Angiopoietin-like-4 and Minimal Change Disease

Gabriel Cara-Fuentes, MD1; Alfons Segarra, MD2; Cecilia Silva-Sanchez, PhD3; Heiman

Wang, BS1; Miguel A. Lanaspa, PhD4; Richard J. Johnson, MD4; Eduardo H. Garin, MD1

From the 1Division of Pediatric Nephrology, Department of Pediatrics, University of Florida,

Gainesville, USA; and the 2 Division of Nephrology, Hospital Vall d'Hebron, Barcelona, Spain;

and the 3Proteomics and Mass Spectrometry, Interdisciplinary Center for Biotechnology

Research, University of Florida, Gainesville, USA; and the 4 Division of Renal Diseases and

Hypertension, Department of Medicine, University of Colorado, Denver, USA.

Address for Correspondence:

Eduardo H. Garin

Division of Nephrology

Department of Pediatrics

University of Florida

1600 SW Archer Rd. HD214

Gainesville, Fl 32610

Tel. No.: 352-273-9180 - Fax No.: 352-392-7107

E-mail: garineh@peds.ufl.edu

1

Abstract

Minimal Change Disease (MCD) is the most common type of nephrotic syndrome in

children. Angiopoietin-like-4 (Angplt4), reported to be expressed by podocytes during relapse,

has been proposed as mediator of proteinuria in MCD. The aim of this study was to evaluate the

role of Angptl4 as a biomarker and/or mediator of proteinuria in MCD. Patients with biopsy-

proven MCD, focal segmental glomerulosclerosis, membranous nephropathy (60, 52 and 52

respectively) and 18 control subjects were included in this study. Urinary and serum Angptl4

were measured by Elisa. Isoelectric point of Angplt4, detected in urine of MCD in relapse, was

determined by 2-D gel electrophoresis. Frozen kidney tissue sections were stained for Angptl4.

Our findings question the proposed roles of Angptl4 as marker and mediator of

proteinuria in MCD. We could not confirm the presence of Angptl4 in glomeruli of MCD in

relapse. The presence of Angptl4 with an acidic pI in the urine in MCD did not support the

hypothesis that hyposialylated molecules result in proteinuria by neutralizing Glomerular

Basement Membrane heparan sulfate negative charges. Finally, elevated Angptl4 levels in urine

was not a marker for MCD because was also found in patients with other glomerulopathies. The

increased urinary Angptl4 levels are likely the result, rather than the cause, of a leaky glomerular

filtration barrier.

Keywords: Angiopoietin-like-4, minimal change disease, nephrotic syndrome, podocyte

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Introduction

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,(1) is thought to be triggered by a circulating factor (s) yet to be

identified. (2) This factor is posited to induce changes in podocytes leading to increased

glomerular permeability to plasma proteins. Historically products from inflammatory cells such

as interleukins were presumed to trigger MCD though evidence supporting such assumption is

still lacking after more than 40 years of research.(3) More recently, microbial products have been

suggested to play an important role in triggering MCD relapse.(4-7) CD80 (4, 6, 8, 9) and

Angiopoietin-like-4 (Angptl4), (10) reported to be expressed by podocytes during relapse, have

been proposed as mediators of proteinuria in MCD.

Human Angptl4 is a 45-65 kDa glycoprotein highly expressed in liver and adipose tissue.

Podocyte Angtpl4 has been proposed to have a key role in the development of proteinuria in

MCD and in certain experimental models of nephrotic syndrome.(10-15) Podocyte Angptl4

overexpression has been reported to neutralize heparan sulfate negative charges in the glomerular

basement membrane (GBM) resulting into proteinuria in animal models of nephrotic syndrome.

(10) Reduction of proteinuria by sialic acid precursor in the experimental model led to Chugh et

al to hypothesize that Angptl4 produced by podocytes in MCD lacks sialic acid and thus when

bound to the glomerular basement membrane proteoglycans decreases GBM anionic charge

3

allowing negative charge molecules such as albumin to cross the capillary wall barrier into the

urinary space.(10)

The aim of this study is to evaluate the role of Angptl4 as a biomarker and/or mediator of

proteinuria in MCD.

Results

Demographics and laboratory tests are shown on Table 1 (see supplementary tables 1-4 for

details).

