The spectrum of primary nephrotic syndrome includes a

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1 In-Depth Review A Proposed Taxonomy for the Podocytopathies: A Reassessment of the Primary Nephrotic Diseases Laura Barisoni,* H. William Schnaper, and Jeffrey B. Kopp *Department of Pathology, New York University School of Medicine, New York, New York; Division of Kidney Diseases, Department of Pediatrics, Feinberg School of Medicine, Northwestern University School of Medicine, Chicago, Illinois; and Kidney Disease Section, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland A spectrum of proteinuric glomerular diseases results from podocyte abnormalities. The understanding of these podocytopathies has greatly expanded in recent years, particularly with the discovery of more than a dozen genetic mutations that are associated with loss of podocyte functional integrity. It is apparent that classification of the podocytopathies on the basis of morphology alone is inadequate to capture fully the complexity of these disorders. Herein is proposed a taxonomy for the podocytopathies that classifies along two dimensions: Histopathology, including podocyte phenotype and glomerular morphology (minimal-change nephropathy, focal segmental glomerulosclerosis, diffuse mesangial sclerosis, and collapsing glomerulopathy), and etiology (idiopathic, genetic, and reactive forms). A more complete understanding of the similarities and differences among podocyte diseases will help the renal pathologist and the nephrologist communicate more effectively about the diagnosis; this in turn will help the nephrologist provide more accurate prognostic information and select the optimal therapy for these often problematic diseases. It is proposed that final diagnosis of the podocytopathies should result from close collaboration between renal pathologists and nephrologists and should whenever possible include three elements: Morphologic entity, etiologic form, and specific pathogenic mechanism or association. Clin J Am Soc Nephrol 2: , doi: /CJN The spectrum of primary nephrotic syndrome includes a variety of causes, presentations, histopathologic findings, and outcomes. These disorders may appear at any age, may be exquisitely sensitive or highly resistant to therapy, and have varied implications for long-term renal function. Past efforts to develop an organized approach to these diseases have largely centered on histopathology. Although these approaches were helpful in defining lesions, for each morphologic entity there is a wide range in response to treatment and outcome. This observation suggests that traditional pathologic description alone is insufficient to classify these disorders. Here, we propose a new taxonomy for podocyte diseases. The word taxonomy was coined by Carl Linnaeus, the 18th century Swedish scientist who proposed the system for naming and classifying organisms that remains in use today. A taxonomy is organized into multiple levels, each of which represents a taxon with one or more elements. The ideal taxonomy separates the elements of each taxon (the taxa) into mutually exclusive, unambiguous, and all-encompassing categories. In practice, a good taxonomy should be simple, easy to remember, and easy to use. Taxonomies provide classification and frequently more: A conceptual framework for analysis, discussion, and hypothesis generation. Our proposed podocyte taxonomy has Published online ahead of print. Publication date available at Address correspondence to: Dr. H. William Schnaper, 303 E. Chicago Avenue, Chicago, IL Phone: ; Fax: ; schnaper@northwestern.edu two dimensions histopathology and etiology and includes additional modifiers related to biomarkers that have been identified through recent progress in the genetics, cell biology, and pathophysiology of glomerular disease. We anticipate that further progress in identifying disease biomarkers will result in improved diagnostic accuracy and enable the nephrologist to administer more precisely targeted therapy that addresses disease mechanisms. Defining the Podocytopathies The diseases that cause nephrotic syndrome can be divided into three categories: Diseases with antibody-mediated mechanisms (e.g., lupus, membranoproliferative glomerulonephritis, and membranous nephropathy), diseases that are associated with metabolic disorders (e.g., diabetes, amyloid, and Fabry disease), and diseases that are caused by abnormal glomerular cell function (1). Current thinking suggests that virtually all cases in this last category begin with podocyte damage or dysfunction. For this reason, these diseases have been termed podocytopathies (2). Efforts to characterize the relationship among the podocytopathies have evolved in the past three decades. Initially, studies that focused on pathologic presentation suggested that minimal-change nephrotic syndrome, focal segmental glomerulosclerosis (FSGS), and mesangioproliferative glomerulonephritis had a common cause and could transition among these different pathologic findings (3). Subsequent iterations recognized that, among the various forms of primary glomerular disease, response Copyright 2007 by the American Society of Nephrology ISSN: /

2 530 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 2: , 2007 to glucocorticoid treatment is the critical determinant of outcome. Thus, different etiologies might cause the same histologic picture, accounting for heterogeneity of clinical course when predicted solely on the basis of histopathology (4,5). However, the nature of the underlying disorders remained unclear. Recent advances in the characterization and treatment of the podocytopathies (reviewed in references [6 9]) have led us to revisit this issue. Our intent is to offer a framework that integrates renal morphology, etiology, and pathogenesis in the diagnosis of the podocytopathies, thereby enabling renal pathologists and nephrologists to investigate the possible molecular and physiologic causes of disease in each individual case. The result should enhance our ability to treat these disorders effectively. Pathogenic Role of the Podocyte Podocytes are postmitotic cells whose function depends on their highly specialized and unique architecture. They are believed to serve at least four distinct functions: Regulation of glomerular permselectivity (10); structural support for the glomerular capillary, cooperating with mesangial cells to resist the distensive force of intracapillary hydraulic pressure (11); remodeling the glomerular basement membrane (GBM), in cooperation with endothelial and mesangial cells (12); and endocytosis of filtered proteins (13). Podocyte function depends on a highly ordered cellular architecture that includes, in part, (1) the slit diaphragm complex; (2) the actin-based cytoskeleton, including associated proteins and