Plasma soluble urokinase receptor levels are increased but do not distinguish primary from secondary focal segmental glomerulosclerosis

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1 & 213 International Society of Nephrology see commentary on page 235 Plasma soluble urokinase receptor levels are increased but do not distinguish primary from secondary focal segmental glomerulosclerosis Jing Huang 1,2,3,4, Gang Liu 1,2,3,4,5, Yi-Miao Zhang 1,2,3,4, Zhao Cui 1,2,3,4, Fang Wang 1,2,3,4, Xiao-Jing Liu 1,2,3,4, Rong Chu 1,2,3,4, Ying Chen 1,2,3,4 and Ming-Hui Zhao 1,2,3,4,5 1 Renal Division, Peking University First Hospital, Beijing, People s Republic of China; 2 Institute of Nephrology, Peking University, Beijing, People s Republic of China; 3 Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, People s Republic of China and 4 Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China, Beijing, People s Republic of China In this study, we measured soluble urokinase receptor levels, a possible permeability factor, in the plasma of patients with primary focal segmental glomerulosclerosis (FSGS) and determined their association with clinical and pathological data in 74 patients with primary FSGS. Healthy donors and patients with minimal change disease, membranous nephropathy, and secondary FSGS were used as controls. The plasma-soluble urokinase receptor levels, measured by commercial ELISA kits, of patients with primary FSGS (median: 2923, interquartile range pg/ml) were significantly higher than those of patients with minimal change disease (median 25 pg/ml), membranous nephropathy (median 229 pg/ml), and normal individuals (median 1739 pg/ml). There was no significant difference in plasma-soluble urokinase receptor levels between the 74 patients with primary and 14 patients with secondary FSGS. The soluble urokinase receptor levels increased in the order of tip variant, to a not otherwise specified variant and a cellular variant. The soluble urokinase receptor levels were significantly but negatively correlated with creatinine clearance at presentation but positively correlated with crescent formation in patients with primary FSGS. During follow-up, receptor levels decreased significantly in patients with complete remission. Thus, plasma-soluble urokinase receptor levels did not differentiate primary and secondary FSGS, and although significantly elevated in FSGS, they showed considerable overlap with other glomerular diseases. Kidney International (213) 84, ; doi:1.138/ki ; published online 27 February 213 Correspondence: Ming-Hui Zhao or Gang Liu, Renal Division, Peking University First Hospital; Institute of Nephrology, Peking University; Key Laboratory of Renal Disease, Ministry of Health of China; Key Laboratory of CKD Prevention and Treatment, Ministry of Education of China; Peking- Tsinghua Center for Life Sciences, Beijing, 134, People s Republic of China. mhzhao@bjmu.edu.cn or liugang23@medmail.com.cn 5 These authors contributed equally to this work. Received 3 June 212; revised 4 December 212; accepted 7 December 212; published online 27 February 213 KEYWORDS: focal segmental glomerulosclerosis (FSGS); nephrotic syndrome; soluble urokinase receptor (supar) Focal segmental glomerulosclerosis (FSGS) is an important cause of steroid-resistant nephrotic syndrome in children and adults. FSGS can be divided into primary and secondary forms. The cause of secondary FSGS may be linked to genetic variations, drug toxicity, viruses, metabolic diseases, and so on. The etiology and pathophysiology of primary FSGS is still unknown. During the past 2 years, a consensus has been reached that the podocyte may have a key role in the initiation and progression of the lesion observed in FSGS 1,2 and the primary FSGS is regarded as a podocytopathy. However, substantial evidences suggest that primary FSGS may not only be a kidney disease, but also a systemic disorder in which podocytes may act as effector cells. It has been found that FSGS may recur in some patients after kidney transplantation, 3 and some patients with FSGS are successfully treated with plasmapheresis. 4 Therefore, a hypothesis that circulating permeability factors may involve in the pathogenesis of FSGS is fairly attractive. 5,6 Recently, Wei et al. 7 studied a group of FSGS patients with kidney transplantation and found that soluble urokinase receptor (supar) might be the most likely pathogenic circulating permeability factor. In our study, we measured plasma supar levels in a variety of primary glomerular diseases including primary FSGS with various pathological variants; their clinical and pathological associations were further analyzed. RESULTS Demographic and clinical characteristics of patients with primary FSGS The median age of the 74 FSGS patients was 29 years, ranging from 13 to 84 years. Of these, 5 were men and 24 were women. Their demographic, clinical, and pathological data are listed in Table 1. Of the 74 patients, 73 had nephrotic syndrome (98.6%), 18 had acute kidney injury (24.3%), and 366 Kidney International (213) 84,

