Clinical Kidney Journal, 2021, vol. 14, no. 2, 482–491 doi: 10.1093/ckj/sfaa110 Advance Access Publication Date: 11 August 2020 CKJ Review CKJ R EV IEW Challenges in primary focal segmental glomerulosclerosis diagnosis: from the diagnostic algorithm to novel biomarkers Conxita Jacobs-Cachá1,2,3, Ander Vergara1,2, Clara Garcı́a-Carro1,2,3, Irene Agraz1,2,3, Nestor Toapanta-Gaibor1,2, Gema Ariceta3,4, Francesc Moreso1,2,3, Daniel Serón1,2,3, Joan López-Hellı́n3,5,6,* and Maria José Soler 1,2,3,* 1 Nephrology Research Group, Vall d’hebrón Institut de Recerca (VHIR), Barcelona, Spain, 2Department of Nephrology, Hospital Universitari Vall d’Hebron, Universitat Autónoma de Barcelona, Barcelona, Spain, 3Red de Investigaciones Renales (RedInRen), Madrid, Spain, 4Department of Paediatric Nephrology, Hospital Universitari Vall d’Hebron. Universitat Autónoma de Barcelona, Barcelona, Spain, 5Department of Biochemistry, Hospital Universitari Vall d’Hebron, Universitat Autónoma de Barcelona, Barcelona, Spain and 6 Biochemistry Research Group, Vall d’hebrón Institut de Recerca (VHIR), Barcelona, Spain Correspondence to: Conxita Jacobs-Cachá; E-mail: conxita.jacobs@vhir.org and Maria José Soler; E-mail: m.soler@vhebron.net; Twitter handle: @PepaSolerR *These authors share credit for senior authorship. ABSTRACT Primary or idiopathic focal segmental glomerulosclerosis (FSGS) is a kidney entity that involves the podocytes, leading to heavy proteinuria and in many cases progresses to end-stage renal disease. Idiopathic FSGS has a bad prognosis, as it involves young individuals who, in a considerably high proportion (15%), are resistant to corticosteroids and other immunosuppressive treatments as well. Moreover, the disease recurs in 30–50% of patients after kidney transplantation, leading to graft function impairment. It is suspected that this relapsing disease is caused by a circulating factor(s) that would permeabilize the glomerular filtration barrier. However, the exact pathologic mechanism is an unsettled issue. Besides its poor outcome, a major concern of primary FSGS is the complexity to confirm the diagnosis, as it can be confused with other variants or secondary forms of FSGS and also with other glomerular diseases, such as minimal change disease. New efforts to optimize the diagnostic approach are arising to improve knowledge in well-defined primary FSGS cohorts of patients. Follow-up of properly classified primary FSGS patients will allow risk stratification for predicting the response to different treatments. In this review we will focus on the diagnostic algorithm used in idiopathic FSGS both in native kidneys and in disease recurrence after kidney transplantation. We will emphasize those potential confusing factors as well as their detection and prevention. In addition, we will also provide an overview of ongoing studies that recruit large cohorts of Received: 12.11.2019; Editorial decision: 4.5.2020 C The Author(s) 2020. Published by Oxford University Press on behalf of ERA-EDTA. V This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 482 Challenges in primary FSGS diagnosis | 483 glomerulopathy patients (Nephrotic Syndrome Study Network and Cure Glomerulonephropathy, among others) and the experimental studies performed to find novel reliable biomarkers to detect primary FSGS. Keywords: biomarkers, diagnosis algorithm, focal segmental glomerulosclerosis, idiopathic nephrotic syndrome, primary FSGS INTRODUCTION genetic forms), treatment of the underlying cause of the disease can reverse or slow down the progression of the disease, Focal and segmental glomerulosclerosis (FSGS) is a histological depending on the extent of established renal damage (i.e. pattern used in clinical practice to define a podocytopathy that angiotensin-converting enzyme inhibitor or angiotensin recep- develops with nephrotic-range proteinuria and segmental oblit- tor blocker treatment for hypertension-caused FSGS or antiviral eration or collapse of glomerular capillary loops with increased treatment for virus-induced FSGS). In contrast, primary FSGS is extracellular matrix in some glomeruli. FSGS is the leading glo- thought to be caused by undefined circulating factors that cause merulopathy responsible for end-stage renal disease (ESRD) in abnormal glomerular permeability and is diagnosed after exclu- the USA [1]. It is the cause of 40% of the nephrotic syndrome in sion of any other identifiable cause of secondary FSGS [4]. This