Minimal change disease and idiopathic FSGS: manifestations of the same disease

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1 PERSPECTIVES OPINION Minimal change disease and idiopathic FSGS: manifestations of the same disease Rutger J. Maas 1, Jeroen K. Deegens 1, Bart Smeets 2, Marcus J. Moeller 3 and Jack F. Wetzels 1 Abstract Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are the key histological findings in patients with idiopathic nephrotic syndrome (INS). Although MCD and idiopathic FSGS are often considered to represent separate entities based on differences in their presenting characteristics, histology and outcomes, little evidence exists for this separation. We propose that MCD and idiopathic FSGS are different manifestations of the same progressive disease. The gradual development of FSGS in patients with non-remitting or relapsing INS has been well documented. Moreover, FSGS is the uniform result of substantial podocyte loss in animal models, and a common feature of virtually all progressive human glomerulopathies. As evidence suggests a common aetiology, the pathogenesis of MCD and idiopathic FSGS should be studied together. In clinical trials, idiopathic FSGS should be considered to represent an advanced stage of disease progression that is less likely to respond to treatment than the earlier stage of disease, which is usually defined as MCD. Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) both underlie idiopathic nephrotic syndrome (INS). In most textbooks, MCD and idiopathic FSGS are described as separate entities 1,2. Here, we propose that they are in fact different histological manifestations of the same disease processes. As the underlying causes of INS are unknown, INS is still a valid disease definition (BOX 1; FIG. 1). INS is a primary podocytopathy, and the underlying abnormality is extensive podocyte foot process effacement on electron microscopy. MCD is currently considered to be a single disease entity, which is characterized by nephrotic syndrome, the absence of any glomerular abnormality on light microscopy, complete remission of proteinuria on corticosteroid treatment, and maintenance of kidney function 1. By contrast, FSGS is not considered to be a single disease entity, but a description of glomerular damage. The morphology and underlying causes of FSGS are heterogeneous 3. Originally, the histological diagnosis of FSGS required the identification of segmental sclerotic lesions in at least one glomerulus 4. More recently, non-sclerotic glomerular lesions became part of the FSGS spectrum 5. Of note, the current morphological classification was defined for all forms of FSGS and does not reflect the underlying aetiology. The term idiopathic FSGS (also known as primary FSGS) was coined to differentiate FSGS with no known cause from secondary forms of FSGS, which are caused by pathogenic events, such as structural and/or functional adaptations owing to nephron loss or mutations in podocyte-associated genes 6,7. Many secondary forms of FSGS are not accompanied by complete podocyte foot process effacement so can be easily differentiated from idiopathic FSGS 8. Compared with MCD, idiopathic FSGS is associated with a higher likelihood of steroid resistance and progression to renal failure 2. In patients with steroid-resistant INS who initially have normal appearing glomeruli on light microscopy but later progress to FSGS, some researchers have suggested that an initial diagnosis of FSGS could have been missed owing to the focal and segmental nature of the FSGS lesion 9,10. Although we agree that sampling error might occur, convincing evidence indicates that FSGS lesions might be absent in the early phase of steroid- resistant INS and the finding of FSGS lesions in repeat biopsy samples reflects disease progression. In the majority of cases, FSGS is generally accepted to be the consequence of podocyte injury, and thus occurs secondary to an underlying glomerular disease process 11,12. We hypothesize that the initial podocyte injury is the critical step; the extent of this injury, the vulnerability of the podocytes, additional factors (such as comorbid hypertension) and the response to therapy determine whether FSGS lesions will develop. In our view, MCD and idiopathic FSGS have the same underlying causes (FIG. 2). Importantly, we do not suggest that a single cause of the initial podocyte injury exists. Moreover, as the development of podocyte injury could be the result of more than one podocytotoxic factor, INS might not represent a single disease entity. The notion of MCD and idiopathic FSGS as separate disease entities has caused confusion and might have impeded major clinical trials. Moreover, dogma can have a large impact on the interpretation of experimental data. If our hypothesis is correct, the therapeutic approach to INS as well as the designs of clinical trials and of basic research need to be modified. Here we review the literature and summarize the evidence that led to our hypothesis. We discuss the similarities and dissimilarities between MCD and idiopathic FSGS based on an overview of histology, animal experiments and clinical and experimental human data (TABLE 1). We conclude that the entity that is currently referred to as idiopathic FSGS is the result of extensive podocyte injury in patients with INS. 768 DECEMBER 2016 VOLUME MacmilanPublishersLimited,partofSpringerNature.Alrightsreserved.