adhesion proteins; (3) an appropriate microenvironment, including the basement membrane (GBM) to which podocytes adhere; and (4) ongoing internal and external biochemical signals that contribute to maintenance of the differentiated state (14). A common feature of podocytopathies is foot process effacement. This finding, however, is seen with many proteinuric diseases and is a nonspecific manifestation of podocyte injury (Table 1). In foot process effacement, the normal three-dimensional interdigitating architecture is lost. The actin-based cytoskeleton usually reorganizes, condensing into a narrow band within the portion of cytoplasm adjacent to the abluminal plasma membrane (facing the GBM). This is typically accompanied by redistribution of the slit diaphragm components to the cytoplasm and the luminal plasma membrane (facing the urinary space) (15). When is foot process effacement reversible, and when is it irreversible? Podocyte effacement can result from the absence of one of the critical architectural components described, or it might represent the rearrangement of slit diaphragm components in response to cytokine stimulation, as has been postulated to occur after stimulation with vascular endothelial growth factor (16). There is much that we do not know. In one model, alterations in the podocyte s microenvironment could result from aberrant cell matrix interaction, leading to podocyte secretion of a pathologic extracellular matrix. The resulting, further impaired cell matrix interaction could foster a vicious cycle that results in further abnormal matrix production (17) and progression from a reversible condition to an irreversible scar. In contrast, in many cases of idiopathic minimalchange nephropathy (MCN), steroid treatment induces remission and restoration of slit diaphragm architecture; this may be due at least in part to direct effects of glucocorticoids to stabilize the podocyte cytoskeleton (18). The reversibility of this lesion may help explain why steroid-sensitive disease carries a better prognosis. At present, most genetic forms of MCN and related podocytopathies manifest irreversible foot process effacement, and they are generally refractory to existing therapies; conceivably, new therapies with molecular chaperones might improve podocyte cytoarchitecture. Thus, the boundary between reversible and irreversible foot process effacement may depend as much on available therapeutics as on intrinsic biology. Podocyte dysfunction can have an idiopathic, genetic, or reactive etiology; the last category involves response to various insults, including mechanical stress, medications, toxins, viral infections, and (as yet unidentified) circulating proteins. The effect of dysfunction that results from any of these etiologies Table 1. Causes of podocyte injury resulting in foot process effacement a Intrinsic Podocyte Stress: Genetic Mutations Nucleus: WT1, PAX2, and LMX1B mutations Cytoskeleton: ACTN4 mutations Slit diaphragm complex: NPHS1, NPHS2, and CD2AP mutations Plasma membrane: ITGB4 and TRPC6 mutations Mitochondria: trna mutations, COQ2 mutations Metabolic: Fabry disease Extracellular matrix: LAMB2 Extrinsic Podocyte Stress Viral: infection or circulating viral protein Toxic: medication (pamidronate, interferon), toxin (puromycin aminonucleoside, Adriamycin) Lymphokine or other host protein: IFN-, IFN-, FSGS permeability factor Mechanical: Podocyte stretch (glomerulomegaly) Acute ischemia associated with thrombotic microangiopathy Immunologic: Immune complex deposition or in situ formation(lupus, IgA nephropathy, membranous nephropathy) Metabolic: Diabetes a From the viewpoint of the podocyte, injury can arise from intrinsic stress (genetic mutations) or extrinsic stress (circulating protein or toxin, mechanical forces, ischemia or viral infection).

3 Clin J Am Soc Nephrol 2: , 2007 Podocytopathy Taxonomy 531 may take three forms, leading to four different glomerular morphologies (Figures 1 and 2). Podocyte Injury with No Change in Podocyte Number In MCN, podocytes exhibit diffuse foot process effacement, with minimal abnormalities on light microscopy. This is typified by what traditionally is called minimal-change disease. It also may include other conditions that are characterized by similar histologic findings but potentially more complex (e.g., specific gene mutation-associated) etiologies. The essential criterion for invoking this form is preservation of podocyte number. Podocyte Injury and Depletion In idiopathic FSGS, the mechanisms of podocyte injury are unknown. Hostetter et al. (19) observed that FSGS is associated with hyperfiltration in a remnant kidney model. Kriz and LeHir (20) suggested that podocyte injury is the initial step toward sclerosis, demonstrating that injured podocytes detach from underlying GBM, either as a consequence of sublethal injury or from apoptotic or necrotic death. The naked GBM contacts the parietal epithelium, forming a synechia; this represents the committed lesion of FSGS. The relationship between these two observations was described by Wiggins et al. (21) in a model of podocyte response characterized by glomerulosclerosis, whereby the podocytes undergo progressive injury through five steps to end with podocytopenia. In this model, during the adaptive phase, podocytes undergo hypertrophy to cover an expanded GBM surface, and the resulting mechanical stretch ultimately induces a shift in podocyte phenotype, favoring structural stability (manifested by actin condensation) over hydraulic conductivity (manifested by fully elaborated slit-diaphragm complexes) (15,21 23). A phase of relative podocytopenia ultimately Figure 1. Three distinct pathways of injury and repair characterize the podocytopathies. First, in minimal-change nephropathy (MCN), podocyte injury is limited to foot process effacement, and podocyte number remains normal. Second, a more severe form of podocyte injury may occur, leading to podocyte detachment and death. This event initiates an injury cascade that results in the segmental scar characteristic of FSGS. Third, podocyte injury may lead to delayed cellular maturation (DMS), dedifferentiation (CG), and proliferation, with either low rates of podocyte proliferation (manifesting as diffuse mesangial sclerosis [DMS]) or high rates of proliferation (manifesting as collapsing glomerulopathy [CG]). Figure 2. Four major patterns of glomerular injury in podocytopathies. MCN: No changes are present on light microscopy. FSGS: FSGS is characterized by segmental solidification of the tuft with accumulation of extracellular matrix. Synechiae form between the tuft and Bowman s capsule. Podocytes are lost in the areas of sclerosis. DMS: Mesangial expansion as a result of accumulated extracellular matrix is the characteristic feature and is accompanied by hypertrophy and mild cobblestone hyperplasia of overlying podocytes. CG: The characteristic features of CG include wrinkling and folding of the glomerular basement membranes (collapse) and proliferation of overlying podocytes forming pseudocrescents. Numerous protein reabsorption droplets are present in the podocyte cytoplasm. Magnifications: 40 in MCN and FSGS; 60 in DMS and CG (Silver stain for all). transitions to a phase of absolute podocytopenia. It is reasonable to speculate that in genetic forms of FSGS, podocyte injury and death are also the underlying pathologic mechanisms of sclerosis. Podocyte Injury and Proliferation Injured podocytes may dedifferentiate and re-enter the cell cycle. Depending on the etiology, proliferation may vary from