adults and 20% in children [2]. As in other kidney diseases, the subtype of FSGS is associated with a poor renal prognosis when incidence of FSGS is generally 1.2- to 1.5-fold higher in men compared with secondary forms [12] and another primary glo- than in women [3]. FSGS can be classified into two main forms: merulonephritis [13] and requires immunosuppressive treat- secondary, which includes genetic, adaptive, infection/inflam- ment. Therefore the distinction between primary and mation and medication-associated FSGS, and primary or idio- secondary forms of FSGS has therapeutic and prognostic impli- pathic FSGS [2, 4]. Primary FSGS is thought to be caused by an cations. As the presence of an FSGS lesion itself in a kidney bi- unknown circulating factor(s) that damages the podocytes and opsy does not offer a precise diagnosis, it remains a clinical consequently permeabilizes the glomerular filtration barrier [2]. diagnosis. Additionally, other diseases may present similar clin- The major concerns of idiopathic FSGS are the poor renal prog- ical and histological findings, such as MCD or focal and global nosis with an absence of response to immunosuppressive ther- glomerulosclerosis (FGGS). Identifying each form properly is apies or relapses and its recurrence after kidney transplantation crucial to avoid unnecessary immunosuppressive-based thera- in ~30–50% of patients, which often leads to renal graft failure peutic approaches and to establish the appropriate treatment [5]. for the FSGS patient. Primary FSGS should be suspected in patients with abrupt FSGS is clinically noticed when patients present with heavy nephrotic proteinuria, severe hypoalbuminaemia, oedema and proteinuria. Depending on the degree of proteinuria, primary or uniform podocyte foot process effacement (FPE) by electron mi- secondary FSGS can be suspected. Patients with primary FSGS croscopy (EM) of a renal biopsy [6]. A careful and detailed medi- usually present with nephrotic-range proteinuria (>3.5 g/day) cal history and clinical examination are needed, as often it is with complete nephrotic syndrome (severe hypoalbuminae- not easy to distinguish between primary and secondary FSGS. mia), often associated with renal insufficiency, hypertension From a practical point of view, it is worth mentioning that pri- and microhaematuria [14]. In contrast, secondary FSGS patients mary FSGS is often confused with other glomerulopathies such usually present a broad range of proteinuria (including as minimal change disease (MCD). In fact, many authors believe subnephrotic and nephrotic range) and in general do not de- that MCD and primary FSGS are the same disease, the second velop complete nephrotic syndrome despite the presence of ne- being a more advanced stage of the first, where glomerular phrotic-range proteinuria. Renal insufficiency is less common lesions can be seen by light microscopy (LM) [7]. Diagnosis of in secondary FSGS and is usually associated with a slow in- primary FSGS is not a straightforward process and therefore crease of proteinuria over time [15]. However, this is not appli- several studies have focused on finding factors and markers cable for genetic FSGS, which may be very aggressive, with that may be helpful for diagnosis of the disease and the detec- early-age onset, even in utero, associated or not with extrarenal tion of FSGS recurrence after kidney transplantation [8–10]. characteristic clinical features, with overall progression to ESRD In this review we discuss the diagnosis algorithm of FSGS in [16]. the native kidney and its relapse after kidney transplantation. Together with clinical and laboratory findings, kidney biopsy In addition, we will review studies focused on new biomarkers is key for disease identification. However, histological injury is for early diagnosis and/or detection of FSGS relapse after kidney difficult to evaluate in single kidney sections since the focal transplant. We will also cover new strategies aimed at identify- sclerotic lesions, which are present in many glomeruli, affect ing accurate biomarkers of disease activity and progression. only 12.5% of the total glomerular volume [17]. Therefore it is es- sential to obtain representative biopsy specimens that include at least 10 glomeruli, both cortical and juxtamedullary, as scle- The primary FSGS diagnosis in the native kidney rotic changes may occur earlier in the latter. Juxtamedullary FSGS is a histological term rather than