2 MCD and FSGS: a continuum Historical background In the early twentieth century, kidney tissue abnormalities in patients with nephrotic syndrome were described based on autopsy material. In 1918, Munk used the term lipoid nephrosis to describe the typical abnormality, which consisted of vacuolar, lipoid changes in the proximal tubules 13. Subsequently, in 1925, Fahr described the presence of sclerotic glomeruli in patients with persistent lipoid nephrosis 14. Most authors, however, attribute the description of the culprit FSGS lesion to Rich. In his seminal paper, published in 1957, Rich described his careful studies of autopsy samples from patients with lipoid nephrosis, the majority of whom died as a result of complications of nephrotic syndrome 15. From his description, it is clear that Rich linked the course of pathological abnormalities in these patients with the gradual clinical transition from the pure nephrotic stage to the one with increasing damage. The extent of the lesions increased with time, as illustrated by the relationship between the extent of the histological changes and the duration of disease. Thus, both Fahr and Rich considered FSGS to be the consequence of non-remitting INS. The introduction of kidney biopsy in the middle of the twentieth century and the development of immunofluorescence and electron microscopy has revolutionized the study of kidney disease. In the 1970s, the observation of FSGS lesions in patients with recent-onset nephrotic syndrome, and the association of this nephrotic syndrome with prednisone resistance, led researchers to conclude that MCD and idiopathic FSGS are separate diseases, and that the term lipoid nephrosis is outdated 9,16,17. The concept of MCD and idiopathic FSGS as distinct entities persists to date 3. FSGS lesions evolve over time Hayslett et al. were among the first authors to note progression from apparently normal glomeruli to sclerotic lesions in a small case series of patients with lipoid nephrosis published in 1969 (REF. 18). Further evidence supporting a transition from MCD to Box 1 Clinicopathological and histological entities in patients with nephrotic syndrome Nephrotic syndrome A combination of symptoms caused by diseases that disrupt the normal permselectivity of the glomerular filtration barrier. Diagnostic features include gross proteinuria and hypoalbuminaemia, often accompanied by oedema and dyslipidaemia. Idiopathic nephrotic syndrome. A clinicopathological entity. Clinical characteristics include nephrotic syndrome of unknown cause, which might be steroid sensitive or steroid resistant. The primary histological abnormality is diffuse podocyte foot process effacement. No immune deposits and no glomerular basement membrane abnormalities are present. On light microscopy, either no glomerular abnormalities or any histological variant of focal segmental glomerulosclerosis might be seen, dependent on the severity and duration of podocyte injury. Minimal change disease* A clinicopathological entity. Clinical features include nephrotic syndrome and complete remission on corticosteroid treatment. Pathological features include normal glomeruli on light microscopy, no immune deposits, and diffuse podocyte foot process effacement on electron microscopy. Focal segmental glomerulosclerosis A histopathological description of glomerular lesions, which might be the consequence of any glomerular disease or other pathogenic event that causes podocyte injury (for example, structural and/or functional adaptations due to loss of nephrons, mutations in podocyte-associated genes or podocytotoxic factors). Idiopathic focal segmental glomerulosclerosis.* Also known as primary focal segmental glomerulosclerosis. In patients with nephrotic syndrome, focal segmental glomerulosclerosis in the context of diffuse foot process effacement and the absence of an underlying cause is considered to be idiopathic. The response to corticosteroid treatment in idiopathic focal segmental glomerulosclerosis is variable. Secondary focal segmental glomerulosclerosis. A clinicopathologic syndrome, which includes proteinuria in association with maladaptive glomerular responses to a reduced number of nephrons or full nephrotic syndrome in the context of a well-known cause of podocyte injury (for example a mutation in a podocyte-associated gene, a viral infection or a podocytotoxic drug) and any morphologic variant of focal segmental glomerulosclerosis on kidney biopsy. *We hypothesize that the distinction between minimal change disease and idiopathic focal segmental glomerulosclerosis is not valid. These entities should, therefore, be studied together as different forms of idiopathic nephrotic syndrome. FSGS was reported by Tejani et al. in 1985 (REF. 19). These researchers analysed renal biopsy samples from 48 children with a typical disease course of steroid-sensitive INS. The first renal biopsies, which were performed because of frequent relapses 1.5 years after disease onset, showed MCD only. Approximately 4.5 years after the first biopsy, a second biopsy was performed in 33 patients who had a continued relapsing disease course. Typical FSGS lesions were reported in 15 (55%) of these patients, most of whom later developed end-stage renal disease. Recurrent FSGS The concept that FSGS always occurs secondary to an underlying glomerular injury, and is thus preceded by a period of INS that is characterized by podocyte foot process effacement only on kidney biopsy, is nicely illustrated by the post- transplantation recurrence of FSGS. In patients with FSGS, a rapid increase in proteinuria often occurs within days or weeks after kidney transplantation 20. This finding is important because the time of disease onset is well defined. Some, but not all, patients with post-transplantation recurrence of FSGS respond to plasmapheresis therapy 21,22. Early and late biopsy samples are often available for transplant