4 532 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 2: , 2007 minimal to widespread. In some cases, after viral infection, for example, podocyte phenotype is aberrant and is associated with glomerular capillary loop collapse. This pattern characterizes collapsing glomerulopathy (CG). In other settings, particularly associated with genetic mutations, podocyte proliferation is less marked, but podocyte phenotype is aberrant and is associated with mesangial accumulation of matrix. This pattern characterizes diffuse mesangial sclerosis (DMS). Relationship among Podocyte Diseases Are MCN, FSGS, DMS, and CG to be considered distinct diseases or distinct clinical syndromes? Idiopathic MCN and idiopathic FSGS do seem to fit as clinicopathologic syndromes, each with variants that in some cases have prognostic implications and may ultimately reflect different patterns of causation. However, particular gene mutations may manifest as different glomerular morphologies; thus, NPHS2 mutations can present as MCN or FSGS (and rarely as DMS) and yet presumably represent a single disease process (24). To provide the widest applicability, it may be useful to consider the morphologies simply as pathologic patterns that help the pathologist and nephrologist define the underlying disease mechanism in any single case. Taxonomy of the Podocytopathies On the basis of our current knowledge, the taxonomy of the podocytopathies is organized along two axes (Table 2): Histopathology, defined by podocyte number and the four patterns of glomerular morphology: MCN, FSGS, DMS, and CG (Figures 1 and 2) Etiology, including idiopathic, genetic, and reactive forms It is expected that, in the future, additional variables (proteomics, transcriptomics, and others) will be added to define better each category and subclass and provide a more reliable basis for selecting therapy and determining prognosis. MCN Definition. MCN is usually characterized by normal histology and extensive podocyte foot process effacement on ultrastructural analysis, accompanied by condensation of the actin-based cytoskeleton against the sole of the podocyte and by microvillous transformation. We use the term minimal-change nephropathy for several reasons. First, it includes what is often called minimal-change nephrotic syndrome or minimal-change disease (25,26). Second, the term presents a more parallel construction with the other diagnostic terms, avoiding a clinical descriptor (nephrosis) that may not be uniformly applicable. Variants. Podocyte injury in MCN may be idiopathic, genetic, or reactive. The major clinical distinction among subgroups of this category is response to glucocorticoid therapy. Idiopathic and reactive forms of MCN seem to be generally steroid sensitive. Steroid-resistant forms have a similar morphology (Figure 3) but worse outcome and likely have distinct etiologies. Idiopathic MCN. Idiopathic MCN usually presents with florid nephrotic syndrome, although it may not always do so, and is typically steroid sensitive, particularly in children. Several variants are described. IgM nephropathy seems to have a clinical outcome similar to typical MCN (27), whereas diffuse mesangial hypercellularity has a worse prognosis (28). Glomerular tip lesion is discussed later as a transcategorical entity. Genetic MCN. Three genetic forms have been identified. Steroid-resistant MCN that presents in infancy or childhood with autosomal recessive inheritance may be caused by mutations in NPHS2, encoding podocin. Autosomal dominant nephrotic syndrome, presenting as FSGS or less commonly as MCN, has been linked to a nearby locus on chromosome 19q, for which the gene remains unidentified (29,30). Recently, MCN was reported in a patient with limb girdle muscular dystrophy type 2B, which is caused by mutations in dysferlin (DYSF) (31). Reactive MCN. Most cases of MCN may be reactive in nature, in view of their association with immunogenic stimuli or malignancy. Medications that are effective in treating MCN may affect the cellular immune system. A variety of extended MHC haplotypes have been associated with recurrent MCN (reviewed in reference [1]). For these and other reasons, cell-mediated immunity has been invoked as an etiologic factor in the development of MCN (32). The molecular pathways that link immune dysregulation to podocyte injury remain to be defined. We lack reliable tools to discriminate among the various forms of MCN, especially to predict those with good response to steroid therapy versus those that are steroid resistant. Regele et al. (33) reported that patients with steroid-sensitive, nephrotic MCN manifest decreased podocyte expression of dystroglycan (an adhesion molecule that is expressed on the abluminal surface of the podocyte). Its expression is preserved in other podocytopathies such as FSGS. In these patients, downregulation of dystroglycan is reversed once foot processes are reconstituted (33). This phenomenon does not necessarily reflect a general effect of the reorganization of the foot processes. Preliminary data indicate that the use of staining for dystroglycan may segregate patients with a diagnosis of MCN at renal biopsy into two groups: Those with a high likelihood of favorable response to corticosteroid therapy, when dystroglycan expression is reduced, and those with a likely poor response, when dystroglycan expression is comparable to normal (L. De Patris et al., unpublished observations, 2006). In contrast to steroid-sensitive MCN, glomeruli of patients with genetically determined, steroid-resistant MCN may have decreased detection of podocytespecific proteins by immunostaining (Figure 3D). FSGS Definition. FSGS is defined as segmental solidification of the glomerular capillary tuft with accumulation of extracellular matrix, often with an adhesion (synechia) between the capillary tuft and Bowman s capsule. Hyalinosis and foam cells also can be present. The common pathophysiologic principle is absolute or relative podocyte depletion; podocyturia also may occur. Positive staining for IgM and C3 may be present by immunofluorescence and is believed to represent macromolecular trapping rather than specific deposition. On ultrastructural analysis, electron-dense material may be found in the mesangium