Urinary and serum Angptl4 in MCD, FSGS and MN

Urinary Angptl4 excretion was increased in patients with massive proteinuria regardless

of the underlying glomerular disease (MCD, focal segmental glomerulosclerosis –FSGS- and

membranous nephropathy –MN-) compared to control subjects (p<0.0001 for each group

(Figure 1). Urinary Angptl4 excretion was significantly elevated in MCD (Figure 1), FSGS and

MN patients (supplementary Figure 1) during relapse compared to the same group of patients

during remission (p< 0.0001 for each group) and to control subjects.

Angptl4 levels in serum from MCD, FSGS and MN patients in relapse were decreased

compared to control subjects though this difference did not reach statistical significance (Figure

2). 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 (supplementary Figure 2a and 2b

respectively).

There was a significant 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

(Supplementary Figure 3 a-c).

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Angptl4, serum albumin and proteinuria

During relapse, patients with MCD had higher degree of proteinuria compared to FSGS

and MN patients (p<0.0001) (Supplementary Figure 4a). The degree of proteinuria was not

statistical different among patients with FSGS and MN during relapse (Supplementary Figure

4a). There was a negative correlation between proteinuria and serum albumin levels in MCD and

MN patients during relapse (p<0.0001 and p 0.001 respectively) (Supplementary Figure 4b and

4c respectively). Such correlation did not reach statistical significance in the FSGS group

(Supplementary Figure 4d).

Urinary Angptl4 excretion in relapse showed correlation with proteinuria in the FSGS

and MN groups during relapse. Such correlation was not found for MCD patients during relapse

(Supplementary Figure 5). No correlation was found between serum Angplt4 levels and

proteinuria in MCD, FSGS and MN patients during relapse (Supplementary Figure 6).

Angptl4 expression in kidney tissue in MCD and other glomerulopathies

We studied the glomerular expression of Angptl4 in patients with MCD and other

glomerulopathies (Figure 3). Angptl4 staining was absent (as in control) or minimal in kidney

tissue from in 4 MCD patients in relapse, 1 patient with membranoproliferative

glomerulonephritis (MPGN) in relapse, 1 patient with recurrent FSGS, and in 2 patients with

class II lupus nephritis during remission. Compared to controls, glomerular Angptl4 was

overexpressed in kidney tissue from 2 MCD patients in remission, 2 patients with recurrent

FSGS, 3 FSGS patients in relapse, 1 patient with class IV lupus nephritis during relapse and 1

MPGN patient during remission.

Isoelectric point and molecular weight of urinary Angptl4.

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Using two-dimensional gel electrophoresis and western blot analysis, urinary Angptl4 in

a MCD patient in relapse was found to have a molecular weight (MW) of approximately 60 kDa

and an isoelectric point (pI) of 5.4 (Figure 4).

Discussion

The present study evaluates Angptl4 as a biomarker and mediator of proteinuria 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 and

variably expressed in subjects with MCD in remission, FSGS and MN, and absent in MCD

subjects in relapse. Evaluation of urinary Angptl4 also found it to not be cationic (pI ~5) which

would be expected if it was hyposialylated. These data suggest Angptl-4 is unlikely to be either a

biomarker or mediator of MCD.

Angptl4 was first posited as a mediator of proteinuria in the nephrotoxic serum (NTS)

model in rats. (10) In this model glomerular Angptl4 was up-regulated, and proteinuria was

reduced in rats that lacked Angptl4 (Angptl4 knockout rats). Subsequently glomerular Angptl4

overexpression was also observed in other animal models of nephrotic syndrome such as the

passive Heymann model of MN,(10, 15) the puromycin aminoglycoside model,(10) the Mna

Buffalo rat,(11) the Adriamycin model in mice (13) and in Zucker Diabetic Fatty rats.(11, 14)

The role of Angptl4 on proteinuria was subsequently evaluated using two transgenic (TG)

rat models that overexpressed systemic and podocyte-Angptl-4 (known as aP2-Angptl4 and

NPHS2-Angptl4 TG rat model respectively). (10) Based on these TG rat models, a dual role for

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Angptl4 in nephrotic syndrome was proposed, in which Angptl4 is secreted by podocytes as a

hyposialylated monomer or low order oligomer that induced neutralization of GBM heparan

sulfate negative charges and proteinuria while circulating Angptl4, produced mainly by adipose

tissue and liver as normosialylated oligomers reduces proteinuria by a presumed interaction with

endothelial cells and also hypertriglyceridemia by blocking lipoprotein lipase. (11) Based on

these studies, the authors proposed the TG rat overexpressing glomerular Angptl4 (NPHS2-

Angptl4 TG rat) as a model for MCD. However, the validity of the NPHS2-Angptl4 TG rat as

MCD model is challenged by the fact that proteinuria occurs slowly over months, and podocyte

foot process effacement was not noted until rats were 3 months old, whereas MCD is usually of

abrupt onset. (10) It is also not known if this model is steroid sensitive (as is the majority of

MCD subjects) or whether the lesion evolves over time to FSGS or remains with a MCD

histology.