2 J Huang et al.: Soluble urokinase receptor in primary FSGS clinical investigation 53 had microscopic hematuria (71.6%). Their median 24-h urine protein was 7.6 (interquartile range (IQR) ) g per 24 h. The mean serum albumin was 21.6±7.2 g/l. The median serum creatinine was 93. (IQR ) mmol/l at presentation. The plasma supar levels in patients with primary FSGS and controls The plasma supar levels of patients with primary FSGS, minimal change disease, membranous nephropathy, secondary FSGS, and normal subjects are shown in Table 2 and Figure 1. The plasma supar levels of patients with primary FSGS (2923, IQR pg/ml) were significantly higher than those of patients with minimal change disease (25, IQR pg/ml, Po.1), membranous nephropathy (229, IQR pg/ml, Po.1), and normal controls (1739, IQR pg/ml, Po.1). There was no significant difference in the plasma supar levels between patients with primary FSGS and those with secondary FSGS (2639, IQR pg/ml, P ¼.171). Of the 14 patients with secondary FSGS, the plasma supar level was 2512 pg/ml for the patient with preeclampsia, 189 pg/ml for the patient with Kimura disease, 2138 and 2333 pg/ml for the two patients with obesity, 382 and 1519 pg/ml for the two patients with Alport syndrome, and 2813 (IQR ) pg/ml for the median Table 1 The demographic and clinical parameters of patients with primary FSGS Parameter n ¼ 74 Age (years; median, range) 29, Gender (male/female) 5/24 Nephrotic syndrome, n (%) 73 (98.6%) Acute kidney injury, n (%) 18 (24.3%) Microscopic hematuria, n (%) 53 (71.6%) 24-h urine protein (g per 24 h; median, IQR) 7.6, Albumin (g/l; mean±s.d.) 21.6±7.2 Serum creatinine at presentation (mmol/l; 93., median, IQR) Percentage of sclerosis in glomeruli 17.9±16.3 (%; mean±s.d.) Percentage of segmental sclerosis in glomeruli 11.1±11.4 (%; mean±s.d.) Percentage of global sclerosis in glomeruli 2.2±5.3 (%; mean±s.d.) Crescent formation, n (%) 11 (14.9%) Abbreviations: FSGS, focal segmental glomerulosclerosis; IQR, interquartile range. plasma supar level of the eight patients without identifiable factors. The plasma supar levels in patients with primary FGGS with different pathological variants We compared the plasma supar levels among different histopathological variants of patients with primary FSGS. As shown in Figure 2, the plasma supar levels increased in the order of tip, not otherwise specified (NOS), and cellular variant (2323, IQR pg/ml; 3216, IQR pg/ml; 327, IQR pg/ml, respectively). As there were only a few cases with perihilar variant, collapsing variant, and advanced FSGS, statistical analysis was not performed for them. The plasma supar levels were 121 and 2755 pg/ml for the two patients with perihilar variant, 3123 pg/ml for the patient with collapsing variant, and 399 and 1337 pg/ml for the two patients with advanced FSGS. 15, 1, 5 Primary FSGS P<.1 Normal controls P<.1 Minimal change disease P<.1 Membranous nephropathy P=.171 Secondary FSGS Figure 1 Plasma soluble urokinase-type plasminogen activator receptor (supar) levels among patients with primary focal segmental glomerulosclerosis (FSGS), normal subjects, and patients with minimal change disease, membranous nephropathy, and secondary FSGS. Table 2 The demographic data and plasma supar levels of patients and controls Primary FSGS Minimal change disease Membranous nephropathy Secondary FSGS Normal controls Numbers of subjects Age (median, range) 29, , , , , Gender (male/female) 5/24 7/7 18/11 5/9 33/23 (median, IQR) 2923, , , , , Abbreviations: FSGS, focal segmental glomerulosclerosis; IQR, interquartile range; supar, urokinase-type plasminogen activator receptor. Kidney International (213) 84,

