1. Blocking Ligand Occupancy of the ␣V␤3 Integrin
Inhibits the Development of Nephropathy in Diabetic
Pigs
Laura A. Maile, Walker H. Busby, Katherine A. Gollahon, William Flowers,
Nikol Garbacik, Stefani Garbacik, Kara Stewart, Timothy Nichols,
Dwight Bellinger, Amit Patel, Paul Dunbar, Matt Medlin, and David Clemmons
Department of Medicine (L.A.M., W.H.B., K.A.G., T.N., D.B., A.P., P.D., M.M., D.C.), University of North
Carolina School of Medicine, Chapel Hill, North Carolina 27599; and Department of Animal Science
(W.F., N.G., S.G., K.S.), North Carolina State University, Raleigh, North Carolina 27695
Hyperglycemia stimulates secretion of ␣V␤3 ligands from vascular cells, including endothelial cells,
resulting in activation of the ␣V␤3 integrin. This study determined whether blocking ligand oc-
cupancy of ␣V␤3 would inhibit the development of diabetic nephropathy. Ten diabetic pigs re-
ceived an F(ab)2 fragment of an antibody directed against the extracellular domain of the ␤3-
subunit, and 10 received a control IgG F(ab)2 for 18 weeks. Nondiabetic pigs excreted 115 Ϯ 50 ␮g
of protein/mg creatinine compared with control F(ab)2-treated diabetic animals (218 Ϯ 57 ␮g/mg),
whereas diabetic animals treated with the anti-␤3 F(ab)2 excreted 119 Ϯ 55 ␮g/mg (P Ͻ .05).
Mesangial volume/glomerular volume increased to 21 Ϯ 2.4% in control-treated diabetic animals
compared with 14 Ϯ 2.8% (P Ͻ .01) in animals treated with active antibody. Diabetic animals
treated with control F(ab)2 had significantly less glomerular podocin staining compared with
nondiabetic animals, and this decrease was attenuated by treatment with anti-␤3 F(ab)2. Glomer-
ular basement membrane thickness was increased in the control, F(ab)2-treated diabetic animals
(212 Ϯ 14 nm) compared with nondiabetic animals (170 Ϯ 8.8 nm), but it was unchanged (159.9 Ϯ
16.4 nm) in animals receiving anti-␤3 F(ab)2. Podocyte foot process width was greater in control,
F(ab)2-treated,animals(502Ϯ34nm)comparedwithanimalstreatedwiththeanti-␤3F(ab)2 (357Ϯ
47 nm, P Ͻ .05). Renal ␤3 tyrosine phosphorylation decreased from 13 934 Ϯ 6437 to 6730 Ϯ 1524
(P Ͻ .01) scanning units in the anti-␤3-treated group. We conclude that administration of an
antibody that inhibits activation of the ␤3-subunit of ␣V␤3 that is induced by hyperglycemia
attenuates proteinuria and early histologic changes of diabetic nephropathy, suggesting that it
may have utility in preventing the progression of this disease complication. (Endocrinology 155:
4665–4675, 2014)
Diabetic nephropathy represents a major cause of end-
stage renal disease (ESRD), and progression to severe
chronic kidney disease occurs in 35% of patients (1). Ag-
gressive lowering of blood glucose prospectively attenu-
ates the subsequent rate of development of ESRD (2, 3),
but the molecular mechanisms by which chronic hyper-
glycemia leads to ESRD are not well defined. Although
activation of the receptor for advanced end glycation
products, stimulation of protein kinase C signaling, aber-
rant activation of the hexosamine pathway, activation of
mitochondrial oxidative stress, and activation of latent
TGF␤ have been investigated (4–9), definitive proof that
attenuation of their activation in animal models will pre-
venttheprogressionofnephropathyhasbeenlimited.One
major limitation is that several animal models, although
developing albuminuria, either do not develop progressive
histologic changes or, if they develop some histologic
changes, these do not progress to the point where glomer-
ISSN Print 0013-7227 ISSN Online 1945-7170
Printed in U.S.A.
Copyright © 2014 by the Endocrine Society
Received April 16, 2014. Accepted August 19, 2014.
First Published Online August 29, 2014
Abbreviations: EM, electron microscopic; ESRD, end-stage renal disease; GBM, glomerular
basement membrane; NS, not significant; PAS, periodic acid Schiff; PFW, podocyte foot
width; RIPA, radioimmunoprecipitation assay; SHP, src homology phosphatase; STZ, strep-
tozotocin; WT-1, Wilms tumor protein.
D I A B E T E S - I N S U L I N - G L U C A G O N - G A S T R O I N T E S T I N A L
doi: 10.1210/en.2014-1318 Endocrinology, December 2014, 155(12):4665–4675 endo.endojournals.org 4665
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2. ular filtration is decreased (10, 11). Therefore, predicting
whether inhibition of specific pathophysiologic changes in
animal models will correlate with successful treatment of
human nephropathy has been difficult.
The role of glomerular capillary wall in the pathogen-
esis of diabetic nephropathy has been a focus of recent
interest. Renal biopsies in humans with type 2 diabetes
have demonstrated that changes in endothelial fenestra-
tions, glycocalyx, and area of podocyte surface in contact
with glomerular capillary wall correlate with the devel-
opment of proteinuria and progressive reduction in cre-
atinine clearance (12). Multiple integrin receptors are ex-
pressed in glomerular endothelium and podocytes,
including ␣V␤3 (13). Our studies have demonstrated that
chronicexposureofvascularendothelialandsmoothmus-
cle cells to hyperglycemia results in stimulation of the syn-
thesis of several ␣V␤3 ligands, including thrombospon-
din, osteopontin, and vitronectin (14). This leads to an
increase in ligand occupancy of ␣V␤3, which in turn stim-
ulates phosphorylation of the ␤3 -subunit (14). Blocking
␤3-subunit phosphorylation has been shown to inhibit
hyperglycemia-induced changes in capillary permeability
in vitro (15). Additionally, inhibition of ␣V␤3 in vascular
endothelial and smooth muscle cells leads to decreased
stimulation of both the MAPK and phosphoinoside-3 ki-
nase pathways in response to IGF-I stimulation (14, 15).