a specific clinical disease. glomeruli can sometimes be missed on the kidney biopsy. FSGS is a lesion resulting from a podocyte injury characterized Furthermore, these samples should be processed in successive by segmental (in parts) and focal (of some) sclerosis of the glo- sections that allow observation of the whole glomerular tuft meruli. Thus the histological finding lends its name to the asso- and evaluation in LM. The histology of FSGS is defined by a seg- ciated clinical disease. It can be classified into secondary FSGS mental increase of the mesangial matrix with obliteration of or primary (idiopathic) FSGS. Secondary FSGS lesions are ob- the capillaries, sclerosis, hyalinosis, segmental scarring and fi- served as an adaptive response to a reduction in nephron mass, nally Bowman’s capsule adhesion to the glomerular tuft. Under genetic mutations, drug consumption or viral infections, among LM, FSGS histological lesions can be classified into five subtypes others (Table 1) [4]. In secondary FSGS cases (except for the according to the Columbia classification [18]: perihilar, tip, 484 | C. Jacobs-Cachá et al. Table 1. Main causes of secondary FSGS (adapted from KDIGO guidelines 2012 for glomerulonephritis [4]) Causes of secondary FSGS Genetic Mutations in the genes coding for crucial podocyte proteins: Mutations in ACTN4 (a-actinin 4), mutations in NPHS1 (nephrin), mutations in NPHS2 (podocin), mutations in WT-1 (Wilms tumor 1), mutations in TRPC6 (transient receptor potential cation channel subfamily C member 6), mutations in SCARB2 (lysosomal integral membrane protein 2), mutations in INF2 (formin), mutations in CD2-associated protein, others: PAX-2 (paired box protein Pax-2), MYO1E (unconventional myosin-Ie), among others [11] Mitochondrial cytopathies Apolipoprotein L1 risk variants: associated to APOL1(apolipoprotein L1) polymorphisms Associated Secondary to viral infections: human immunodeficiency virus, parvovirus B19, hepatitis virus B and C, cy- with infectious diseases tomegalovirus, Epstein–Barr virus, varicella zoster Secondary to parasite or bacterial infections: malaria, syphilis, toxoplasmosis Medication Medication or drug consumption induced: heroin, interferon-a, lithium, pamidronate/alendronate, ana- bolic steroids Adaptive structural–functional Reduced kidney mass: oligomeganephronia, unilateral kidney agenesis, kidney dysplasia,cortical necro- responses likely due sis, reflux nephropathy, surgical kidney ablation, chronic allograft nephropathy, any advanced kidney to hypertrophy disease with reduction in functioning nephrons or hyperfiltration Initially normal kidney mass: diabetes mellitus, hypertension, obesity, cyanotic congenital heart disease, sickle cell anaemia Malignancy Associated mainly with lymphoma Non-specific pattern of FSGS Produced secondary to the scarring due to the presence of other glomerulopathies: focal proliferative glo- merulonephritis (immunoglobulin A nephropathy, lupus nephritis, pauci-immune focal necrotizing and crescentic glomerulonephritis), hereditary nephritis (Alport syndrome), membranous glomerulop- athy, thrombotic microangiopathy, associated with apolipoprotein L1 (ApoL1) polymorphisms collapsing, cellular and not otherwise specified (NOS). NOS is routine whole-exome sequencing has allowed not only the de- the most common form in diverse series [19]. Prognosis may scription of >30 genes related to steroid resistance in nephrotic also be associated with the histological subtype. The tip variant syndrome [11, 27, 28], but also discarding of other kidney dis- has shown the highest rates of remission, while the collapsing eases with proteinuria and FSGS, but from a totally different variant is related to poor prognosis [20]. Of note, initial stages aetiology (i.e. Alport syndrome [29, 30], kidney dysplasia [31] or are only detectable by electronic microscopy where a degree of congenital abnormalities of the kidney and the urinary tract [32, diffuse podocyte FPE should be observed. Generalized diffuse 33]). A positive genetic test will focus the therapy on an antipro- FPE (>80% of the analysed podocitary surface) can be associated teinuric and symptomatic treatment, avoiding exposure to im- with primary FSGS or MCD, while segmental and <80% FPE is munosuppression, but a negative result does not fully exclude usually related to secondary FSGS [21, 22]. FPE assessment using mutations not previously reported. transmission electronic microscopy