3 J Huang et al.: Soluble urokinase receptor in primary FSGS 15, 1, , Tip variant 3216, NOS variant Cellular variant 327, Figure 2 The plasma soluble urokinase-type plasminogen activator receptor (supar) levels in primary focal segmental glomerulosclerosis (FSGS) patients with different pathological variants. NOS, not otherwise specified. The associations between plasma supar levels and clinical and pathological data of patients with primary FSGS In our study, it is of great interest to note that: (1) patients with tubular atrophy X5% had higher plasma supar levels than those with tubule atrophy o5% ((3835, IQR ) pg/ml vs. (2574, IQR ) pg/ml, P ¼.14)); (2) patients with interstitial fibrosis X5% had higher plasma supar levels than those with interstitial fibrosis o5% ((3311, IQR ) pg/ml vs. (2566, IQR ) pg/ml, P ¼.42); and (3) patients with crescent formation had higher plasma supar levels than those without crescents ((3833, IQR ) pg/ml vs. (2628, IQR ) pg/ml, P ¼.12). The details are listed in Table 3. Furthermore, the plasma supar levels in the patients with primary FSGS were negatively correlated with creatinine clearance (r ¼.34, P ¼.3; Figure 3a) and associated with crescent formation (P ¼.46; Table 4). The details are listed in Table 5. As molecular weights of supar fragments ranged from 22 to 45 kda, these molecules could pass through the glomerular filtration barrier. To investigate whether the elevated plasma supar was independent of renal function, we performed a multivariate linear regression analysis. The results showed that, even after adjusting for renal function, the difference of supar levels between patients with primary FSGS and patients with minimal change disease (P ¼.1) and patients with membranous nephropathy (P ¼.3) were still significant; the difference of supar levels between patients with Table 3 Associations between plasma supar levels and pathological data of patients with primary FSGS Patient group No. of patients Level of supar (pg/ml; median, IQR) P-value Patients with tubular atrophy B þ , þþ , Patients with interstitial fibrosis B þ , þþ , Patients with interstitial infiltration , þ , þþ , Patients without crescents , Patients with crescents , 399 1,35 Abbreviations: FSGS, focal segmental glomerulosclerosis; IQR, interquartile range; supar, urokinase-type plasminogen activator receptor. primary FSGS and patients with secondary FSGS (P ¼.8) was still not significant. This indicated that the elevated supar was independent of renal function. Second, correlation analysis for the individual glomerular disease showed that supar level and creatinine clearance were not correlated significantly in patients with minimal change disease (r ¼.27, P ¼.478; Figure 3b), patients with membranous nephropathy (r ¼.9, P ¼.965; Figure 3c), and patients with secondary FSGS (r ¼.376, P ¼.197; Figure 3d). The change of plasma supar levels at presentation and during follow-up of patients with primary FSGS During follow-up with a mean duration of 78. (IQR ) weeks, we collected plasma samples of 24 patients with therapeutic responses. There were 11 patients achieving complete remission, 5 patients achieving partial remission, and 8 patients were treatment failure. At presentation, the mean plasma supar levels of the three groups were not significantly different (2583 (IQR ) pg/ml; 2537 (IQR ) pg/ml; and 324 (IQR ) pg/ml, respectively). Interestingly, in comparison of the plasma supar levels at presentation with that of follow-up, the plasma supar level of patients with complete remission decreased significantly (2583 (IQR ) pg/ml vs (IQR ) pg/ml, P ¼.41; Figure 4a), whereas for patients with partial remission it was comparable (2537 (IQR ) pg/ml vs. 254 (IQR ) pg/ml, P ¼.138; Figure 4b); however, for patients with treatment failure, the plasma supar level increased significantly (324 (IQR ) pg/ml vs (IQR ) pg/ml, P ¼.36; Figure 4c). In the comparison of the estimated glomerular filtration rate at presentation with that of followup, we performed correlation analysis between change of supar levels and change of estimated glomerular filtration rate in patients with complete remission (r ¼.267, P ¼.488), patients with partial remission (r ¼.2, P ¼.8), 368 Kidney International (213) 84,

4 J Huang et al.: Soluble urokinase receptor in primary FSGS clinical investigation a b c d r=.34 P=.3 1, 15, r =.27 P=.478 1, 15, r =.9 P=.965 supar levels (pg/ml) supar levels (pg/ml) supar levels (pg/ml) supar levels (pg/ml) 1, 15, r=.376 P=.197 Figure 3 The correlations between plasma soluble urokinase-type plasminogen activator receptor (supar) level and creatinine clearance rate for the individual glomerular disease. (a) Patients with primary focal segmental glomerulosclerosis (FSGS), (b) patients with minimal change disease, (c) patients with membranous nephropathy, and (d) patients with secondary FSGS. 1, 15, Table 4 The number of patients with primary FSGS with and without crescents