 years is recommended in patients who relapse despite cyclophosphamide [35 37] orinpeople who wish to avoid cyclophosphamide due to potential infertility issues. Mycophenolate ( mg/1.73 m 2 ) twice daily for 1 2 years is suggested in patients who are intolerant to corticosteroids, cyclophosphamide or CNIs. Beyond KDIGO Debate about the use of corticosteroid-sparing agents involves at least three main issues: the efficacy of the potential drug to be used, the need to maintain the therapy for a protracted time and side effects. In the pre-rituximab era, the choice of the steroid-sparing agent used to treat SSNS/SDNS was based on the amount of steroid that the child needed to maintain remission and on the superior benefits of cyclosporine/tacrolimus as compared with cyclophosphimide and mycophenolate to lower proteinuria [34, 35, 38]. Little data support the use of levamisole as a low-dose steroid therapy [39]; the drug is not available in many countries, limiting its importance. As a result, a CNI is recommended when the prednisone dose is >0.7 mg/kg/day and an antiproliferative agent is used when the prednisone dose is between 0.3 and 0.7 mg/kg/day. Whether prednisone should be continued for months at doses <0.3 mg/kg/day or replaced by an antiproliferative agent remains under debate. The development of new approaches based on anti-cd20 monoclonal antibodies has substantially modified therapy in cases of protracted multidrug dependence and is now modifying the approach to cases of simple steroid dependence. In the last 6 7 years, small case series and observational prospective studies [40] have reported data on the successful use of anti-cd20 monoclonal antibodies to minimize steroid use in nephrotic syndrome [41]. Two clinical trials have been conducted in children with nephrotic syndrome who were dependent on the combined use of steroids and CNIs; one [42] was designed to test the non-inferiority of rituximab versus standard therapy to maintain remission and the other [43] tested the superiority of rituximab versus standard therapy to prevent relapses. In the former study, 54 children maintained in remission with prednisone and CNIs were stratified based on signs of drug toxicity related to steroids and/or CNIs and randomized to continue the combination therapy alone or to receive one (if toxicity was absent) or two (if toxicity was present) doses of rituximab (375 mg/m 2 each). After 30 days, standard therapies were tapered off in both arms. In the second trial, 47 children were maintained in remission with a variable combination therapy of steroids, cyclosporine and mycophenolate and/or were randomized to receive one rituximab infusion (375 mg/m 2 ) or placebo. They were followed for 1 year, during which oral drugs were tapered off. Both trials showed that rituximab is effective and safe in maintaining remission and preventing relapses without other drugs for 9 12 months on average. A more recent randomized trial [44] showed a longer duration of steroid-free remission following one rituximab infusion in children dependent on high-dose steroid alone (0.7 mg/kg/day). It is important to note the alternative treatment in these patients was based on either cyclophosphimide or cyclosporine, and the reduced dose of steroids suggests that rituximab is a candidate for the early treatment of nephrotic syndrome [45]. ONGOING CLINICAL TRIALS With the exception of the initial steroid scheme in children with nephrotic syndrome (B1), the strength of the KDIGO guideline recommendations is low (C2 D), largely because evidence is based on small clinical trials. A few new studies are now being conducted, especially in SDNS (ClinicalTrials.gov). Some address the selection of the antiproliferative agent in children, i.e. mycophenolate mofetil versus prednisone (NCT ) or cyclophosphamide (NCT ). Similar studies address the same question in adults (NCT i10 P. Ravani et al.

