Vascular endothelial growth factor expression and glomerular endothelial cell loss in the remnant kidney model

Darren J. Kelly, Claire Hepper, Leonard L. Wu, Alison J. Cox and Richard E. Gilbert

Department of Medicine, University of Melbourne, St Vincent's Hospital, Fitzroy, Australia



   Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Background. Vascular endothelial growth factor (VEGF) is constitutively expressed in the glomerulus where it may have a role in the maintenance of capillary endothelial cell integrity. The present study sought to examine changes in VEGF expression in a model of progressive renal disease and to assess the effects of angiotensin converting enzyme (ACE) inhibition.

Methods. Subtotal nephrectomized (STNx) rats were randomly assigned to receive vehicle (n=10) or the ACE inhibitor perindopril (8 mg/l drinking water) for 12 weeks duration (n=10). Sham-operated rats were used as controls (n=10). Glomerular capillary endothelial cell density was evaluated by immunostaining for the pan-endothelial cell marker RECA-1 and VEGF expression was assessed by quantitative in situ hybridization.

Results. In STNx rats glomerular capillary endothelial cell density was reduced to 19% that of sham rats (P<0.01) with a concomitant reduction in glomerular VEGF expression, also to 19% of sham rats (P<0.01). Perindopril treatment was associated with normalization of both capillary endothelial cell density and glomerular VEGF mRNA.

Conclusions. Reduction in glomerular VEGF expression is a feature of the renal pathology that follows subtotal nephrectomy. In the context of the known functions of this growth factor, these findings suggest that diminution in VEGF may contribute to the demonstrated loss of glomerular endothelium that develops in this model of progressive renal disease.

Keywords: angiotensin converting enzyme; endothelium; glomerulosclerosis; renin–angiotensin system; vascular endothelial growth factor



   Introduction
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The integrity of the glomerular capillary tuft is vital to the kidney's primary function of plasma filtration. Progressive capillary loss, with the obliteration of the microvasculature, frequently accompanies fibrosis as a characteristic feature of chronic renal disease [1]. Indeed, capillary loss correlates closely with declining function in a range of kidney diseases [2] and may represent the endothelial component of a common adverse response to renal insult. However, while much attention has been focussed on the mechanisms underlying the development of renal fibrosis, the pathogenesis of microvascular injury has only begun to be unravelled with recent studies suggesting a central role for vascular endothelial growth factor (VEGF) in endothelial cell survival and repair [1,3].

Blockade of the renin–angiotensin system (RAS) has repeatedly been shown to have renoprotective actions in a range of human and experimental renal diseases [4,5]. While these effects were initially viewed to result from attenuation of angiotensin II-mediated effects on intraglomerular pressure [6], studies conducted over the past decade have highlighted that other mechanisms may also contribute. These include, among others, angiotensin II-mediated stimulation of transforming growth factor-ß (TGF-ß) expression and the induction of macrophage chemotaxis [7]. However, the effects of angiotensin II on the expression of VEGF have been more controversial [8,9].

The aims of the present studies were therefore 2-fold. First, we sought to examine the effects of progressive renal disease on VEGF expression and glomerular endothelial cell integrity. Secondly, we also sought to determine the effects of angiotensin converting enzyme (ACE) inhibition on these parameters.



   Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Animals
Thirty male Sprague–Dawley rats weighing 200–250 g were randomized to three groups of 10 animals each. Anaesthesia was achieved by the i.p. administration of pentobarbital (7 mg/100 g body weight, Boehringer Ingelheim, Artarmon, NSW, Australia). The control group underwent sham surgery consisting of laparotomy and manipulation of both kidneys before wound closure. The other 20 rats all underwent subtotal nephrectomy (STNx) performed by right subcapsular nephrectomy and infarction of approximately two-thirds of the left kidney by selective ligation of two of three to four extrarenal branches of the left renal artery. Animals were then randomly assigned to two groups: STNx alone or STNx with the ACE inhibitor perindopril (8 mg/l drinking water, Servier, Neuilly, France). Rats were housed in a temperature (22°C) controlled room with ad libitum access to commercial standard rat chow (Norco Co-Operative Ltd., Lismore, NSW, Australia) and water during the entire study. Rats from each group were sacrificed at 12 weeks post-surgery. At death the remnant (left) kidney was then sliced sagitally and one half immersion fixed in 10% neutral-buffered formalin for in situ hybridization and the other half fixed in 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4) for immunohistochemistry. All tissues were subsequently embedded in paraffin. All experiments adhered to the guidelines of the Animal Welfare and Ethics Committee of St Vincent's Hospital and the National Health and Medical Research Foundation of Australia.