is to date the only estab- lished way to analyse the podocyte morphology, but it is far from being a standard method, as it is technically complex, Relapse of FSGS after kidney transplantation requires ability and time and, moreover, deals with a geometric One of the major concerns of primary FSGS is that the disease bias derived from physical sectioning [23]. Therefore, novel can recur after kidney transplantation and it seems to be related high-resolution microscopy techniques are in development to to the permeabilization activity of the FSGS circulating factor(s). improve visualization of the glomerular filtration barrier com- Post-transplant FSGS recurrence happens in 30–50% of pounds [23, 24]. patients and it can occur immediately or months to years after When the onset of proteinuria with nephrotic syndrome is transplantation [5, 34]. The known risk factors for FSGS recur- noted in paediatric patients, the diagnostic approach is very dif- rence include younger patients, those who progress to ESRD ferent. A kidney biopsy is discouraged as a first-line diagnostic within 3 years of diagnosis, a history of recurrence in a prior procedure. When a child presents with proteinuria associated allograft and patients with higher proteinuria levels pre- with nephrotic syndrome, treatment with steroids is started. transplantation [35]. Recurrent primary FSGS presents with ne- The majority of patients are steroid-sensitive (85%) [25, 26] phrotic-range proteinuria and frequently has a rapid onset in and a kidney biopsy is normally not performed unless the pa- the early post-transplant period. Patients usually have symp- tient develops steroid resistance. In case of steroid resistance, a toms and signs of nephrotic syndrome, such as oedema, hypo- kidney biopsy and genetic testing are indicated. About 15% of albuminaemia and hyperlipidaemia, together with some degree the paediatric patients with nephrotic syndrome are corticoste- of graft dysfunction. Nevertheless, the presence of proteinuria roid resistant and, of these, 60% do not respond to any other is enough to raise the suspicion of FSGS recurrence in patients therapeutic option. FSGS is detected in the kidney biopsy in that do not develop the full clinical picture. The definitive diag- >50% of steroid-resistant paediatric patients [26]. Moreover, nosis is performed by allograft biopsy, where the characteristic most of the genetic forms of FSGS appear during childhood and features of FSGS are identical to FSGS in the native kidney, al- are mainly associated with corticosteroid resistance. Mutations though in the initial phases the histological findings are charac- in the nephrin (NPHS-1), Wilms tumour 1 (WT-1) and podocin terized by a normal kidney under the optical microscope and (NPHS-2) genes can explain 70% of the studied cases of podocyte FPE using EM [36]. The treatment for FSGS recurrence, corticosteroid-resistant nephrotic syndrome [26]. Furthermore, as in native kidney idiopathic FSGS patients, is not standardized Challenges in primary FSGS diagnosis | 485 Table 2. Characteristics of various forms and diseases included in the differential diagnosis of FSGS Characteristics Primary FSGS Secondary FSGS Genetic FSGS MCD Clinical history Acute onset of nephrotic Risk factors are present, Family history of FSGS Acute onset of nephrotic syndrome without such as obesity, drug disease (although fre- syndrome without risk factors or previ- consumption, vesi- quently there are not risk factors or previ- ous renal disease coureteral reflux, re- familiar records); pro- ous renal disease history nal agenesis or teinuria or nephrotic history reduced nephron syndrome with onset mass or viral in early childhood or infection adolescence Laboratory findings Nephrotic syndrome: Non-nephrotic or ne- Childhood-onset genetic More rapid onset of ne- peripheral oedema, phrotic-range pro- FSGS: usually ne- phrotic syndrome; hypoalbuminaemia teinuria, without phrotic syndrome is peripherial oedema, and >3.5 g of protein- nephrotic syndrome; present; adolescence hypoalbuminaemia uria in 24-h urine; normal serum albu- or adult-onset genetic and >3.5 g of protein- haematuria is min levels FSGS: proteinuria uria in 24-h urine common without nephrotic syndrome Pathological findings LM: segmental areas of sclerosis, partial capillary collapse and hyaline depositsa LM: normal glomeruli IF: none or few immune deposits in sclerotic lesions IF: negative positive to IgM and