recipients, enabling studies of the time course of morphological changes. Canaud et al. noted that in early post-transplantation biopsy samples no glomerular abnormalities were visible on light microscopy, whereas electron microscopy showed extensive foot process effacement 23. We validated these findings in a study of 43 biopsy samples from 16 patients with recurrent FSGS, and observed a clear relationship between histology and time after FSGS recurrence (R. J. Maas, unpublished observation). In early biopsy samples (<1 month post-transplantation) no glomerular abnormalities were observed on light microscopy, but extensive podocyte foot process effacement was observed in all samples on electron microscopy. The percentage of biopsy samples with foot process effacement but no other glomerular abnormalities decreased progressively with longer follow up (45% after 1 3 months, 33% after 3 12 months, and 13% after >12 months). Post-transplantation FSGS recurrence is characterized by early-onset proteinuria despite treatment with high-dose corticosteroids and other immuno suppressive drugs. This disease course clearly differs from that of MCD in the native kidney, which is NATURE REVIEWS NEPHROLOGY VOLUME 12 DECEMBER

3 steroid sensitive by definition. Similar to MCD, however, glomeruli appear normal on light microscopy in early post- transplantation FSGS. Although FSGS lesions might be missed in some patients owing to sampling error, especially in those with few lesions and low numbers of glomeruli, initially normal glomeruli in early post-transplantation FSGS has been consistently reported throughout the literature, including in samples with large numbers of glomeruli 24. Several explanations exist for the observed difference in steroid responsiveness between MCD and post- transplantation FSGS with normal appearing glomeruli. First, the underlying disease process might have evolved between the time of initial diagnosis in the native kidney and the time of transplantation, resulting in an accumulation of circulating permeability factors and increased severity of immunological abnormalities. Second, a single renal allograft obviously has a lower total number of nephrons at baseline than two native kidneys, and this reduced nephron number predisposes the graft to glomerular hypertrophy. Third, podocytes in allograft kidneys might display increased vulnerability owing to transplantationrelated preservation reperfusion injury 25. These factors might partially explain why the parietal epithelial cell activation marker CD44 is expressed on the glomerular tuft in patients with early post-transplant recurrence of FSGS, but not in patients with native kidney MCD 26. A study that investigated CD44 expression in patients with INS reported steroid-induced complete remission of proteinuria in at least five patients with CD44 staining, indicating that CD44 is not a marker of steroid-resistant INS 27. Morphological variants of FSGS In 2004 a group of renal pathologists proposed a standardized pathological classification system for FSGS, which was based entirely on light microscopic evaluation 5. In this classification, non-sclerotic lesions termed the tip and collapsing variants are considered to be forms of FSGS. Although the classification system was intended to enable greater insight into the pathogenesis of FSGS, it may have unintentionally contributed to the notion that FSGS is an individual disease, or even that several forms of idiopathic FSGS exist. As outlined below, the majority of the existing data do not support this view. In general, individual histological variants are not unique to any specific cause or subgroup of FSGS. For example, Initiating event Idiopathic nephrotic syndrome Histology Clinical outcome Idiopathic FSGS Minimal change disease in patients with membranous nephropathy, FSGS not otherwise specified is the predominant variant, but tip, collapsing and perihilar variants have also been reported 28,29. Similarly, among patients with HIV-associated nephropathy the disease in which collapsing FSGS was originally described 30 several variants have been observed 31. Repeated allograft biopsies in patients with recurrent FSGS have revealed all types of FSGS lesions ( tip, collapsing and not otherwise specified ) at late time points after transplantation 23,24,32. In general, a change from no light microscopic abnormalities to the presence of FSGS variants has been observed in sequential biopsy samples, with the not otherwise specified variant as a final stage 23,24. A change from FSGS variants to no light microscopic abnormalities has not been reported. Causes of MCD and idiopathic FSGS Insights from animal models INS is characterized by sudden-onset, heavy proteinuria and, in most cases, a chronic disease course with remissions, relapses and, in a subgroup of patients, eventual progression to kidney failure. Notably none of the available animal models of nephrotic syndrome follow this disease course. Moreover, the pathogenesis of INS might be multifactorial, whereas the animal models are mostly characterized by a single cause of podocyte injury. Despite these shortcomings, A B C Nephrotic syndrome Podocyte injury Foot process effacement Normal glomeruli Remission Relapse FSGS Steroid resistance Figure 1 Schematic representation of idiopathic nephrotic syndrome, minimal change disease and idiopathic focal segmental glomerulosclerosis (FSGS). Idiopathic nephrotic syndrome is caused by unknown initiating events (A, B and C) that lead to podocyte damage. Clinical features include gross proteinuria and hypoalbuminaemia, often accompanied by oedema and dyslipidaemia. The disease is histologically defined by complete podocyte foot process effacement with no immune deposits and no glomerular basement alterations. On light microscopy, either normal appearing glomeruli or FSGS might be visible. Disease outcomes are unpredictable at diagnosis, and