Childhood nephrotic syndrome current and future therapies

Transcription

1 Childhood nephrotic syndrome current and future therapies Larry A. Greenbaum, Rainer Benndorf and William E. Smoyer Abstract The introduction of corticosteroids more than 50 years ago dramatically improved the prognosis of children with nephrotic syndrome. Corticosteroids remain the standard initial treatment for children with this disease, but a considerable proportion of patients do not respond and are therefore at risk of progressing to end-stage renal disease. Because of this risk, new therapeutic strategies are needed for steroid-resistant nephrotic syndrome. These strategies have historically focused on identifying effective alternative immunosuppressive agents, such as ciclosporin and tacrolimus, yet evidence now indicates that nephrotic syndrome results from podocyte dysfunction. Even conventional immunosuppressive agents, such as glucocorticoids and ciclosporin, directly affect podocyte structure and function, challenging the immune theory of the pathogenesis of childhood nephrotic syndrome in which disease is caused by T cells. This Review summarizes the currently available treatments for childhood nephrotic syndrome, and discusses selected novel pathways in podocytes that could be targeted for the development of next-generation treatments for children with this syndrome. Greenbaum, L. A. et al. Nat. Rev. Nephrol. 8, (2012); published online 12 June 2012; doi: /nrneph Introduction Although edema and proteinuria have been recognized clinically for more than 2,000 years, the initial description of children with nephrotic syndrome has been credited to Roelans in 1484, 1 whereas the term nephrotic syndrome was coined in 1929 by Henry Christian. 2 Prior to the advent of glucocorticoids or antibiotics, the mortality of children with nephrotic syndrome was very high (~67%). 3 The mortality rate first dropped markedly in 1939 to ~42% following the introduction of sulfonamides, and to ~35% in 1944 following the introduction of penicillin. 3 Mortality fell even more dramatically in the 1950s to ~9% after the introduction of adrenocorticotropic hormone (ACTH) and cortisone, which caused a marked reduction in proteinuria in many patients. Children with corticosteroid-resistant disease have increased short-term mortality and are at a greater risk of mortality from end-stage renal disease than are those who do respond to treatment. 4,5 Although glucocorticoids have continued as the mainstay of therapy for nephrotic syndrome for more than 50 years, neither the target cell nor the mechanism of action of these agents in nephrotic syndrome has been clearly determined. Furthermore, although the majority of treatments found to be effective in treating nephrotic syndrome have been immunosuppressive, a minority have been thought to act by nonimmune mechanisms, and a few actually through immunostimulation. Unfortunately, however, the only therapy approved by the FDA for the treatment of nephrotic syndrome is ACTH (H.P. Acthar Competing interests The authors declare no competing interests. gel, Questcor Pharmaceuticals, NY, USA; repository corticotropin injection). One challenge in the treatment of childhood nephrotic syndrome is that it is clearly not a single disease; indeed, multiple genetic mutations have now been reported to cause nephrotic syndrome in children. 6 8 Nevertheless, one or more circulating factors (for example, soluble urokinase receptor) seem to cause nephrotic syndrome in a subset of children and adults. 9,10 The majority of patients with genetic forms of childhood nephrotic syndrome are resistant to current immunosuppressive treatments, including corticosteroids. 11,12 The clinical response to treatment with corticosteroids separates children with nephrotic syndrome into two groups: a corticosteroidresistant group (steroid-resistant nephrotic syndrome; SRNS), in which patients have a high risk of developing kidney failure, and a corticosteroid-sensitive group (steroid-sensitive nephrotic syndrome; SSNS), in which patients risk morbidity from relapses and cortico steroid exposure. The extent of overlap in the pathogenesis of childhood nephrotic syndrome between these two groups remains unclear. Interestingly, the specific medications currently employed in patients who are steroidresistant and those who are steroid-sensitive are virtually identical, albeit with different indications and dosing strategies. Hence, this Review discusses both groups, but places greater emphasis on the more challenging steroid-resistant group. Extensive research over the past decade has highlighted the crucial importance of the podocyte as a site of cellular injury in nephrotic syndrome. 13,14 These studies have revealed that numerous podocyte intracellular Emory University, 2015 Uppergate Drive, Atlanta, GA 30322, USA (L. A. Greenbaum). Center for Clinical and Translational Research, The Research Institute at Nationwide Children s Hospital, 700 Children s Drive, Columbus, OH 43205, USA (R. Benndorf, W. E. Smoyer). Correspondence to: W. E. Smoyer william.smoyer@ nationwidechildrens.org NATURE REVIEWS NEPHROLOGY VOLUME 8 AUGUST

2 Key points Corticosteroids have been used to treat childhood nephrotic syndrome for more than 50 years but a minority of patients, especially those with focal segmental glomerulosclerosis, are resistant to this treatment Evidence now suggests that childhood nephrotic syndrome is attributable to podocyte dysfunction; many medications used to treat childhood nephrotic syndrome target the immune system, but also directly affect podocytes Several currently available agents developed for other diseases, such as diabetes, are now being considered for treatment of steroid-resistant childhood nephrotic syndrome Ongoing research has identified a number of pathways occuring in podocytes that may be potential targets for future therapies to treat steroid-resistant childhood nephrotic syndrome proteins and molecular pathways that regulate podocyte structure and function have crucial roles in the development of nephrotic syndrome and the response of patients to therapy. This finding has led to research into exciting new therapeutic approaches for nephrotic syndrome in which these specific pathways might be manipulated to produce more effective and less toxic treatments. This Review provides a summary of the currently available therapies for nephrotic syndrome and describes some of the available alternative therapies that are actively being tested. Selected novel pathways that have been found to mediate podocyte injury are also highlighted. Manipulation of these pathways might offer the potential to develop better targeted and more effective therapies for nephrotic syndrome in the future. Currently available treatments Oral glucocorticoids Minimal-change disease (MCD) is the most common cause of nephrotic syndrome during childhood 15 and corticosteroids induce remission in more than 90% of children with MCD. 16 By contrast, the majority of children with the second most common etiology focal segmental glomerulosclerosis (FSGS) 15 do not respond to oral glucocorticoids. 