Furthermore,itinhibitsIGF-I-stimulatedcellproliferation
and capillary tube formation (15, 16). Because IGF-I and
␣V␤3 activation have been implicated in the pathogenesis
of diabetic nephropathy, we determined whether block-
ade of the ␣V␤3 integrin using a monoclonal antibody
would result in inhibition of albuminuria and the histo-
logic changes that occur in response to chronic hypergly-
cemia in diabetic pigs.
Materials and Methods
All reagents were obtained from Sigma unless stated otherwise.
Purification of the F(ab)2 fragment of the anti-C ␤3
monoclonal antibody (C-loop) and control IgG
Balb/c mice were immunized using a peptide immunogen
(amino acids 177–184 of the human ␤3-subunit) (Table 1) that
was conjugated to keyhole limpet hemocyanin. Monoclonal an-
tibody-producing clones were prepared and clones selected as
described (17). The antibody-producing cells were grown in
RPMI 1640 medium containing low IgG serum (Gibco), 10-
␮g/mL IL-6, 5mM glutamine, penicillin (100 U/mL), and strep-
tomycin (100 ␮g/mL). After achieving a density of 2 ϫ 105
cells/
mL, they were transferred to roller bottles and maintained at that
density by adding fresh medium every 2 days until the volume
reached 600 mL. Sufficient media were collected to purify the
antibody used here. Medium was concentrated by ammonium
sulfate purification and then purified over a protein G Sepharose
column. The purified material (3 g/L) was cleaved using the Ficin
cross-linked to agarose (100 mg/L; Pierce, Thermo Fisher Sci-
entific, Inc). After 96 hours at 37°C, the digested IgG was applied
to a protein-A Sepharose column to remove the noncleaved in-
tact IgG and Fc fragment. The material that was not retained
contained the F(ab)2 fragment, and it was further purified by
protein G Sepharose and an aliquot analyzed by SDS-PAGE fol-
lowed by immunoblotting for IgG and silver staining. A single
band (molecular weight 120 kDa) was detected. Immunoblot-
ting with an anti-Fc antibody showed that greater than 99% of
the Fc fragment had been removed and less than 0.2% intact IgG
remained. The F(ab)2 fragment from the control, mouse IgG
(Sigma) was prepared using Ficin digestion and the same chro-
matographic procedures. The F(ab)2 fragments were quantified
using an antigen capture-based ELISA; 96-well plates were
coated with the immunogen peptides (50 ␮g/mL) conjugated to
BSA. After washing with 0.05% Tween and blocking with 2%
BSA, the test samples were added and incubated for 1 hour at
22°C, and an alkaline phosphatase conjugated secondary anti-
body, goat antimouse (Jackson ImmunoResearch) was added
followed by diethanolamine developer containing Nitro phenyl
phosphate for 15 minutes. The results quantified using spec-
trometry at 405 nm. The specificity of the antibody was deter-
Table 1. Antibodies Used
Peptide/
Protein
Target
Antigen
Sequence
(if known)
Name of
Antibody
Manufacturer,
Catalog Number,
and/or Name of
Individual Providing
the Antibody
Species Raised
in; Monoclonal or
Polyclonal
Dilution
Used
DOI or
Publication
Data
␤3 Integrin CYDMKTTC Anti-␤3 monoclonal antibody (C-loop) David Clemmons Mouse monoclonal IgG 0.5 mg/kg
␤3 Integrin CYDMKTTC Anti-␤3 F(ab)2 David Clemmons Mouse monoclonal IgG 0.5 mg/kg or 2 ␮g/mL
Mouse IgG Mouse IgG Control, mouse IgG Sigma N/A 0.5 mg/kg
Mouse IgG Mouse IgG Control F(ab)2 Sigma N/A 0.5 mg/kg
CNLEENDHCNPKYIEFPISEARI Anti-␤3 antibody (R2949) David Clemmons Rabbit polyclonal 1:500
p-Tyr antibody (PY99) Santa Cruz Biotechnology,
Inc (sc-7020)
Mouse monoclonal IgG2b 1:1000
WT-1 antibody (C-19) Santa Cruz Biotechnology,
Inc (sc-192)
Rabbit polyclonal 1:1000
Peroxidase-AffiniPure goat
antirabbit IgG (H ϩ L)
Jackson ImmunoResearch
(111-035-003)
Goat polyclonal 1:20 000
Peroxidase-AffiniPure goat
antimouse IgG (H ϩ L)
Jackson ImmunoResearch
(115-035-003)
Goat polyclonal 1:10 000
4666 Maile et al ␣V␤3 and Diabetic Nephropathy Endocrinology, December 2014, 155(12):4665–4675
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3. mined by analyzing its binding to whole-cell lysates using West-
ern immunoblotting. The results showed that a single band was
detected that corresponded to the estimated molecular weight of
the ␤3-subunit.
Testing the purified anti-␤3 F(ab)2 for bioactivity
Human umbilical vein endothelial cells were isolated and
maintained as described (15). Cultures were grown in medium
containing 25mM glucose for 72 hours and then incubated with
serum-free medium containing 0.2% BSA overnight. Control
IgG F(ab)2 or the anti-C-loop ␤3 F(ab)2 (10nM) was added for
4 hours. Cultures were lysed in modified radioimmunoprecipi-
tationassay(RIPA)buffer,lysateswerethenincubatedovernight
with an anti-␤3 antibody (1:500 dilution) and immune com-
plexes precipitated with protein-A Sepharose. Pellets were re-
suspended in 45 ␮L of Laemmli sample buffer with 0.2M di-
thiothreitol, and proteins were immunoblotted using an
antiphosphotyrosine antibody (PY-99; Santa Cruz Biotechnol-
ogy, Inc).
