The soluble urokinase receptor is not a clinical marker for focal segmental glomerulosclerosis

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1 & 214 International Society of Nephrology see commentary on page 499 The soluble urokinase receptor is not a clinical marker for focal segmental glomerulosclerosis Björn Meijers 1,2, Rutger J.H. Maas 3, Ben Sprangers 1,2, Kathleen Claes 1,2, Ruben Poesen 2, Bert Bammens 1,2, Maarten Naesens 1,2, Jeroen K.J. Deegens 3, Ruth Dietrich 4, Markus Storr 4, Jack F.M. Wetzels 3, Pieter Evenepoel 1,2 and Dirk Kuypers 1,2 1 Department of Nephrology, UZ Leuven, Leuven, Belgium; 2 Department of Microbiology and Immunology, KU Leuven, Leuven, Belgium; 3 Department of Nephrology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands and 4 Department of Research and Development, Gambro Dialysatoren GmbH, Hechingen, Germany The soluble urokinase receptor (supar) promotes proteinuria and induces focal segmental glomerulosclerosis (FSGS)-like lesions in mice. A serum supar concentration cutoff of 3 pg/ml has been proposed as a clinical biomarker for patients with FSGS. Interestingly, several studies in patients with glomerulopathy found an inverse correlation between the estimated glomerular filtration rate (egfr) and supar. As patients with FSGS present at different egfrs, we studied the relationship between egfr and supar in a cohort of 476 non-fsgs patients and 54 patients with biopsy-proven idiopathic FSGS. In the non-fsgs patients, egfr was the strongest significant determinant of supar. The proposed cutoff for supar in FSGS patients was exceeded in 17%, 39%, and 88% in patients with egfrs of more than 6, 45 6, and 3 45 ml/min per 1.73 m 2, respectively. In patients with egfr of o3 ml/min per 1.73 m 2, supar exceeded the cutoff in 95% of patients. Levels of supar in patients with idiopathic FSGS overlapped with non-fsgs controls and for any given egfr did not discriminate FSGS cases from non-fsgs controls. In the overall cohort, there was a negative association between idiopathic FSGS and supar, and idiopathic FSGS was not an independent predictor of FSGS concentration over 3 pg/ml. Thus, this study does not support an absolute, egfr-independent, supar concentration cutoff as a biomarker for underlying FSGS pathology and questions the validity of relative, egfrdependent supar cutoff values. Kidney International (214) 85, ; doi:1.138/ki ; published online 8 January 214 KEYWORDS: biomarker; chronic kidney disease; focal segmental glomerulosclerosis; soluble urokinase receptor Correspondence: Björn Meijers, Department of Nephrology, Laboratory of Nephrology, UZ Leuven, Herestraat 49, B-3 Leuven, Belgium or Department of Microbiology and Immunology, KU Leuven, Herestraat 49, B-3 Leuven, Belgium. bjorn.meijers@uzleuven.be Received 26 April 213; revised 24 September 213; accepted 1 October 213; published online 8 January 214 Idiopathic focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome in both children and adults, and despite intensive treatment it will progress to endstage renal disease in 4% of affected patients. FSGS frequently recurs after transplantation, and recurrences have been associated with decreased allograft survival. The pathogenesis of FSGS is incompletely resolved; however, evidence suggests that it involves immune cell dysfunction, secretion of a circulating factor, and podocyte maladaptation. The existence of a circulating vascular permeability factor has been postulated based on rapid recurrence of proteinuria following kidney transplantation, 1,2 the occurrence of a transient nephrotic syndrome in a newborn to a women affected by FSGS, 3 the beneficial effect of immunoadsorption and/or plasma exchange, 4 and the ability of FSGS patient serum to induce albuminuria in rats. 5 In a recent publication, Wei et al. 6 provide ample evidence that serum soluble urokinase receptor (supar) is a circulating factor that may cause FSGS. The urokinase receptor (upar; encoded by PLAUR), apart from being the cellular receptor for urokinase, is a versatile receptor affecting migration, adhesion, differentiation, and proliferation through intracellular signaling. 7 upar consists of three internally disulphide-bonded domains (D1, D2, and D3) anchored to the cell surface by a glycosyl phosphatidylinositol bridge. supar is released from the plasma membrane by cleavage of the glycosyl phosphatidylinositol anchor. 7 Induction of podocyte upar signaling leads to foot process effacement and proteinuria via the activation of b3 integrin. 8 In a similar manner, circulating supar also activates podocyte b3 integrin, causing foot process effacement, proteinuria, and FSGS-like glomerulopathy. supar-induced glomerular disease can be blocked by neutralizing supar antibodies. 6 In the original report, supar was elevated in patients with FSGS, but not in patients with minimal-change nephrotic syndrome, membranous nephropathy, or preeclampsia. 6 The highest serum supar levels were found in sera from patients with FSGS who developed post-transplant proteinuria recurrence. Using an supar cutoff value of 3 pg/ml, circulating supar levels were elevated in 84.3 and 55.3% of 636 Kidney International (214) 85,

