Recurrence of idiopathic focal segmental glomerulosclerosis (FSGS) following kidney transplantation occurs in a large percentage of patients. Accurate prediction of recurrence and elucidation of its pathogenesis are major therapeutic goals. To detect differential proteins related to FSGS recurrence, proteomic analysis was performed on plasma and urine samples from 35 transplanted idiopathic FSGS patients, divided into relapsing and nonrelapsing. Several proteins were detected increased in urine of relapsing FSGS patients, including a high molecular weight form of apolipoprotein A-I, named ApoA-Ib, found exclusively in relapsing patients. This finding was verified by Western blot individually in the 35 patients and validated in an independent group of 40 patients with relapsing or nonrelapsing FSGS, plus two additional groups: FSGS-unrelated patients showing different proteinuria levels (n = 30), and familial FSGS transplanted patients (n = 14). In the total of 119 patients studied, the ApoA-Ib form was detected in 13 of the 14 relapsing FSGS patients, and in one of the 61 nonrelapsing patients. Only one of the 30 patients with FSGS-unrelated proteinuria tested positive for ApoA-Ib, and was not detected in familial patients. Urinary ApoA-Ib is associated with relapses in idiopathic FSGS and warrants additional investigation to determine its usefulness as biomarker of relapse following transplantation.
protein AMBP (precursor of alpha-1-microglobulin, bikunin and trypstatin)
modified apolipoprotein A-I
familial FSGS transplanted patients
focal segmental glomerulosclerosis
mass to charge ratio
non-relapsing FSGS transplanted patients
patients with proteinuria unrelated to FSGS
relapsing FSGS transplanted patients
retinol binding protein
Focal segmental glomerulosclerosis (FSGS) is now understood as a group of diseases that produce progressive glomerular scarring and often, nephrotic syndrome. Severe cases may be unresponsive to treatment and progress to end-stage renal failure . The secondary forms of FSGS have known causes (mutations in specific podocyte genes, viral infection induced, drug toxicity or adaptation of the kidney to work overload), whereas the primary or idiopathic form is caused by a circulating factor of unknown nature, such as the proposed suPAR [2, 3].
A serious problem related to FSGS is frequent relapse of the condition following kidney transplantation, sometimes a few hours after organ replacement . Disease recurrence often leads to graft loss, requiring a new transplant. The probability of survival of the new graft is lower than the first; hence, repeated recurrence and kidney transplantation is not uncommon in these patients. Specific peritransplantation therapeutic measures have been proposed to reduce the risk of relapse, with variable success [4, 5].
FSGS is usually diagnosed by histological findings in a biopsy from a steroid-resistant nephrotic syndrome patient. Nonetheless, biopsy is an invasive procedure that may fail to detect FSGS lesions in early, prescarring stages, as in an immediate posttransplant relapse, making difficult to establish the precise diagnosis .
Identification of proteins useful as biomarkers that will enable prompt diagnosis and prognosis of recurrent FSGS would be beneficial for these patients and might additionally provide new insights into the pathogenic mechanisms of the idiopathic type. Thus, this study was designed to investigate blood and urine differential proteins specific of relapse in kidney transplant recipients with idiopathic FSGS. We found that a form of apolipoprotein A-I (ApoA-I) is present almost exclusively in urine of relapsing idiopathic FSGS patients.
This study was planned in three steps: (1) identification of differential proteins in a test group of selected patients using proteomics; (2) verification of the findings individually in the same test group using immunoblot; and (3) validation on an independent group of patients, plus other patient groups with similar diseases using immunoblot. The protocol was approved by the hospital ethics committee (PR-IR 103/2008), and informed consent was obtained from all participants. The study was conducted in accordance with the principles of the Declaration of Helsinki.
Differential protein identification by proteomics was done on pooled plasma and urine samples from two groups of patients with histological diagnoses of idiopathic FSGS who had received a kidney transplant, excluding cases with genetic base or familial history of FSGS. The relapsing group (R) included eight patients and the nonrelapsing group (NR) comprised 27 patients. Verification was carried out on the same samples, but individually.