might include steroid-induced proteinuria remission, potentially followed by relapse, or (primary or secondary) corticosteroid resistance. Patients with persistent proteinuria ultimately develop FSGS. Minimal change disease and idiopathic FSGS are manifestations of idiopathic nephrotic syndrome that are defined by histology and/or clinical outcomes; minimal change disease is characterized by foot process effacement, normal glomeruli and steroid sensitivity, whereas idiopathic FSGS is characterized by foot process effacement and FSGS lesions. these models have provided valuable insights into the consequences of podocyte loss, and have demonstrated that overlap exists between the MCD and FSGS phenotypes. Models of FSGS. Podocyte injury can be induced by injection of antibodies or toxic agents that are directed against endogenous or ectopically expressed podocyte molecules Among the oldest and most widely used models of FSGS are the adriamycin and puromycin aminonucleoside (PAN) nephrosis models 35,36. In the PAN model, FSGS develops after injection of a single high dose of PAN 38. In rats, injection of PAN induced a rapid onset of nephrotic-range proteinuria and the loss of almost 30% of podocytes 39. No sclerotic lesions were observed on days 5 7 after PAN injection, but adhesions to the Bowman capsule were present by day 11, and sclerotic lesions by day 45. The relationship between podocyte depletion and glomerulosclerosis was validated by Wharram et al. using a transgenic rat model that enabled specific ablation of podocytes using the human diphtheria toxin 34. The researchers demonstrated that the severity of lesions correlated with the degree of podocyte loss. Depletion of more than 20% of podocytes induced sustained proteinuria and the development of FSGS, whereas depletion of less than 20% of podocytes resulted in transient proteinuria, mesangial expansion 770 DECEMBER 2016 VOLUME 12

4 and little or no glomerular tuft adhesion. In both the diphtheria toxin and PAN models, the temporal sequence of events was proteinuria and extensive foot process effacement, followed by a reduction in podocyte number, the formation of early adhesions to the Bowman capsule, and finally the appearance of glomerulosclerosis. A similar course of events has been observed in the Thy1.1 transgenic mouse model of FSGS 37. These findings demonstrate that FSGS is the consequence of a critical amount of podocyte depletion, and explain why FSGS lesions are not observed in early post-transplantation FSGS. Buffalo/Mna rats spontaneously develop proteinuria and FSGS lesions at the age of 2 months. In this model, early recurrence of proteinuria without evidence of rejection has been observed after transplantation of a healthy rat kidney 40. By contrast, when a diseased Buffalo/Mna kidney was transplanted into a healthy rat, regression of proteinuria occurred 41. These observations provide experimental support for the concept of a circulating permeability factor that might underlie the development of INS and be associated with post-transplantation recurrence of proteinuria. New models of FSGS are under development. For example, an inducible transgenic mouse model of podocyte Rac1 expression has been reported 42. Rac1 is a small intracellular GTPase and its activation is associated with actin cytoskeleton Likelihood of remission Steroid sensitivity Disease recurrence in allograft MCD Transplantation reorganization, podocyte motility and foot process effacement 43. In the transgenic mice, low-level, short-term Rac1 activation caused transient proteinuria and reversible foot process effacement, whereas intense and prolonged Rac1 activation caused non-remitting proteinuria and the development of FSGS. In line with our hypothesis, this model demonstrates that the same initiating event might underlie either transient proteinuria and complete histological recovery (that is, MCD), or non-remitting proteinuria with progressive glomerular abnormalities (that is, FSGS), depending on the intensity and duration of the podocyte injury. Models of MCD. A model of MCD requires acute and selective nephrotic-range proteinuria, foot process effacement and steroid sensitivity. However, all of the available models of chronic and heavy proteinuria eventually develop FSGS. Models used for MCD research have, therefore, mostly been characterized by transient podocyte injury. Models in which transient proteinuria can be induced include the adriamycin, PAN, aminopeptidase A, protein overload, neuraminidase and lipopolysaccharide models The most widely used model of MCD is the PAN model. Although a single high-dose injection or repeated injections of PAN result in FSGS, a single low-dose injection results in selective and Tip variant FSGS NOS ESRD Intensity of podocyte injury Point of no return Collapsing variant Figure 2 Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are manifestations of the same disease. MCD is characterized by podocyte injury, which results in foot process effacement and proteinuria. If the initial injury is severe, focal segmental glomerulosclerosis (FSGS) lesions might develop. These lesions are characterized by parietal epithelial cell proliferation (resulting in the tip and collapsing FSGS variants). In the initial phase, the disease is steroid sensitive apart from in patients with the most severe injury. With longer duration of proteinuria or multiple relapses, ongoing loss of podocytes occurs. This process contributes to loss of steroid sensitivity and reduced likelihood of remission. When more than 30 40% of podocytes are lost, a point of no return is reached and progression to end-stage renal disease (ESRD) is inevitable. After transplantation, disease recurrence in the allograft manifests with proteinuria, podocyte foot process effacement and ultimately the development of FSGS lesions. NOS, not otherwise specified variant. Podocyte depletion transient