17 A child presenting with idiopathic nephrotic syndrome who does not enter remission following 1 month of corticosteroid therapy is typically classi fied as having SRNS. 18 Evidence from adults, however, suggests that a prolonged (3 6-month) course of oral glucocorticoids improves their response rate. 19,20 In children with SSNS, compelling evidence from a systematic review indicates that a longer course of initial cortico steroids (minimum of 3 months) decreases the likelihood of subsequent relapses. 21 Moreover, the dose of oral cortico steroids might also influence the number of relapses. 22 The efficacy of glucocorticoids in idiopathic nephrotic syndrome might be attributable to the immuno suppressive effects of these agents, their direct action on the podocyte, or, potentially, a combination of these effects. 23 Intravenous glucocorticoids High-dose intravenous glucocorticoids can sometimes induce remission of proteinuria in children with SRNS An 18-month protocol of intravenous glucocorticoids, with or without the alkylating agent cyclophosphamide, is effective in some children with SRNS With this approach, all patients receive high doses of intra venous methylprednisolone, whereas cyclophosphamide is added only for patients who do not respond to intravenous methylprednisolone after the first 10 weeks, or who relapse as the frequency of intravenous methylprednisolone administration is reduced Intravenous glucocorticoids may also improve the response of children with SRNS when combined with ciclosporin. 31 Cytotoxic drugs Cytotoxic agents are known to deplete cells of the immune system, yet the mechanism of action of these drugs in nephrotic syndrome is currently unknown. Although cyclophosphamide is used more commonly in children with frequently relapsing nephrotic syndrome (FRNS; more than four relapses in a 12-month period or two relapses in the initial 6 months) or steroid-dependent nephrotic syndrome (SDNS; relapses during steroid taper or within 2 weeks of discontinuation), 32 it is also used for treating children with SRNS. In a randomized study of 32 children with SRNS, intravenous cyclophosphamide was inferior to ciclosporin in inducing remission. 33 Cyclophosphamide had greater efficacy in children with SSNS who had polymorphisms in glutathione Stransferase (which is important for cyclophosphamide metabolism) than in those who do not express these varients, potentially as a result of slower drug metabolism. 34,35 An alternative cytotoxic agent is chlorambucil, but a meta-analysis revealed an increased risk of seizures and severe bacterial infections with chlorambucil when compared with cyclophosphamide. 36 Mycophenolate mofetil and mizoribine Mycophenolate mofetil has been used for the management of a variety of glomerular diseases, including lupus nephritis and IgA nephropathy, 37,38 but its mechanism of action in nephrotic syndrome remains unknown. Mycophenolate mofetil inhibits lymphocyte DNA synthesis and proliferation through the inhibition of inosine monophosphate dehydrogenase a key enzyme in purine biosynthesis. 39 This inhibition is restricted to T cells and B cells, however, as all other cell types have a salvage pathway through which purines can still be synthesized in the presence of mycophenolate mofetil. 40 In one randomized trial involving 33 adults with FSGS, mycophenolate mofetil and low-dose prednisone was as effective as standard oral corticosteroids in inducing remission. 41 In a 6 month open-label study, use of mycophenolate mofetil in adults with FSGS who were resistant to other treatments resulted in a reduction in proteinuria in almost 50% of patients, but did not achieve complete remission in any. 42 A number of uncontrolled studies have reported a good response to mycophenolate mofetil in children with SRNS or FRNS. 43,44 Indeed, in a retrospective study of 52 children who were resistant to corticosteroids and cyclophosphamide, 59.5% had either a complete or a partial remission in response to mycophenolate mofetil. 45 Nevertheless, the response rate to mycophenolate mofetil in children is quite variable. 46, AUGUST 2012 VOLUME 8

3 Mizoribine, which also inhibits inosine monophosphate dehydrogenase, has been used to treat patients with nephrotic syndrome, mostly in Japan. The majority of case series describe its use in children with SDNS or FRNS, 48 but it has also been used in children with SRNS. 49 Ciclosporin and tacrolimus Ciclosporin prevents T cell activation through inhibition of calcineurin-induced IL2 gene expression a crucial early event in T cell activation. 50 However, ciclosporin also stabilizes the podocyte actin cytoskeleton, which might partly explain its effects in nephrotic syndrome. 51 The efficacy of ciclosporin in SRNS has been demonstrated in placebo-controlled studies in both adults and children. 52,53 In a pediatric study, 12 children in the ciclosporin arm completed the 6 month protocol; four children had a complete remission and eight had a partial response. By contrast, only two of 12 patients in the placebo arm had a partial response and none had a complete remission. 52 As mentioned earlier, in a non randomized trial, 65 children with SRNS (45 with MCD; 20 with FSGS) were treated with a combination of ciclosporin and prednisone, with 27 children (41%) achieving complete remission. 54 In a randomized study published in 2008, ciclosporin was superior to intra venous cyclophosphamide in children with SRNS. 33 The efficacy of ciclosporin might be enhanced when combined with intravenous glucocorticoids. 31 The combination of mycophenolate mofetil and high-dose oral dexamethasone was compared with ciclosporin in a randomized clinical trial published in Although the remission rate was higher in the ciclosporin group than in the mycophenolate and dexamethasone group, this trend did not reach statistical significance. The most common adverse effects of ciclosporin are hypertension, mild increases in serum creatinine levels, hyperkalemia, gingival hyperplasia, and hyper trichosis. However, one of the major drawbacks of the use of ciclosporin is the risk of nephrotoxicity, which manifests as interstitial fibrosis, often in a striped pattern that is reflective of the interstitial vascularization. Indeed in a study of children with MCD, tubulointerstitial lesions attributed to ciclosporin were found in two of 18 (11%) of children with MCD treated with ciclosporin for less than 2 years, but increased to 11 of 19 (58%) of those who continued on this medication beyond 2 years. 