Chugh’s group has previously reported increased glomerular Angptl4 by

immunohistochemistry in 5 patients with MCD compared to an unknown number of controls, all

from kidneys prior to transplantation.(10) It is unclear whether these MCD patients were in

relapse or remission. Similarly, Li et al also reported glomerular Angplt4 expression, by

immunostaining, in a non-specified number of MCD and MN patients during active nephrotic

syndrome, but they did not include normal controls or MCD patients in remission.(13) In

contrast, we found increased expression of Angptl4 compared to controls in 2 MCD patients

during remission, in 5 out of 6 patients with active FSGS, in 1 MPGN and 1 class IV lupus

nephritis patients during relapse and in 1 MPGN patient with mild proteinuria (Figure 3). The

reason for the difference in findings for Angptl4 in biopsies of MCD between our group and the

two prior studies is not clear. Like the others, we used a polyclonal antibody against Angptl4

7

containing the N-terminus domain (see supplementary table 5 for details). As D’Agati

comments regarding CD80 glomerular staining, another controversial subject,

immunohistochemistry is a tricky technique subject to numerous variables including “quantity of

antigen, type and duration of fixation and image exposure time and contrast”.(16)

We found increased urinary excretion of Angptl4 in patients with massive proteinuria

regardless of the underlying glomerular 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).(13) However, the significance of their data is unclear because

urinary Angptl4 was not standardized to urinary creatinine, and 1 of the 5 mPGN patients had no

proteinuria and another patient had non-nephrotic proteinuria whereas all MCD patients had

massive proteinuria. Moreover, authors did not provide the number of patients analyzed in each

group nor data on control subjects. In addition, Li et al found an increase urinary Angptl4

expression, by western blot, in patients with MCD, FSGS and MN with proteinuria compared to

that observed in mPGN patients, again consistent with a relationship of Angptl4 urinary

excretion to proteinuria rather than to MCD per se. (13)

The most likely source of Angptl4 detected in urine in MCD is freely filtered circulating

Angptl4 during the nephrotic stage. The molecular weight of Angptl4 found in urine of MCD in

relapse is ~50 kDa which is the molecular weight of Angptl4 found in the serum of these patients

(see below) and normal controls. This molecule can be freely filtered through a leaky glomerular

filtration barrier (GFB) as observed with other molecules with MW less than 100 kDa.(17)

Chugh’s group also found Angptl4 “fragments” (50-70 kDa) in urine from 1 MCD and 4 FSGS

patients in relapse as well as Angptl4 oligomers (~220 kDa) in urine from 4 MCD in relapse.(10)

8

Neither Li’s nor our group detected oligomers in the urine of MCD patients during relapse. The

lack of correlation between urinary and serum Angplt4 levels in MCD patients in our study is

likely due to the fact that the majority of circulating Angplt4 molecules presents as oligomers of

high molecular weight preventing its free passage through the GBM.(18)

Elevated Angptl4 plasma levels were reported by Chugh’s group in patients with

nephrotic syndrome due to MCD, FSGS, MN and crescentic glomerulonephritis compared to

control subjects.(11) Their data showed large variability (range from 49 to 710 ng/ml and

mean±SD 260.3±215 ng/ml in MCD patients compared to a range of 24 to 119 ng/ml and

mean±SD of 59.6±22.1 ng/ml in controls). In contrast, we found lower serum Angplt4 levels in

patients with MCD, FSGS and MN during relapse compared to controls which is consistent with

the increased urinary angiopoietin excretion observed in these patients. This opposite results

cannot be explained by the use of different source of antibodies as Chugh’s as our group used

goat antihuman polyclonal antibodies raised against same aminoacids (aa) (aa 26-406) (see

supplementary table 6). Thus, both antibodies should theoretically recognize the full Angptl-4

molecule. Furthermore, the mean plasma Angptl4 reported by Chugh et al in control subjects is

2-10 times higher than that published by other authors using same antibodies against Angptl4 (aa

26-406). (19-23) The mean serum levels of normal control subjects observed in our study is

rather similar to the ones described by these other authors. These contrasting results cannot be

explained because we did not fast our patients because no difference was seen between results in

our control patients when compared to levels observed in normal controls of other studies (see

supplementary table 6).

