Vasohibin-1 and diabetic nephropathy
Vasohibin-1, a negative feedback regulator of angiogenesis, ameliorates renal
alterations in a mouse model of diabetic nephropathy
Tatsuyo Nasu1, Yohei Maeshima1, Masaru Kinomura1, Kumiko Hirokoshi1, Katsuyuki
Tanabe1, Hitoshi Sugiyama1, Hikaru Sonoda2, Yasufumi Sato3 and Hirofumi Makino1.
1
Department of Medicine and Clinical Science, Okayama University Graduate School of
Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan; 2Discovery
Research Laboratories, Shionogi, Osaka, Japan and 3Department of Vascular Biology,
Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Japan.
Online-Only Appendix
1
Vasohibin-1 and diabetic nephropathy
Research design and methods
Adenoviral vectors. Adenoviral vectors were expanded in human embryonic
kidney cell line 293 and purified by cesium chloride ultracentrifugation as described
previously (1). The purified viruses were dialyzed against phosphate-buffered saline
(PBS) with 10% glycerol and stored at –70°C until use. The viral concentration and the
viral titer were determined as previously described (2).
Induction of diabetes and experimental protocols. The optimal viral titer for
the present experiments was determined following preliminary in vivo experiments with
various titers of AdhVASH-1, and the titer as described above was utilized since we
could confirm the increase of VASH-1 levels in sera after 2 weeks as detected by the
immunoblots (data not shown).
Individual 24-hr urine sample collections were performed using metabolic cages,
and the body weight was measured. Non-fasting blood samples were drawn from the
retro-orbital venous plexus using heparinized capillary tubes under anesthesia at the
time of sacrifice. Kidney weight was measured just after sacrifice.
Blood and urine examination. Serum and urinary creatinine levels were
measured by the enzymatic colorimetric method as described (3). Urinary albumin
concentration was measured by nephelometry (Organon Teknika-Cappel, Durham, NC)
using anti-mouse albumin antibody (ICN Pharmaceuticals, Aurora, OH) as previously
described (3).
Measurement of blood pressure. Arterial blood pressure was measured before
sacrifice using a programmable sphygmomanometer (BP-98A; Softron, Tokyo, Japan)
by the tail-cuff method as described previously (4).
Histological Analysis. Mean glomerular tuft volume (GV) was determined from
the mean glomerular cross-sectional tuft area (GA) as described previously (3; 5; 6).
Twenty glomeruli from each cortical area were observed, images were taken and
analyzed by using Lumina Vision software (Mitani, Fukui, Japan) to determine the
mean GA. GV was calculated as GV = /k x (GA)3/2, with  = 1.38, the shape coefficient
for spheres and k = 1.1, a size distribution coefficient (5).
Mesangial matrix index was defined as the proportion of the glomerular tuft
occupied by the mesangial matrix excluding nuclei. The mesangial matrix areas of 20
glomeruli in each kidney were analyzed and averaged. The mesangial areas were
selected using Photoshop software (Adobe Systems Inc., San Jose, CA), followed by
analysis using Lumina Vision.
Immunohistochemistry. For immunohistochemistry of CD31 and type IV
collagen, frozen sections (4-m) were fixed in acetone. Then, sections were blocked
with 10% normal goat serum (Sigma) followed by incubation with rat anti-mouse CD31
monoclonal antibody (Pharmingen, San Diego, CA) or polyclonal rabbit anti-mouse
type IV collagen antibody (Chemicon International, Inc., Temecula, CA) overnight.
Sections were then washed, and incubated with Alexa fluor 546-labeled goat anti-rat
IgG (Invitrogen, CD31) or Alexa fluor 488-labeled anti-rabbit IgG (Invitrogen, type IV
collagen) for 30 min at room temperature. After washing in PBS, sections were
observed by a confocal laser fluorescence microscope (LSM-510; Carl Zeiss, Jena,
Germany). The immunoreactivity of glomerular CD31 or type IV collagen was
quantified as follows; color images were obtained as TIF files by LSM-510. The
brightness of each image file was uniformly enhanced and analysis using Lumina
2
Vasohibin-1 and diabetic nephropathy
Vision. Image files (TIFF) were inverted and opened in gray scale mode. Type IV
collagen or CD31 indices were calculated using the following formula, {[X (density) x
positive area (m2)]/ glomerular total area (m2)}, where the staining density is
indicated by a number from 0 to 256 in gray scale. In regard to peritubular capillary
(PTC) density, the number of CD31+ peritubular capillaries in each high power field
was determined. The PTC density of 20 high power fields in each kidney were analyzed
and averaged.
