The authors have declared that no competing interests exist.
Minimal Change Disease (MCD) is the most common type of nephrotic syndrome in children. Angiopoietin-like-4 (Angplt4) has been proposed as mediator of proteinuria in MCD. The aim of this study was to evaluate the role of Angptl4 as a biomarker in MCD.
Patients with biopsy-proven primary MCD, focal segmental glomerulosclerosis, membranous nephropathy (60, 52 and 52 respectively) and 18 control subjects had urinary and serum Angptl4 measured by Elisa. Frozen kidney tissue sections were stained for Angptl4.
Angptl4 was not identified in glomeruli of MCD patients in relapse. Urinary Angptl4 levels were elevated in MCD in relapse as well as in patients with massive proteinuria due to other glomerular diseases.
Neither serum nor urine Angptl4 appear to be good biomarkers in MCD. Elevated urinary Angptl4 n glomerular disease appears to reflect the degree of proteinuria rather than any specific disease.
Nephrotic syndrome is defined by the presence of massive proteinuria, hypoalbuminemia, edema and hypercholesterolemia. Minimal Change Disease (MCD), the most common type of nephrotic syndrome in children [
Human Angiopoietin-like-4 (Angptl4) is a 45–65 kDa glycoprotein highly expressed in liver and adipose tissue. Podocyte Angtpl4 has been proposed to have a role in the development of proteinuria in MCD [
The aim of this study is to evaluate the role of Angptl4 as a biomarker in MCD.
All patients had either biopsy-proven primary MCD, focal segmental glomerulosclerosis (FSGS) or membranous nephropathy (MN) [
164 patients (18 children and 146 adults) with biopsy proven MCD, FSGS or MN and 18 control subjects were included in this study (
Groups→ | MCD |
FSGS |
MN |
Control |
|||
---|---|---|---|---|---|---|---|
Variable | Relapse | Remission | Relapse | Remission | Relapse | Remission | |
44 | 26 | 36 | 16 | 36 | 16 | 18 | |
30.7±20.2 | 20.2±16.1 | 48±12.7 | 44.1±12.2 | 54.1±13 | 51.4±9.1 | 13.3±3.9 | |
26/18 | 14/12 | 28/8 | 12/4 | 23/13 | 10/6 | 12/6 | |
2.2±0.5 | 4.1±0.3 |
2.9±0.6 | 3.8±0.4 | 2.5±0.5 | 3.3±0.2 |
4.7±0.2 |
|
12±3.5 |
NA |
6.6±3.5 |
NA | 7.4±3.1 |
NA | NA | |
7.4±5.4 |
0.3±0.4 |
6.6±3.4 |
0.7±0.6 | 6.4±4.7 |
0.8±0.6 | 0.06±0.03 |
|
>300 mg/dl |
Negative |
NA | NA | NA | NA | Negative |
|
0.7±0.2 |
0.6±0.3 |
1.1±0.3 | 1.4±0.7 |
1±0.3 | 1±0.2 |
0.6±1.9 |
aMCD minimal change disease,
bFSGS focal segmental glomerulosclerosis,
cMN membranous nephropathy,
dn refers to the number of patients,
eNA not available, data expressed as mean±SD.
Children and adult subjects were followed at the University of Florida and at Vall d’Hebron Hospital in Barcelona (Spain) respectively. The study was performed according to the Declaration of Helsinki and approved by the Institutional Review Board of the University of Florida (IRB protocol#481–2008) and Vall d’Hebron Hospital [(AC/I(AG)158/2013(3822)]. Written informed consents were obtained from all participants and from caretakers/guardians of minors. Written assents were also obtained when indicated.
Blood samples from patients and control subjects were centrifuged for 5 minutes at 2400 RPM and serum collected and stored at −80°C. Urine samples from the above mentioned patients and controls were also stored at -80°C until samples were analyzed.
Angplt4 levels were measured using a commercially available Enzyme-Linked Immunosorbent Assay (ELISA) (Sigma-Aldrich, catalog number RAB0017). Serum Angplt4 levels were expressed as ng/ml and urinary Angptl4 as ng/g of creatinine.
Proteinuria, measured by autoanalyzer, was expressed as grams of protein in 24 hour urine collection and as protein to creatinine ratio in random urine samples.
Cryosections of kidney biopsy from patients were fixed with pre-cooled ice old acetone for 10 min. The sections were wash twice with PBS and then incubated with block solution (5% goat serum and 5% donkey serum in PBS) for 30 min. Afterwards, the sections were incubated with anti-synaptopodin antibody (American Research Products, INC) at room temperature for 1 hour, followed by rabbit polyclonal anti-Angptl4 antibody (Catalog number#sc-66806, Santa Cruz, 1:50) for 1 hour. After 3 times of PBS wash, each for 5 minutes, the sections were incubated with chicken anti-mouse 488 and chicken anti-rabbit 594 Alexa Fluor secondary antibodies (Thermo Fisher, 1:1000) for 1 hour. After 3 times of PBS wash, 5 minutes each, the sections were incubated with DAPI for counterstaining. The images were then taken and analyzed with confocal microscopy (Leica). Clinical data of patients whose renal tissue was stained are provided in
Statistical analysis was performed using the statistical software GraphPad Prism 6. The non-parametrical tests, Kruskal–Wallis and Mann–Whitney U, were applied to evaluate differences between the groups. Spearman and Pearson correlation coefficient were calculated to determine statistical correlation between two numerical variables as indicated.