occasionally to C3 EM: usually diffuse EM: usually segmental EM: either diffuse or EM: diffuse (>80%) (>80%) podocyte FPE (<80%) podocyte FPE segmental podocyte podocyte FPE FPE a Depending on the location of the lesions, tip and perihiliar variants are distinguished. Cellular and collapsing variant show their own characteristics. If not a quality biopsy, glomeruli may seem normal. IF, immunofluorescence. and the results are variable [37]. Kidney transplanted patients conjunction of different factors that damage the glomerular fil- are usually already under a tacrolimus-based immunosuppres- tration barrier. Furthermore, it is not clear what determines the sive treatment, therefore, to manage the FSGS recurrence, a response to immunosuppressive treatment and if there are dif- change to cyclosporine can be performed as a strategy to de- ferent variants of primary FSGS. Finally, although genomic crease proteinuria [38]. Currently, early plasmapheresis treat- analysis has allowed the description of many genetic mutations ment combined with intensified immunosuppression is the related to FSGS, it is possible that still unidentified genetic alter- most common treatment choice for primary FSGS recurrence ations are responsible for adult FSGS onset, a fact that would [39]. However, the results obtained with this treatment ap- lead to a mistaken diagnosis. For all these reasons, during the proach are variable and, in addition, these treatments are not last decades, different groups have tried to find different bio- exempt from toxicity [34, 40, 41]. markers to help us recognize and understand the pathways in- De novo FSGS may also occur in the transplanted kidney volved in primary FSGS. among patients who did not have FSGS as a cause of ESRD in the native kidney. This is often detected >12 months after transplantation and is associated with variable amounts of pro- Towards a better understanding of FSGS teinuria (including the nephrotic range), hypertension and pro- FSGS is a complex pathology not only because of the difficulties gressive deterioration of renal allograft function. Compensatory in diagnosis mentioned earlier, but also due to its low preva- glomerular hyperfiltration in residual nephrons caused lence and the lack of clinical tools for its risk stratification, pre- by nephron loss or low nephron number in the transplanted diction of remission, treatment selection and monitoring of kidney (size discrepancies between the graft and the recipient) drug response. In addition, FSGS may show a slow progression, has been implicated in the pathogenesis of de novo FSGS. thus requiring years of follow-up to prove the effectiveness of Therefore hypertension, diabetes, allograft rejection, an intervention. Therefore it has been necessary to join efforts immunosuppressive-induced allograft damage, BK polyomavi- and collect data from patients from different hospitals to obtain rus or parvovirus B19 infection and any other condition leading large cohorts to study the pathologic mechanism (among to a loss of renal mass can be involved in the pathogenesis of de others) of FSGS and the response to different treatment novo FSGS [42]. De novo FSGS should be closely monitored in approaches. Several registries have been created to this end, patients with unknown aetiology of ESRD in the native kidney, such as the Nephrotic Syndrome Study Network (NEPTUNE) and as primary FSGS recurrence cannot be totally excluded. Cure Glomerulonephropathy (CureGN). Despite features that allow us to discern primary FSGS and The NEPTUNE is a prospective observational study that be- its relapses from other variants of FSGS and other renal diseases gan in 2010 and will recruit 450 paediatric and adult patients (Table 2), none of these findings is pathognomonic and identify- not only with FSGS, but also with MCD and membranous glo- ing primary FSGS continues to be challenging. It is important to merulonephritis (MGN) at the time of the first renal biopsy clini- highlight again that FSGS is a histological pattern rather than a cally indicated for proteinuria. The enrolment includes 18 specific disease and that not all causes leading to podocyte clinical centres in the USA and Canada and aims to find changes damage have been elucidated. It is unknown whether primary in urinary protein excretion and renal function and to assess FSGS is the consequence of one circulating factor or the the outcomes in life quality, development of new-onset 486 | C. Jacobs-Cachá et al. diabetes, malignancies, infections, thromboembolic events, of the M-type phospholipase A2 receptor [68] and thrombospon- hospitalization, acute kidney injury and death [43]. From the din type-1 domain-containing 7A [69] as the target antigens in NEPTUNE study cohort, a scoring strategy for renal biopsies has most patients with primary MGN, studies of galactose-deficient been designed to standardize morphological analysis. This scor- IgA1 and antiglycan response in IgAN [70] and associating ing method provides a multilevel annotation of the glomeruli of mutations in apolipoprotein L1 to the development of kidney a whole-slide image for multidimensional reconstruction. This disease in patients of African ancestry [71, 72]. But although the method allows simultaneous review and scoring of the same number of genetic mutations identified in patients with FSGS glomeruli on each digital slide [44, 45]. The purpose of the has also expanded in the last decade [73], the causal mecha- authors is to redefine histopathological classification and link nism of the primary FSGS form is still unknown. The registry these novel criteria to clinical and molecular phenotypes of glo- data such as that obtained with the NEPTUNE or the CureGN merular diseases [46–48]. Renal tissue and compartment- cohorts will most probably provide a platform for genetic testing specific genetic profiles obtained by genome-wide meesenger and the identification and validation of new biomarkers. These RNA (mRNA) expression data [49–51] will be performed using large studies may be helpful to better understand and manage the samples of the NEPTUNE cohort with the aim to obtain mo- primary FSGS as well as other glomerulonephritis. lecular scoring data that will facilitate multidisciplinary and transdisciplinary research exploration along with the genotype– phenotype continuum. Other relevant data that have also been Novel biomarkers to diagnose primary FSGS obtained thanks to study of the NEPTUNE cohort are the poor As mentioned before, sometimes it is difficult to distinguish be- correlation that exists between the spot urine urinary protein:- tween different forms of FSGS and even to differentiate it from creatinine ratio (which is usually used to estimate 24-h protein- other glomerular diseases. The major challenge in FSGS diagno- uria) with the real 24-h urine protein excretion in glomerular sis is early detection of the primary or idiopathic forms in order diseases [52, 53], new statistical models to improve longitudinal to decide the best therapeutic option. Several approaches have CKD outcome biomarkers analysis [54] and the relevant factors been used in order to improve our understanding of the disease that could predict nephrotic syndrome remission [55]. and help in diagnosis and management (Figure 1). Another of the biggest studies ongoing is the CureGN, that As the disease is mainly a glomerular dysfunction, efforts began recruitment in 2014 and plans to enrol 2400 children have been made to establish which proteins are differentially and adults with MCD, FSGS, MGN or immunoglobulin A ne- expressed in glomerular cells of primary FSGS patients that phropathy (IgAN) (including IgA vasculitis). Patients are in- could potentially be used as markers. In 2003, gene expression cluded if they have a first kidney biopsy–proven diagnosis analysis of common podocyte proteins (ACTN4, GLEPP-1, WT-1, within the last 5 years and, once enrolled, a prospective synaptopodin, dystroglycan, nephrin, podoplanin and podocin) follow-up is performed under a common protocol. Patients of microdissected glomeruli suggested that the podocin:synap- with ESRD and those with secondary causes of glomerular topodin expression ratio may be useful to distinguish FSGS disease are excluded. At least 30% of the cohort is expected from MCD and nephrotic syndrome, although not from MGN to be paediatric. This large cohort will target a racially, ethni- [74]. CD44þ staining in the glomerular parietal cells has also cally and geographically diverse population with glomerular been related to FSGS. Although initially it seemed that it was disease, which contrasts with other studies that included specifically associated to primary FSGS forms [75, 76], it has also more homogeneous patients [56]. The hypothesis of the been detected in secondary forms [77], suggesting that CD44 ex- authors of the CureGN is that different disease mechanisms pression in the parietal