2 B Meijers et al.: supar in CKD clinical investigation patients in two patient cohorts of biopsy-proven FSGS, compared with 6% of controls (Po.1). These studies suggest the potential role of supar as an independent biomarker of FSGS. 9 Multivariate analysis revealed an inverse correlation of supar with estimated glomerular filtration rate (egfr), in agreement with several single-center reports A similar association has been observed in patients admitted to the intensive care unit. 14 This may be attributed to reduced clearance of supar and/or overproduction. The relative importance of kidney function to supar concentrations in chronic kidney disease (CKD) patients has not been investigated in depth. We therefore studied supar concentrations in a large cohort of patients with non-fsgs CKD (the Leuven mild-to-moderate CKD cohort) to study the relationship between egfr and supar concentrations. In addition, we evaluated supar levels in patients with biopsyproven idiopathic FSGS to test the validity of supar as an FSGS biomarker. RESULTS We determined supar concentrations in 476 patients (Table 1) with known non-fsgs CKD (controls), and in 54 patients with biopsy-proven FSGS (cases: 44 active disease, 1 remission), using the human upar enzyme-linked immune sorbent assay (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany). Compared with control patients, patients with FSGS were younger and had higher egfr. supar concentrations were similar between FSGS cases and CKD controls. We first studied explanatory variables for supar concentrations in non-fsgs CKD patients (controls). As supar concentrations showed a non-normal distribution, supar concentrations were log-transformed for linear regression analyses. In multivariate analysis, supar concentrations were higher in female individuals (P ¼.3). A direct association was observed with age (Po.1) and C-reactive protein (P ¼.9), whereas albumin (Po.1) and the egfr (Po.1) were inversely associated, with the latter having the highest partial coefficient of determination (R 2 ). In multivariate analysis, proteinuria was not an independent determinant of supar concentrations (Table 2). As egfr was the strongest determinant, we stratified supar according to egfr (Figure 1a). The percentage of patients with supar concentrations exceeding the proposed cutoff for FSGS of 3 pg/ml rises significantly from 17% in those with egfr of 46 ml/min per 1.73 m 2 to 39 and 88% for those with egfr of 45 6 and 3 45 ml/min per 1.73 m 2, respectively. In patients with egfr of o3 ml/min per 1.73 m 2, supar concentrations exceeded the proposed cutoff in 95% of patients. As patients with FSGS present at different egfrs, we then plotted biopsy-proven FSGS patients and non-fsgs CKD controls on supar per egfr graphs, irrespective of proteinuria (Figure 1b), those with 24-h proteinuria exceeding 1 g (Figure 1c), and only those with 24-h proteinuria exceeding 3 g (Figure 1d). On these graphs, FSGS cases are interspersed with non-fsgs controls and, for any given egfr, are not identifiable by higher supar concentrations, questioning the use of any supar cutoff value to be used as a biomarker for FSGS. We compared patients with steroidresistant and steroid-sensitive idiopathic FSGS (Table 3). supar levels were equal, providing additional evidence that supar does not distinguish between steroid-sensitive and steroid-resistant FSGS. Finally, we performed multivariate analysis in the overall cohort, combining data of FSGS cases with active disease and non-fsgs controls with study determinants of supar (Table 4). In multivariable analysis, with supar as the continuous parameter, primary FSGS was negatively associated with supar concentrations (P ¼.4). Furthermore, in the analysis with dichotomized supar Table 2 Variables related to supar concentrations in non-fsgs patients Univariate Multivariate Variable b R 2 P b P Age o.1.4 o.1 Sex Diabetes.26.6 o.1 egfr o.1.19 o.1 Proteinuria CRP.16.8 o Albumin o.1.19 o.1 Hb.8.12 o.1 Phosphate o.1 Model R 2.55 Abbreviations: CRP, c-reactive protein (log-transformed); egfr, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; Hb, hemoglobin; supar, soluble urokinase receptor. Table 1 Patient demographics Variable controls FSGS active disease FSGS remission Active FSGS versus controls FSGS remissions versus controls Active FSGS versus remissions Patients (n) Age (years) 64 (51 75) 47 (33 6) 43 (39 7) o Sex (male/female (%)) 26/216 (54.6/45.4) 31/13 (7/3) 5/5 (5/5) Serum creatinine (mg/dl) 1.79 ( ) 1.22 ( ) 1.11 ( ) egfr (ml/min per 1.73 m 2 ) 33.2 ( ) 62.5 ( ) 57.7 ( ) o Serum albumin (g/l) 44.9 ( ) 23. ( ) 43.6 ( ) o.1.7 o.1 Proteinuria (g/g creatinine).3 (.1.9) 1.6 ( ).5 (.1 1.7) o.1 1. o ( ) 3772 ( ) 2684 ( ) Abbreviations: CKD, chronic kidney disease; egfr, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; supar, soluble urokinase receptor. Kidney International (214) 85,