proteinuria, which is associated with podocyte foot process effacement. Furthermore, this model exhibits partial responsiveness to steroids The dose-dependent development of FSGS lesions in the PAN model, and in other models of transient proteinuria, support the hypothesis that MCD and idiopathic FSGS might represent different manifestations of the same injury, which are related to the severity of podocyte loss 52. As nephritogenic serum causes upregulation of podocyte angio poietinlike 4 (Angplt4) expression 53, a transgenic rat model that overexpresses podocyte Angptl4 has been generated 51. These rats develop selective proteinuria from the age of 1 month. Although they have been described as a model of MCD, some dissimilarity with the MCD disease course exist. In contrast to patients with MCD, proteinuria in the transgenic rats is of gradual onset and relatively mild. Furthermore, the development of FSGS lesions in these rats cannot be excluded as they have not been monitored beyond the development of complete foot process effacement, which occurs at 5 months of age. Finally, unlike patients with MCD, the Angptl4 transgenic rat is not corticosteroid sensitive; this discrepancy has been attributed to the fact that transgenic expression of podocyte Angptl4 is not driven by the native Angptl4 promoter 54. The Angplt4-overexpression model clearly differs from human MCD, and does not provide evidence that MCD and idiopathic FSGS are separate entities. In summary, all of the available models of FSGS and MCD are based on induction of podocyte injury and subsequent podocyte loss. Consistent with our hypothesis, during the initial disease phase only foot process effacement similar to MCD is observed, whereas persistent podocyte loss and/or proteinuria result in the development of FSGS. Circulating permeability factors Serum circulating factors. Strong evidence suggests that the pathogenesis of INS involves a circulating factor that disturbs podocyte function and increases glomerular permeability 55. Several candidates for the FSGS permeability factor have been proposed, including cardiotrophin-like cytokine 1 and soluble urokinase receptor (supar) 56,57. In MCD, attempts to identify a circulating factor have focused on cytokines 58 60, but an isoform of haemopexin has also been suggested NATURE REVIEWS NEPHROLOGY VOLUME 12 DECEMBER

5 as a possible culprit 61. To date, no single circulating permeability factor that underlies INS has been conclusively identified 55. Most studies have concentrated on identifying a specific circulating factor in either MCD or FSGS; few have compared MCD with FSGS. Although an initial report suggested that levels of supar were higher in patients with FSGS than in those with MCD 56, we and others have demonstrated that supar levels are mostly dependent on renal function After correction for renal function, supar levels did not significantly differ between patients with MCD and those with FSGS 66. Thus, the possibility exists that the same permeability factor is responsible for MCD and FSGS. Podocyte-secreted factors. Although a role of podocyte Angptl4 expression in the pathogenesis of MCD has been suggested based on experimental data 51, limited data are available regarding human Angptl4 expression. Angptl4 staining has been reported in biopsy samples from patients with MCD, but samples from patients with FSGS have not been analysed 51. Although urine samples from patients with MCD had a distinctive pattern of Angptl4 oligomers compared with those from patients with FSGS or membranous nephropathy, this result might be unreliable because many of the patients with FSGS or membranous Table 1 Evidence for and against the hypothesis that MCD and idiopathic FSGS are separate entities Biopsy findings For In steroid-resistant INS with normal appearing glomeruli, FSGS is likely missed owing to sampling error 9,10,7 Later biopsy samples always show FSGS 18,19 Against Recurrent FSGS is characterized by an initial phase with diffuse podocyte foot process effacement and normal appearing glomeruli on light microscopy, even in biopsy samples with many glomeruli 23,24 Findings in later biopsy samples are compatible with the concept that FSGS develops over time 18,19 Animal models NA Following an initial phase with only diffuse podocyte foot process effacement, all of the available animal models of persistent proteinuria develop FSGS 33,35 37 ; in models of toxic damage to podocytes, FSGS development is dose-dependent 34,37,39 Circulating factors Histological markers Immune pathogenesis Genetic causes Steroid responses FSGS is caused by a circulating permeability factor that is associated with progressive decline in renal function and recurrence of proteinuria after transplantation 56 ; MCD is not associated with loss of renal function Serum supar concentration is elevated in FSGS compared with MCD; high levels of supar were associated with post-transplantation FSGS recurrence 56 Angptl4 expression was upregulated in the glomeruli of patients with MCD; these patients had a distinctive pattern of Angptl4 oligomers in their urine, which was not seen in patients with FSGS 51 Patients with MCD can be differentiated from those with FSGS by urinary B7 1 excretion 90,91 Decreased glomerular α dystroglycan staining has been documented in MCD; expression of α dystroglycan was normal in idiopathic FSGS 98,99 The ratio of podocin to synaptopodin mrna expression distinguished patients with MCD from those with early-stage idiopathic FSGS and a steroid-resistant disease course 101 Many genes are differentially expressed in the glomeruli of patients with MCD versus those with idiopathic FSGS 102,104 Evidence suggests that abnormal T cell function underlies MCD 68,71 ; this mechanism has not been reported in FSGS FSGS often has genetic causes 75,77 80 ; no genetic causes of MCD have been identified MCD is steroid sensitive, whereas FSGS is steroid