5 Clin J Am Soc Nephrol 2: , 2007 Podocytopathy Taxonomy 533 Table 2. Taxonomy of the podocytopathies Podocytopathy Idiopathic Forms Genetic Forms Reactive Forms Minimal-change nephropathy Idiopathic MCN steroid-sensitive steroid-resistant Nonsyndromic NPHS2 Syndromic DYSF (limb-girdle muscular dystrophy 2B) Reactive MCN Hodgkin disease Immunogenic stimuli Medication-associated: nonsteroidal anti-inflammatory agents, gold, penicillamine, lithium, IFN- and -, pamidronate Postadaptive FSGS Reduced nephron mass: renal dysplasia, surgical renal mass reduction, reflux nephropathy, chronic interstitial nephritis Initially normal nephron mass: obesity, increased muscle mass, sickle cell anemia, cyanotic congenital heart disease, hypertension b Medication-associated FSGS: cyclosporine, tacrolimus, IFN-, lithium, pamidronate Focal segmental glomerulosclerosis Idiopathic FSGS Nonsyndromic NPHS1 NPHS2 NPHS2 ACTN4 CD2AP TRPC6 WT1 mtdna trna Leu PLCE1 Syndromic WT1 (Frasier) mt DNA trna Leu (MELAS) PAX2 (renal-coloboma syndrome with oligomeganephronia) LMX1B (nail-patella) COQ2 ITGB4 COL4A3, A4, A5 (Alport) GLA (Fabry) Nonsyndromic NPHS1 (congenital nephrotic syndrome, Finnish type) WT1 NPHS2 PLCE1 Syndromic LAMB2 (Pierson) WT1 (Denys-Drash) Nonsyndromic COQ2 Syndromic action myoclonus-renal failure Diffuse mesangial sclerosis Idiopathic DMS Collapsing glomerulopathy Idiopathic CG Infection-associated CG viruses (HIV-1, parvovirus B19 b,cmv b ) other (Loa loa filiariasis b, visceral leishmaniasis b, Mycobacterium tuberculosis b ) Disease associations: Adult Still disease b, thrombotic microangiopathy, multiple myeloma b Medication-associated CG: IFN-, pamidronate a The primary podocytopathies are organized into four morphologic patterns and three etiologic categories. Named syndromes are shown. ACTN4, -actinin-4; FSGS, focal segmental glomerulosclerosis; CD2AP, CD2-associated protein; CMV, cytomegalovirus; COL4, type IV collagen; COQ2, co-enzyme Q synthetase 2; DYSF, dysferlin; FSGS, focal segmental glomerulosclerosis; GLA, galactosidase A; ITGB4, 4-integrin; LAMB2, laminin 2 chain; LMX1B, Lim homeobox transcription factor 1B; MELAS, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes; NPHS1, nephrin; NPHS2, podocin; PAX2, paired homeobox protein 2; PLCE1, phospholipase C 1; TRPC6, transient receptor potential cation channel, member 6; WT1, Wilms tumor-1. b Disease associations for which causation has not been clearly established.

6 534 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 2: , 2007 Figure 3. Steroid-resistant MCN. (A) Steroid-resistant MCN, shown here, resembles on light microscopy steroid-sensitive forms and normal kidney (Silver stain). (B) On electron microscopy, extensive foot process effacement is invariably seen, as well as podocyte microvillous transformation (cytoplasmic projections into the urinary space). (C) Podocin is expressed in normal kidney podocytes (immunofluorescence). (D) In a patient with steroid-resistant MCN, no staining for podocin was detected (immunofluorescence). Magnification, 40 in A, C, and D; 2500 in B. and in the subendothelial compartment, consistent with hyalinosis. Variants. Podocyte injury in FSGS can be idiopathic, genetic, or reactive (postadaptive FSGS or medication-associated FSGS). Idiopathic FSGS. Idiopathic FSGS is the most common variant of FSGS and likely includes various etiologic forms that will ultimately be recognized as distinct entities, including new genetic forms and recurrent FSGS associated with a circulating permeability factor. The Columbia classification defines morphologic criteria for idiopathic FSGS, using five categories: Collapsing variant, tip lesion, perihilar variant, cellular variant, and not otherwise specified (NOS) (34). Our taxonomy does not focus on these different morphologic variants but considers them in our discussion to help the reader understand how we have incorporated these and other histologic definitions into our approach. Tip lesion is discussed further as a transcategorical entity. In our taxonomy, collapsing FSGS is believed to have a different pathogenetic mechanism. It is included as CG and is discussed further. Cellular variant, as originally defined by Schwartz et al. (35), included biopsies with segmental scar and either podocyte or endocapillary proliferation. The cellular FSGS variant in the Columbia classification includes cases with endocapillary proliferation and/or podocyte proliferation but without glomerular collapse. A recent study indicated that, whereas some cases of the cellular lesion progress extremely rapidly, behaving clinically like more aggressive forms of podocytopathies such as CG, the overall percentage of patients who progress to renal failure is similar for the cellular and NOS categories of the Columbia criteria (36). This observation raises the possibility that there may be heterogeneity among patients with the cellular lesion. Genetic FSGS. Patients may manifest genetic FSGS as part of a syndrome or in renal-limited (nonsyndromic) disease (Table 2). Genetic mutations that are responsible for syndromic FSGS include mutations in GBM proteins such as the mutated COL4 genes, encoding collagen IV chains, in Alport syndrome (37); transcription factors that are critical for podocyte differentiation, such as WT1, encoding the Wilms tumor 1 protein (38), and LMX1B, encoding a homeobox protein, in nail-patella syndrome (39); metabolic disorders (GLA, encoding -galactosidase A in Fabry disease) (40) and mitochondriopathies (mitochondrial trna mutations that causing MELAS [mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes] syndrome or isolated FSGS [41], and COQ2 mutations [42]). Genetic mutations that are responsible for renallimited FSGS include those that encode proteins in the actinbased cytoskeleton complex or the slit diaphragm complex and adhesive proteins (reviewed in reference [6]). Most of the genetic forms of FSGS listed in Table 2 have