55 An alternative calcineurin inhibitor is tacrolimus, although differences exist in its mechanism of action when compared with ciclosporin. 56 Tacrolimus and ciclosporin have similar adverse effects but tacrolimus is seldom associated with the cosmetic adverse effects of hirsutism and gingival hyperplasia that affect the quality of life of many patients using ciclosporin. Tacrolimus has been used effectively in treating children with SRNS In one study, the relapse rate was lower with tacrolimus than with ciclosporin. 61 Plasmapheresis Many reports have described the use of plasmapheresis in the treatment of recurrent nephrotic syndrome following kidney transplantation in patients with FSGS This strategy aims to remove a suspected soluble substance, such as a cytokine, which mediates FSGS recurrence. 9 Interestingly, few studies have reported on the use of plasmapheresis in nephrotic syndrome occurring in native kidneys, 64,66 68 which is potentially related to the perceived invasive nature of plasmapheresis. Of note, however, most alternative therapies for nephrotic syndrome, such as cytotoxic agents and calcineurin inhibitors, also have considerable adverse effects. New approaches with available drugs In addition to the therapies for nephrotic syndrome described earlier, several drugs developed for treating other diseases are now being used to treat patients with nephrotic syndrome. Four of the most prominent drugs rituximab, galactose, adalimumab, and thiazolidinediones are discussed below. Rituximab Rituximab is a chimeric monoclonal antibody that depletes CD20 + B cells and was originally developed to treat B cell non-hodgkin lymphoma and chronic lympho cytic leukemia. 69 In the past several years, rituximab has also been used to treat rheumatoid arthritis, microscopic polyangiitis and granulomatosis with polyangiitis (Wegener) (FDA approval in 2011), posttransplant lymphoproliferative disorder, lupus nephritis, membranous nephropathy, recurrence of nephrotic syndrome in patients with FSGS following transplantation, 72 as well as nephrotic syndrome Although reports of the use of rituximab to treat nephrotic syndrome have increased in the past 5 years, most have been anecdotal and in children Importantly, no prospective studies have directly compared rituximab with other treatments for nephrotic syndrome, making it difficult to compare its efficacy and toxicity against other currently available therapies. A number of studies describe the use of rituximab to treat SDNS. A randomized study in 54 children with SDNS reported that adding rituximab to glucocorticoids and calcineurin inhibitors enabled reduction in the doses of both medications, while yielding similar or better shortterm (3-month) outcomes than standard dosing without rituximab. 84 In a retrospective comparison of 23 children with SDNS, rituximab and tacrolimus were similarly effective in reducing relapse rates and glucocorticoid exposure. 85 In another retrospective study of 30 children with SDNS treated with repeated doses of rituximab to maintain depressed CD19 levels for at least 15 months, long-term remission (~17 months) following complete CD19 recovery was noted in 19 (63%) of patients. 86 In addition, a retrospective review of long-term outcomes for 37 children with SDNS found that 375 mg/m 2 given weekly for 1 4 courses resulted in a sustained remission in 26 children (70%) for 12 months and, among 29 children followed for more than 2 years, 12 (41%) remained in remission. 87 A randomized, double-blind, placebocontrolled, multicenter trial comparing rituximab with placebo infusions in children with SDNS is currently underway in Japan. 88 NATURE REVIEWS NEPHROLOGY VOLUME 8 AUGUST

4 a Number of children SDNS/FRNS SRNS Post-tx NS 375 mg/m mg/m 2 1,125 mg/m 2 Good initial response Type of NS 1,200 mg/m 2 1,500 mg/m 2 1,875 mg/m Poor initial response SDNS/FRNS SRNS Post-tx NS Type of NS Figure 1 Total rituximab dose does not seem to correlate with initial clinical response. The total rituximab dose based on the initial clinical response is shown for 70 children with SDNS and/or FRNS, SRNS, and post-tx NS. a Good initial response (no or mild proteinuria and serum albumin >30 g/l); b poor initial response (marked proteinuria and serum albumin <30 g/l). Individual doses were mg/m 2 and patients received as many as four doses, with doses given every 1 2 weeks. Abbreviations: FRNS, frequently recurring nephrotic syndrome; NS, nephrotic syndrome; SDNS, steroid-dependent nephrotic syndrome; SRNS, steroid-resistant nephrotic syndrome; tx, transplantation. Permission obtained from Springer Prytula, A. et al. Pediatr. Nephrol. 25, (2010). b Rituximab has been reported to be effective in SRNS, including in children with either MCD or FSGS. The largest series reported to date is a cohort study that included 33 children with SRNS who received 2 4 doses of rituximab. 75 At 6 months after the last dose of rituximab, nine (27%) children had entered complete remission, seven (21%) had experienced a partial remission, and 17 (51%) had had no response. Notably, the median time to response was 32 days (8 60 days), which provides valuable new information for clinical practice on how long this therapy should be continued before seeking an alternative approach. This favorable response to rituximab contrasts with the results of a randomized trial in which 31 children who were resistant to corticosteroids and calcineurin inhibitors were continued on a calcineurin inhibitor and low-dose cortico steroids. 89 Half of these children also received two doses of rituximab. After 3 months, no statistical difference in protein uria was found between the two groups, arguing against any additional benefit of rituximab in this patient population. Given the growing interest and use of rituximab for SRNS, it is important to note that almost all reports in SRNS are in children who had already failed multiple other therapies. In this light, the currently available reports might present a negative bias against the potential efficacy of rituximab in SRNS. This bias could be attributable either to the selection of only those patients who would have been resistant to any therapy, or to the possible effects of disease progression resulting from delayed treatment or alterations induced by prior ineffective therapies. These uncertainties highlight the potential value of a prospective, randomized, controlled trial to clarify the relative efficacy and toxicity of rituximab compared with the other currently available therapies. The most appropriate dosing for rituximab in childhood nephrotic syndrome is not known. The most common dose used has been 375 mg/m 2 weekly, and most treatments have been within the range of 1 4 weeks. 