Induction of diabetes and antibody administration
Male Yorkshire pigs (ages 7–9 wk), obtained from North
Carolina State University, were maintained according to guide
for Laboratory Animals National Institutes of Health publica-
tion number 85/2320. To induce hyperglycemia, animals re-
ceived streptozotocin (STZ), 50 mg/kg iv daily, for 3 days. One
week after STZ infusion, fasting glucose rose from 93 Ϯ 19 to
372 Ϯ 43 mg/dL in animals that subsequently received control
IgG and from 95 Ϯ 9 to 391 Ϯ 46 mg/dL in the animals who went
on to receive the active anti-C-loop ␤3 F(ab)2. One week after
STZ infusion, the animals were placed on a high-fat diet con-
taining 1% cholesterol, 20% beef tallow, and cholic acid for 4
weeks. Cholesterol was measured 3 times (4, 8, and 12 wk) dur-
ing the study and dietary composition adjusted accordingly to
maintain serum cholesterol levels more than 500 mg/dL. Cho-
lesterol content of the feed was varied based on individual cho-
lesterol measurements (cholesterol content varied between 0.5%
and 2%).
After 2 weeks of hyperglycemia plus high-fat diet, 10 animals
received either the purified anti-␤3 F(ab)2 (0.5 mg/kg) and 10
received (0.5 mg/kg) of control IgG F(ab)2 every 72 hours for 18
weeks.
All animals were weighed daily and were observed for food
intake. Blood was obtained twice daily for capillary glucose mea-
surements. Neutral protamine Hagedorn insulin was adminis-
tered twice daily sc (2–45 U per injection) to maintain glucose
between 350 and 500 mg/dL.
For control, nondiabetic animals, kidney and urine samples
from control, nondiabetic male pigs were obtained from differ-
ent animals in the same herd. The animals were of similar age and
weight as compared with the diabetic animals. These animals
were not part of the main study. Tissue was harvested using the
same protocol by the same personnel.
Preparation of kidney for histologic or biochemical
analyses
After euthanasia, the left kidney was clamped, removed, and
immediately perfused with saline/heparin, followed by PBS and
then fixed by perfusion with formalin (10%). The right kidney
was also removed, dissected, and pieces of cortex were either
snap frozen or fixed in 2% paraformaldehyde and 2.5% glutar-
aldehyde in 0.1M phosphate buffer.
Preparation of kidney for electron microscopic
(EM) analysis
After fixation, the kidney sections were rinsed with PBS fol-
lowed by postfixation with 1% osmium tetroxide in 0.1M Na2
PO4 (pH 7). The samples were dehydrated through a series of
graded ethyl alcohols from 70% to 100% and then passed
through, 2 changes of 100% propylene oxide, and finally into a
50:50 mixture of propylene oxide and the embedding resin (Em-
bed 812; Electron Microscopy Sciences). Fresh 100% embed-
ding media were added overnight, changed, and then maintained
for 12–18 hours at 60°C to allow polymerization. Resin blocks
were thick sectioned at 1–2 ␮m using an Ultracut UCT (Leica)
and stained with Toluidine blue. The appropriate blocks were
thin sectioned using a diamond knife (Diatome; Electron Mi-
croscopy Sciences) at 70–90 nm (silver to pale gold using color
interference), and sections were placed on nickel mesh grids.
After drying, the sections were stained with the heavy metal
uranyl acetate for contrast. The grids were viewed on a Tecnai
BioTwin (FEI). Digital images are taken with an AMT charged
coupled device camera.
EM image analysis
The glomerular basement membrane (GBM) thickness was deter-
minedbytheorthogonalinterceptmethod(18).A200-by200-nmgrid
was overlayed on each image (using ImageJ). The GBM width was
measured, on a line orthogonal to the edge of the peripheral GBM, at
each point where the grid intersected the endothelial side of the mem-
brane to the outer lining of the lamina rara external underneath the
cytoplasmic membrane of the epithelial foot process. The harmonical
mean was then calculated:
Th (harmonical mean) ϭ (8/3⌸) ϭ 106
/M ϫ 1h
1h ϭ n/͚(1/L) (ϭ harmonic mean apparent thickness)
Where n, number of measurements made in tissue; L, GBM in
absolute units; M, final magnification.
Glomeruli comprising a total of 14 images, and at least 200
measurements were used and the mean GBM thickness was cal-
culated for each animal (18, 19).
The average podocyte foot width (PFW) was calculated
from 20 images at ϫ9000. The length of GBM in each image
was measured, and then discrete separate podocyte foot pro-
cesses making contact with the GBM were counted. PFW was
calculated as (⌺ GBM length/⌺ foot processes) and expressed
in nm (20).
Preparation of kidney sections for histological
analysis
Formalin fixed kidney was placed in a tissue cassette. Kidney
sections were embedded in paraffin, and 10 ϫ 3- to 4-␮m serial
sections were prepared.
Two slides (with 2 sections of kidney/slide) were then stained
with either hematoxylin and eosin or periodic acid Schiff (PAS).
Protocols from the Animal Models of Diabetic Complications
Consortium were used.
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4. Anti-Wilms tumor protein (WT-1) and podocin
staining of kidney sections
One slide from each animal (prepared from frozen tissue) was
stained with an anti-WT-1 antibody as a marker of podocytes
(21) according to the following protocol: Animal Models of Di-
abetic Complications Consortium Protocols (http://www.
diacomp.org/). Briefly, slides were stained with the anti-WT-1
antibody (C-19; Santa Cruz Biotechnology, Inc) and antibody
binding visualized with the Vectastain ABC kit (Vector Labora-
tories). In addition, 1 slide for each animal was stained with
NPHS2 anti-Podocin (Abcam ab82108) at 1:1000 and antirab-
bit-horseradish peroxidase at 1:250 and developed with di-
aminobenzidine reagents. To determine whether the antibody
treatment altered proliferative changes that occur in the tubu-
lointerstitium, 1 slide from each animal was stained with Picro-
sirius red. Paraffin-embedded sections were deparaffinized and
then stained with Picrosirius red phosphomolybodic acid hy-
drate (Polysciences), then rinsed in 70% ethanol followed by
100% ethanol and xylene (22).