Double immunofluorescent staining was performed as previously described (7;
8). Briefly, frozen sections (4-m) were fixed in cold (-20°C) methanol for 20 min and
then air dried. The kidney sections were then blocked with Protein Block Serum-Free
(Dako Cytomation Inc, CA). Sections were incubated with primary antibodies, rabbit
anti-mouse VASH-1 (9), rat anti-mouse CD31 (Pharmingen) or mouse anti--smooth
muscle actin (SMA; Sigma, MO, USA) at 4°C overnight. Subsequently, sections were
washed three times in PBS and incubated with Alexa Fluor 488-labeled donkey anti
rabbit IgG (Invitrogen, VASH-1), Alexa Fluor 546-labeled goat anti-rat IgG (Invitrogen,
CD31) or Alexa Fluor 546-labeled goat anti-mouse IgG (Invitrogen, -SMA) at room
temperature for 1 hour. Nuclei were stained with DAPI (Chemicon). After three
washes with PBS, Permafluor (Beckman Coulter, Inc. Galway, Ireland) was applied and
sections were observed under a BIOZERO fluorescent microscope BZ-800 (Keyence,
Osaka, Japan) and images were obtained. Normal rat and mouse IgG were used as
negative controls.
Glomerular accumulation of monocytes/macrophages was determined by
immunohistochemistry using rat anti-mouse F4/80 antibody (Serotec, Oxford, UK).
Frozen sections were fixed in acetone for 10 min and exposed to H2O2 to eliminate
endogenous peroxidase activity. The kidney sections were then blocked with 10% goat
serum for 30min, and incubated with primary antibody for 60min. The sections were
washed with PBS and exposed to secondary antibody, HRP-labeled goat anti-rat IgG
(Chemicon) for 1 hour. Diaminobenzidine was used as a chromogen. The number of
F4/80-positive cells was determined by observing more than 20 glomeruli from each
section. All slides were counterstained with hematoxylin. Normal rat IgG was used as
a negative control.
RNA Extraction and quantitative real-time polymerase chain reaction (realtime PCR). Kidneys from each mouse were homogenized and total RNA was extracted
using RNeasy Midi Kit (Qiagen, Chatsworth, CA) and stored at -80°C until use. Total
RNA was subjected to RT with poly-d (T) primers or random primers and reverse
transcriptase (GeneAmp RNA PCR Kit; Applied Biosystems, Foster City, CA).
Quantitative real-time PCR was used to quantify the mRNA levels of MCP-1 and TGFb1, and the amount of 18s rRNA. cDNA was diluted 1:50 with autoclaved deionized
water. For the detection of MCP-1 and TGF-1 mRNA levels, 5 l of the diluted
cDNA was added to the Lightcycler-Mastermix, 1 M of specific primer, 3 mM of
MgCl2 and 2 l of Master SYBR Green. For detecting the level of 18s rRNA, 5 l of
the diluted cDNA was added to the Lightcycler-Mastermix, 2 M of specific primer, 3
mM of MgCl2 and 2 l of SYBR Premix Ex Taq (Takara Bio, Japan). These reaction
mixtures were filled up to a final volume of 20 l with water. PCR reactions were
carried out in a real-time PCR cycler (Lightcycler; Roche Diagnostics). The program
3
Vasohibin-1 and diabetic nephropathy
was optimized and performed finally as denaturation at 95°C for 10 min followed by 40
cycles of amplification (18s rRNA; 95°C for 5 s; 60°C for 20 s, MCP-1; 95°C for 10 s;
62°C for 10 s; 72°C for 6 s, TGF-1; 95°C for 10 s; 61°C for 15 s; 72°C for 11 s,
respectively). The temperature ramp rate was 20°C/s. At the end of each extension
step, the fluorescence was measured to quantitate the PCR products. After completion
of the PCR, the melting curve of the product was measured by temperature gradient
from 65 to 95°C at 0.1 or 0.2°C/s with continuous fluorescence monitoring to produce a
melting profile of the primers. The amount of PCR products was normalized with 18s
rRNA to determine the relative expression ratio for MCP-1 or TGF-1 mRNA in
relation to 18s rRNA. The following oligonucleotide primers specific for mouse MCP1, TGF-1 and 18s rRNA were used: MCP-1, 5’-AAG CTGTAGTTTTTGTC ACC-3’
(forward) and 5’-GGGCAGATGCAGTTTTAA-3’ (reverse); TGF-b1, 5’-AACAA
CGCCATCTATCAG-3’ (forward) and 5’-TATTCCGTCTCCTTGGTT-3’ (reverse);
18s rRNA, 5’-ACTCAACACGGGAAACCTCA-3’ (forward) and 5’-AACCA
GACAAATC GCTCCAC-3’ (reverse). Four independent experiments were performed.