Chugh et al suggested that cationic (hyposialylated) Angplt4 might induce proteinuria in

this disease by neutralizing heparan sulfate negative charges, allowing the trans-capillary

9

movement to the Bowman space of negative charged molecules such as albumin. Consistent

with this hypothesis, he detected cationic (high pI) Angplt4 fragments from 1 MCD and 1 FSGS

patient and neutral pI oligomers in the MCD patient.(10) The molecular size and pI of Angptl-4

detected in urine is similar to that initially described, by the same group, in plasma of MCD

patients, so these “high pI” molecules found in urine most likely represent circulating Angptl4.

(10) In contrast, in our study, and Li et al’s study, (13) the urinary Angptl4 had a pI < 7. Thus,

our studies do not support a role for Angptl4 as a mechanism for neutralizing negatively charged

heparan sulfate in the GBM.

The hypothesis that MCD is due to a neutralization of heparan sulfate in the GBM has not

been confirmed in recent studies. Isolated rat GBM demonstrate little or no charge selectivity.

(24) Sieving curves measured in isolated GBM for Ficoll and its anionic derivative, Ficoll

sulfate, are indistinguishable.(25) Moreover, treating the isolated GBM with heparatinase to

remove heparan sulfate proteoglycan or adding protamine to neutralize GBM polyanions, had

little effect on the sieving of Bovine Serum Albumin. (26)

In transgenic mice models characterized by the absence of agrin and perlecan, despite a

significant reduction in the GBM anionic charge, mice did not develop proteinuria and the

fractional clearance of an anionic tracer was unchanged. (27) In a similar study, Chen et al.

knocked out Ext1 gene expression in podocytes mice halting polymerization of heparan sulfate

glycosaminoglycans of the proteoglycan core proteins secreted by podocytes.(28) The mice

showed foot process effacement and a significant decrease in heparan sulfate

glycosaminoglycans by immunohistochemical analysis but only mild albuminuria. Finally, Van

den Hoven et al. used mice overexpressing heparanase and evaluated the expression of different

heparan sulfate domains in the kidney with anti-heparan sulfate antibodies.(29)

10

Glycosaminoglycan-associated anionic sites were reduced about fivefold in the glomerular

basement membrane of transgenic mice, whereas glomerular ultrastructure and protein excretion

remained normal.

It is currently thought that is the cellular layers rather than the GBM responsible for the

glomerular charge selective barrier.(24) Therefore, based on the current concepts of glomerular

charge selective barrier and the role of GBM proteoglycans as charge barrier, the hypothesis

presented by Chugh of neutralization of GBM proteoglycans negative charges by binding of

hyposialylated Angplt4 to GBM is unlikely to explain the development of abrupt and massive

proteinuria seen in MCD patients.

In summary, our findings question the proposed roles of Angptl4 as marker and mediator

of proteinuria in MCD. We could not confirm the presence of Angptl4 in glomeruli of MCD in

relapse. The presence of Angptl4 with an acidic pI in the urine in MCD does not support the

hypothesis that hyposialylated molecules result in proteinuria by neutralizing GBM heparan

sulfate negative charges. Finally, elevated Angptl4 levels in urine is not a marker for MCD

because is also found in patients with other glomerulopathies. The increased urinary Angptl4

levels are likely the result rather than the cause, of a leaky glomerular filtration barrier.

Methods

Definitions. All patients included in this study had either biopsy-proven MCD, FSGS or

MN. Primary MCD was defined according to the established criteria by the International Study

for Kidney Diseases in Children.(30) Primary FSGS was defined according to the presence of

focal and segmental consolidation of the glomerular tuft with either negative

immunofluorescence or only segmental IgM or minimal C3 staining, and no electron dense

11

deposits on electron microscopy.(31) Primary MN was diagnosed based on renal biopsy findings

and lack of known secondary causes of MN. Secondary causes of MCD (Hodgkin’s lymphoma),

FSGS (HIV, drugs, hyperfiltration) and MN (hepatitis B, hepatitis C, HIV and systemic lupus

erythematous) were excluded by history, physical exam and laboratory tests. Genetic testing was

not obtained in patients included in this study.