74,75,77,84 In addition, most children have complete B cell depletion after only one dose. 90 Moreover, in the largest retrospective study to date (70 children: 28 with SDNS and/or FRNS, 27 with SRNS and 15 with post-transplantation nephrotic syndrome), the total rituximab dose did not seem to correlate with the initial clinical response to therapy (Figure 1). 74 Such findings, although far from conclusive, suggest that two doses of rituximab (with the second dose 1 2 weeks after the first), or perhaps even a single dose, is adequate to obtain the desired clinical benefits from this drug in children with nephrotic syndrome. The appropriate timing for repeat dosing of rituximab in childhood nephrotic syndrome has also not been determined. In children with SRNS, the majority of those who respond do so within 2 months, although, as previously noted, the response is not clearly related to the total dose (Table 1). After successful induction of remission, nephrotic syndrome often recurs, but the time to relapse in SRNS has been highly variable, typically after 5 9 months (Table 2). 74,75 Relapses have often, but not always, been associated with repopulation of CD20 + B cells Some children, however, remain in remission despite B cell recovery, whereas others respond to rituximab despite an absence of B cell depletion. 74,75 As a result, repeat dosing strategies have varied between those based on improvement or full recovery of CD20 + B cell numbers and those based strictly on clinical relapse of nephrotic syndrome, regardless of B cell numbers. Despite the lack of evidence to date demonstrating superior efficacy of rituximab, the reports of it inducing remission of nephrotic syndrome have led to a reevaluation of the inter-relationship between nephrotic syndrome and the immune system. Although most historical evidence has suggested involvement of T cells, the effectiveness of rituximab in nephrotic syndrome implies a potential role of B cells in the development of nephrotic syndrome. However, the novel finding in 2011 that rituximab binds directly to an acid sphingomyelinaselike phosphodiesterase 3b (SMPDL3B) on the surface of podocytes suggests an even more direct mechanism of action for this drug in nephrotic syndrome. 72 In this report, the binding of rituximab to this off-target podocyte protein prevented the downregulation of acid sphingo myelinase activity in cultured podocytes induced by sera from adults with recurrent FSGS, as well as restored actin stress fiber formation and podocyte 448 AUGUST 2012 VOLUME 8

5 Table 1 Timing of clinical response following treatment of SRNS with rituximab Patients in remission (n) Weekly dose Time of response Study mg/m 2 for 4 weeks <3 months Suri et al. (2008) mg/m 2 for 4 weeks 2 8 weeks (median: 4 weeks) Bagga et al. (2007) mg/m 2 for 1 week 1 month Kurosu et al. (2009) ,000 mg for 2 weeks* 2 weeks/4 months* Peters et al. (2008) mg/m 2 for 1 week 2 weeks Smith (2007) mg/m 2 for 1 week 1 month/8 months Nakayama et al. (2008) mg/m 2 for 1 week 1 month/4 months Nakayama et al. (2008) 82 *Adult patient: proteinuria decreased from 10 g per day to 2 3 g per day in 2 weeks and to <1 g per day at 4 months. Partial response in <1 month (albumin >25 g/l) and complete response (protein/creatinine ratio <0.2 mg/mg) within 8 months. Partial response in <1 month (albumin >25 g/l) and complete response (protein/creatinine ratio <0.2 mg/mg) within 4 months. Abbreviation: SRNS, steroid-resistant nephrotic syndrome. Table 2 Timing of relapse following treatment of SRNS with rituximab* Patients (n) Weekly dose Time of relapse B cells present at relapse mg/m 2 for 4 weeks 6 months Yes Sharma & Filler (2009) mg/m 2 for 4 weeks None (30 weeks) N/A Bagga et al. (2007) mg/m 2 for 4 weeks 6 months ND Bagga et al. (2007) mg/m 2 for 4 weeks None (38 weeks) N/A Bagga et al. (2007) mg/m 2 for 4 weeks None (14 weeks) N/A Bagga et al. (2007) mg/m 2 for 1 week None (6 months) N/A Kurosu et al. (2009) ,000 mg for 2 weeks 13 months Yes Peters et al. (2008) 80 2 ND None (6 months and 13 months) ND Prytula et al. (2010) 74 9 ND 1 16 months (median 5 months) ND Prytula et al. (2010) 74 Study mg/m 2 for 1 week None (10 months) N/A Smith (2007) mg/m 2 for 1 week None (14 months) N/A Nakayama et al. (2008) mg/m 2 for 1 week 8 months Yes Nakayama et al. (2008) mg/m 2 for 1 week 4 months No Kari et al. (2011) 77 *Patients were on variable immunosuppression following rituximab treatment. Patient received a second dose of rituximab at 6 months while still in remission owing to an increase in B cells. He remains in remission 6 months after 2 nd dose. Adult patient: proteinuria decreased from 10 g per day to 2 3 g per day in 2 weeks and to <1 g per day at 4 months. Abbreviations: N/A, not applicable; ND, not determined; SRNS, steroid-resistant nephrotic syndrome. viability. 72 As potentially important as this finding has been, the mechanistic role of SMPDL3B in the regulation of podocyte structure and function remains to be determined. 92 Galactose Galactose has been proposed as a novel treatment for nephrotic syndrome based on a study that demonstrated that galactose binds the FSGS permeability factor with high affinity and is able to alter glomerular permeability in vitro, as well as decrease this permeability activity after intravenous administration to patients. 94 Subsequently, a case report in a single adult with nephrotic syndrome who was resistant to multiple immunosuppressants and plasmapheresis demonstrated that administration of oral galactose induced remission of nephrotic syndrome, in correlation with a reduction in permeability activity (Figure 2). 95 In 2011, two children with SRNS who were also resistant to tacrolimus were reported to have partial remissions following treatment with oral galactose. 96 Based in part on these findings, galactose has now been added to the Novel Therapies in Resistant Focal Segmental Glomerulosclerosis (FONT2) clinical trial for steroid-resistant FSGS, 97 and is also being evaluated in a prospective clinical trial in children with SRNS. 98 Adalimumab Adalimumab is a monoclonal anti-tumor necrosis factor (TNF) antibody, and it functions by binding TNF and preventing it from activating TNF receptors. The use of this agent in nephrotic syndrome stems from the reported roles of TNF in nephrotic syndrome and fibrosis. 99,100 To date, no reports have documented the use of adalimumab in nephrotic syndrome, although its efficacy when compared with galactose in the treatment of children with steroid-resistant FSGS is currently being tested as part of the FONT2 clinical trial noted above. 97 Anecdotal reports, however, have shown associations between adalimumab use and both remission and induction of nephrotic syndrome. 101,102 Dosing strategies for the use of adalimumab in nephrotic syndrome were developed from phase I pharmacokinetic studies performed during the FONT1 clinical trial, and no serious adverse events were noted in the 10 patients studied. 97,103 Thus, although the potential NATURE REVIEWS NEPHROLOGY VOLUME 8 AUGUST