7 Clin J Am Soc Nephrol 2: , 2007 Podocytopathy Taxonomy 535 Mendelian inheritance, including autosomal recessive, autosomal dominant, and sex-linked patterns. Exceptions include WT1 and mitochondrial gene mutations. To date, each of the mutations that are associated with FSGS has involved genes that are expressed in podocytes. This finding underscores the centrality of podocyte injury in the etiology of FSGS. The distinction between genetic FSGS and idiopathic FSGS will continue to shift as new Mendelian forms are discovered. Recently, association studies showed that certain single-nucleotide polymorphisms or haplotypes are associated with increased risk for sporadic FSGS (43); it is likely that many such variants will be found. These findings need extensive replication in diverse populations before they have clinical utility. Genomic profiling is likely to enter medical practice in the next decade, in support of personalized medicine. This profiling will likely include many of the Mendelian genetic FSGS variants and may include genetic mutations that increase FSGS risk. The mutations that most commonly are associated with podocytopathies are in NPHS2, encoding podocin (44). NPHS2- associated renal disease follows autosomal recessive inheritance, with homozygous or compound heterozygous mutations, and accounts for approximately 20% of pediatric FSGS or steroid-resistant nephrotic syndrome (45). Most of the affected patients develop the disease during childhood, but onset occurs up to the fourth decade (46). These patients present with a range of morphologic patterns of glomerular injury, including MCN, diffuse mesangial hypercellularity, and FSGS. Genetic testing is important, because these patients are consistently resistant to glucocorticoid therapy and show limited response to other immunosuppressive therapy (24). Antibodies against podocin are available, and their use should be included in the evaluation of renal biopsies in patients with suspected genetic podocytopathy. Patients with NPHS2 mutations show heterogeneity in podocin expression, including either absence or abnormal cytoplasmic staining. Two different available antibodies recognize the N-terminal and C-terminal domains, respectively, of podocin. Because mutations may occur in any portion of the gene, the pattern of staining may vary between the two antibodies when both are used in the same biopsy (47). Autosomal dominant, familial FSGS typically presents in adulthood, although earlier onset has been described. Three genetic loci have been identified: ACTN4 encoding -actinin-4 on chromosome 19q13 (48), an unidentified gene near the same location (49), and TRPC6, encoding transient receptor potential cation channel 6 (30). Patients with ACTN4 mutations, reported in five families, tend to present late in life and to progress to ESRD slowly. In biopsies that are taken from patients with these mutations, foot process effacement is patchy, which challenges the belief that the extent of foot process effacement is a reliable indicator of genetic or idiopathic FSGS compared with secondary forms of FSGS. Patients with TRPC6 mutations, reported in six families, present at ages 20 to 50 yr and progress more rapidly to ESRD. Patients with mutations mapped to chromosome 19q13 present with morphologic heterogeneity that is similar to that seen with NPHS2 mutations, ranging from steroid-resistant, apparent MCN through mesangial hypercellularity and FSGS (29,49). These data underscore that morphologic criteria are not sufficient to characterize a disease fully. WT1 mutations of have been described in patients with Frasier syndrome but also in genetically female patients with isolated FSGS (50). Most WT1 mutations are felt to be new mutations rather than inherited mutations. It is generally believed that genetic forms of FSGS do not respond to steroid therapy. Recently, however, nontruncating mutations of PLCE1, encoding phospholipase C 1, were found in patients with steroid-sensitive FSGS and also in patients with intermittent proteinuria that resolves spontaneously (51). Reactive FSGS. Reactive FSGS may develop through postadaptive mechanisms or may be associated with certain medications. Postadaptive FSGS, which may also include hyalinosis, begins when the glomerulus undergoes an adaptive response that is characterized by glomerular hyperperfusion and hyperfiltration, leading to glomerulomegaly (52). These events occur in the presence of either reduced renal mass (e.g., renal dysplasia, surgical renal mass reduction, reflux nephropathy, chronic interstitial nephritis) or initially normal renal mass (obesity, sickle cell anemia, or cyanotic congenital heart disease). In the former setting, glomerular hyperperfusion is compensatory; in the latter setting, the mechanisms that are responsible for hyperperfusion are unknown. After a period of months (rats) to years (humans), postadaptive FSGS emerges. It is believed that, in response to glomerular capillary hypertension and glomerulomegaly, podocytes undergo hypertrophy and, as noted, continue to provide structural support to the capillary loop at the expense of hydraulic conductivity. Alternative but less accurate terms for postadaptive FSGS include secondary FSGS, which has been extended beyond the podocytopathies to other conditions, including scarring as a result of glomerulonephritis, and hyperfiltration FSGS, which may mislead by implying that hyperfiltration has been established as the central pathogenic process, rather than glomerulomegaly. The distinction between idiopathic FSGS and postadaptive FSGS seems to have clinical relevance, because patients with postadaptive FSGS may have sizable reductions in proteinuria with conservative therapy (53) and the role of immunosuppressive therapy is unproved. Clinically, the diagnosis of postadaptive FSGS is influenced by the presence of appropriate history. This approach is understandable, but it risks errors such as classifying a case of idiopathic FSGS in a morbidly obese individual as being postadaptive. In general, patients with postadaptive FSGS have higher serum albumin concentrations (despite significant proteinuria) and lesser degrees of foot process effacement. Perhaps related to these findings, they also are less likely to manifest florid nephrotic syndrome. However, these markers may not be reliable in individual cases. Arguably the most reliable criterion is the presence of glomerulomegaly (Figure 4), although this determination requires direct measurement of the maximal glomerular diameter observed on multiple sections (54). As an approximation, the glomerulus is considered to be enlarged when the diameter exceeds 50% of the field of view using a 40 objective. Problems with both approaches are the small number of glomeruli available on clinical renal