resistant Circulating factors have been implicated in both MCD and FSGS 55 ; they have not been identified but might be identical in MCD and FSGS this hypothesis is supported by the observation that posttransplantation recurrence of FSGS occurs in patients with initially steroid-sensitive INS who develop secondary steroid resistance 125 Elevated serum supar concentration has not been validated as a biomarker of FSGS; supar levels were not elevated in patients with FSGS after correction for renal function Glomerular Angptl4 expression was not investigated in patients with FSGS; urinary tests were performed in a small group of patients, and two of the four patients with FSGS were not nephrotic 51 ; the results have not yet been validated Elevated urinary B7 1 excretion in MCD has not been validated; patients with FSGS may also have strong B7 1 expression 93 Among patients with INS, dystroglycan staining did not predict proteinuria remission in response to therapy 99 ; differential expression of α dystroglycan between MCD and idiopathic FSGS was not confirmed in a subsequent study 100 Differences in the ratio of podocin to synaptopodin mrna expression in patients with MCD and idiopathic FSGS have not been validated; studies of glomerular mrna expression profiles in idiopathic FSGS 104,102 and MCD 102 confirm that podocyte stress and parietal epithelial cell activation are involved in the development of FSGS lesions 103,105 The reported gene profiles are compatible with different stages of disease progression rather than differences in the aetiology of MCD and idiopathic FSGS 102,104 T cell function in MCD and idiopathic FSGS has not been systematically compared; data from a humanized mouse model suggest that CD34 + peripheral blood mononuclear cells are involved in the pathogenesis of both MCD and idiopathic FSGS 74 Genetic FSGS often has clear features of secondary FSGS with a gradual increase in proteinuria and incomplete podocyte foot process effacement 77,79 ; no genetic causes of idiopathic steroid-sensitive FSGS have been reported Some patients with MCD might have relapsing or secondary steroid-resistant disease resulting in FSGS, whereas some patients with FSGS might have complete remission on corticosteroid treatment 120,122,123 FSGS, focal segmental glomerulosclerosis; INS, idiopathic nephrotic syndrome; MCD, minimal change disease; NA, not applicable; supar, soluble urokinase receptor. 772 DECEMBER 2016 VOLUME 12

6 nephropathy had subnephrotic proteinuria, whereas all of the patients with MCD had nephrotic proteinuria. Validation of these findings in a large, well-described cohort is still required. The available data on human podocyte Angptl4 expression are, therefore, insufficient to support the view that MCD and idiopathic FSGS are separate diseases. Immune pathogenesis Several decades ago, INS was proposed to result from a systemic abnormality of T lymphocytes that leads to the production of cytokines that directly or indirectly disrupt glomerular function 67. Although the precise T lymphocyte abnormalities remain unidentified, clinical and experimental evidence support this hypothesis. In children with steroid-sensitive nephrotic syndrome, relapses were associated with a decrease in the levels of natural regulatory T (T reg ) cells, and altered cytokine production 68,69. Similarly, in the Buffalo/Mna rat, an increase in the frequency of natural T reg cells (as a result of cell transfer or treatment with a derivate of deoxyspergualin) induced proteinuria remission 70. Altered expression of the intracellular proteins NFRKB and NF κb might underlie disturbed T cell function in MCD 60,71. Although many studies have addressed differences between disease relapse and remission, and some have compared steroid-sensitive and steroid-resistant nephrotic syndrome 72,73, no study has systematically investigated differences in the immunological phenotypes of MCD and FSGS. Of note, data from a humanized mouse model suggested an identical cellular source of steroid-sensitive nephrotic syndrome and post-transplantation INS recurrence 74. In this study, the researchers transplanted CD34 + stem cells or CD34 peripheral blood mononuclear cells from patients with INS into severely immunocompromised mice. They observed proteinuria and complete foot process effacement 3 4 weeks after injection of CD34 + cells, but not after injection of CD34 cells. Importantly, identical results were obtained using cells from a patient with MCD and cells from patients with post-transplantation recurrence of FSGS, suggesting a shared pathological process. Genetic causes FSGS can be caused by mutations in single genes that have crucial roles in podocyte function. Recessive mutations in NPHS1, NPHS2, LAMB2 and WT1 predominate in patients with disease onset in the first year of life 75. Of note, no FSGS lesions were described in fetuses with congenital nephrotic syndrome of the Finnish type 76. Rather, the characteristic renal pathology consisted of dilated proximal tubules. Genetic forms of FSGS that present in adults mostly have dominant inheritance, caused by mutations in TRPC6, ACTN4, INF2, ANLN or other yet unidentified genes In many cases, these patients have proteinuria without full nephrotic syndrome. Genetic causes are reportedly rare in adult patients with non-familial FSGS An exception might be young adults (aged years) with steroid-resistant FSGS; single-gene causes have been reported in >20% of these patients 84. No single genetic causes of MCD have been identified 85. Genetic forms of nephrotic syndrome are typically steroid-resistant. Remarkably, recessive EMP2 mutations can cause either steroid-sensitive or steroid-resistant nephrotic syndrome 86. Podocyte EMP2 regulates caveolin 1, which is involved in podocyte albumin endocytosis. In a zebrafish model, knockdown of EMP2 resulted in upregulation of caveolin 1 and podocyte injury, which was partially restored