A relapse was diagnosed in patients with proteinuria >3 g/day or in patients with proteinuria <3 g/day and FSGS histology during the first year posttransplant.
The validation samples came from an independent set of patients. The relapsing group (R) was composed by six patients and the non-relapsing (NR) by 34 patients. In addition to R and NR, two new groups were included in the validation set to check the specificity of the marker: group F consisted of 14 transplanted patients with familial FSGS, and group P contained 30 patients with proteinuria unrelated to FSGS, including 17 transplant recipients and 13 nontransplanted patients.
Fifteen milliliters of urine were concentrated by ultrafiltration (Amicon Ultra 3K, cut-off of 3 kDa) to a volume of 500 μL. Protein concentration was determined using the bicinchoninic acid method (Pierce BCA; Thermo-Fisher Scientific, Rockford, IL, USA).
It was done as described previously . Briefly, plasma or concentrated urine were applied to a 24-cm immobilized pH gradient strip (Immobiline DryStrip 3–10L; Amersham Biosciences Europe, Freiburg, Germany) and focused in an IPGphor system (Amersham Biosciences Europe). Focused strips were equilibrated sequentially with reducing and alkylating SDS buffer, placed on a 16 × 24 cm SDS-PAGE gel and run in an Ettan Dalt 6 system (Amersham Biosciences Europe). Gels were stained with colloidal Coomassie, digitized (calibrated densitometer GS800 Bio-Rad, Hercules, CA, USA), and analyzed (PDQuest 6.2, Bio-Rad Laboratories).
Differential spots were excised, digested with trypsin, and analyzed on an Ultraflex TOF-TOF (Bruker Daltonics, Bremen, Germany). Calibrated spectra were processed using FlexAnalysis 2.2 software (Bruker Daltonics). Peak lists were generated using the signals in the 800–4000 mass:charge ratio (m/z) range, with a signal:noise threshold of >3. Keratin and trypsin autolysis peptides were removed from the peak list.
Differential spots were analyzed on an Esquire HCT IT mass spectrometer (Bruker Daltonics) coupled to a nano-HPLC (Ultimate; LC Packings, the Netherlands). Trypsin digested samples were loaded onto a 75-mm id, 15-cm PepMap nanoseparation column (LC Packings). Peptides were eluted by an acetonitrile gradient through a PicoTip emitter nanospray needle (New Objective, Woburn, MA, USA) onto the ionization source. MS/MS fragmentation (1.9 s, 100–2800 m/z) was performed on two of the most intense ions. The peak list was created with DataAnalysis 3.4 software (Bruker Daltonics).
Proteins were identified using Mascot v.2.2.04 (Matrix Science, London, UK) to search the International Protein Index (IPI)-Human 3.26 database, with a mass tolerance <100 ppm for MALDI-TOF and 1.5 Da (precursor) and 0.5 Da (fragment) for MS/MS, allowing two missed cleavages for trypsin (cysteine carbamidomethylation, fixed and methionine oxidation, variable). The positive identification criteria were, for MALDI-TOF, statistical significance of the score, and for MS/MS spectra, individual MASCOT score for each MS/MS spectrum higher than the corresponding identity threshold score.
Western blots were done in SDS-PAGE gels with tris-glycine-SDS buffer. In the absence of a reliable loading control for urine, protein concentration was accurately determined as described above, and a fixed amount of urinary protein was used per well. Normal plasma served as the positive control. Molecular weight markers (Prestained Broad Range or Precision Plus; BioRad) were used in each experiment. Proteins were transferred to PVDF membranes, probed with anti ApoA-I (goat polyclonal PAB9966 Abnova, Jhongli, Taiwan plus HRP-antigoat secondary antibody P0449 Dako, Glostrup, Denmark). Proteins were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech and detector LAS3000, Fujifilm, Tokyo) and quantified (QuantityOne, Bio-Rad).