Demographics and laboratory tests are shown on
Urinary Angptl4 excretion was increased in patients with massive proteinuria regardless of underlying glomerular disease (MCD, FSGS and MN) compared to control subjects (p<0.0001 for each group (
(A) Urinary Angptl4 in MCD, FSGS, MN patients and control subjects. (B) Urinary Angptl4 in FSGS and MN patients during relapse and remission. MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients, data expressed as mean±SD.
Serum Angptl4 levels were decreased in MCD, FSGS and MN patients in relapse compared to control subjects, although this difference did not reach statistical significance (
Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients, data expressed as mean±SD.
No statistical differences were observed in serum Angptl4 levels among patients with MCD, FSGS and MN patients during relapse compared to the same group of patients during remission and in those patients during remission compared to controls (
There was a positive correlation between urinary and serum Angptl4 levels in MN patients in relapse (p 0.02) but not in the MCD and FSGS groups during relapse (
During relapse, patients with MCD had a higher level of proteinuria compared to FSGS and MN patients (p<0.0001) (
Urinary Angptl4 excretion in relapse correlated with proteinuria in the FSGS and MN groups. This positive correlation was not found for MCD patients during relapse (
Angptl4 staining was absent (as in control) in glomeruli from 4 MCD patients in relapse, 1 patient with membranoproliferative glomerulonephritis (MPGN) in relapse, and 1 of 6 with FSGS and heavy proteinuria (
Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, LN lupus nephritis, MPGN membranoproliferative glomerulonephritis, n refers to the number of patients, Green, blue and red stains represent Angptl4, DAPI and synaptopodin respectively. Yellow lines represent scale bars. Magnification x200.
Two patients with class II lupus nephritis in remission showed minimal staining for glomerular Angptl4. Compared to controls, glomerular Angptl4 was overexpressed in kidney tissue from 2 MCD patients in remission, 5 patients with FSGS and proteinuria, 1 patient with class IV lupus nephritis during relapse and 1 MPGN patient during remission.
We evaluated Angptl4 as a biomarker in MCD. The primary finding was that the serum and urinary excretion of Angptl4 were not specific for MCD, and there was no correlation of either serum or urine Angptl4 with proteinuria in MCD patients. Glomerular Angptl4 immunohistochemistry was negative in control subjects, variably expressed in subjects with MCD in remission, FSGS and MN, and absent in MCD subjects in relapse. Thus, this study could not document urine or serum Angptl- 4 to be a biomarker of MCD.
One group previously reported increased glomerular Angptl4 by immunohistochemistry in 5 patients with MCD compared to controls, all from kidneys prior to transplantation [
Elevated plasma Angptl4 levels has been reported in patients with nephrotic syndrome due to MCD, FSGS, MN and crescentic glomerulonephritis compared to control subjects [
We documented marked elevations in urine Angptl4 in patients with massive proteinuria regardless of the underlying disease (MCD, FSGS and MN) compared to the same group of patients during remission and to normal controls. Li et al reported higher urinary Angptl4 levels in MCD in relapse compared to five patients with mesangial proliferative glomerulonephritis (mPGN) of which two of the latter had absent or mild proteinuria [
Chugh’s group has presented experimental evidence supporting a role for podocyte Angptl4 as a mediator of proteinuria in MCD and has suggested that it acts by altering the charge barrier of the glomerular capillary wall. Our data do not prove or disprove this hypothesis, although we would suggest that the variable expression of Angptl4 that we observed in glomeruli may reflect nonspecific binding of Angptl4 to glomerular structures, such as binding to extracellular matrix [
In summary, we could not confirm the presence of Angptl4 in glomeruli of MCD patients in relapse. Elevated urinary Angptl4 is not a specific marker for MCD and more likely reflects nephrotic range proteinuria.
Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients, data expressed as mean±SD.
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Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients.
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MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients.
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Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients.
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Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MN membranous nephropathy, n refers to the number of patients.
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MCD minimal change disease, UPC urine protein to creatinine ratio, Angptl4 angiopoietin-like-4, F female, M male, NA not available, * excluded from mean as it represents protein detected by dipstick, Negative refers to negative protein by dipstick, SD standard deviations, IQ interquartile 25–75% percentile, data presented as mean±SD and median (IQ) when data were not normally distributed, † non normally distributed data.
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FSGS focal segmental glomerulosclerosis, UPC urine protein to creatinine ratio, Angptl4 angiopoietin-like-4, F female, M male, NA not available, SD standard deviations, IQ interquartile 25–75% percentile, data presented as mean±SD and median (IQ) when data were not normally distributed, † non normally distributed data.
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MN membranous nephropathy, UPC urine protein to creatinine ratio, Angptl4 angiopoietin-like-4, F female, M male, NA not available, SD standard deviations, IQ interquartile 25–75% percentile, data presented as mean±SD and median (IQ) when data were not normally distributed, † non normally distributed data.
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UPC urine protein to creatinine ratio, Angptl4 angiopoietin-like-4, F female, M male, NA not available, SD standard deviations, IQ interquartile 25–75% percentile, negative refers to negative protein by dipstick, data presented as mean±SD and median (IQ) when data were not normally distributed, † non normally distributed data.
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Angptl4 angiopoietin-like-4, MCD minimal change disease, FSGS focal segmental glomerulosclerosis, MPGN membranoproliferative glomerulonephritis, * individuals not included in any of the studied groups. Data from these individuals were not included in
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Angptl4 angiopoietin-like-4, MW molecular weight, kDa kilodalton, aa aminoacids.
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Angptl4 angiopoietin-like-4, aa aminoacids, y year-old, N refers to the number of patients, SD standard deviation, SEM standard error of mean, IQ interquartile, * “Elisa assay virtually identical to the Duoset Elisa Angptl4 offered commercially by R&D (DY3485)”.
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