5 and NCT ), while other trials compare tacrolimus with a classical steroid regimen (NCT and NCT ). Data from the adults are expected to be available very soon. A clinical trial comparing the effect of mizoribine versus cyclophosphamide in adults with several forms of nephrotic syndrome resistant to steroids is under way, including cases with MCD and FSGS (NCT ). Finally, trials are being conducted that are testing more innovative approaches, including adrenocorticotropic hormone (ACTH) and anti- CD20 monoclonal antibodies. ACTH (corticotropin) is an alternative to steroids for the protracted treatment of more resistant forms of nephrotic syndrome and could be utilized in SDNS. Results deriving from a small series of patients with membranous nephropathy and FSGS treated with synthetic corticotropin (synacthen) form the basis of its potential efficacy in SDNS [46]. Although synacthen is not available worldwide, two studies are currently in progress to verify the effects of ACTHAR gel, a natural, highly purified porcine ACTH: the first study is being conducted in children with frequent relapses of nephrotic syndrome (NCT ) and the second in adults (NCT ). The efficacy of rituximab is being tested in various forms of nephrotic syndrome. Two clinical trials are currently in progressinchildrenwithsdnscomparingtheefficacy of two rituximab infusions of 375 m 2 given at a week s interval compared with CNIs (NCT ; NCT ). A new humanized anti-cd20 monoclonal antibody, Ofatumumab (2F2, HuMax-CD20, Genmaband GlaxoSmithKline), has been developed and is under study to test its hypothesized greater efficacy in determining B cell death [47]. Ofatumumab recognizes a distinct membrane epitope in the human CD20 molecule, allowing tighter binding between the antigen and antibody to the B cell membrane. Two reports in small cohorts of children with nephrotic syndrome resistant to all treatments indicated that ofatumumab may induce remission if given in high doses (i.e. six infusions of 2 g/1.73 m 2 each) [48]; a low dose (i.e. two pulses of 300 and 700 mg/1.73 m 2 given 2 weeks apart) may be partially effective in patients with normal renal function [49]. Two clinical trials are now testing the efficacy of ofatumumab versus placebo in children with resistant forms of nephrotic syndrome and versus rituximab in children with multidrug dependence (NCT and NCT ). FUTURE DIRECTIONS The development of new drugs for MCD and, more generally, for nephrotic syndrome is strictly dependent on our increased understanding of the mechanisms of the disease. Several drugs directed towards modifying specific molecules that are potentially involved in MCD are available and are now being tested in clinical trials (see the Ongoing Clinical Trials section). Anti-CD80 and -CD40 inhibitors are two examples. The CD80 inhibitor abatacept is licensed for use in renal transplantation. Abatacept is a fusion protein, composed of the Fragment crystallizable (Fc) of an Immunoglobulin G1 (IgG1) fused with the extracellular domain of CTLA4, that interacts with the T cell antigen CD80, preventing its interaction with CTLA4 and thus blocking the second signal of the adaptive immune response. Abatacept has been utilized in patients with FSGS in an attempt to reduce post-transplant recurrence, but the results are conflicting [50, 51]. However, it is important to stress that available data from human beings implicate CD80 in MCD and not in FSGS [13]. A pilot study to evaluate the safety and efficacy of abatacept (given every 28 days for four cycles) in adults and children with both FSGS and MCD and active proteinuria (implying steroid resistance) is now in progress (NCT ). A monoclonal anti-cd40 antibody inhibitor has also been developed (ASKP1240) and is under evaluation in clinical trials for severe psoriasis and organ rejection after kidney transplantation. TNF-α and the two correlate molecules TNFR1 and 2 represent a second group of potential targets. Although case reports support the efficacy of anti-tnf-α agents in nephrotic syndrome, there is also evidence that the same agents can cause membranous nephropathy, highlighting a potentially serious side effect and emphasizing the need for clinical trials in this area. The US Food and Drug Administration recently approved the use of adalimumab, a human monoclonal antibody anti-tnf-α with anti-inflammatory properties, for treatment of rheumatoid arthritis and ankylosing spondylitis. A small study in 10 patients (with few affected by FSGS) showed a decrease of proteinuria in one patient and small changes in three others; proteinuria did not change or increased in two other cases. Results so far are too weak to justify a clinical trial [52]. Two potential new drugs for nephrotic syndrome are retinoids and the sialic acid precursor ManNAc. A non-randomized study seeking to evaluate the effect of retinoids given for at least 6 months to patients with MCD, FSGS and with the HIVassociated variant, is now in progress and is expected to complete in January 2018 (NCT ). The use of ManNAc is supported by the demonstration that ManNAc, a precursor of sialic acid, increases sialylation of Angptl4, a secretory glycoprotein that is essential for maintenance of the negative charge of the GBM. A non-randomized study is now testing whether simple manipulation of GBM sialylation is sufficient to reverse proteinuria in all kinds of nephrotic syndromes, including patients with MCD, FSGS and membranous nephropathy (NCT ). Study completion is expected by the end of Other drugs are now under evaluation in phase II III clinical trials for FSGS, and more generally for drug-resistant nephrotic syndrome. They have been reported in Table 1, in consideration of the fact that cases of clear success could be of interest regarding treatment of MCD. CONCLUSIONS Classical therapy, based on steroids and CNIs, remains the cornerstone of MCD treatment, and several trials are now seeking to improve the selection of antiproliferative agents. Studies are focusing on mycophenolate mofetil versus both prednisone and cyclophosphamide in children and on tacrolimus versus prednisone in adults. The objective is to reduce the side effects of steroids. Anti-CD20 antibodies were introduced into clinical practice only a few years ago and now represent a i11