6 35 Prednisone 1 mg/kg Prednisone 5 mg Cyclophosphamide Mycophenolate mofetil Proteinuria (g/24h) Ciclosporin Plasmapheresis Galactose Serum creatinine (μmol/l) Follow-up period (months) Figure 2 Oral galactose induces remission of proteinuria in an adult with nephrotic syndrome resistant to multiple immunosuppressive agents. The clinical courses of proteinuria and serum creatinine are shown during various immunosuppressive regimens, before and following introduction of oral galactose. Doses: cyclophosphamide (initial course 125 mg daily [1.5 mg/kg]; 2 nd course up to 100 mg daily); mycophenolate mofetil 500 mg twice daily; plasmapheresis three times weekly and ultimately once every 2 weeks; ciclosporin 50 mg twice daily, increased to 75 mg twice daily; ciclosporin (2 nd course) 175 mg twice daily; oral galactose 10 g twice daily then 15 g twice daily. Permission obtained from Oxford University Press De Smet, E., Rioux, J. P., Ammann, H., Deziel, C. & Querin, S. Nephrol. Dial. Transplant. 24, (2009). 0 for adalimumab in the treatment of nephrotic syndrome is still unknown, both its efficacy and toxicity in steroidresistant FSGS are currently being evaluated, and should be available in the near future. Thiazolidinediones Thiazolidinediones are approved by the FDA for the treatment of type 2 diabetes mellitus. Thiazolidinediones are ligands of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ), which is known to modulate many metabolic and nonmetabolic processes, including adipogenesis, glucose homeostasis, inflammatory responses and apoptosis. 104 In the past few years, the beneficial effects of thiazolidinediones on the kidneys have also been reported. In addition to improvement of chronic kidney disease resulting from metabolic syndrome, thiazolidinediones reduced proteinuria, microalbuminuria, podocyte injury, vascular injury, inflammation and fibrosis in both diabetic nephro pathy and nondiabetic glomerulosclerosis in mouse and rat models, as well as in humans The thiazolidinedione pioglitazone protected against progression of puro mycin aminonucleoside (PAN)-induced glomerulosclerosis in vivo and against injury of cultured podocytes in vitro. 106,110 In addition, the thiazolidinedione rosiglitazone attenuated proteinuria and glomerulosclerosis in doxorubicin-induced FSGS in rats. 111 A meta-analysis in 2010 concluded that thiazolidinediones markedly decreased albuminuria and proteinuria in patients with diabetes mellitus, potentially through direct renal protective effects. 108 The renoprotective effects of thiazolidinediones result, at least in part, from their direct action on podocytes, seen in both cultured podocytes and in PAN-induced nephrotic syndrome in rats. 110,112 Thiazolidinediones induced complex responses in podocytes that may involve the canonical pathway (that is, PPARγ binding to responsive DNA elements), off-target pathways such as activation of the glucocorticoid receptor, and nongenomic mechanisms independent of DNA binding, such as modulation of mitogen-activated protein kinase (MAPK) activity (Figure 3). Among the known molecular effects of the thiazolidinediones is the capacity of these agents to decrease the glomerular production of transforming growth factor (TGF) β, 113 a key mediator of renal injury. 109 Of potential relevance to nephrotic syndrome, cultured podocytes treated with thiazolidinediones mimicked some podocyte responses to glucocorticoids, or had additive effects with them. 112 This partial overlap of the molecular actions of thiazolidinediones and glucocorticoids on podocytes might account for some of the beneficial effects of thiazolidinediones in 450 AUGUST 2012 VOLUME 8

7 Thiazolidinediones Plasma membrane Nongenomic pathway Off-target pathway Canonical pathway Modifiers ERK Glucocorticoid receptor Co-activators PPARγ RXR PPARγ Target genes FABP4 PLIN2 LPL ADIPOQ CEBPA Biological response Adipocyte differentiation Increased lipid storage Antidiabetic effects p38 FKBP5 Decreased PPRE TSC22D3 inflammation JNK Podocyte protection GRE Nucleus Figure 3 Schematic of the canonical and alternate modes of action of thiazolidinediones. Traditionally, thiazolidinediones bind to their receptor, PPARγ, which dimerizes with the nuclear receptor RXR and acts on peroxisome proliferator response elements, together with co-activators, to promote the transcription of responsive genes (canonical pathway). Thiazolidinediones can also act on the glucocorticoid receptor directly (off-target pathway). In this off-target pathway, thiazolidinediones might imperfectly bind to and stimulate the glucocorticoid receptor through phosphorylation and nuclear translocation, possibly resulting in the expression of glucocorticoid-responsive genes. Thiazolidinediones may also modulate the glucocorticoid receptor pathway indirectly by binding to PPARγ, resulting in subsequent interaction between the two receptors directly or through the involvement of common cofactors. Thiazolidinediones may also deactivate mitogen-activated protein kinases via a nongenomic pathway, which subsequently modifies other cellular systems including the phosphorylation of the glucocorticoid receptor and PPARγ. Figure modified from the Nuclear Receptor Resource ( with the permission of Dr J. Vanden Heuvel, University Park, PA, USA. Abbreviations: GRE, glucocorticoid response element; PPARγ, peroxisome proliferator-activated receptor γ; PPRE, peroxisome proliferator response element; RXR, retinoid X receptor. nephrotic syndrome. Moreover, it raises the possibility that use of these agents as therapy in nephrotic syndrome might enable a reduction in glucocorticoid exposure or improvement in their efficacy. In summary, thiazolidinediones slow the progression of glomerular diseases, and have also been shown to directly protect podocytes. Such findings suggest that thiazolidinediones might, therefore, have considerable potential as a therapy for nephrotic syndrome, even though the mechanisms of action are only partially understood. Potential future treatments p38 MAPK, MK2 and PKCα signaling The major families of MAPK typically translate extracellular stimuli (including adverse environmental factors) to intracellular responses. The p38 MAPK pathway has crucial roles in inflammation, differentiation, senescence, tumorigenesis, and apoptosis, as well as in a variety of renal diseases. 