Image analysis of histologic sections
Antipodocin-, Picrosirius red-, and PAS-stained slides were
digitized at a magnification of ϫ20 using a Nikon Eclipse 80i
with Surveyor mosaic imaging software (Confocal and Multi-
phon Imaging Core, Department of Neurosciences, University of
North Carolina). Bitmap images were converted to TIFF images
using Adobe Photoshop. TIFF images were then opened in Im-
ageScope. The entire kidney section was scanned, and individual
glomeruli were captured (1/image) to obtain at least 50 glomer-
uli/animal. Only glomeruli with the stalk region of the glomer-
ulus were selected. This assumed a bisected glomerulus. Individ-
ual glomerular images were opened in ImageJ. The area of the
glomerulus within the capsule was recorded, then the area of the
mesangial matrix was recorded. Fifty glomeruli were analyzed
per animal. For each glomerulus, the next calculations were
performed:
Percent of the glomerulus occupied by mesangial matrix
% ϭ [(area of the mesangial matrix)/
(area of the glomerulus)] ϫ 100
The volume of the glomerulus occupied by mesangial matrix
was also estimated. The area of the glomerulus was assumed to
be a circle. The radius of the glomerulus was used to calculate an
estimated volume of the glomerulus using the equation:
V ϭ
4
3
␲r3
The volume of the mesangial matrix was calculated by cal-
culating the area (as above). The mesangial matrix area was then
assumed to be circular, and the radius was calculated from the
area allowing an estimate of mesangial matrix volume to be
calculated as described for the glomerulus (23).
Mesangial matrix expansion was then defined as the per-
centage of the cross-sectional area of the glomerular tuft made
up by the mesangium calculated by the formula (Vv(mes/
glom). The average of all 50 glomeruli was used to generate a
single data point for each pig. The data points for each pig
within a group were averaged to determine the final data point
for that group.
After podocin staining, nonoverlapping sections were
scanned at ϫ4 magnification on the MSL BX61 using the Retiga
camera. ϫ4 images were exported to ImageJ. To determine the
podocin signal intensity, the background staining intensity (the
parenchyma excluding any area without tissue or with glomer-
uli) was calculated. Next, the pixel intensity for each individual
glomerulus was calculated using at least 20 glomeruli/animal.
The background signal was subtracted to derive a change in
intensity. The average change in intensity was calculated for each
animal to derive 1 data point/animal. The results are expressed
as relative pixel intensity over background.
Isolation of glomeruli from pig kidneys
Kidneys were removed from control pigs under anesthesia
then cut into 5-mm pieces, placed into 15 mL of PBS, washed 3
times, and then applied to a 190-␮m metal filter and ground
through the mesh. The 190-␮m filter was removed, and this
process was repeated using a 104-␮m filter and then a 73-␮m
filter. The tissue that remained was collected and washed, cen-
trifuged,andresuspendedinmedium.Thesuspensionwasplated
on a collagen coated dish and incubated at 37°C. After 4–5 days,
glomeruli attached and culture outgrowths were visible. After
7–9 days, cells were trypsinized, centrifuged, and then resus-
pended in RPMI 1640 supplemented containing 1% fetal bovine
serum and passed over a 40-␮m pore cell strainer to remove
residual glomerular cores consisting of mesangial and endothe-
lial cells. The cells were replated and grown to a density of 700
000/25-cm2
flask. The medium for some cultures was supple-
mented to 25mM glucose. At confluence, cells were incubated in
serum-free medium (maintaining glucose levels according to cul-
ture medium condition) overnight and exposed to the anti-␤3
F(ab)2 (2 ␮g/mL) for 4 hours.
Biochemical analysis of glomerular lysates and
kidney homogenates
Cells obtained from the glomerular isolation were lysed in mod-
ified RIPA buffer, and kidneys were homogenized in RIPA and
centrifuged to generate a clarified lysate. Lysates (200 ␮g of total
protein) were immunoprecipitated with an anti-␤3 antibody
(R2949)followedbyseparationbySDS-PAGE.Phosphorylationof
␤3wasvisualizedbyimmunoblottingwithanantiphosphotyrosine
antibody(PY-99;SantaCruzBiotechnology,Inc).Imagesweredig-
itized, and relative ␤3 phosphorylation was quantified using Im-
ageJ.Equalamountsofproteinfromeachkidneyhomogenatewere
also separated by SDS-PAGE (8%), and total ␤3 protein was visu-
alizedbyWesternimmunoblottingwiththeanti-␤3antibodyR294
(14). ␤3 phosphorylation was expressed as average arbitrary scan-
ning units for each treatment group.
Urine
Urine was collected directly from the bladder, placed on ice,
centrifuged, and frozen at Ϫ20°C before analysis. The University
of North Carolina Animal Clinical Chemistry and Gene Expres-
sion Laboratories performed the tests for total protein and cre-
atinine using an Automatic Chemical Analyzer (Johnson &
Johnson’s VT350).
Blood glucose
Blood glucose was measured in capillary blood samples. A
lancet was used to prick the area at the base of the ears to obtain
4668 Maile et al ␣V␤3 and Diabetic Nephropathy Endocrinology, December 2014, 155(12):4665–4675
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5. blood for the glucose readings. A Precision Xtra glucometer with
appropriateglucosetestingstripswasusedthroughoutthestudy.
Serum cholesterol
Blood was drawn using a venipuncture into serum separating
tubes. Blood was allowed to clot and then centrifuged to obtain
serum, which was frozen at Ϫ20°C until analysis. Serum cho-
lesterol was measured using a VetScan VS2 Blood Chemical
Analyzer.
ELISA for measuring drug levels
An antigen capture ELISA was used to measure drug levels in
serum samples as described previously (17).
