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.
1Department 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
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 heparinizedcapillary 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 wasmeasured 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 beforesacrifice 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 andtype 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, SanDiego, 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 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 (DakoCytomationInc,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 1hour. 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 usingrat 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 (real-time PCR). Kidneys from each mouse were homogenized and total RNA was extracted usingRNeasy 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 TGF-b1, 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, 5l of the diluted cDNA was added to the Lightcycler-Mastermix, 1M of specific primer, 3 mM of MgCl2 and 2 l of MasterSYBR Green. For detecting the level of 18s rRNA, 5l of the diluted cDNA was added to the Lightcycler-Mastermix, 2M of specific primer, 3 mM of MgCl2 and 2 l of SYBRPremix Ex Taq (Takara Bio, Japan). These reaction mixtures were filled up to a final volume of 20l with water. PCR reactions were carried out in a real-time PCR cycler (Lightcycler; Roche Diagnostics). The program 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). Thetemperature 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 MCP-1, TGF-1 and 18s rRNA were used: MCP-1,5’-AAG CTGTAGTTTTTGTCACC-3’ (forward) and 5’-GGGCAGATGCAGTTTTAA-3’ (reverse);TGF-b1, 5’-AACAACGCCATCTATCAG-3’ (forward) and 5’-TATTCCGTCTCCTTGGTT-3’ (reverse);18s rRNA, 5’-ACTCAACACGGGAAACCTCA-3’ (forward) and5’-AACCA GACAAATCGCTCCAC-3’ (reverse). Four independent experiments were performed.
Immunoblot. Briefly, kidneys or liverswere 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 4C, supernatant was collected and stored at -80C until use. Total protein concentration was determined by using DC-protein determinationsystem (Bio-Rad Laboratories,Inc.) using bovine serum albumin (BSA) as a standard. Samples were processed for SDS-PAGE 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) orpolyclonal rabbit anti-mouse VASH-1 (9)antibodies at 4C. After incubation with HRP-labeled secondary antibodies for 1hour, 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 examinedand 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), 100U/mL penicillin and 100g/mL streptomycin at 37C. After subconfluence, cells were starved for 24hr by incubating them in DMEM containing 0.4% FCS. Quiescent cells were incubated with 5.5mM normal glucose (NG), NG with 19.5mM mannitol (NG/Manni), 25mM high glucose with PBS-DTT buffer (HG/N0), HG with 1nM recombinant VASH-1 (HG/V1), 10 nMrecombinant VASH-1 (HG/V10) or 20 nMrecombinant VASH-1 (HG/V20) for 24hr. 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 24hr by incubating them in CS-C complete medium supplemented with 19.4mM D-glucose and0.5% FBS. Quiescent cells were incubated with 1nM recombinant human VEGF165(R&D Systems) for 0, 2, 5, 10 or 15min. 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; 5.5mM NG, NG with 24.5mM mannitol (NG/Manni), 30 mM HG with PBS-DTT buffer (HG/N0), HG with 1nM recombinant VASH-1 (HG/V1), 10 nMrecombinant VASH-1 (HG/V10) or 20 nMrecombinant 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-diabetic0.077 ± 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-diabetic1.0 ± 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 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 ofendogenous mouse VASH-1 in kidney. Additionally, the protein levels of endogenous VASH-1 in the renal cortex in experimental groups wereexamined 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 endothelium-derived 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 53:1831-1840, 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-6-methoxy-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 up-regulated 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
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
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.
Appendix Fig. 2. Immunoblot analysis of mouse Vasohibin-1 (mVASH-1). Immunoblots for mVASH-1and actin are shown. In each lane, 50g 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.
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