in different ranges of supar levels Groups stratified by median No. of patients without crescents No. of patients with crescents P-value G1, n (%) 35 (94.6%) 2 (5.4%).46 G2, n (%) 28 (75.7%) 9 (24.3%) Abbreviations: FSGS, focal segmental glomerulosclerosis; supar, urokinase-type plasminogen activator receptor. Table 5 Correlations between the levels of plasma supar and clinical and pathological parameters in patients with primary FSGS Clinical parameter r value P-value ESR (mm/h) Hemoglobin (g/l) Urine protein (g per 24 h) Albumin (g/l) Serum creatinine (mmol/l) Abbreviations: ESR, erythrocyte sedimentation rate; FSGS, focal segmental glomerulosclerosis; supar, urokinase-type plasminogen activator receptor. and patients with treatment failure (r ¼.143, P ¼.787). These indicated lack of significant correlations between changes in supar levels and changes in estimated glomerular filtration rates. DISCUSSION FSGS is a major pathologic type of steroid-resistant nephrotic syndrome of children and adults and a major cause of endstage renal disease. 8 It occurred in a primary form of unknown causes or secondary to many other conditions, including drug toxicity, viruses, metabolic disorders, and so on. For several decades, many evidences revealed that there might be causative circulating factors in patients with primary FSGS. Recently, Wei et al. 7 revealed that serum supar was a possible candidate that might cause primary FSGS. In this study, we aimed to validate the association of the newly found circulating factor supar in patients with primary FSGS and to further analyze its clinical and pathological significance in a Chinese cohort of patients with primary FSGS. The upar is a glycosylphosphatidylinositol-anchored protein that could release to plasma as soluble upar after being cleaved of the glycosylphosphatidylinositol anchor. 9,1 In addition to regulation of proteolysis, supar initiates signaling transduction in cooperation with other transmembrane proteins such as integrins, caveolin, and G protein coupled receptors that promote cell proliferation, invasion, motility, and survival. 1 Wei et al. 7 showed that plasma supar levels increased in patients with primary FSGS with renal transplantation compared with patients with minimal change disease, membranous nephropathy, and preeclampsia. Patients with higher plasma supar levels before transplantation had increased risks for the recurrence of FSGS after transplantation. They found that supar levels in nearly 71.4% of patients with primary FSGS were higher than the cutoff value. In this study of 74 patients with primary FSGS, their plasma supar levels were significantly higher than that of normal subjects and patients with minimal change disease and membranous nephropathy. Recently, Maas et al. 11 in a letter reported that the median serum supar level of their 11 patients with primary FSGS was not elevated compared with those with secondary FSGS and minimal change disease. However, the authors indicated that the discrepancy could not exclude selection bias because of the limited number of patients. In their letter, there was no detail of biopsy confirmation of the cases with FSGS and no clarification of the criteria used to distinguish primary from secondary FSGS in detail. Our study indicated that, at least in our patients, the increase of plasma supar levels might be specific for some patients with primary FSGS. However, part of the patients with primary FSGS did not have elevated supar levels; we could not distinguish these patients from other glomerular disease depending on supar levels only. Wei et al. 7 had demonstrated that supar caused proteinuria and FSGS in mouse models. Zhang et al. 12 had reported that amiloride, a potassium-sparing diuretic, could inhibit upar expression and activation of b3 integrin of Kidney International (213) 84,

5 J Huang et al.: Soluble urokinase receptor in primary FSGS 12,5 P= P= P=.36 1, At presentation Follow-up At presentation Follow-up At presentation Follow-up Figure 4 The changes in plasma soluble urokinase-type plasminogen activator receptor (supar) level in patients with primary focal segmental glomerulosclerosis (FSGS) during follow-up. (a) Patients with complete remission, (b) patients with partial remission, and (c) patients with treatment failure. podocytes in vitro and in 5/6 nephrectomy rat models in vivo, which showed that amiloride could reduce proteinuria and glomerular sclerosis. In our study, we compared the plasma supar levels between patients with primary FSGS and secondary FSGS. Both of them