Statistical analysis
GraphPad Prism (GraphPad Software) was used for all sta-
tistical analyses. The statistical significance of the differences
between 2 treatment groups was compared using the Mann-
Whitney test, with P Ͻ .05 being considered significant. For the
comparison of 3 groups, the Kruskal-Wallis test was used to
determine whether a difference between any treatment group
was present, and P Ͻ .05 was also considered significant. If
significant, then direct comparisons between 2 groups were per-
formed using the Mann-Whitney test. Data shown are mean Ϯ
SEM unless otherwise stated.
Results
Study animal summary
Bodyweightwascomparableinbothtreatmentgroups,
and it increased progressively with time, such that there
was no difference between the control F(ab)2 treated and
the animals treated with anti-␤3 F(ab)2 (Table 2). Cho-
lesterol values were significantly elevated after 4 weeks on
diet and continued to increase during the first 12 weeks.
There were no significant differences between the 2 treat-
ment groups in cholesterol at any time point (Table 2).
One week after STZ administration, mean glucose was
increased and it remained elevated, and mean glucose val-
ues for all determinations were comparable between the 2
groups (P ϭ not significant [NS]) (Table 2).
Drug levels
Antibody levels were determined 4 weeks of treatment.
Administration of the anti-␤3-F(ab)2 monoclonal anti-
body achieved peak plasma concentrations of 3.0 Ϯ 0.4
␮g/mL 4 hours after injection and declined to 0.2 Ϯ 0.2
␮g/mL at 72 hours after injection.
Proteinuria
Kidney weight in the group that received the anti-␤3
F(ab)2 was 250 Ϯ 18 g as compared with 287 Ϯ 12 g in the
animals that received control F(ab)2 (P ϭ NS). Urinary
protein at the end of the treatment interval is shown in
Figure 1 (mean Ϯ SEM). The animals receiving control
F(ab)2 had a mean value of 218 Ϯ 57-␮g protein/mg cre-
Table 2. Changes in Metabolic Parameters During the Study
Start
Start
Injection Week 4 Week 8 Week 12 Week 15 Week 18 Entire Study
Glucose
(mg/dL)
(C) 97.3 Ϯ 18.7 419 Ϯ 74 397 Ϯ 69 375 Ϯ 69 351 Ϯ 87 372 Ϯ 67 384 Ϯ 66 383 Ϯ 70
(T) 95.2 Ϯ 17.0 388 Ϯ 93 412 Ϯ 62 368 Ϯ 86 362 Ϯ 65 393 Ϯ 44 396 Ϯ 51 387 Ϯ 81
Cholesterol
(mg/dL)
(C) 292 Ϯ 38 444 Ϯ 71 899 Ϯ 294 1022 Ϯ 318 ND 538 Ϯ 161 726 Ϯ 208
(T) 284 Ϯ 33 464 Ϯ 96 868 Ϯ 270 995 Ϯ 246 ND 473 Ϯ 137 700 Ϯ 185
Weight (kg)
(C) 27.6 Ϯ 3.1 42.0 Ϯ 6.5 67.1 Ϯ 12.6 89.2 Ϯ 13.0 99.5 Ϯ 14.5 123 Ϯ 15.0 153 Ϯ 13.1 106.4 Ϯ 13.7
(T) 28.0 Ϯ 3.2 42.4 Ϯ 5.4 70.5 Ϯ 13.3 92.3 Ϯ 13.4 105 Ϯ 12.4 129 Ϯ 12.0 151 Ϯ 14.9 109.6 Ϯ 12.2
SI unit conversion: glucose (mmol/L) ϭ ϫ 0.5555, cholesterol (mmol/L) ϭ ϫ 0.0259. Mean Ϯ SD. ND, not determined; C, control; T, treated.
Figure 1. Urinary protein excretion (␮g/mg creatinine) after 18 weeks
of treatment. Mean Ϯ SEM, diabetic ϩ anti-␤3, n ϭ 10; diabetic ϩ
control IgG F(ab)2, n ϭ 10; nondiabetic, n ϭ 5. *, P Ͻ .05 when
diabetic control F(ab)2 is compared with anti-␤3 F(ab)2 or nondiabetic.
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6. atinine. The diabetic animals that received the anti-␤3
F(ab)2 active antibody had a mean value of 119 Ϯ 45
␮g/mg (P Ͻ .05 when the anti-␤3 F(ab)2-treated group is
compared with the control F(ab)2 animals). The nondia-
betic animals had a mean value of 115 Ϯ 50 ␮g/mg.
Kidney histology
The mesangial index (the percentage of the glomerulus
that stains PAS positive) was greater, eg, 35 Ϯ 5.2%, in the
diabetic animals receiving control F(ab)2 compared with
the diabetic animals that received the anti-␤3 F(ab)2,
27.4 Ϯ 3.8% (P Ͻ .02) (Figure 2). This change was also
seen when mesangial volume was quantified. Mesangial
volumeexpressedaspercentageofglomerularvolumewas
significantly greater (21 Ϯ 2.4%) in the diabetic animals
that received control F(ab)2 compared with control, non-
diabetic pigs (13 Ϯ 1.5%) (P Ͻ .05). This change was not
present in the animals that received anti-␤3 F(ab)2 14 Ϯ
2.1% (P Ͻ .02) compared with diabetic IgG animals (Fig-
ure 2B). Representative glomerular sections are shown in
Figure 2C.
Podocyte number and podocin staining
Sections that were stained with the WT-1 antibody
showed fewer podocytes in the diabetic animals treated
with IgG F(ab)2 compared with control animals. This dif-
ference was attenuated with the anti-␤3 F(ab)2 (Figure
3A). To quantify changes in podocytes, podocin staining
intensity was determined. The entire glomerular area was
calculated, and podocin pixel intensity was expressed as
change in podocin intensity compared with background.
Nondiabetic animals had a pixel intensity of 32.0 Ϯ 0.3 U.