3 B Meijers et al.: supar in CKD 12, P <.1 12, FSGS, remission > < egfr (ml/min per 1.73 m 2 ) egfr (ml/min per 1.73 m 2 ) 12, FSGS, remission 12, egfr (ml/min per 1.73 m 2 ) egfr (ml/min per 1.73 m 2 ) Figure 1 Circulating soluble urokinase receptor (supar) in patients with chronic kidney disease (CKD). (a) supar concentrations according to strata of estimated glomerular filtration rate (egfr). (b d) supar concentrations plotted as a function of the egfr for non-focal segmental glomerulosclerosis (FSGS) controls (black dots), patients with active FSGS (red dots), and FSGS in remission (green dots). Data are plotted for all patients (a, b) for patients with 24-h proteinuria more than 1 g, (c) and for patients with a 24-h proteinuria exceeding 3 g. Table 3 Steroid-resistant and steroid-sensitive FSGS in the Nijmegen cohort Variable Steroid resistant Steroid sensitive P Patients (n) a Age (years) 5 (22 62) 47 (34 58).6 Sex (male/female) 12/3 17/6 1. Serum creatinine (mg/dl) 1.39 ( ) 1.3 ( ).1 egfr (ml/min per 1.73 m 2 ) 62. ( ) 74.7 ( ).6 Serum albumin (g/l) 23 (13 27) 22 (2 26).2 Proteinuria (g/g creatinine) 9.5 ( ) 1.4 ( ) ( ) 3489 ( ).9 Abbreviations: CKD, chronic kidney disease; egfr, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; IQR, interquartile range; supar, soluble urokinase receptor. Data expressed as median (IQR). a Steroid responsiveness could not be determined in one patient with recently diagnosed FSGS. values, FSGS was not an independent determinant (P ¼ 1.) of supar concentrations above the proposed 3 pg/ml cutoff. DISCUSSION The current study questions the value of supar as clinical biomarker for FSGS. We demonstrate that egfr is a potent determinant of supar. Consequently, supar concentrations exceed the proposed threshold of 3 pg/ml in the large majority of patients with advanced CKD. The causes for the accumulation of supar in patients at reduced glomerular filtration are incompletely understood. Urinary excretion of supar has been demonstrated. supar is a circulating protein Table 4 Variables related to supar concentrations in the overall study supar concentration supar 43 pg/ml Variable b P b P Age.5 o.1.5 o.1 Sex egfr.8 o.1.8 o.1 CRP Albumin.18 o FSGS Model R 2.5 Model R 2.44 Abbreviations: CRP, c-reactive protein (log-transformed); egfr, estimated glomerular filtration rate; FSGS, focal segmental glomerulosclerosis; supar, soluble urokinase receptor. ranging from 2 to 5 kda, depending on the degree of glycosylation and proteolytic cleavage. 6,7 As the molecularweight cutoff for glomerular filtration is thought to be around 3 5 kda, 15 loss of glomerular filtration is expected to result in rising supar concentrations. 16 To what extent glycosylation affects glomerular filtration of supar is unclear. The degree of supar glycosylation in patients with CKD has not been studied to date. In addition, the tubular handling that is, whether and to what extent supar is absorbed by the tubuli remains to be studied. Renal handling may not even explain the observed wide dispersion in serum concentrations, especially in patients with low egfr (Figure 1a). Contrary to the published cohorts of FSGS patients, we did observe a direct gradual relationship with C-reactive protein, pointing toward a role 638 Kidney International (214) 85,

4 B Meijers et al.: supar in CKD clinical investigation for inflammation. Our multivariate model only predicted slightly more than half of the variance in supar concentrations, indicating a role for as yet unidentified determinants. A key question is whether accumulation of supar in patients with progressive CKD is of clinical relevance. Ample evidence suggests that supar affects the podocyte through integrin signaling. Given the versatile roles of upar as a signaling orchestrator, it is tempting to speculate that the accumulation of supar is in the causal chain of extrarenal manifestations of CKD. Clinical or experimental data to support a pathophysiological role of supar in CKD have not been published to date. Although the number of patients with primary FSGS was considerably lower than that of control non-fsgs patients, the cohort is still of reasonable size, and it is unlikely that clinically relevant differences will be observed in larger studies. In any case, in future studies, control patients should be selected not solely based on histopathological diagnosis but should also be matched for demographic and biochemical parameters including the egfr. In conclusion, our study does not support the use of any absolute that is, egfr-independent supar concentration cutoff as a biomarker for FSGS. Our data, moreover, question the validity of relative that is, egfr-dependent supar cutoff values. PATIENTS AND METHODS The Leuven mild-to-moderate CKD study (clinicaltrials.gov NCT441623) is a prospective cohort to study uremic retention solutes, including protein-bound uremic solutes. 