had glomerular sclerotic lesions in renal histopathology. Although the difference was not statistically significant, further analysis showed that the median supar levels of secondary FSGS patients with hemodynamic-related secondary factors, such as preeclampsia, obesity, and Alport syndrome, were significant lower than that of patients with primary FSGS. However, 8 out of the 14 patients diagnosed as secondary FSGS without identifiable factors due to nondiffuse effacement of foot process had elevated supar levels that were comparable with primary FSGS. We could not exclude the possibility that those secondary FSGS without identifiable factors might be an early stage or a special variant of primary FSGS. These need further investigation. Therefore, our results indicated that the increase of plasma supar in patients with primary FSGS might not be the consequence of glomerular sclerotic lesions. This finding further supported the speculation that the elevation of plasma supar level might be specific for some patients with primary FSGS. Until now, the relationship between minimal change disease and primary FSGS is still controversial; whether minimal change disease and primary FSGS represent different diseases or a continuum of one disease with different phenotypes is still unknown. There have been many studies focused on differentiation of the two glomerular diseases, and some investigators claimed several biomarkers that might facilitate the differentiation, including proteins expressed on podocytes and molecules excreted into urine, but none of these biomarkers were confirmed reliable. In this study, we found that the plasma supar levels, in part, of our patients with primary FSGS were higher than that in patients with minimal change disease and membranous nephropathy, which indicated that the elevation of plasma supar might be related to the pathogenesis of primary FSGS. In our study, we found that patients with tip variant had the lowest plasma supar level compared with the NOS and cellular variants. In renal histopathology, patients with severe interstitial fibrosis and tubular atrophy had higher plasma supar levels. Previous studies had demonstrated that the 5-year renal survival in patients with tip variant was significantly better as compared with patients with NOS and perihilar variants. 2 The degree of interstitial fibrosis was correlated with the value of initial serum creatinine and was predictive of the occurrence of end-stage renal disease. 21 Therefore, we speculated that the plasma supar might induce severe pathological lesions in the kidney. Furthermore, the plasma supar levels were associated with crescent formation in patients with primary FSGS at presentation. Previous studies had reported that uparinduced podocyte damage and proteinuria were dependent on the activation of b3 integrin on podocytes in kidney, which was associated with cell dynamics in vivo. 7,12,22 It was suggested that the supar bound to b3 integrin, induced podocyte damage, and then, by some unknown proinflammatory mechanisms, resulted in the formation of crescents. Although Maas et al. 11 in their letter had indicated that supar could not predict steroid responsiveness in patients with FSGS, they did not observe the changes of plasma supar levels during follow-up. In our study, with a limited number of patients with follow-up data, we found that the plasma supar level at presentation was not associated with therapeutic response, which was consistent with the study of Maas et al. 11 However, we found that in patients with complete remission, the plasma supar levels decreased significantly, but in patients with partial remission and treatment failure, the plasma supar levels remained unchanged or even elevated. These findings indicated again that supar might be involved in the pathogenesis of primary FSGS. However, our current study had a limited number of patients with follow-up data, and a further study with a larger cohort is needed to validate this result. 37 Kidney International (213) 84,