Staining was significantly decreased (eg, 12.2 Ϯ 3.5) in the
diabetic animals that received control F(ab)2 (P Ͻ .03)
(Figure 3B). In contrast, the diabetic animals that received
anti-␤3 F(ab)2 had a significantly greater level of podocyte
staining 31.7 Ϯ 3.4 compared with the IgG, F(ab)2-treated
animals (P Ͻ .03). To determine whether the antibody had
an effect on tubulointerstitial changes, kidney sections
were stained for type I and III collagen. The kidneys from
the diabetic animals that received the control F(ab)2
showed increased staining of the tubular basement mem-
brane, Bowman’s capsule, and in several areas of the in-
terstitium compared with control kidney sections (Figure
4A). Staining intensity was 4.92 Ϯ 0.71 U in the diabetic
animals and 0.097 Ϯ 0.06 in the nondiabetic pigs. It was
significantly less in the animals that received the anti-␤3
F(ab)2 (2.9 Ϯ 0.54 U), compared with diabetic animals
that received control F(ab)2 (P Ͻ .05) (Figure 4B).
Figure 2. Mesangial changes. The results were obtained from digital images generated using PAS-stained kidney sections. In each case, at least
20 individual glomeruli from each animal were measured to obtain 1 data point/animal. A, Comparison of the mesangial matrix area as a
percentage of the total glomerular area. B, Comparison of the mesangial volume as a percentage of the glomerular volume. A and B, Results are
the mean Ϯ SEM. Diabetic ϩ anti-␤3 F(ab)2, n ϭ 5; diabetic IgG F(ab)2, n ϭ 6; nondiabetic, n ϭ 2 (A). *, P Ͻ .05 when diabetic IgG F(ab)2
treatment is compared with nondiabetic control or diabetic ϩ anti-␤3 F(ab)2 treatment. B, *, P Ͻ .05 when diabetic IgG F(ab)2 is compared with
nondiabetic and **, P Ͻ .02 when it is compared with anti-␤3 F(ab)2. C, Representative PAS-stained images.
4670 Maile et al ␣V␤3 and Diabetic Nephropathy Endocrinology, December 2014, 155(12):4665–4675
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7. GBM thickness and PFW
Nondiabetic animals showed an average GBM thick-
ness of 182 Ϯ 7.1 nm (harmonic mean), whereas it was
significantly greater (204.4 Ϯ 5.1 nm) in the diabetic an-
imals who received control F(ab)2 (P Ͻ .05) (Figure 5A).
In contrast, GMB thickness was 187.4 Ϯ 6.4 nm in the
group that received the anti-␤3 F(ab)2, which was not
statistically different from nondiabetic animals and was
significantly decreased compared with the animals that
received IgG (P Ͻ .01). An electron micrograph showing
these differences in a representative kidney is shown in
Figure 4A. PFW was also significantly greater in the dia-
betic animals treated with control anti-␤3 F(ab)2 (503 Ϯ
15 nm) compared with nondiabetic animals 251 Ϯ 4.0 nm
(P Ͻ .005) (Figure 5B). Treatment with anti-␤3 F(ab)2
yielded a significantly lower value of 359 Ϯ 21.2 (P Ͻ .01,
compared with diabetic, control F(ab)2-treated animals;
and P ϭ NS compared with nondiabetic pigs).
Demonstration of inhibition of target activation
Glomerular cell cultures were prepared from pig kid-
neys. The cultures were enriched in podocytes as shown by
the WT staining a marker of podocytes (Figure 6A, lower
panel) (21). The cells were maintained in 5mM or 25mM
glucose and then exposed to the anti-␤3 or control anti-
body, and tyrosine phosphorylation of the ␤3-subunit of
␣V␤3 integrin was quantified. ␤3 phosphorylation was
increased in the cultures exposed to 25mM glucose com-
pared with cultures maintained in 5mM glucose (30%
increase). The anti-␤3 F(ab)2 inhibited ␤3 phosphoryla-
tion in both culture additions (Figure 6A, upper panel).
The results shown are representative of 3 independent ex-
periments. The scanning units were 5mM (1802 Ϯ 520),
5mM ϩ ␤3 AB (459 Ϯ 104), 25mM (2873 Ϯ 308), and
25mM ϩ ␤3 AB (865 Ϯ 250) (P Ͻ .01) when 25mM ␤3
AB is compared with 25mM alone. When lysates from the
kidneys from diabetic control F(ab)2-treated animals were
analyzed, ␤3 phosphorylation was 13 934 Ϯ 6437 scan-
ning units (n ϭ 10), whereas tissue obtained from the
animalsthatreceivedtheanti-␤3F(ab)2 (nϭ8)hadavalue
of 6730 Ϯ 1524 (P Ͻ .01) (Figure 6B).
Figure 3. A, Representative images with arrows indicating
podocytes stained with WT-1 are shown in the top panel. B, Data
obtained from digital images that were generated using
antipodocin antibody. In each case, 20 glomeruli/pig were
measured. *, P Ͻ .01 when diabetic control IgG F(ab)2-treated
animals are compared with nondiabetic or diabetic animals
receiving anti-␤3 F(ab)2. The values represent the total staining
intensity minus background intensity. The data shown in B are
mean Ϯ SEM from diabetic ϩ anti-␤3 F(ab)2, n ϭ 5; diabetic ϩ
anti-IgG F(ab)2, n ϭ 5; nondiabetic, control, n ϭ 3 animals/group.
*, P Ͻ .05 when IgG F(ab)2 is compared with control nondiabetic or
anti-␤3 AB.