Results were first analyzed by ANOVA. A post hoc test (Tukey-HSD) was applied to determine the influence of individual factors. When ANOVA requirements were not met (variance homogeneity and normality of the sample assessed by standardized skewness and kurtosis), the nonparametric Kruskal–Wallis test was used. When comparing two groups, Student t-test and Mann–Whitney U-test were used respectively. Homogeneity of qualitative variables was tested with the chi-square test. Statistical analyses were done with StatGraphics (Manugistics Inc., Herndon, VA, USA).
There were no statistically significant differences between the test and validation patients for R and NR groups, but for proteinuria and plasma creatinine of the NR group (Table 1). Clinical data for each group are shown in the Table 2. Individual data are presented in the Tables S1 and S2.
Table 1. Test and validation patients. Clinical data comparison
Data expressed as mean ± SEM.
R = relapsing FSGS; NR = non-relapsing FSGS; t = Student t-test; u = Mann–Whitney U-test.
R, urine protein
6.390 ± 1.264
9.213 ± 3.868
n = 8
n = 6
5.51 ± 0.41
5.59 ± 0.33
n = 8
n = 6
3.21 ± 0.18
3.30 ± 0.24
n = 8
n = 6
1.87 ± 0.35
2.83 ± 0.69
n = 8
n = 6
NR, urine protein
0.115 ± 0.012
0.481 ± 0.081
p < 0.0002 u
n = 26
n = 34
7.340 ± 0.089
7.049 ± 0.177
n = 23
n = 31
4.429 ± 0.074
4.208 ± 0.095
n = 24
n = 30
1.270 ± 0.081
1.821 ± 0.191
p < 0.04 u
n = 26
n = 34
Table 2. Clinical data by group
Data expressed as mean ± SEM.
Different from NR and F.
Different from NR, F and P.
R = relapsing FSGS; NR = nonrelapsing; FSGS F = familial FSGS; P = patients with FSGS-unrelated proteinuria; Statistical analyses: A = ANOVA; KW = Kruskal–Wallis.
Identification of differential proteins in test patients
Proteomic analyses of plasma samples did not detect differences between relapsing and nonrelapsing FSGS groups. However, important differences were observed in urine (Figure 1). To exclusively detect proteins from kidney, or plasma proteins modified by the kidney in urine, the analysis software did not take into account protein spots that were also present in plasma, thus eliminating proteins that leak directly from blood through the damaged glomerular barrier. The software detected 41 differential spots (Figure 1). Mass spectrometry identified seven different proteins in 33 of these spots (Figure 1; Table S3). Five of these proteins (RET4, A1AT, AMBP, HEMO and TTHY) had been previously associated to unspecific proteinuria or kidney damage, remaining zinc-alpha-2-glycoprotein (ZAG) and ApoA-I as candidates for further analysis.
Although ZAG was found only in one spot, six differential ApoA-I spots were detected in urine from relapsing patients. The Figure 2 shows the differential ApoA-I spots, plus the nondifferential spot SSP3217, which corresponds to the regular ApoA-I found in plasma. The most intense differential ApoA-I form detected was spot SSP3304 (Figure 2B), which had a higher molecular weight than the regular form (spot SSP3217).
Verification of ZAG in test patients
Individual Western blot analysis of the urine protein (20 μg) from test patients detected specific ZAG bands in both R and NR groups. After quantifying the intensity of the specific ZAG band, ANOVA analysis showed that relapsing patients had higher ZAG than nonrelapsing patients (p < 0.0001).
Verification of ApoA-Ib in test patients
Sixty micrograms of urinary protein were used to determine ApoA-I by Western blot individually in the test patients. All the R patients showed an ApoA-I band with an apparent molecular weight higher than the control blood sample, whereas it was absent in all the NR patients (Figure 3A). These results were consistent with the proteomic findings: the higher molecular weight ApoA-I band observed in Western blot corresponded to the higher molecular weight spot detected in 2DE (SSP3304). We named ApoA-Ib to this urinary form of ApoA-I, exclusive of the relapsing patients and detected by both proteomic and Western blot analysis.