114,115 p38 MAPK signaling is largely transmitted through phosphorylation and activation of MAPKactivated protein kinase 2 (MAPKAPK2, also known as MK2), a direct downstream substrate of p38 MAPK. 116 Pro-apoptotic p38 MAPK signaling has been implicated both in experimental models of renal injury and in human glomerulopathies. In rodent models of crescentic glomerulonephritis, activation of p38 MAPK was found in podocytes and other glomerular cells after disease induction, and inhibition of this signaling pathway was effective in reducing the severity of glomerular injury In histological studies from adults with a variety of glomerular diseases, increased expression or activation of p38 MAPK isoforms in renal cells was reported and correlated with kidney dysfunction and histopathology, suggesting that p38 MAPK activation has an important role in human glomerulonephritis. 120,121 Similarly, increased p38 MAPK activation was observed in podocytes in biopsy samples from adults with various forms of nephrotic syndrome. 119 A study using cultured podocytes demonstrated that both p38 MAPK and MK2 are involved in cell injury, and that inhibition of either protein kinase protects podocytes from PAN-induced injury. 122 Exposure of podocytes in vivo or in vitro to physiologically relevant concentrations of serum albumin also induced injury, and this response involved p38 MAPK and TGF β. 122,123 The various forms of protein kinase C (PKC) also have a role in both glomerular and tubular function. 124,125 In podocytes, different PKC isoforms seem to serve specific functions. For example, deletion of atypical PKCλ/ι caused mislocalization of the slit diaphragm, podocyte polarity defects and nephrotic syndrome in mice, 126 whereas the loss of PKCα had podocyte- protective effects in mice after diabetic injury. 127 Compared with a wildtype podocyte cell line, a PKCα-deficient podocyte cell line exhibited enhanced activation of the antiapoptotic NATURE REVIEWS NEPHROLOGY VOLUME 8 AUGUST

8 TGF-β P PKCα p38 MAPK MK2 Apoptosis Renal injury TGF-β receptor Inhibitor (in clinical trials) Inhibitor (in clinical trials) Inhibitor (experimental) Figure 4 Following TGF-β binding, the TGF-β receptor is activated, resulting in downstream activation of proapoptotic p38 MAPK and MK2 signaling via activation of PKC. Accordingly, inhibition of PKCα, p38 MAPK, or MK2 would inhibit pro-apoptotic signaling in podocytes at different levels of the same pathway. Abbreviations: MAPK, mitogen-activated protein kinase; MK2, MAPK-activated protein kinase 2; PKC, protein kinase C; TGF-β, transforming growth factor β. PI3K/AKT, ERK1/2 and Smad pathways after stimulation with TGF β, whereas pro-apoptotic p38 MAPK signaling was reduced. 128 In this study, PKCα was found to interact with the TGF β receptor 1, resulting in promotion of its endocytosis with subsequent consequences for receptor recycling and degradation. Thus, PKCα seems to be a regulator of podocyte survival and proteinuria, and might have an important role in several glomerular diseases. In this light, PKCα inhibition is a potential strategy to protect podocytes in glomerular disease. 128 p38 MAPK activation in podocytes is downstream of PKCα, and both protein kinases are activated by TGF β. 129 Thus, inhibition of PKCα, p38 MAPK, or MK2 would affect the same signaling pathway, although at different levels (Figure 4), and such inhibition seems to be an exciting potential strategy to treat nephrotic syndrome in both children and adults. Indeed, research is already underway to develop inhibitors (including antisense oligonucleotides) with specificity towards these target protein kinases. 122, Inhibitors of p38 MAPK and PKC are at the most advanced developmental stage and have been tested in clinical trials, albeit in the treatment of patients with conditions other than nephrotic syndrome. 134,135 Notch signaling Similar to MAPK signaling, Notch signaling regulates multiple cellular processes including development, differentiation, proliferation and apoptosis in a cell-contextspecific manner. 136 Notch signaling involves receptors, ligands, modifiers, and transcription factors. Following Jagged or Delta-like (DLL) protein ligand binding, the intracellular domain of the Notch receptor (Notch-ICD) is cleaved off by a γ secretase and subsequently translocated to the nucleus where it binds to the transcriptional repressor RBP J (Box 1). 136 Notch signaling seems to be crucial for cell differentiation in nephrogenesis. In this light it was not entirely surprising when abnormally increased Notch signaling was found in the glomeruli of patients with FSGS and diabetic nephropathy, and also in rodent models of glomerular disease Niranjan and co-workers 138 also demonstrated that transgenic mice conditionally overexpressing Notch-ICD in mature podocytes developed proteinuria and glomerulosclerosis, in association with p53 activation and podocyte apoptosis. Conversely, genetic deletion of downstream Rbpj in the podocytes of mice with diabetes protected against glomerular proteinuria, as did pharmacologic inhibition of the upstream γ secretase in rats with PAN-induced proteinuria. 138 In a similar study, ectopic expression of Notch-ICD in embryonic podocytes in mice was associated with proteinuria and glomerulosclerosis. 140 Both studies also suggested that abnormally increased Notch signaling is associated with dedifferentiation of podocytes. Notably, abnormally increased Notch signaling was associated with both apoptosis 138 and cell cycle reentry, 140 which in turn were associated with a loss of mature podocyte characteristics. The Notch-mediated inappropriate proliferation of podocytes might even result in cell death by mitotic catastrophe. 141 In the same study, 141 Notch signaling was also shown to promote the proliferation of podocyte progenitors (parietal cells) and differentiation towards the podocyte lineage. Indeed, the severity of glomerular injury has been suggested to depend on Notch-regulated balance between podocyte death and regeneration provided by progenitor cells. Taken together, Notch signaling seems to have a key role during nephrogenesis, but is largely silenced thereafter, and reactivation of Notch signaling in podocytes is associated with renal injury. In this situation, inhibition of the Notch pathway in mature podocytes thus presents a new opportunity to treat the podocyte injury associated with nephrotic syndrome, both in children and in adults. Potentially druggable steps for inhibition of Notch signaling include inhibition of the γ secretase-mediated cleavage of the Notch receptor, or inhibition of ligand binding with selective antibodies. 142 Of particular interest is that γ secretase inhibitors have already been developed and used in clinical trials, albeit only so far for the treatment of cancer. 143 Furthermore, in patients whom have already lost podocytes following glomerular injury, stimulation of Notch signaling in progenitor cells may be indicated as a future therapeutic approach, with the goal to support their proliferation and differentiation into mature podocytes. Before this concept can be applied to patients, however, we need to expand our knowledge on the regulation of proliferation, death and (de)differentiation of progenitor cells and podocytes, both in health and disease. Targeting IL 13 Numerous studies have suggested the involvement of various cytokines in the pathogenesis of nephrotic 452 AUGUST 2012 VOLUME 8