Figure 4. A, Representative images of kidney sections stained for type
I and III collagen. B, Results obtained from digital images were
generated. The values represent total staining intensity minus
background intensity. The values represent the mean Ϯ SD of 1 slide/
animal in each group. *, P Ͻ .05 when the values for anti-IgG F(ab)2
treatment is compared with anti-␤3 F(ab)2 or control animals.
doi: 10.1210/en.2014-1318 endo.endojournals.org 4671
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8. Discussion
The results demonstrate that administration of the anti-␤3
antibody inhibited activation of the ␣V␤3 integrin within
the kidney as assessed by inhibition of tyrosine phosphor-
ylation. Because stimulation of ␤3 tyrosine phosphoryla-
tion has been linked to cellular dysfunction in the presence
of hyperglycemia, this result suggests that the antibody
was active in the doses that were administered to these
animals (15, 16). This was confirmed by demonstrating
that the antibody prevented the development of protein-
uria that occurred in the animals who received control IgG
F(ab)2. Multiple histological changes, including GBM
thickening and podocyte effacement, which have been as-
sociated with the development of diabetic nephropathy,
were inhibited. These results strongly support 2 major
conclusions. The first is that the porcine model of diabetic
Figure 5. The data shown were obtained from digital images from
electronmicrographs. A, GBM thickening. The bar graph shows the
harmonic mean Ϯ SEM obtained from glomeruli comprising a total of
14 images and at least 200 measurements to yield 1 data point/animal.
B, PFW. Twenty images were analyzed per animal. The data shown are
mean Ϯ SEM, diabetic ϩ anti-␤3 F(ab)2, n ϭ 5; diabetic ϩ IgG F(ab)2,
n ϭ 5; nondiabetic, n ϭ 8 animals/group. *, P Ͻ .05 when control IgG
F(ab)2-treated animals are compared with nondiabetic or diabetic
animals receiving the anti-␤3 F(ab)2.
Figure 6. A, Podocyte-enriched cultures were isolated from the
glomeruli of control kidneys and were maintained in medium
containing 5mM or 25mM glucose. After overnight incubation in
serum-free medium (with the glucose levels held constant), the
cultures were treated either with control F(ab)2 or anti-␤3 F(ab)2 for 4
hours before lysis. Lysates were immunoprecipitated (IP) (upper panel)
with an anti-␤3 antibody before separation by SDS-PAGE and
immunoblotted (IB) using an antiphosphotyrosine antibody (p-Tyr) or
immunoblotted directly with a ␤3 antibody (middle panel) or anti-WT-
1 antibody (lower panel). The results represent 3 independent
experiments. The scanning units were 5mM (1802 Ϯ 520), 5mM ϩ ␤3
AB (459 Ϯ 104), 25mM (2873 Ϯ 308), and 25mM ϩ ␤3 AB (865 Ϯ
250). P Ͻ .01 when anti-␤3 AB ϩ is compared with hyperglycemia
alone. B, Kidney homogenates were prepared as described previously
in Materials and Methods; 0.1 mg of protein was loaded in each lane
shown in the lower panel. The samples were analyzed as in A. A
representative blot is shown. The bar graph represents the mean Ϯ
SEM of diabetic animals treated with control F(ab)2 (n ϭ 10) and
diabetic pigs treated with anti-␤3 F(ab)2 (n ϭ 8). *, P Ͻ .01.
4672 Maile et al ␣V␤3 and Diabetic Nephropathy Endocrinology, December 2014, 155(12):4665–4675
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9. nephropathy may be useful for evaluating early changes
that occur during the course of this disease as manifested
by increased albuminuria, GBM thickening, and podocyte
effacement. The second is that inhibition of ␣V␤3 integrin
activation is associated with attenuation of these changes.
Because hyperglycemia induces increased synthesis or de-
creased degradation of ␣V␤3 ligands (14, 15), leading to
increasedligandoccupancyof␣V␤3andstimulationof␤3
tyrosine phosphorylation, our results demonstrate that in-
hibition of ligand occupancy with subsequent inhibition
of ␤3 tyrosine phosphorylation is functioning to limit sev-
eral pathophysiologic changes that occur in diabetic
kidneys.
Integrins have been shown to be present in multiple
glomerular cell types (13, 24). Two cell types that have
been proposed to be important for the development of
diabetic nephropathy (eg, glomerular endothelium and
podocytes) express ␣V␤3 (25). Recently, the changes in
glomerular endothelium in response to chronic hypergly-
cemia have been a focus of research interest in the patho-
genesis of diabetic nephropathy. Using renal biopsies,
Weil et al (12) showed that loss of endothelial fenestration
and damage to the glycocalynx occurred early in the
courseofthediseaseandthatthesechangescorrelatedwell
with the development of proteinuria and the decline in
glomerular filtration rate. These authors concluded that
these changes were more directly linked to glomerular en-
dothelial injury rather than to podocyte effacement, al-
though they pointed out that the 2 processes may be
linked. Other studies have concluded that endothelial in-
jury and podocyte effacement or detachment are probably
linked (26). Based on these findings, it is reasonable to
conclude that a strategy that limits glomerular endothelial
damage would inhibit the development of proteinuria.
Therefore, we conclude that this monoclonal antibody is
functioning by inhibiting the changes in glomerular endo-
thelium that lead to altered barrier function.
Podocyte injury has also been proposed as a major eti-
ologic factor in diabetic nephropathy (27). Abnormal
podocyte contact and foot process widening that occur
with hyperglycemia are associated with subsequent podo-
cyte apoptosis, and there are significant correlations be-
tween various stages of diabetic nephropathy and podo-
cyte foot process widening or detachment (12, 28).
Podocytes express ␣V␤3 (28), and indirect inhibition of
␣V␤3 by blockade of urokinase plasminogen type I acti-
vation receptor-induced activation resulted in decreased
proteinuria (29). In this study, we noted early changes in
podocyte foot process widening that were not present in
the kidneys from animals that received the anti-␤3. Fur-
thermore, podocytes in culture were responsive to high
glucose, which stimulated increased ␤3 phosphorylation,
and the antibody inhibited this increase. Therefore, it is
possible that the antibody is also functioning by inhibiting
pathophysiologic changes in podocyte function, and
thereby stabilizing barrier function. Tuberlointerstitial
proliferative changes have also been proposed to be an
important cause of declining renal function in diabetes. In
response to hyperglycemia, proximal tubular cells secrete
excess collagen, which is deposited in the basement mem-
branes, and this leads to myofibroblast proliferation and
endothelial destabilization (30). Our findings show that
the inhibition of ␣V␤3 was associated with decreased col-
lagen in the tubular basement membrane and the intersti-
tium. This result suggests that the antibody also inhibited
these pathophysiologic events.