Validation of ZAG in independent patients
ZAG was determined by Western blot in the validation patients. ANOVA showed statistical differences (p < 0.0001) between the groups, but post hoc test indicated that groups R (1892 ± 259 mean ± sem, arbitrary intensity units) and P (1981 ± 811) were similar among them and significantly higher than groups NR (475 ± 163) and F (837 ± 474), whose results were also similar. ZAG findings did not enable differentiation between relapsing FSGS patients and those with proteinuria unrelated to FSGS, indicating that ZAG was not specifically related to FSGS relapses after transplantation.
Validation of ApoA-Ib in independent patients
The urinary ApoA-Ib band was observed in all the validation relapsing patients but one, patient R112, who had low proteinuria (1 g/day) and had undergone plasmapheresis treatment (Figure 3A). Furthermore, ApoA-Ib was detected in only one of the 34 nonrelapsing FSGS patients studied (NR45). Some patients showed more than one ApoA-I band, probably corresponding to the other isoforms detected on proteomic analysis (e.g. R107; Figure 3A). The 14 patients in the familial FSGS group did not show the ApoA-Ib band, nor did the 30 patients with proteinuria unrelated to FSGS (Figure 3B), with the exception of one case (P186). There was no significant relationship between proteinuria and presence of ApoA-Ib in urine (two-way ANOVA; p = 0.4625 for ApoA-I, p < 0.0001 for the patient group). Faint ApoA-I bands were observed in some patients with FSGS-unrelated proteinuria when the blot was overexposed (Figure 3B), but with a molecular weight lower than that of the ApoA-Ib. The ApoAI-b form was not detected in plasma of relapsing patients, only the regular plasma form of ApoA-I (Figure 3C).
Specificity of ApoA-Ib in FSGS relapses
ApoA-Ib results in all patients are summarized in Figure 4. There were no significant differences in the percentage of ApoA-Ib positive cases between NR, F and P groups (chi-square test; p = 0.4351), but the relapsing group (R) showed a significantly higher percentage of positive patients than all the other groups studied (chi-square test; p < 0.00001). The appearance of ApoA-Ib in urine achieved a sensitivity of 92.8% and a specificity of 98.1% in respect to the clinical diagnosis of FSGS relapse, calculated for all the patients studied in the test and validation sets.
Proteomic analysis detected seven differential proteins found exclusively in urine of relapsing idiopathic FSGS patients. From these, five proteins (A1AT, AMBP, RET4, TTHY and HEMO) were discarded for further studies because they are not specifically associated to relapsing FSGS, as reported previously: urinary A1AT increases in acute kidney injury  and nephrotic syndrome of diverse origin . Urinary AMBP is associated with impaired tubular function , glomerulopathies  and renal transplantation . Impaired tubular function increases urinary RET4 , and urinary TTHY and HEMO increase in glomerulopathies of diverse origin .
To avoid the bias caused by the different proteinuria levels, the same fixed amount of urinary protein was used in Western blots for all patients. The group of FSGS-unrelated proteinuria patients, with levels of plasma albumin and proteinuria similar to R group, was added to check whether the differential proteins were artifacts caused by the proteinuria. This P group included patients with proteinuria of different cause, with different selectivity levels.
ZAG is involved in lipid mobilization . It is elevated in urine during energy-demanding conditions , but decreases in acute kidney injury . However, on individual quantification in the validation set, ZAG levels were similar to those found in patients with FSGS-unrelated proteinuria, thus lacking the specific link to FSGS.
ApoA-I is a small protein (28 kDa), component of the HDL particles and absent in the urine of healthy subjects and most patients with glomerular proteinuria [17-19]. The presence of ApoA-I in urine is associated to postrenal proteinuria or hematuria . Accordingly, urinary ApoA-I has been detected in patients with bladder cancer using proteomic techniques [19, 21]. Our proteomic analysis did not detect regular ApoA-I differentially expressed in urine, but several modified ApoA-I forms. One of them, ApoA-Ib, was consistently present in urine from relapsing FSGS patients, whereas it was absent in all the other patients, including the FSGS-unrelated proteinuria, and it was not found in plasma. ApoA-Ib was a heavier form of ApoA-I, and the molecular weight increase could correspond to posttranslational changes of the regular ApoA-I form, such as the previously described acylation  or glycation . Although most nonrelapsing and familial FSGS transplanted patients showed no ApoA-I bands at all, some patients with proteinuria unrelated to FSGS showed lower molecular weight forms of ApoA-I, but lacked the ApoA-Ib band.