by corticosteroids 87. Rare mutations in single podocyteassociated genes cannot account for the majority of patients with INS and do not explain the development of MCD or the progression to FSGS. However, we think that susceptibility genes might be involved and could partly determine the pathway of the disease process by contributing to the severity and time course of podocyte loss. For example, childhood steroid-sensitive nephrotic syndrome has been associated with HLA DQA1 missense coding variants, suggesting a role of immune derangement in the pathogenesis of this disease 88. Histological markers of MCD and FSGS Podocyte B7 1 B7 1 was originally identified as a co stimulatory receptor on antigen presenting cells 89 ; podocyte B7 1 expression was first demonstrated in experimental models of nephrotic syndrome 48. Garin et al. reported that podocyte-specific B7 1 expression was highly specific for MCD and absent in idiopathic FSGS. This difference in B7 1 expression could be demonstrated non-invasively by elevated urinary B7 1 levels in patients with MCD but not in those with idiopathic FSGS 90,91. The researchers hypothesized that urinary B7 1 levels might differentiate steroid-sensitive from steroid-resistant cases of INS, indicating progression to FSGS. However, elevated urinary B7 1 levels were also observed in patients with steroid-resistant forms of INS who had no glomerular abnormalities on light microscopy 92, indicating that urinary B7 1 is not a specific marker of MCD. With regard to histology, glomerular B7 1 immunostaining was demonstrated in patients with MCD, idiopathic FSGS and other glomerulopathies 93. Importantly, this study also reported therapeutic efficacy of the B7 1 blocking agent abatacept in patients with post-transplant INS recurrence, suggesting that B7 1 immunostaining might be a biomarker for abatacept efficacy. Unfortunately, other researchers have failed to identify podocyte B7 1 immunostaining in any form of INS, despite the use of various techniques Moreover, the therapeutic efficacy of abatacept in post- transplantation INS has not been confirmed 95,97. Interestingly, the only other published example of temporary abatacept efficacy was observed in a patient with MCD, indicating that the potential efficacy of abatacept might not be specific for post-transplantation FSGS recurrence, but could be a common feature of some patients with INS 97. Thus, in vivo detection of podocyte B7 1 expression has not been validated, and no evidence that this expression is unique to either MCD or FSGS exists. Dystroglycan A complex of α and β dystroglycans anchors the podocyte to the glomerular basement membrane. Disruption of dystroglycan, particularly of the α subunit, has been reported to be a feature of MCD that is absent in FSGS 98. Decreased α dystroglycan staining has been reported in patients with MCD compared with levels in patients with FSGS lesions 99. The staining pattern did not, however, predict outcome (proteinuria remission or progression) in either patient group. Moreover, we found no difference in α dystroglycan expression between patients with MCD and those with idiopathic FSGS 100. Gene expression profiles An early study that investigated the glomerular expression of podocyte-specific genes in biopsy samples from patients with MCD (n = 13) or idiopathic FSGS (n = 9) reported a trend towards lower podocin expression in FSGS 101. In addition, the ratio of podocin to synaptopodin was lower in FSGS samples than in MCD samples; response to treatment was not reported in this study. In an independent cohort of six patients with early idiopathic FSGS or MCD on biopsy, the podocin to synaptopodin ratio NATURE REVIEWS NEPHROLOGY VOLUME 12 DECEMBER

7 distinguished between a steroid-resistant and steroid-responsive disease course. The results of this study might be relevant for defining patient subgroups with a high risk of progression, but they have not been validated in large, prospective series. In future studies, podocyte mrna expression should be assessed in relation to glomerular volume and podocyte count, standard pathological evaluation and treatment outcomes. Hodgin et al. performed unbiased mrna expression analysis on laser-dissected glomeruli from 21 patients with MCD, classic FSGS or collapsing FSGS (including 19 patients with INS and two without full nephrotic syndrome) and nine control renal biopsy samples from patients without glomerular disease 102. They reported decreased expression of many podocyteassociated genes in the patients with FSGS. By contrast, many genes that indicate parietal epithelial cell proliferation were overexpressed in patients with FSGS, supporting the essential contribution of activated parietal cells to the formation of FSGS lesions 103. Consistent with the findings of a previous study that compared gene expression in FSGS samples and normal glomeruli 104, COL1A1, SOX9 and SPP1, which encodes osteopontin, were among the major upregulated genes. Podocytes express osteopontin in response to stress 105, whereas SOX9 is an established mediator of extracellular matrix deposition in fibrosis 106. Overall, mrna expression studies suggest that podocyte damage and upregulation of genes associated with the development of glomerulosclerosis (such as involvement of parietal epithelial cells) occurs in idiopathic FSGS but not in MCD. However, these observations do not point to different aetiologies, but rather seem compatible with different stages of disease progression. Factors driving disease progression Clinical factors A report of the International Study of Kidney Disease in Children described 521 children with INS 107. MCD was diagnosed in 398 (76%) and FSGS in 36 (7%) of these children. Notably, patients with FSGS were older (50% aged >6 years versus 20% aged >6 years), were more often hypertensive (49% versus 21%), and more often had non-selective proteinuria (57% versus 15%) and