Immunoblot.
Briefly, kidneys or livers were homogenized in
radioimmunoprecipitation assay (RIPA) Lysis buffer (Santa Cruz Biotechnology, Inc.
CA) at 4°C. Similarly, cultured mesangial cells were lysed using RIPA buffer as
previously described (6; 10). After centrifugation at 13,000 rpm for 30 min at 4C,
supernatant was collected and stored at -80C until use. Total protein concentration was
determined by using DC-protein determination system (Bio-Rad Laboratories, Inc.)
using bovine serum albumin (BSA) as a standard. Samples were processed for SDSPAGE and proteins were electrotransferred onto nitrocellulose membrane with iBlot
Dry Blotting System (Invitrogen). The membranes were blocked with 5% nonfat dry
milk in 1X TBS (0.1% Tween-20) for 1 hr, incubated overnight with polyclonal rabbit
anti-mouse TGF-1/2/3 (Santa Cruz), polyclonal rabbit anti-VEGF-A, anti-VEGFR2
(Santa Cruz), anti-VEGFR2 phosphospecific antibody (Invitrogen), hamster anti-mouse
MCP-1 (BioLegend, San Diego, CA), anti-RAGE (R&D Systems, Inc. Minneapolis,
MN), monoclonal anti-human VASH-1 (2) or polyclonal rabbit anti-mouse VASH-1 (9)
antibodies at 4C. After incubation with HRP-labeled secondary antibodies for 1 hour,
signals were detected with ECL system (Amersham). Membranes were re-probed with
rabbit polyclonal anti-actin antibodies (Bio-Rad) to serve as controls for equal loading.
The density of each band was determined by using Image J software, and expressed as a
value relative to the density of corresponding band obtained from actin immunoblot. In
some experiments, the levels of phosphorylated VEGFR2 (pVEGFR2) were examined
and expressed as a value relative to the density of corresponding band obtained from
total VEGFR2 immunoblot.
Cell culture. Primary murine mesangial cells (MES13) were purchased from the
ATCC (Rockville, MD). Mesangial cells in these studies were used between the 10th
and 20th passage.
Characteristics of mesangial cells were confirmed by
immunoreactivity for actin and desmin and lack of staining for factor VIII as previously
described (11). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM;
Sigma, St. Louis, MO) containing 10% fetal calf serum (FCS; Cansera International Inc.,
Canada), 100 U/mL penicillin and 100 g/mL streptomycin at 37C. After
subconfluence, cells were starved for 24 hr by incubating them in DMEM containing
4
Vasohibin-1 and diabetic nephropathy
0.4% FCS. Quiescent cells were incubated with 5.5 mM normal glucose (NG), NG with
19.5 mM mannitol (NG/Manni), 25 mM high glucose with PBS-DTT buffer (HG/N0),
HG with 1 nM recombinant VASH-1 (HG/V1), 10 nM recombinant VASH-1 (HG/V10)
or 20 nM recombinant VASH-1 (HG/V20) for 24 hr. They then were harvested and
subjected to Western blot analysis.