6 Table 1. Principal studies on MCD and FSGS registered with the ClinicalTrials.gov or EUdraCT registries Drug 1 Drug 2 Design Identifier Phase Age (years) Start PI MCD/FSGS Cyclophosph amide MMF RCT NCT < V. Baudouin, France MMF Steroid RCT NCT < H. J. Choi, Korea Mycophenolic acid Steroid RCT NCT > P. Remy, France Mycophenolic acid Steroid RCT NCT > C. W. Tang, Hong Kong Tacrolimus Steroid RCT NCT > M. Griffith, UK Tacrolimus Steroid RCT NCT > Astellas Mizoribine Cyclophosph RCT NCT > Asai Kasei ACTHAR Single arm NCT > S. Lee, USA ACTHAR Single arm NCT > G. Apple, USA ACTHAR Placebo RCT NCT < L. Greenbaum Rituximab Placebo RCT NCT < V. Guigoins, France Rituximab Placebo RCT NCT < H. G. Kang, Korea Rituximab MMF RCT NCT < W. Smoyer, USA L. Greenbaum Rituximab Tacrolimus RCT NCT < B. Basu, India Rituximab Steroid RCT EUdraCT < G. M. Ghiggeri, India Ofatumumab Rituximab RCT NCT < G. M. Ghiggeri, India Retinoids Single arm NCT > J. B. Kopp, USA DEX-M74 Single arm NCT > J. B. Kopp, USA Abatacept Placebo RCT NCT > Bristol-Myers FSGS/NS Galactose Single arm NCT > H. Trachtman,USA Fresolimumab Placebo RCT NCT > Genzyme Losmapimod Single arm NCT > GSK Liposorber Single arm NCT > M. J. Somers,USA Dapagliflozin Single arm NCT > D. Cherney, Canada Mesenchymal Stem Cell Single arm NCT < H. Gourabi, Iran Only ongoing studies that had been updated in 2015 and had still not been published by mid-2016 have been included. Most studies focused on a single drug utilized in both MCD and FSGS and, more generally, were utilized in nephrotic syndrome with reduced sensitivity to common multidrug approaches. MMF, mycophenolate mofetil; NS, nephrotic syndrome unresponsive to drugs; PI, principal investigator; RCT, randomized controlled trial. practical option as steroid- and calcineurin-sparing agents in those patients that are multidrug dependent. Their use has also been anticipated in those MCD patients with simple steroid dependence, who represent the best targets of this new therapy; humanized anti-cd20 may improve and potentiate the efficacy of this approach. New drugs targeting molecules potentially involved in the pathogenesis of MCD are in development. However, the evolution of such treatments is limited by a lack of clear knowledge regarding the specific mechanisms behind the disease s development. Two crucial issues are the definition of the burden between MCD and FSGS and a lack of animal models that are specific for MCD. Drugs designed to inhibit the adaptive immune response have failed to resolve FSGS but are still under evaluation for MCD. New molecules implementing the sieving properties of the podocyte GBM unit may succeed where others have failed. Clinical trials currently in progress should furnish a proper solution to what appears to be a solvable problem. ACKNOWLEDGEMENTS Istituto Giannina Gaslini provided logistic and financial support. This work was supported by the Italian Ministry of Health Ricerca Corrente and contributions derived from Cinque per mille dell IRPEF. The authors acknowledge the support of the Renal Child Foundation, Fondazione La Nuova Speranza ( Progetto integrato per la definizione dei meccanismi implicati nella glomerulo sclerosi focale ). REFERENCES 1. Habib R. Editorial: focal glomerular sclerosis. Kidney Int 1973; 4: Hayslett JP, Krassner LS, Bensch KG et al. Progression of lipoid nephrosis to renal insufficiency. N Engl J Med 1969; 281: Tejani A, Phadke K, Nicastri A et al. Efficacy of cyclophosphamide in steroid-sensitive childhood nephrotic syndrome with different morphological lesions. Nephron 1985; 41: Ahmad H, Tejani A. Predictive value of repeat renal biopsies in children with nephrotic syndrome. Nephron 2000; 84: Trompeter RS, Lloyd BW, Hicks J et al. Long-term outcome for children with minimal-change nephrotic syndrome. Lancet 1985; 1: Bertelli R, Bonanni A, Di Donato A et al. Regulatory T cells and minimal change nephropathy: in the midst of a complex network. Clin Exp Immunol 2016; 183: Bertani T, Poggi A, Pozzoni R et al. Adriamycin-induced nephrotic syndrome in rats: sequence of pathologic events. Lab Invest 1982; 46: Ghiggeri GM, Cercignani G, Ginevri F et al. Puromycin aminonucleoside metabolism by glomeruli and glomerular epithelial cells in vitro. Kidney Int 1991; 40: Le Berre L, Godfrin Y, Gunther E et al. Extrarenal effects on the pathogenesis and relapse of idiopathic nephrotic syndrome in Buffalo/Mna rats. J Clin Invest 2002; 109: Reiser J, von Gersdorff G, Loos M et al. Induction of B7-1 in podocytes is associated with nephrotic syndrome. J Clin Invest 2004; 113: i12 P. Ravani et al.

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BMC Nephrol 2015; 16: 111 Received for publication: ; Accepted in revised form: i13