17 Prevalent CKD patients, followed up at the nephrology outpatient clinic of the University Hospitals Leuven, 18 years of age or older, were eligible for inclusion. Data on baseline demographics and cause of kidney disease were collected at the time of informed consent. The study was performed according to the Declaration of Helsinki and approved by the ethics committee of the University Hospitals Leuven. Informed consent was obtained from all patients. We enriched the number of patients with biopsy-proven primary FSGS taking samples from the Leuven renal research Biobank. Approval for measurement of supar on samples of the Leuven renal research Biobank was granted by the medical ethics committee of the University Hospitals Leuven. In Radboud University Nijmegen Medical Centre, patients with proteinuria are evaluated using a standard protocol. 18 In brief, patients are seen after an overnight fast. Blood pressure and body weight are measured and serum and urine are collected for clinical laboratory tests. In addition, aliquots of serum and urine are stored at 7 1C for research on prognostic factors. The local medical ethics committee has given approval for the protocol and collection of follow-up data from patients. All patients have given written informed consent. For the current study, we selected stored serum samples obtained from 39 patients with FSGS, including 11 patients with previously reported supar levels. 1 Patients aged X18 years with biopsy-proven primary FSGS were included. Patients were classified as primary FSGS based on the following criteria: all patients had nephrotic syndrome (serum albumin p3 g/l, proteinuria X3.5 g/24 h). Secondary causes of FSGS (hereditary, HIV, drugs, adaptive structural functional responses mediated by hyperfiltration, malignancy, other glomerular diseases) were excluded by detailed diagnostic work-up, including medical history and family history, physical examination, kidney imaging, and kidney pathology, including electron microscopy studies. In the absence of a positive family history, genetic testing was not routinely performed, as currently known FSGS-associated mutations are rare in adults with isolated FSGS. 19 Outcome was defined by response to steroid treatment. Patients with steroid-sensitive FSGS had at least 5% reduction in peak proteinuria and attained subnephrotic levels (o3.5 g per 24 h) on steroid treatment. 2 Patients with steroidresistant FSGS did not meet the criteria for steroid-sensitive FSGS after 16 weeks of high-dose steroid therapy (prednisone dose equivalent of 1 mg/kg/day). Serum supar has been proposed as a risk marker for post-transplant FSGS recurrence. 6 As recurrence typically occurs in steroid-resistant patients, 21 we performed additional analyses in this subgroup. Creatinine was measured using an isotope dilution mass spectrometry-traceable method. egfr was calculated using the CKD-EPI equation. 22 In both centers, measurement of supar was performed using the Human upar Quantikine enzyme-linked immune sorbent assay kit (DUP, R&D systems). All samples were measured twice, and the mean supar concentrations were used. Reproducibility of the assay was excellent, with the mean coefficient of variation in individual patients of 2.43%. Data are expressed as mean (standard deviation) for normally distributed variables or median (interquartile range) for nonnormally distributed variables. Differences were tested using parametric analysis of variance, Kruskal Wallis test, or w 2 square test as appropriate. For multivariate analysis, we used a stepwise approach, with P include o.2 and P exclude 4.5. All statistical analyses were performed using SAS (version 9.2, the SAS institute, Cary, NC). DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS RJHM is supported by a grant of the Dutch Kidney Foundation (NSN OW8). RP is the recipient of a Ph.D. fellowship of the Research Foundation Flanders (FWO grant 11E9813N). RD and MS are employees of Gambro Dialysatoren GmbH, Hechingen, Germany. REFERENCES 1. Ponticelli C. Recurrence of focal segmental glomerular sclerosis (FSGS) after renal transplantation. Nephrol Dial Transplant 21; 25: Hariharan S, Adams MB, Brennan DC et al. Recurrent and de novo glomerular disease after renal transplantation: a report from Renal Allograft Disease Registry (RADR). Transplantation 1999; 68: Lagrue G, Branellec A, Niaudet P et al. Transmission of nephrotic syndrome to two neonates. Spontaneous regression. Presse Med 1991; 2: Dantal J, Godfrin Y, Koll R et al. Antihuman immunoglobulin affinity immunoadsorption strongly decreases proteinuria in patients with relapsing nephrotic syndrome. J Am Soc Nephrol 1998; 9: Zimmerman SW. 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