Relapse was defined as the presence of proteinuria (urinary protein creatinine ratio > 2.0

mg/mg or 3 + or greater by using the tetrabromophenol-citrate buffer colorimetric qualitative test

–dipstick- or >3 grams of protein in 24 hour urine collection), serum albumin ≤3.5 g/d, and

edema. Complete remission was defined as negative proteinuria by dipstick or by urinary protein

creatinine ratio <0.2 mg/mg. Patients with urinary protein creatinine ratio greater than 0.2 but

lower than 2 were included in the remission group as they were considered in early remission

based on subsequent clinical course.

Patients. A total of 164 patients (18 children and 146 adults) with biopsy proven MCD,

FSGS and MN and 18 control subjects were included in this study (see table 1 and

supplementary tables 1-4 for details). Sixty patients had MCD (18 children and 42 adults) of

which 44 were studied during relapse and 26 during remission. Ten patients were studied during

both relapse and remission. Fifty-two patients had primary FSGS as their underlying glomerular

disease. Thirty-six and sixteen patients were studied in relapse and in remission respectively. Of

the 52 patients with primary MN, 36 were studied during relapse and 16 during remission.

Eighteen subjects, aged 5 to 19 years, served as control. These individuals were seen at the

University of Florida- Pediatric Nephrology Outpatient Clinic because of essential hypertension

(n=8), microscopic hematuria (n=2), nephrogenic diabetes insipidus (n=2), urinary tract infection

(n=1), solitary kidney (n=1), urinary incontinence (n=1), simple kidney cyst (n=1),

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hypoaldosteronism (n=1) and enuresis (n=1). None of these control subjects had any known

immunological disorder.

All children included in this study were followed at the University of Florida whereas all

adults were followed at Vall d’Hebron Hospital in Barcelona, Spain. The study was performed

according to the Declaration of Helsinki and approved by the Institutional Review Boards of

both Institutions. Informed consents, and assent when indicated, were obtained from all

participants.

Serum and urine collection. Blood samples from MCD, FSGS, MN patients and from

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. All samples (except for 24-hour urine collection samples) were

analyzed at the University of Florida.

Serum and urinary angiopoietin like-4. 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.

Serum albumin and urine protein and creatinine. Levels were measured by

Autoanalyzer. Proteinuria was measured as grams of protein in 24 hour urine collection and as

protein to creatinine ratio in random urine samples in 70 and 45 patients, respectively, studied

during relapse.

13

Reducing 2D-PAGE of protein concentrated from urine sample. Proteins were

concentrated form urine samples from 1 MCD patient in relapse using a ProteoSpin™ Urine

Protein Concentration Maxi Kit (Norgen Biotek; Thorold, ON, Canada). An aliquot of

concentrated protein was precipitated with acetone and pellet was solubilized in 2D buffer (8 M

urea, 2 M thiourea, 4% CHAPS, 40 mM DTTand 2% of IPGbuffer pH 3-10). Protein

concentration was determined by EZQ protein quantitation assay (Thermo scientific). A total of

100 µg of protein were loaded on Immobiline DryStrip 7cm 3-10 NL (GE healthcare life

sciences; Piscataway, NJ) and isoelectrofocused on an IPGphor 3 unit. 2D-PAGE was run on a

4-12% Bis-Tris ZOOM gel using the NuPAGE system.

Western Blot. Immediately after protein electrophoresis, gel was transferred to a PDFV

membrane on a semidry transfer unit using 0.18 mA/cm2 for 2 hours. Membrane was blocked

with 5% non-fat milk and then incubated at 4°Celsius overnight with primary Angptl-4 antibody

(catalog number#AF3485) at a dilution of 1:2000. After washing, the membrane was incubated

with mouse anti-goat IgG-HRP, as secondary antibody, at a dilution of 1:5000 (catalog

number#sc-2354, Santa Cruz Biotechnology. The blot was visualized using GE healthcare

Amersham ECL Western Blotting Detection Reagents (catalog number#RPN2109; GE

Healthcare, Piscataway Township, NJ). After western blot, picture was analyzed in SameSpot

software (Totallab, UK) to calculate molecular weights and isoelectric points.

Immunohistochemistry of Angptl4 of human kidney tissue specimens. 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-

14

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 supplementary table 7.

Statistical analysis. Data graphics and statistical analysis were 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. P < 0.05 was regarded as statistically significant. D’Agostino-Pearson

omnibus test was used to assess data distribution. Data were expressed as mean±standard

deviation (SD) for normally distributed data and median +/- interquartile 25%-75% (IQ) when

data were not normally distributed.

Disclosure

Authors declare no conflict of interest.

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