6 J Huang et al.: Soluble urokinase receptor in primary FSGS clinical investigation Regarding the fact that elevated supar levels could be identified in a few patients with other glomerular diseases and the wide spectrum of the assay results in the primary FSGS category, there were several possible reasons: first, FSGS is a histologic feature based on light microscopic examination with sufficient glomeruli, and therefore primary FSGS patients with limited glomeruli might be misdiagnosed as minimal change disease; second, concomitant infections and inflammation may result in the release of supar; third, the structure or subunits of supar in patients with primary FSGS and infection-induced supar might be not the same and might have different biological functions, although detectable using the same immunological methods. These needed further investigations to establish more specific methods to identify primary FSGS and to differentiate primary FSGS from other glomerular diseases. In conclusion, circulating supar was specifically elevated, in part, in patients with primary FSGS and might be associated with severe renal damage. Further studies with a larger cohort of patients with primary FSGS are needed to validate the importance of supar in patients with primary FSGS. MATERIALS AND METHODS Patients According to the definition of primary FSGS in the Columbia classification, patients with primary FSGS with complete clinical and pathological data, diagnosed in Peking University First Hospital between January 26 and January 212, were enrolled in this study. FSGS secondary to other primary glomerular diseases, such as IgA nephropathy, lupus nephritis, pauci-immune glomerulonephritis, and membranous nephropathy, were excluded. The pathological variants of the 74 patients with primary FSGS include 23 tip variants, 25 NOS variants, 21 cellular variants, 2 perihilar variants, 1 collapsing variant, and 2 advanced FSGS. The clinical and pathological data were collected at the time of presentation. Of the 74 patients, 24 patients also had follow-up data. Patients with nephrotic syndrome, defined as urinary protein excretion X3.5 g per 24 h with serum albumin o3 g/l were treated with corticosteroid combined with immunosuppressive agents including cyclophosphamide and cyclosporine A. Oral prednisone commenced at 1 mg/kg per day for up to weeks and then with subsequent tapering, oral cyclophosphamide at mg/kg per day for 3 months or cyclosporine A at 2 3 mg/kg per day with a trough concentration of B1 15 mg/ml for 6 12 months. All patients were treated with angiotensin-converting enzyme inhibitors and/or angiotensin receptor blockers. For evaluation of therapeutic response of patients with nephrotic syndrome, complete remission was defined as proteinuria p.15 g per 24 h with stable serum creatinine (no more than 25% increase in serum level from baseline). Partial remission was defined as proteinuria o3.5 g/24 h but 4.15 g per 24 h, with stable renal function in patients presenting with nephrotic syndrome. Treatment failure was defined as not reaching the criteria of partial remission. Moreover, 14 patients with minimal change disease, 29 patients with membranous nephropathy, 14 patients with secondary FSGS, and 56 age- and gender-matched normal subjects were used as disease and normal controls. According to Hepinstall s Pathology of the Kidney (6th Edition), 24 the pathologic diagnosis of secondary FSGS requires that a glomerular lesion falls within the morphologic spectrum of FSGS by light microscope but had segmental or less severe degree of foot process effacement, with or without clinically identifiable causes of FSGS. The 14 patients with secondary FSGS include 2 tip variants, 1 NOS variants, and 2 perihilar variant according to light microscopy. Of the 14 patients with secondary FSGS, 1 was with pre-eclampsia, 1 with Kimura disease, 2 with obesity, 2 with Alport syndrome, and 8 without identifiable factors. Informed consent was obtained from each patient for tissue and blood sampling. The study was in compliance with the Declaration of Helsinki Principles. Renal histopathology Renal biopsy was performed at the time of diagnosis. Renal specimens were evaluated with direct immunofluorescence, light, and electron microscopy, and were forwarded to two pathologists. Both pathologists examined the biopsies separately, being blinded to each other as well as patients clinical data. Differences in diagnosis between the two pathologists were resolved by re-reviewing the biopsies and coming to a consensus. For direct immunofluorescence, IgG, IgM, IgA, C3c, C1q, fibrinogen, and albumin were detected by fluorescein isothiocyanate conjugated antibodies (Dako, Copenhagen, Denmark) on frozen tissues. The fluorescence intensity was determined using a semiquantitative scale of to 4 þ. For light microscopy, paraffin sections were stained with hematoxylin and eosin, periodic acid Schiff, periodic acid-silver methenamine, and Masson s trichrome. For electron microscopy, in brief, the tissue was fixed in 2.5% glutaraldehyde and 1% osmium tetroxide, then dehydrated in graded acetone, and embedded in Epon 812 (Emicron, Shanghai, China). Ultrathin sections were cut at a thickness of 8 nm and placed on nickel grids. Then, ultrathin sections were stained with uranyl acetate and examined by a transmission electron microscope JEM-123 (JEOL, Tokyo, Japan). The renal biopsy findings were categorized according to the Columbia FSGS classification system. 