8 536 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 2: , 2007 Figure 4. Postadaptive FSGS. (A) Large glomerulus with segmental solidification of the tuft and hyalinosis (arrows). (B) Very large glomerulus without sclerosis within the same biopsy (glomerulomegaly). Magnification, 20 (hematoxylin and eosin). biopsies (approximately 50 glomeruli are required for reliable determination of diameter) and the need for a sample that includes both superficial and juxtamedullary nephrons. Thus, specific molecular markers to distinguish postadaptive FSGS are needed. Although hypertension is commonly cited as a cause of postadaptive FSGS, this association was challenged recently (55). DMS Definition. DMS is defined by mesangial expansion as a result of accumulated extracellular matrix protein, together with podocyte hypertrophy and mild hyperplasia. In contrast to the FSGS category, lesions that are assigned to DMS show relatively dedifferentiated or less-differentiated podocyte phenotype. In contrast to the CG category, although the podocytes show increased expression of proliferation markers (e.g., Ki67), rates of proliferation are low. DMS Variants. DMS occurs in a syndromic form and a sporadic form, both of which tend to be refractory to therapy. Many DMS cases have a genetic basis. Congenital DMS is associated with WT1 mutations (Denys-Drash syndrome) (56) and mutations of LAMB2, encoding laminin 2 chain (Pierson syndrome) (57,58). In the former case, immunostaining reveals markedly reduced expression of WT-1 in podocyte nuclei with simultaneous increased expression of PAX2, a WT-1 target gene. In both forms, there is increased expression of proliferative markers, such as Ki67, and increased podocyte expression of cytokeratin, reflecting a dedifferentiated phenotype. The expression of other podocyte maturity markers such as synaptopodin, nephrin, and -actinin-4 is preserved, and negative staining for these markers is observed only when glomerulosclerosis develops, as a result of podocyte loss (59). These forms, as well as the recently described familial form of DMS that is caused by truncated mutation of PLCE1, are associated with podocyte developmental arrest. Rarely, patients with NPHS2 mutations may also present at the renal biopsy with a morphologic picture that resembles mesangial sclerosis (60). Another form of congenital renal disease that falls into this category is congenital nephrotic syndrome of Finnish type (CNF). CNF is caused by homozygous or compound heterozygous mutations in NPHS1, encoding nephrin (6). In contrast to other DMS variants, in CNF, the earliest histologic finding is not only mesangial expansion but also mesangial cell hypercellularity. Podocyte phenotype is altered and nephrin is not expressed, but the expression of other podocyte proteins such as zona occludens 1 (ZO-1) seems to be maintained (61). In the early phases of the disease, podocyte proliferative rate is low. Podocyte detachment and podocyturia also are detected, and it is intriguing to speculate that this may represent the mechanism of progression toward sclerosis. In fact, the disease rapidly progresses, and subsequent changes include glomerulosclerosis, Bowman space dilation, and tubulointerstitial abnormalities, including tubular microcysts (62). CG Definition. The Columbia classification defines collapsing FSGS as the presence of segmental capillary tuft collapse (wrinkling and folding) in at least one glomerulus (Figure 5), in association with podocyte hypertrophy and/or hyperplasia (34). The term collapsing glomerulopathy seems more appropriate than FSGS, because the lesion is defined by pseudocrescent formation and by collapse of the capillary loops rather than accumulation of extracellular matrix; glomerulosclerosis is a later manifestation (63). Importantly, CG differs from FSGS in exhibiting podocyte proliferation rather than podocyte depletion, and the actin cytoskeleton may not appear condensed but rather absent. On ultrastructural analysis, podocytes resemble immature precursor cells, with a cuboidal shape and loss of primary processes and foot processes. Exuberant podocyte hyperplasia seems to generate the apparent pseudocrescents within Bowman s space (64,65). Recently, this theory was challenged, and it has been suggested that cells that migrate from Bowman s capsule participate in pseudocrescent formation (66). This difference of opinion derives from the anatomic observation that visceral and parietal cells represent distinct populations that meet only at the point of reflection of the glomerular tuft into the Bowman s capsule. However, bridging of epithelial cells from Bowman s capsule to the glomerular tuft now seems to be a common event even under nonpathologic

9 Clin J Am Soc Nephrol 2: , 2007 Podocytopathy Taxonomy 537 Figure 5. Collapsing glomerulopathy. (A) Transplant biopsy in a patient with CG secondary to thrombotic microangiopathy. Segmental collapse and pseudocrescent formation are present (arrows) (Silver stain). (B) The proliferative marker Ki67 is detected in podocyte nuclei in this 2-yr-old patient with CG associated with COQ2 mutation (immunoperoxidase). (C) Electron microscopy shows podocytes overlying mildly wrinkled glomerular basement membrane (GBM). The podocytes have lost both primary processes andfoot processes (arrows). Podocytes are separated from the original GBM by newly formed extracellular matrix (*). (D) Dedifferentiated podocytes re-express other proteins that are normally expressed only during development, such as cytokeratin (immunoperoxidase). Magnifications: 40 in A; 60 in B and D; 5000 in C. circumstances (67). The difficulty in proving that one cell rather than the other is the central participant in the disease comes from the fact that these cells change their phenotype during disease states. Furthermore, a new subset of cells has been identified along the inner surface of the Bowman s capsule. Some of these cells express podocyte markers (67), and others express adult stem cell markers (68). These findings suggest not only that bridging of cells that migrate from the inner side of the Bowman s capsule is not a pathologic event but also that cells other than parietal epithelial cells, such as migrated podocytes or stem cells, could participate in pseudocrescent formation. Ongoing studies will clarify the exact nature of bridging cells in pathologic states. Variants. CG may be idiopathic, genetic, or reactive in etiology. Idiopathic CG. Idiopathic CG is characterized by a dysregulated phenotype, manifested by loss of maturity markers in areas of collapse (synaptopodin, podocalyxin, GLEPP1, and CALLA) (69) and re-expression of immaturity markers (PAX2 and cytokeratin) (70). Immature podocytes re-enter the cell cycle and proliferate, expressing Ki67 (Figure 5B). Moreover, podocytes lose the expression of WT-1, a transcription factor that is normally expressed by podocytes beginning in fetal development and continuing through adulthood. Loss of its expression indicates that the dedifferentiated podocyte manifests functional phenotypic changes. Significantly, podocytes in seemingly normal glomeruli demonstrate loss of synaptopodin and WT-1 expression, suggesting that dedifferentiation and dysregulation precede capillary collapse. Genetic CG. Families with arthritis (71) or neurologic disease (72) and CG have been reported, although the responsible genes have not been identified. Recently, mutations in COQ2, encoding a gene that is involved in ubiquinone synthesis, have been associated with CG (F. Diomedi Camassei et al., unpublished observations, 2006) as well as FSGS (42). These findings suggest that mitochondrial dysfunction may result in podocyte loss (FSGS) or podocyte proliferation (CG); the responsible pathways are not known. Reactive CG. The morphologic features of podocyte hypertrophy were initially described in association with HIV infection (HIV-associated nephropathy) (27), and later an idiopathic form of CG was recognized (63). Both forms share a similar altered podocyte phenotype (69). More etiologic factors have been added to the list of possible causes of CG, including infections, medications, and acute ischemia associated with thrombotic microangiopathy (Table 2) (73). The biologic mech-