Primary human glomerular endothelial cells (GEC) were obtained from Applied
Cell Biology Research Institute (Kirkland, WA) and cultured in CS-C complete medium
supplemented with 19.4 mM D-glucose, 10% FCS and EGM-2 signaleQuots (Clonetics)
on 6-well plates coated with Attachment Factor (Cell Systems) in a 5% CO2 incubator at
37°C following manufacturer’s instruction. GECs in these studies were used between
the 4-8th passages. After subconfluence, cells were starved for 24 hr by incubating
them in CS-C complete medium supplemented with 19.4mM D-glucose and 0.5% FBS.
Quiescent cells were incubated with 1 nM recombinant human VEGF165 (R&D
Systems) for 0, 2, 5, 10 or 15 min. In some experiments, cells were also incubated with
10 nM recombinant VASH-1 in the presence of 1 nM recombinant human VEGF165. In
other sets of experiments, quiescent cells were incubated under the following condition
for 24 hr; 5.5 mM NG, NG with 24.5 mM mannitol (NG/Manni), 30 mM HG with PBSDTT buffer (HG/N0), HG with 1 nM recombinant VASH-1 (HG/V1), 10 nM
recombinant VASH-1 (HG/V10) or 20 nM recombinant VASH-1 (HG/V20). They then
were harvested and subjected to Western blot analysis.
Results
Serum and hepatic levels of VASH-1 following adenoviral transfer (Figure
1A). The AdhVASH-1-injected diabetic mice exhibited significantly elevated serum
VASH-1 levels compared to the AdLacZ injection at 4 weeks after the initial injections
as detected by the immunoblot and densitometry (AdLacZ/diabetic 1.00 ± 0.08;
AdhVASH-1/diabetic 2.99 ± 0.32, arbitrary units, P < 0.01). Similarly, hepatic
expression of VASH-1 was significantly elevated in the AdhVASH-1-injected diabetic
mice compared to the AdLacZ-injected animals (AdLacZ/diabetic 1.00 ± 0.19;
AdhVASH-1/diabetic 17.49 ± 0.51, VASH-1/actin ratio, P < 0.01).
Changes in serum creatinine, creatinine clearance (Ccr) and urinary albumin
excretion. Serum creatinine levels did not significantly differ among the experimental
groups (non-diabetic 0.077 ± 0.01, vehicle/diabetic 0.080 ± 0.016, AdLacZ/diabetic
0.077 ± 0.010; AdhVASH-1/diabetic 0.073 ± 0.013, mg/dl). Although control diabetic
mice showed a marked elevation of Ccr and urinary albumin/creatinine ratio (UACR),
AdhVASH-1 suppressed STZ-induced increase of Ccr (non-diabetic 0.32 ± 0.06;
vehicle/diabetic 0.88 ± 0.09; AdLacZ/diabetic 0.93 ± 0.09; AdhVASH-1/diabetic 0.58 ±
0.05, ml/min per 100 g body weight) and UACR (non-diabetic 1.0 ± 0.0,
vehicle/diabetic 40.5 ± 5.6; AdLacZ/diabetic 40.0 ± 1.2; AdhVASH-1/diabetic 25.3 ±
6.2, g albumin/mg creatinine), at 4 weeks after the initial injection of adenoviral
vectors.
Histology and morphometric analysis. Systemic administration of adenoviral
vectors did not exhibit any pathological alterations in the liver or the heart of diabetic
mice (data not shown).
Immunohistochemical analysis of CD31(+) endothelial area. Additionally, we
investigated peritubular capillary (PTC) density in a similar manner, and no significant
5
Vasohibin-1 and diabetic nephropathy
differences were observed among experimental groups (non-diabetic 5.34 ± 0.29;
vehicle/diabetic 5.94 ± 0.30; AdLacZ/diabetic 5.91 ± 0.31; AdhVASH-1/diabetic 5.84 ±
0.28, CD31+PTC vessel number/high power field; data not shown).
Localization and the levels of endogenous mouse VASH-1 in kidney.
Additionally, the protein levels of endogenous VASH-1 in the renal cortex in
experimental groups were examined by immunoblot assay. The level of VASH-1 was
not significantly different among non-diabetic and diabetic experimental groups (data
not shown).