cells is related to a common FSGS devel- can result in similar pathologic and clinical phenotypes but opment mechanism. Furthermore, kidney tissue microRNA show very different disease development and that the histo- (miRNA) expression has also been analysed in FSGS patients. pathology per se does not adequately define disease course Although several miRNAs are upregulated in glomeruli of pri- and response to therapy for all individuals within a given di- mary FSGS patients [78], it seems that miRNA-193a is relevant agnosis. The identification and characterization of the under- for development of the disease, as it decreases the expression lying mechanisms will have a broad influence on diagnostic of WT-1, which compromises the podocyte function [79]. classification, accurate prognostication, definition of patient miRNA-193a is also increased in urinary exosomes of FSGS cohorts for clinical trials and the indication of individualized patients when compared with MCD patients [80], thus this therapies. Awaiting results in terms of diagnosis improve- miRNA could be suitable to detect primary FSGS, although this ment and disease management, data from this cohort regard- needs further validation. ing health-related quality of life [57] and cardiovascular Although study of the proteins specifically expressed in disease risk in paediatric patients [58] are available. Most of FSGS in the kidney tissue has helped in understanding the dis- the patients in the NEPTUNE study meet the inclusion criteria ease, to date the clue to better diagnose idiopathic FSGS has not of the CureGN study; this will allow us follow patients for been found. Therefore a specific non-invasive biomarker would years and to obtain long-term longitudinal information, be very useful to discriminate different forms of FSGS. which is very important in low-prevalence pathologies. Both Biomarkers have been searched both in blood and in urine sam- NEPTUNE and CureGN data intend to complement relevant ples. The search for plasma biomarkers has been basically fo- work from previous large registry studies that have provided cused on the permeabilizing factor. Research on the putative important data on risk factors for disease progression [25, 59– plasma factor began about two decades ago [81, 82], and al- 67]. It is worthy of mention that the PodoNet, a paediatric co- though some candidates have been proposed [83, 84], it has not hort study that recruited steroid-resistant nephrotic syn- been firmly demonstrated that any of them causes idiopathic drome patients, has provided valuable information regarding FSGS. Certainly, one of the most promising proposed circulating kidney histology, genetic defects, treatment response and factors was soluble urokinase receptor (suPAR), which was outcomes of this complex clinical entity [25]. found to be increased in the serum of FSGS patients [8]. In a pre- Many authors agree that recent advances have led to the dis- vious work, Wei et al. [85] showed that the urokinase receptor covery of biomarkers of glomerular disease, including discovery could be involved in the FPE of podocytes via activation of the Challenges in primary FSGS diagnosis | 487 FIGURE 1: Milestones for primary FSGS biomarker identification. Timeline of research studies focused on finding putative biomarkers to detect primary FSGS in blood, kidney tissue and urine. ab3-integrin pathway in a urokinase plasminogen activator re- FSGS patients induces the expression of several specific genes ceptor (uPAR) knockout mouse model [85]. Therefore they hy- upon cultured podocytes and the authors suggest that this pothesized that maybe the soluble uPAR (suPAR) could be the could be used to distinguish FSGS recurrence from other types FSGS causal circulating factor. suPAR levels were measured in of renal disease. As an example, analysis of interleukin-1b gene serum samples of 78 subjects with FSGS, 25 with MCD, 16 with expression induced by serum of recurrent FSGS patients shows MN, 7 with pre-eclampsia and in 22 healthy individuals. Blood >80% sensitivity and specificity to discriminate relapsing FSGS suPAR levels were higher in the FSGS patient group than in the patients from other nephropathies [100]. Although the study rest of the studied groups and, moreover, suPAR levels were was elegantly designed, it represents an indirect method on cul- also higher in the FSGS patients that relapsed after kidney tured podocytes, cells that need several days