10 538 Clinical Journal of the American Society of Nephrology Clin J Am Soc Nephrol 2: , 2007 anisms for these associations remain to be determined. Several mechanisms have been suggested to be involved, including intrinsic injury of podocytes (e.g., intracellular expression of a viral genome, dysregulation of vascular endothelial growth factor expression) or extrinsic injury (dysregulation of the immune system, release of cytokines from infected cells in the circulation or renal parenchyma, ischemia, and toxic damage) (reviewed in reference [73]). Transcategorical Entities Glomerular Tip Lesion Glomerular tip lesion has been a controversial subject since its description by Howie in 1984 (74). It is situated at the portion of the glomerular tuft located adjacent to the origin of the proximal convoluted tubule and consists of a collection of intracapillary foam cells or accumulation of the extracellular matrix, overlaid by hypertrophic podocytes bridging toward the Bowman s capsule or the most proximal portion of the tubule. Some cases are associated with FSGS, whereas in other instances, tip lesions are the only morphologic abnormalities in glomeruli that otherwise appear normal on light microscopy. It has been suggested that the later cases may be part of the MCN spectrum of diseases. Against this proposal is the definition of MCN itself, if we accept that for a diagnosis of MCN all glomeruli should appear normal on light microscopy. However, such cases very often behave clinically like MCN. Tip lesion has been included in the Columbia classification as a variant of FSGS, regardless of the presence of segmental sclerosis in the biopsy. It also has been described in other proteinuric conditions, including membranous nephropathy (where it is present in 64% of cases) (75), postinfectious glomerulonephritis (76), and diabetic nephropathy (77). On the basis of these observations, Haas et al. (78) argued that the tip lesion is a response to prolonged heavy proteinuria and does not represent a specific disease entity. The prognosis in FSGS tip lesion may be more benign than in other morphologic forms of FSGS (79,80), leading some authorities to postulate that it is an intermediate form between MCN and FSGS. However, given these data, it would seem at least as likely that it is a distinct process that is overlaid onto the various podocytopathy categories and other proteinuric states. Diffuse Mesangial Hypercellularity Diffuse mesangial hypercellularity is an uncommon form of relatively steroid-resistant nephrotic syndrome. The glomerular morphology resembles MCN or FSGS, with superimposed mesangial hypercellularity. Genetic forms have been associated with NPHS2 mutations and the locus on chromosome 19q13 (29). Patients with idiopathic forms of diffuse mesangial hypercellularity are at increased risk for recurrent nephrotic syndrome after renal transplantation, which makes the clinical recognition of the disorder important (81). C1q Nephropathy C1q nephropathy was first described by Jennette and Hipp (82) as the presence of mesangial C1q staining, either dominant or co-dominant with IgG, IgM, and/or C3, and the lack of serologic or clinical findings of lupus. The immune deposits are predominantly mesangial, although occasionally the deposits extend into glomerular capillary loops. C1q and other members of the newly recognized C1q/TNF superfamily share a structurally similar globular domain. C1q functions as a major link between innate and acquired immunity (83). Human genetic C1q deficiency is associated with lupus, presumably by impairing apoptotic cell clearance and thereby exposing the immune system to nuclear and cytoplasmic antigens. The pathogenesis of C1q nephropathy, particularly in relation to podocyte injury, remains uncertain. Some authors have suggested that the disease fits best among the podocytopathies (84,85). Because C1q deposits are found in nonpodocytopathic glomerular disease (82), however, it is not clear whether this entity should belong to the podocytopathies or be considered as part of the complement-mediated glomerulopathies. Limitations in Classifying the Podocytopathies It is increasingly apparent that morphology-based classification of the podocytopathies is insufficient and must be coupled with an understanding of how each morphology has idiopathic, genetic, and reactive forms. Morphology-based classification schemes have a number of important limitations that the current taxonomy tries to address. The ideal disease taxonomy is composed of taxa that represent distinct diseases. Particular genetic mutations may present with diverse histologic appearances, underscoring the limitations of morphology alone in determining a diagnosis. In the ideal disease taxonomy, patients progress through various stages of a particular disease but generally do not progress from one disease to another. Some cases of MCN on first biopsy may manifest FSGS on subsequent biopsy. There are several ways to understand this observation, including sampling error (FSGS was missed on the first biopsy) and disease progression. The recent findings that steroid-resistant MCN and FSGS may share preserved dystroglycan expression and certain genetic mutations are consistent with either model. Morphologic approaches have not solved this quandary, but molecular analysis may do so in the future. Morphology-based diagnosis depends in part on the absence of particular findings, and this is problematic, because the absence of a feature does not exclude that the feature is present elsewhere in unsampled tissue. Future diagnostic advances, using molecular markers and other approaches, will likely reduce the diagnostic uncertainty. The finding of the presence of multiple glomerular morphologies on one renal biopsy remains problematic (86). It is not immediately obvious how a biopsy with different glomeruli showing tip lesion, perihilar sclerosis, and glomerular collapse with podocyte hyperplasia should be classified, either from a biologic or a clinical standpoint. Longitudinal studies to evaluate clinical outcome will help determine the most useful diagnostic approaches.