Strategies that target activation of ␣V␤3 have been an-
alyzed in other systems in which there is endothelial dys-
function (31). Tumstatin, a peptide derived from type IV
collagen, which binds to ␣V␤3 and inhibits ligand-stim-
ulated activation (32), inhibited glomerular hypertrophy,
albuminuria,increasesinglomerularendothelialcellnum-
ber, and renal vascular endothelial growth factor expres-
sion in diabetic mice (33). Furthermore, extracellular
concentrations of ␣V␤3 ligands, osteopontin, and throm-
bospondin are increased in diabetic kidneys in vivo (34,
35). Taken together with our findings, it is likely that the
anti-␤3 antibody is blocking the changes that are induced
by these ␣V␤3 ligands.
Our previous studies showed that in both vascular
smooth muscle and endothelial cells, there is a cooperative
interaction between signaling through the IGF-I receptor
and ␣V␤3 in the presence of hyperglycemia (14–16).
Phospho-␤3 recruits src homology phosphatase (SHP)-2
to the plasma membrane (36). After IGF-I receptor acti-
vation, it directly phosphorylates SHPS-1 (37), leading to
SHP-2 recruitment to phospho-SHPS-1, which is neces-
sary to stimulate activation of the PI-3 and MAPK path-
ways (38). Stimulation of these pathways has been linked
to endothelial dysfunction in diabetes (15, 16).
Hyperglycemia also stimulates ␤3 tyrosine phosphor-
ylation in retinal endothelial cells and loss of maintenance
of endothelial tight junctions leading to increased vascular
permeability to macromolecules (15). Hyperglycemia also
sensitizes endothelium to stimulation of dextran perme-
ation in response to IGF-I. Exposure to the anti-␤3 anti-
body restored normal endothelial permeability and IGF-I
responsiveness in vitro (15). Activation of this pathway
has been shown to be linked to increased retinal capillary
permeability in diabetic rats. Using an antibody that dis-
rupted the ability of hyperglycemia to enhance cellular
responsiveness to IGF-I, we showed that the increase in
retinal capillary permeability induced by hyperglycemia
was completely inhibited (16). Hyperglycemia has been
doi: 10.1210/en.2014-1318 endo.endojournals.org 4673
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10. shown by other investigators to enhance IGF-I down-
stream signaling in diabetic kidneys, even though IGF-I
receptor activation is not increased. This suggests that in
diabetes, there is increased renal sensitivity to IGF-I stim-
ulation (39). Therefore, although we did not quantify
changes in IGF-I signaling in this animal model, we con-
clude based on previous results that it is likely that the
antibody inhibited IGF-I signaling in renal endothelial
cells in the diabetic animals.
Our model also used hyperglycemia and diet-induced
hypercholesterolemia. Analysis of the diabetes complica-
tion control trial showed that hypercholesterolemia is as-
sociated with an increased albumin excretion rate (40).
Experimental animal models using mice, rats, and pigs
have shown that high-fat feeding to diabetic animals is
associated with accelerated development of albuminuria,
mesangial matrix expansion, and glomerular hypertrophy
(41–44). Although the mechanism is unknown, studies
have shown that increased oxidized low density lipopro-
tein induces podocyte retraction and albumin permeabil-
ity (45). Our studies have shown that glucose oxidized low
density lipoprotein activates the ␣V␤3 signaling pathway
in vascular cells in a manner that is functionally similar to
hyperglycemia (46), suggesting that hyperlipidemia may
also play a role in loss of endothelial-podocyte barrier
function in this system.
In summary, the results of this study show that hyper-
lipidemic pigs with type 1 diabetes develop early changes
in loss of barrier function and histopathologic changes
that are consistent with diabetic nephropathy. The
changes are accompanied by activation of the ␣V␤3 in-
tegrin. Blocking ␣V␤3 integrin ligand occupancy leads to
prevention of albuminuria and the early histologic
changes of diabetic nephropathy. The results suggest that
this antibody may have efficacy in preventing diabetic ne-
phropathy progression.
Acknowledgments
We thank Ms Laura Lindsey for her help in preparing the man-
uscript. The C-loop ␤3 monoclonal antibody was prepared by
the Immunology Core Facility at the University of North Caro-
lina. Surveyor images were taken at the Confocal and Mulitphon
Imaging Core, Department of Neurosciences, University of
North Carolina. We also thank Dr Vincent H. Gattone II and Ms
Caroline Miller for EM preparation, sectioning, and image cap-
ture (University of Indiana, Department of Anatomy and Cell
Biology). Urinary analysis was performed at the University of
North Carolina Animal Clinical Chemistry and Gene Expression
core facility by Dr Hyung-Suk Kim. Robin Raymer, Elizabeth
Merricks, and Kent Passingham assisted with care of diabetic
pigs. Areeg Rehman and Amyn Murji assistance with data gen-
eration and analysis. Histological slides were prepared by Ms
Carolyn Suitt at The Center for Gastrointestinal Biology and
Disease Histology.
Address all correspondence and requests for reprints to:
David Clemmons, MD, Department of Medicine, University of
North Carolina School of Medicine, 8024 Burnett-Womack CB
7170, Chapel Hill, NC 27599. E-mail: endo@med.unc.edu.
This work was supported by the National Institutes of Health
Grant HL084857.
Disclosure Summary: L.A.M. and D.C. have equity interest in
Vascular Pharmaceuticals and are inventors on US07723483.
W.H.B., K.A.G., W.F., N.G., S.G., K.S., T.N., D.B., A.P., P.D.,
M.M. have nothing to disclose.
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