Renal function
Body weight was measured weekly. Plasma urea and creatinine were measured by autoanalyser (Beckman Instrumentals, Palo Alto, CA) at the beginning and end of the study. Glomerular filtration rate (GFR) was measured prior to death by a single shot Tc99m-DTPA clearance. Systolic blood pressure was measured in conscious rats using an occlusive tail-cuff plethysmograph attached to a pneumatic pulse transducer (Narco Bio-system Inc., Houston, TX). Before death, rats were housed in metabolic cages for 24 h for subsequent measurement of urinary protein excretion using Coomassie Brilliant Blue method.

Renal structure
The glomerulus was considered as the area internal to and including Bowman's capsule. In 3 µm kidney sections stained with PAS, 50–80 glomeruli from rats were examined in a masked protocol. The degree of sclerosis in each glomerulus was subjectively graded on a scale of 0 to 4; Grade 0, normal; Grade 1, sclerotic area up to 25% (minimal); Grade 2, sclerotic area 25–50% (moderate); Grade 3, sclerotic area 50–75% (moderate to severe) and Grade 4, sclerotic area 75–100% (severe). Glomerulosclerosis was defined as glomerular basement membrane thickening, mesangial hypertrophy and capillary occlusion. A glomerulosclerotic index (GSI) was then calculated using the formula: Go


(001)
where Fi is the per cent of glomeruli in the rat with a given score (i).

Immunohistochemistry
Glomerular capillary endothelial cell density was evaluated by immunostaining with RECA-1, a pan-endothelial cell-specific monoclonal antibody. Three micron sections were placed into histosol to remove the paraffin wax, hydrated in graded ethanol and immersed in tap water before being incubated for 20 min with normal goat serum (NGS) diluted 1:10 with 0.1 M PBS at pH 7.4. Sections were then incubated for 18 h at 4°C with RECA-1 (Serotec, Oxford, UK). Sections incubated with 1:10 NGS instead of the primary antiserum served as the negative control. After thorough washing with PBS (3x5 min changes), the sections were flooded with a solution of 5% hydrogen peroxide, rinsed with PBS (2x5 min) and incubated with biotinylated goat anti-mouse IgG (Dakopatts, Glostrup, Denmark) diluted 1:200 with PBS. Sections were rinsed with PBS (2x5 min) and incubated with an avidin–biotin peroxidase complex (Vector, Burlingame, CA) diluted 1:200 with PBS. Following rinsing with PBS (2x5 min), sections were incubated with 0.05% diaminobenzidine and 0.05% hydrogen peroxide (Pierce, Rockford, IL) in PBS at pH 7.6 for 1–3 min, rinsed in tap water for 5 min, counterstained in Mayer's haematoxylin, differentiated in Scott's tap water, dehydrated, cleared and mounted in Depex.

The magnitude of RECA-1 immunostaining was quantified using image analysis as described previously [10]. Briefly, the glomerulus, as defined previously, was outlined by interactive tracing. Images were then captured using a BX50 Olympus microscope attached to a Fujix HC-2000 digital camera and a Pentium III IBM computer. The colour range for RECA-1 positive cells (brown on immunoperoxidase labelled sections) were selected and image analysis was performed using a chromogen-separating technique [10]. The proportional glomerular area showing positive RECA-1 immunostaining was measured from three sections per rat (n=6/group), providing in excess of 50 glomeruli/treatment group.