11 Clin J Am Soc Nephrol 2: , 2007 Podocytopathy Taxonomy 539 A major limitation shared by all of the current classifications of podocyte diseases, including this one, is the uncertainty as to whether diverse diagnostic pathologists will manifest high interobserver reliability. We have tried to point out that formulating a clinically useful diagnosis requires more than morphology. It is essential to define new markers of glomerulopathies that will clarify areas of confusion and that will serve as new bases for therapeutic intervention. In 1900, David Hilbert remarked in an address to the International Congress of Mathematicians, Every real advance goes hand in hand with the invention of sharper tools and simpler methods which at the same time assist in understanding earlier theories and cast aside older more complicated developments (87). The current approaches to glomerular diagnosis, including the one proposed here, will become simpler and more logical as sharper molecular tools are developed, resulting in more precise, reproducible, and clinically informative diagnoses. Formulating a Diagnosis How, then, should pathologist and nephrologist approach the classification and diagnosis of the podocytopathies? First, the nephrologist should supply information concerning the clinical history and family history to the renal pathologist. Second, in addition to routine renal biopsy studies, the pathologist should stain for molecular markers of podocyte phenotype in certain instances. In the future, transcriptional profiling using RNA extracted from glomeruli may prove useful in characterizing podocyte phenotype (88). We propose that final diagnosis of the podocytopathies requires close collaboration between renal pathologists and nephrologists and will often be an iterative process, as additional information is obtained. The final diagnosis may include up to four elements: Disease entity, pathologic variant, etiologic form, and specific pathogenic mechanism or association. Such diagnostic approaches will be refined by the development of tools to enhance the standard morphologic description with cellular markers. Examples of this interactive, iterative approach to diagnosis are given next. Note that the specificity of the diagnosis may evolve over time, as additional test results are obtained, and that in some cases the final report will include two or more possible diagnoses. Examples of MCN Diagnosis MCN, idiopathic MCN, associated with Hodgkin disease MCN; reduced podocin staining, consider NPHS2 mutation (pathologic diagnosis) MCN, NPHS2 (podocin) mutation-associated (R138Q/ R138Q homozygosity) Examples of FSGS Diagnosis FSGS (glomerulomegaly present), likely postadaptive etiology, obesity associated FSGS, associated with lithium use FSGS, mitochondriopathy associated (consider trna Leu or COQ2 mutations) Example of DMS Diagnosis DMS, reduced WT-1 staining, consider WT1 mutation (pathologic diagnosis) DMS, WT1 mutation R394W DMS, CNF, NPHS1 mutation Fin-major Examples of CG Diagnosis CG, HIV-associated CG, thrombotic microangiopathy associated, consistent with tacrolimus toxicity Conclusion Recent advances in podocyte biology and in elucidating the genetic basis of podocyte disorders have provided an opportunity to reconsider how clinicians and scientists think about the podocytopathies. We believe that the major contribution of this taxonomy to renal biopsy diagnosis is that it seeks to combine glomerular morphology (assessed by light and electron microscopy), podocyte molecular phenotype, genetic mutations, extrarenal manifestations, and clinical associations. As with any proposed schema for classifying lesions, it will require testing and refinement as its clinical utility is assessed. Improved diagnosis is important for the clinical care of patients and is essential for patients who enroll in clinical trials, because treatments that work for one variant and etiologic form may work less well or not at all for other forms. Therefore, distinction between forms that are characterized by podocyte proliferation versus podocytopenia or by genetic defects versus reactive processes is critical for a more specific therapeutic approach. It is intriguing to speculate about the potential use of proliferative cytokines or stem cell therapy in podocytopenic forms of the disease and of antiproliferative drugs in proliferation-driven glomerulopathies. MCN, FSGS, DMS, and CG seem to manifest distinct combinations of podocyte protein expression and cell activity, yet some genetic mutations are associated with two or more disease entities, at least upon the initial diagnosis. This observation highlights the complexity of the task at hand. Indeed, as our understanding of disease mechanisms improves, biomarkers may prove to override traditional morphologic analysis to a greater degree than that used in our proposal. For instance, what we currently would classify as MCN with NPHS2 mutation, likely steroid resistant, as shown in Figure 3, could manifest FSGS on a subsequent biopsy. Perhaps in such a case, the use of the morphologic descriptor alone could delay appropriate treatment or enhance the potential for toxicity from futile therapy. Determining the final glomerular diagnosis requires close collaboration among nephrologists and pathologists. The proposed taxonomy represents a working classification that attempts to incorporate the current knowledge base. Ongoing modifications to this classification will be required as our understanding of human genetics, podocyte biology, and glomerular pathophysiology continues to advance. Disclosures None.