23 Patients with any of the structural manifestations of FSGS were entered into the registry. Pathologic findings in the glomeruli, tubules, interstitial compartments, and vessels were described. The scoring of interstitial fibrosis and tubular atrophy was approached by renal pathologists using a modified previously reported system, and it was as follows: 21,25,26, normal; þ, 5% of the acreage of interstitium affected; and þþ, X5% of the acreage of interstitium affected in each specimen. The scoring of interstitium inflammatory cell infiltration was as follows: 21,25,26, normal; þ, 5% of the acreage of interstitium affected; and þþ, X5% of the acreage of interstitium affected in each specimen. Differences in scoring between the pathologists were resolved by re-reviewing the biopsies and thus reaching a consensus. Sample collection Plasma samples of patients were collected at the day of renal biopsy using disodium-edta as anticoagulant. The plasma samples from 56 age- and gender-matched healthy donors were collected as normal controls. The plasma was collected immediately after centrifugation at 2 g for 15 min at 4 1C. Then, the plasma was stored in aliquots at 8 1C until use. Repeated freeze/thaw cycles were avoided. Plasma samples from the 24 patients with follow-up were also collected and stored until use. Kidney International (213) 84,

7 J Huang et al.: Soluble urokinase receptor in primary FSGS Quantification of plasma supar We detected the concentration of plasma supar in human subjects using Quantikine Human upar Immunoassay (R&D Systems, Minneapolis, MN), following the manufacturer s protocol. In brief, the principle of the assay was a five-step procedure: (1) 96-well polystyrene microplates were precoated with a mouse monoclonal antibody against upar; (2) plasma samples were diluted to 1:5 and added to each well and incubated for 2 h at room temperature; (3) after incubation and washing, horseradish peroxidase conjugated polyclonal antibodies against upar were added and incubated for 2 h at room temperature; (4) after washing, substrate solution was added to each well and incubated for 3 min at room temperature while protecting from light; and (5) stop solution was added to each well and then the absorbance was recorded using an ELISA reader (Bio-Rad Laboratories, Philadelphia, PA) at 45/57 nm. The supar level of each sample was calculated using Curve expert 1.3 (Curve Expert Software, Chattanooga, TN). Statistical analysis Statistical analysis was performed using statistical software SPSS 16. (SPSS, Chicago, IL). Comparison of quantitative parameters was assessed using the nonparametric test between two nonnormally distributed variables or one normally with one nonnormally distributed variable. Comparison of paired variables was assessed using paired samples nonparametric test (for data that were not normally distributed). Spearman s correlation test was used to measure the correlation between two nonnormally distributed variables or one normally with one nonnormally distributed variable. A linear regression analysis was performed to investigate whether the elevated plasma supar was independent of renal function. In this model, supar levels were used as dependent variables, and creatinine clearance rate and groups of individual glomerular disease (including primary FSGS (reference), minimal change disease, membranous nephropathy, and secondary FSGS) were all included as independent variables. All statistical analyses were two tailed and Po.5 was considered as significant. Bonferroni correction was used for multiple comparisons. DISCLOSURE All the authors declared no competing interests. ACKNOWLEDGMENTS The technical support by Ying Zhang and Ji-chuan Liu was greatly appreciated. This work was supported by a grant of Chinese 973 Project (no. 212CB51772), National Natural Science Foundation of China to Innovation Research Group (no ). REFERENCES 1. Wharram BL, Goyal M, Wiggins JE et al. Podocyte depletion causes glomerulosclerosis: diphtheria toxin induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene. J Am Soc Nephrol 25; 16: Kriz W. 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