haematuria (48% versus 23%) than those with MCD. The presence of haematuria might suggest the presence of underlying genetic abnormalities in the glomerular basement membrane rather than INS 108. These findings are consistent with those of an earlier study and literature review which reported that patients with FSGS were older and more hypertensive than those with MCD 109. These data suggest that specific subgroups of patients have an increased risk of developing FSGS lesions. Glomerular hypertrophy Further insight in the relationship between MCD versus idiopathic FSGS, podocyte susceptibility and haemodynamic effects can be derived from detailed quantitative evaluation of glomerular morphology. Fogo et al. evaluated adults and children with MCD or idiopathic FSGS 110. In both age groups, glomerular area was significantly larger in patients with FSGS than in those with MCD. Subsequently, these researchers studied the initial biopsy samples of 42 children who were diagnosed with MCD, of whom 10 developed FSGS in later biopsy samples and 32 had the typical course of MCD with maintenance of normal renal function. The mean glomerular area was 76% greater among patients who progressed to FSGS than in those with persistent MCD. Other studies have confirmed that glomeruli are larger in patients with idiopathic FSGS than in those with MCD, and that larger glomerular volume is associated with steroid resistance 111,112. An association between glomerular volume and progression to FSGS has also been observed in membranous nephropathy 29, and the prognostic significance of parameters related to glomerular volume has been confirmed in patients with IgA nephropathy 113. Podocytopenia Glomerular hypertrophy predisposes to functional podocytopenia (that is, a relative lack of podocytes). A study of kidneys obtained at autopsy from patients without kidney disease linked glomerular hypertrophy to decreased podocyte density 114, which fits with reports of glomerular hypertrophy in steroid- resistant INS Traditionally, selectivity of proteinuria has been used as a prognostic indicator of steroid sensitivity 115. Highly selective proteinuria indicates that relatively low amounts of high-molecular weight proteins are lost, implying less damage to the glomerular filter than in non-selective proteinuria. Non-selective proteinuria has also been shown to correlate with reduced numbers of podocytes per glomerular tuft area 116. Moreover, patients with nephrotic syndrome may experience a continuous loss of podocytes. Non-invasive measurement of podocin mrna in urinary sediments has been used to monitor podocyte loss longitudinally. In general, patients with idiopathic FSGS and progressive decline in renal function had high levels of urinary podocin mrna (100 fold increase compared with healthy control samples) 117. Proteinuria remission induced by various forms of immunosuppressive treatment was possible in patients with MCD or idiopathic FSGS despite heavy podocyte loss. Response to corticosteroids Several observational studies have reported clinical outcomes in patients with either MCD or idiopathic FSGS In all series, corticosteroid-induced proteinuria remission was associated with good outcomes, independent of the histological picture. The incidence of complete remission was, however, substantially reduced when FSGS lesions were present. Although idiopathic FSGS is generally assumed to represent an individual disease entity that does not respond to steroids, we propose that some forms of FSGS might arise from INS that does not respond well to steroids despite initially normal glomeruli. Prolonged nephrotic-range proteinuria (possibly together with additional factors such as hypertension) might trigger FSGS in this setting. Histopathological phenotypes are insufficient predictors of steroid response in INS. Unsurprisingly, current guidelines advise similar initial treatments for MCD and idiopathic FSGS, whereas second-line immunosuppressive treatment depends on initial steroid responsiveness rather than histology 124. Steroid-sensitive INS, which is associated with MCD, might progress to steroid-resistant INS, which is associated with FSGS 18,19,125. Likewise, steroid resistance and progressive renal failure might develop if MCD is not treated 126. Children with INS who develop secondary steroid resistance have a >90% risk of post- transplantation proteinuria recurrence 125. Steroid resistance and FSGS might develop in patients with initial MCD and persistent or repeated podocyte injury, which results in a vicious cycle of podocyte loss after a critical number of podocytes have been depleted 127. Conclusions The available data suggest that MCD and idiopathic FSGS are different phenotypes of the same disease, which is best described as INS (FIG. 2). MCD and idiopathic FSGS represent a gradual overlap in clinical characteristics, treatment responses and 774 DECEMBER 2016 VOLUME 12

8 outcomes. Clinicopathological studies and experimental models support our hypothesis that idiopathic FSGS is the consequence of extensive podocyte injury in patients with INS, and that the same initiating factors are responsible for both MCD and idiopathic FSGS. Potential differences in the pathogenesis or selective triggers of MCD and idiopathic FSGS have not been validated. We suggest that in future studies, the underlying causes of MCD and idiopathic FSGS should be investigated together under the definition of INS. In clinical trials, idiopathic FSGS should be considered to be an advanced stage of MCD. Response to treatment is, therefore, less likely in patients with idiopathic FSGS than in those with MCD. Prognostic markers for risk stratification should be sought in both MCD and idiopathic FSGS. In particular, the prognostic value of podocyte number and glomerular volume should be investigated in kidney biopsy studies 128. 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