References
1. Kanegae Y, Lee G, Sato Y, Tanaka M, Nakai M, Sakaki T, Sugano S, Saito I:
Efficient gene activation in mammalian cells by using recombinant adenovirus
expressing site-specific Cre recombinase. Nucleic Acids Res 23:3816-3821, 1995
2. Watanabe K, Hasegawa Y, Yamashita H, Shimizu K, Ding Y, Abe M, Ohta H,
Imagawa K, Hojo K, Maki H, Sonoda H, Sato Y: Vasohibin as an endotheliumderived negative feedback regulator of angiogenesis. J Clin Invest 114:898-907, 2004
3. Yamamoto Y, Maeshima Y, Kitayama H, Kitamura S, Takazawa Y, Sugiyama H,
Yamasaki Y, Makino H: Tumstatin Peptide, an inhibitor of angiogenesis, prevents
glomerular hypertrophy in the early stage of diabetic nephropathy. Diabetes 53:18311840, 2004
4. Hashimoto N, Maeshima Y, Satoh M, Odawara M, Sugiyama H, Kashihara N,
Matsubara H, Yamasaki Y, Makino H: Overexpression of Angiotensin Type 2
Receptor Ameliorates Glomerular Injury in mouse remnant kidney model. Am J
Physiol Renal Physiol, 2003
5. Weibel ER: Stereological Methods. In Practical Methods for Biological
Morphometry London, Academic, 1979, p. 51-57
6. Ichinose K, Maeshima Y, Yamamoto Y, Kinomura M, Hirokoshi K, Kitayama H,
Takazawa Y, Sugiyama H, Yamasaki Y, Agata N, Makino H: 2-(8-hydroxy-6methoxy-1-oxo-1h-2-benzopyran-3-yl) propionic acid, an inhibitor of angiogenesis,
ameliorates renal alterations in obese type 2 diabetic mice. Diabetes 55:1232-1242,
2006
7. Ichinose K, Maeshima Y, Yamamoto Y, Kitayama H, Takazawa Y, Hirokoshi K,
Sugiyama H, Yamasaki Y, Eguchi K, Makino H: Anti-angiogenic endostatin peptide
ameliorates renal alterations in the early stage of type 1 diabetic nephropathy model.
Diabetes 54:2891-2903, 2005
8. Tanabe K, Maeshima Y, Ichinose K, Kitayama H, Takazawa Y, Hirokoshi K,
Kinomura M, Sugiyama H, Makino H: Endostatin peptide, an inhibitor of
angiogenesis, prevents the progression of peritoneal sclerosis in a mouse
experimental model. Kidney Int 71:227-238, 2007
9. Shen J, Yang X, Xiao WH, Hackett SF, Sato Y, Campochiaro PA: Vasohibin is upregulated by VEGF in the retina and suppresses VEGF receptor 2 and retinal
neovascularization. Faseb J 20:723-725, 2006
10. Maeshima Y, Sudhakar A, Lively JC, Ueki K, Kharbanda S, Kahn CR, Sonenberg
N, Hynes RO, Kalluri R: Tumstatin, an endothelial cell-specific inhibitor of protein
synthesis. Science 295:140-143., 2002
6
Vasohibin-1 and diabetic nephropathy
11. Maeshima Y, Kashihara N, Yasuda T, Sugiyama H, Sekikawa T, Okamoto K,
Kanao K, Watanabe Y, Kanwar YS, Makino H: Inhibition of mesangial cell
proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo. J Clin Invest
101:2589-2597., 1998
7
Vasohibin-1 and diabetic nephropathy
Appendix Fig. 1. CD31+ peritubular capillary (PTC) number. The number of CD31+
PTC was quantitated as described in ‘RESEARCH DESIGN AND METHODS’. Slight
increase in CD31+ PTC number was observed in diabetic group without any intergroup
differences. n = 5 for each group. N, nondiabetic control; Ve, diabetic mice treated
with vehicle buffer; LacZ, diabetic mice treated with AdLacZ; Vas, diabetic mice
treated with AdhVASH-1. Each column consists of means ± SE.
8
Vasohibin-1 and diabetic nephropathy
Appendix Fig. 2.
Immunoblot analysis of mouse Vasohibin-1 (mVASH-1).
Immunoblots for mVASH-1and actin are shown. In each lane, 50 g protein obtained
from renal cortex was loaded. Each band was scanned and subjected to densitometry.
Lower graph: Intensities of mVASH-1 protein relative to actin are shown. n = 5 for
each group. Each column consists of means ± SE.
9