9 syndrome Although no single cytokine has been shown to have a direct causative role in nephrotic syndrome, several studies have suggested a potentially important role for IL 13. For example, IL13 expression was reported to be elevated in CD4 + and CD8 + T cells in children with SSNS during a relapse. 150 Similarly, IL 13 serum levels and peripheral blood mononuclear cell IL13 mrna levels were found to be increased in children with SRNS when compared with levels in healthy individuals, and both IL 13 gene and protein expression was reduced by methylprednisolone therapy. 151 By contrast, in another study, treatment with prednisolone resulted in a further increase in IL 13 levels, but only in those children with SSNS. 152 In rat studies, IL 13 protein overexpression led to renal injury that resembled minimal-change nephrotic syndrome, thus providing further support for a potentially causative role of IL 13 in nephrotic syndrome. 153 In summary, growing evidence suggests that inhibition of IL 13 is a potentially effective therapy for, at least, SSNS. Fortunately, an IL 13 antibody (lebrikizumab) has already been developed and demonstrated to be effective for the treatment of asthma. 154 Thus, treatment of nephrotic syndrome using lebrikizumab or alternative IL 13 inhibitors represents yet another possible future therapeutic approach for nephrotic syndrome. Suppressing the unfolded protein response Stress-induced disturbance of protein folding in the endoplasmic reticulum, called the unfolded protein response (UPR), is recognized as the underlying pathologic mechanism of a growing number of acquired and inherited diseases, including neurologic and muscular degeneration, cardiac diseases, cancer, immune and inflammatory disorders, and diabetes. 155 The UPR is a complex signaling program of stress adaption by maintaining protein folding homeostasis. These processes involve the induction of chaperones, attenuation of translation, and activation of protein degradation. If folding homeostasis cannot be maintained because of severe stress, programs are activated that initiate auto phagy and/or apoptosis. This cellular stress response has already been recognized as having a pathogenic role in some forms of nephrotic syndrome. In kidney biopsy samples from adults with nephrotic syndrome associated with FSGS, crescentic glomerulonephritis, membranous glomerulonephritis, and membranoproliferative glomerulonephritis, indicators of the UPR, such as heat shock 70 kda protein 5 (HSPA5, also known as GRP78) and DNA-damage-inducible transcript 3 (DDIT3, also known as GADD 153), were found to be upregulated when compared with patients with MCD, whereas the apoptosis regulator Bcl 2 was downregulated. 156 Moreover, in glomeruli from rats with PAN-induced nephrotic syndrome, the expression of HSPA5 was upregulated, together with increased phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eif2α; another indicator of the UPR), suggesting involvement of this response in renal injury. 157 Notably, the UPR was regulated by the mammalian target of rapamycin (mtor) complex 1 (mtorc1), and proteinuria in this model was prevented by inhibition Box 1 Notch signaling In mammals, there are four Notch (Notch 1 4) and five ligand (Jagged1, Jagged2, DLL1, DLL3, DLL4) genes, all containing transmembrane domains such that ligand-receptor signaling occurs between adjacent cells. 195 During ontogeny, nephrogenesis includes stimulation of the cap mesenchyme, probably by the terminal branches of the ureteric buds, to form renal vesicles (stage I nephrons). By elongation, these vesicles develop into S shaped bodies (stage II nephrons). The proximal (lower) parts of these S shaped bodies then give rise to the Bowman s capsules and glomeruli, whereas the rest of the S shaped bodies elongate to form, via stage III nephrons, the mature stage IV nephrons that include the proximal tubules, the loops of Henle, and the distal convoluted tubules. 196 Notch signaling seems to be crucial for cell differentiation in nephrogenesis, as the different stages of nephron segmentation are characterized by specific expression patterns of the various Notch genes and their downstream signaling molecules. 136, For example, Notch1, Notch2, DLL1, and Jagged1 are expressed in renal vesicles and their derivatives, whereas Notch3 and Notch4 are expressed in the distal parts of S shaped bodies and in endothelial cells, respectively. Interestingly, Notch2, but not Notch1, seems to have a nonredundant role in nephron segmentation, as a genetic analysis revealed that only Notch2 is required for the differentiation of the proximal nephron. 200 Functional studies also demonstrated a specific role for Notch signaling in nephrogenesis. 197,201 Treatment in vitro of cultured mouse metanephroi from day E12.5 embryos with a γ secretase inhibitor resulted in a severe deficiency in glomerular podocytes. By contrast, treatment of metanephroi from day E14.5 embryos did not affect the formation of podocyte clusters. Thus, it seems that Notch signaling is required for podocyte formation only when S shaped bodies are forming, but not beyond the S shaped bodies stage, and that its suppression may be necessary for the terminal differentiation of podocytes. of mtorc1 with everolimus a derivative of the currently clinically available mtor inhibitor, sirolimus. In addition, indicators of the UPR were also induced in glomeruli of rats with passive Heymann nephritis. 158 The UPR has also been suggested to have a role in some inherited forms of nephrotic syndrome, caused by mutations in nephrin, podocin and α actinin ,160 In vitro, expression of mouse mutant Lys256Glu α actinin 4 (corresponding to human Lys255Glu) in podocytes induced endoplasmic reticulum stress, as indicated by induction of HSP90B1 and DDIT3 expression, with simultaneous impairment of the ubiquitin proteasome system, formation of aggresomes (which is triggered by many mutant proteins), and induction of apoptosis. 161 Taken together, these findings indicate that the UPR has an important role in the development of both acquired and inherited forms of nephrotic syndrome. Thus, future therapeutic approaches could target stabilization of folding homeostasis in podocytes. Approaches might include enhancement of protein chaperoning or enhancement of degradation capacities, by increasing the expression of relevant chaperones or proteasome system activity, respectively. Alternative approaches might include the use of low-molecular mass compounds that reduce misfolding and protein aggregation, such as sodium 4 phenylbutyrate 162 or ( )-epigallocatechin 3-gallate, 163 which is a secondary plant metabolite. Maintaining redox homeostasis Oxidative stress occurs in both children and adults with various kidney diseases, including glomerulonephritis, acute kidney injury, chronic kidney disease, diabetic nephropathy, and nephrotic syndrome, possibly in NATURE REVIEWS NEPHROLOGY VOLUME 8 AUGUST