to differentiate transplantation. In addition, the authors demonstrated that the (from 7 to 14 days) [101], hence it would not allow a fast diagno- uPAR recombinant form injected into a knockout mouse model sis of FSGS relapse, making it difficult to use in current clinical induced proteinuria, reinforcing the idea of suPAR is the plasma practice. Finally, plasma miRNAs have also been explored as po- circulating factor related to FSGS [8]. Unfortunately, later studies tential biomarkers to detect primary FSGS and several of these were unable to demonstrate the same results in independent have been associated to the disease [102–104]. cohorts [86–88], nor could it be firmly demonstrated that it was Urine has also been exploited as a biomarker source. In 2007, the cause of the disease [89, 90]. Moreover, plasma suPAR levels Varghese et al. [105] performed two-dimensional electrophoresis have also been found to be elevated in several extrarenal pa- (2DE) of urine samples from several groups of patients (FSGS, thologies that can potentially be concomitant to FSGS, such as IgAN, MGN and diabetic nephropathy) with the aim of finding cancer or inflammatory disorders, among others [91–95]. Recent biomarkers to distinguish glomerulopathies. They quantified studies suggest that both urinary levels of suPAR [96, 97] and the relative abundance of each spot (that represents a protein) uPAR detection in kidney biopsies [98] may be useful in the di- and, based on these data, they designed a prediction algorithm. agnosis of primary FSGS, but further investigation is needed. Although this study was not focused on FSGS, it revealed that Anti-CD40 blood levels have also been associated with FSGS, variations in the urinary proteome could be useful to discrimi- with 78% accuracy to predict post-transplant FSGS recurrence nate kidney diseases with proteinuria. Similarly, in a rat model [99]; however, further studies are required to confirm these of induced FSGS, a serial analysis of urine samples using 2DE results. Recently it has been shown that plasma of relapsing revealed that the proteomic profile changed along the course of 488 | C. Jacobs-Cachá et al. the disease and that some proteins appeared before the scle- professional meetings from AstraZeneca, Mundipharma, rotic lesions, suggesting that they could be useful as early bio- Boehringer Ingelheim, Astellas, Menarini, Novartis, Esteve, markers [106]. To our knowledge, the first and only urinary Sanofi, Chiesi and Novo Nordisk. G.A. has participated in advi- biomarker that has been specifically associated with post- sory boards and educational activities for Alexion transplantation recurrent FSGS is apolipoprotein A-Ib (apoA-Ib), Pharmaceuticals, Chiesi, Advicenne, Kyowa-Kirim and a modified form of apoA-I, described by Lopez-Hellı́n et al. in Recordati Rare Diseases. She has received honoraria for re- 2013 [107]. Results from two cohorts [10, 107] have shown that search activities from those companies and from Oxthera and the presence of this form in urine allows the detection of recur- travel support for professional meetings from Alexion rent FSGS patients with high specificity and sensitivity (>87% Pharmaceuticals, Advicenne, Kyowa-Kirim and Recordati Rare and >90%, respectively). ApoA-Ib also has a potential prognostic Diseases. M.J.S. has received speaker fees or travel support from value, as it can be found in urine before the FSGS recurrence epi- Otsuka, Menarini, AstraZeneca, Boehringer Ingelheim, Janssen, sodes [10]. Although the exact role of apoA-Ib in FSGS is un- Mundipharma, Novartis, Eli Lilly, Esteve and Novo Nordisk. known, other authors have also found increased levels of apoA- I [108] or the presence of high molecular weight forms of apoA-I [109] in urine of FSGS patients, reinforcing the idea that either apoA-I or the mechanisms that modify this lipoprotein are in- REFERENCES volved in the pathogenic mechanism of this disease [110]. The 1. D’Agati VD, Kaskel FJ, Falk RJ. Focal segmental glomerulo- potential role of this biomarker to discriminate primary FSGS in sclerosis. N Engl J Med 2011; 365: 2398–2411 native kidney is currently being pursued, but preliminary 2. Rosenberg AZ, Kopp JB. Focal segmental glomerulosclero- results obtained with idiopathic FSGS patients before kidney sis. Clin J Am Soc Nephrol 2017; 12: 502–517 transplantation pointed out that the detection of apoA-Ib in na- 3. 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