We found only three discrepant values in the 119 patients studied. The two false positive values may be the result of a putative lack of sensitivity of the clinical diagnosis. The glomerular scarring is preceded by an undetermined period of action of the permeability factor, with active proteinuria but no detectable histological injury. This fact, together with the focal nature of the disease, indicate that a negative biopsy does not preclude the existence of idiopathic FSGS . In our patients, several clinical diagnosis of relapse with positive ApoA-Ib were accompanied by biopsies without lesions of FSGS, indicating that the appearance of ApoA-Ib precedes the histological damage and suggesting that ApoA-Ib could be useful to increase the sensitivity of the clinical diagnosis.
There is no evidence to determine whether ApoA-Ib is a consequence of relapsing FSGS or is related to its pathogenesis, but there are indications on the implication of HDL or their component in FSGS. In a preliminary study in a pediatric population, we detected a differential form of ApoA-II, other component of HDL, in plasma of idiopathic FSGS patients, but not in genetic ones . It has been demonstrated that components of HDL inhibit glomerular albumin permeability induced by serum from patients with FSGS . Genetic variants of HDL components have been associated to idiopathic FSGS, such as ApoE  or paraoxonase , and specially the apolipoprotein L1 . This would result in altered HDL particles, which have been implicated in the pathogenesis of FSGS .
The findings of this study might be clinically relevant. ApoA-Ib can be detected in urine of relapsing FSGS patients, thereby enabling differentiation from other types of proteinuria and facilitating specific therapy. Another possibility worthy of further study is the predictive capacity of ApoA-Ib: early detection of ApoA-Ib in urine of transplanted FSGS patients could allow prediction of relapse before the clinical manifestations are evident, allowing initiation of prompt preventive treatment to curb or even preclude relapse of the disease. Lastly, our findings provide new data supporting the relationship between HDL particles and idiopathic FSGS.
This study was funded by Astellas Pharma, Fundación Mutua Madrileña and Fundación Renal Iñigo Alvarez de Toledo. The authors wish to thank Dr. D. Seron and Dr. F.J. Moreso for the critical reading of the manuscript, and Celine L. Cavallo for the text correction.
GREAT group (Spanish Group of new projects in transplantation): Carme Cantarell (Hospital Vall d'Hebron, Barcelona), Lluis Guirado (Fundació Puigvert, Barcelona), Miguel Angel Gentil (Hospital Virgen del Rocio, Sevilla), Juan C. Ruiz San Millan (Hospital Marques de Valdecilla, Santander) Carlos Jiménez (Hospital Universitario La Paz, Madrid), Jaime Sanchez-Plumed (Hospital La Fe, Valencia), Sofia Zárraga (Hospital de Cruces, Barakaldo), Javier Paul (Hospital Miguel Servet, Zaragoza), Ana Sanchez Fructuoso (Hospital Clínico, Madrid), Vicens Torregrosa (Hospital Clínic, Barcelona), Ricardo Lauzurica (Hospital Germans Trias i Pujol, Badalona), Angel Alonso (Hospital Juan Canalejo, A Coruña), Ernesto Gómez (Hospital Central de Asturias, Oviedo), Ana Fernández (Hospital Ramón y Cajal, Madrid), Auxiliadora Mazuecos (Hospital Puerta del Mar, Cadiz), Domingo Hernández, Dolores Burgos (Hospital Carlos Haya, Malaga), Alberto Rodriguez (Hospital Reina Sofia, Cordoba), Antonio Osuna (Hospital Virgen de las Nieves, Granada), Antonio Franco (Hospital General, Alicante), Luisa Jimeno (Hospital Virgen de la Arrixaca, Murcia), Aurelio Rodríguez (Hospital Universitario de Canarias, Tenerife).
The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.