10 conjunction with hyperlipidemia Reactive oxygen species (ROS) have been suggested to have a role in the pathogenesis of nephrotic syndrome through actions such as impairing the integrity of the glomerular basement membrane or reducing podocyte proteoglycan de novo synthesis. 171 PAN treatment in vivo and in vitro increases ROS concentrations, lipid peroxidation, and DNA damage, which all contribute to oxidative injury in podocytes. 172,173 In response, podocytes induce antioxidative enzymes, apparently in an attempt to maintain redox homeostasis. 174 Oxidative injury of podocytes in nephrotic syndrome might also be caused indirectly through increased exposure to oxidized serum albumin. 175 Albumin is the most abundant protein in serum, and is susceptible to oxidation in oxidative stress situations, which can result in alterations in its structure and possibly its function. 176,177 Serum albumin is also known to be more oxidized in patients with FSGS than in healthy individuals. 177,178 Following endocytotic uptake and transport through podocytes, such oxidized serum albumin can potentially injure podocytes in nephrotic syndrome. 175 The above findings suggest that oxidative stress is a common mechanism in various types of progressive renal injury, including nephrotic syndrome. Thus, developing improved, or more targeted, strategies to reduce podocyte oxidative stress and/or regulate redox homeostasis would be an auspicious approach to attenuate podocyte injury in nephrotic syndrome in both children and adults. Indeed, a few reports using the radical scavenger edaravone or dietary supplementation with the antioxidants probucol and vitamin E in a rat model of PAN-induced nephrosis 179,180 or children 181 with nephrotic syndrome have provided modest evidence in support of this approach. Treating genetic forms of nephrotic syndrome The term idiopathic nephrotic syndrome implies that the mechanistic cause for disease is unknown. However, since the first identification of a mutation in the podocyte protein nephrin, 182 research has identified multiple mutations that can cause nephrotic syndrome. 7,8,183 This new knowledge enables a more functionally relevant grouping of the causes of nephrotic syndrome (such as podocytopathies for mutations directly affecting podocyte structure or function, or channelopathies for mutations affecting an ion channel such as TRPC6). 184 Eventually, we fully expect that this new knowledge will translate into new therapeutic approaches to treat nephrotic syndrome. Examples of affected genes include NPHS1, NPHS2, CD2AP, PLCE1, LAMB2, ACTN4, TRPC6, INF2, and ARHGAP24, with mutations causing non syndromic forms of nephrotic syndrome Mutations in these genes collectively account for the majority of all cases in the first year of life and a sizeable minority of cases of childhood nephrotic syndrome. 188,189 The genetic cases of nephrotic syndrome are generally characterized by earlier onset proteinuria presenting in infancy and throughout childhood, resistance to steroids and other treatments, and an unremitting clinical course progressing to end-stage renal disease. The identification of genetic causes of nephrotic syndrome enables clinicians to provide prognostic information and genetic counseling to affected patients and families. Moreover, patients can potentially avoid ineffective, toxic therapies such as glucocorticoids. Most importantly, understanding the mechanism of genetic forms of nephrotic syndrome might enable the development of specific treatment strategies. An informative example is the role of the transient receptor potential cation channel TRPC6 in podocytes in nephrotic syndrome. Sustained excessive calcium influx through this channel has been reported to be a common pathway in many glomerulopathies that originate in podocytes, with downstream consequences of NFAT-dependent transcription and activation of NF κb signaling. 190,191 A number of nephrotic syndrome-associated mutations in the TRPC6 gene are now known, and most of them seem to increase receptor activity compared with the wild-type receptor. 191 Interestingly, ciclosporin can downregulate TRPC6 mrna expression in podocytes by nonimmune mechanisms, 192,193 and this approach may be effective in patients with mutations resulting in elevated TRPC6 activity. Thus, pharmacologic inhibition of TRPC6 expression or activity may be a realistic goal to ameliorate the adverse consequences of such gain-of-function mutations. Strategies for treating other genetic etiologies of nephrotic syndrome remain to be determined. Conclusions For more than 50 years, glucocorticoids have been the mainstay of therapy for children with nephrotic syndrome. Despite this, neither their target cell nor the mechanism of action in nephrotic syndrome is clearly known. For those children who develop SRNS, a variety of alternative drugs have been used. However, most of these drugs are only partially effective, and many have marked adverse effects. Moreover, failure to respond clinically to either glucocorticoids or these alternative treatments results in a greatly increased risk of end-stage renal disease (>50% within 4 years of diagnosis of SRNS). 194 In this context, new therapies to treat childhood nephrotic syndrome are urgently required. Several currently available drugs developed for other purposes are now being employed in the treatment of nephrotic syndrome, and have the potential to become standard therapies in the future. In addition, the recognition of the crucial role of podocyte injury in nephrotic syndrome has led to many new studies that have identified several important molecular pathways that are able to regulate podocyte injury. Development of drugs able to regulate these pathways offers hope for the availability of targeted and effective treatments for nephrotic syndrome in the future. Review criteria This Review was based on a search of the PubMed database using a combination of search terms that included nephrotic syndrome, focal segmental glomerulosclerosis, children, minimal change disease, and treatment. No date or language restrictions were placed on the search. 454 AUGUST 2012 VOLUME 8