In situ hybridization
In situ hybridization was performed using a cDNA encoding mouse VEGF164 (gift of Dr Steven Stacker, Ludwig Institute for Cancer Research, Melbourne, Australia). The fragment containing the entire open reading frame of VEGF was cloned into pGEM 4Z (Promega, Maddison, WI) and linearized with HindIII to produce an antisense riboprobe using T7 RNA polymerase. In situ hybridization was then performed on 4 µm paraffin sections using 33P-labelled antisense riboprobe. In brief, tissue sections were dewaxed in histosol, hydrated through graded ethanol and immersed in distilled water. Sections were then washed in 0.1 M PBS, pH 7.4 and hybridized with 33P-labelled antisense and sense-specific probes (5x105 c.p.m./25 ml hybridization buffer) which were added to hybridization buffer (300 mM NaCl, 10 mM Tris–HCl, pH 7.5, 10 mM Na2HPO4, pH 6.8, 5 mM EDTA, pH 8.0, 1x Denhardt's solution, 0.8 mg yeast RNA/ml, 50% deionized formamide and 10% dextran sulphate), heated to 85°C and 25 ml added to the sections. Coverslips were placed on the sections and the slides incubated in a humidified chamber (50% formamide) at 60°C for 14–16 h. Slides were then washed in 2x SSC (0.3 M NaCl, 0.33 M Na3C6H5O7.2H2O) containing 50% formamide at 50°C to remove the coverslips. The slides were again washed with 2x SSC, 50% formamide for a further 1 h at 55°C. Sections were then rinsed three times in RNase buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA, pH 8.0, 0.5 M NaCl) at 37°C and treated with 150 mg RNase A/ml in RNase buffer for a further 1 h at 37°C, then washed with 2x SSC at 55°C for 45 min. Finally, sections were dehydrated through graded ethanol, air dried and exposed to Kodak Biomax MR Autoradiography film for 4 days at room temperature. Slides were coated with Ilford K5 emulsion (Ilford, 1:1 with distilled water), stored with desiccant at room temperature for 21 days, developed in Ilford phenisol, fixed in Ilford Hypam and stained with H&E.

Quantitative autoradiography
Quantitative in situ hybridization, which permits the assessment of gene expression equivalent to northern blot analysis was used to determine the magnitude of gene expression. Quantification was performed using two methods: autoradiographic film denistometry and grain counting of emulsion-coated sections using established methods, as reported previously by our group [11].

Film densitometry of autoradiographic images obtained by in situ hybridization was performed by computer-assisted image analysis as described previously [12] using a Micro Computer Imaging Device (MCID, Imaging Research, St Catherine's, Ontario, Canada). With this method quantification of transcript is based on the changes in X-ray film density that follows exposure to the radioactive emissions of radiolabelled VEGF mRNA. In situ autoradiographic images were placed on a uniformly illuminating fluorescent light box (Northern Light Precision Luminator Model C60, Image Research, Ontario, Canada) and captured using a video camera (Sony Video Camera Module CCD, Japan) connected to an IBM AT computer with a 512x512 pixel array imaging board with 256 grey levels. Following appropriate calibration by constructing a curve of optical density vs radioactivity quantification of digitalized autoradiographic images was performed using MCID software. Data were expressed as optical density per cm2 relative to control kidneys (Relative Optical Density, ROD).

In addition to film densitometry, grain counting of emulsioncoated sections was also used to quantify gene expression and to explore the cell-specific sources of transcript within the kidney. With this method exposure of the photographic emulsion to radiolabelled VEGF mRNA leads to the formation of silver grains that can then be quantified. In brief, light microscopic images viewed through a 20x objective lens were captured and digitized using a Fujix HC-2000 digital camera (Fuji, Tokyo, Japan). The outline of 50 glomeruli as defined by interactive tracing was used to assess glomerular gene expression in each kidney section. Regional gene expression was quantitatively measured to determine the proportion of each area occupied by autoradiographic grains as described previously [12], using computerized image analysis (Analytical Imaging Station, Imaging Research Inc.).

All sections were cut in a uniform manner in the mid-saggital plane, hybridized to their respective probes in the same experiment and analysed in duplicate under identical conditions. All analyses were performed with the observer masked to the animal study group.

Statistics
Data are expressed as means±SEM unless otherwise stated. Statistical significance was determined by ANOVA with a Fishers post-hoc comparison. Because of its skewed distribution, proteinuria was analysed using log transformed data and are represented as geometric means x/÷ tolerance factors. Data derived from the glomerulosclerosis index were not normally distributed and were expressed as median (range) and analysed using the Kruskal–Wallis test. Analyses were performed using Statview II + Graphics package (Abacus Concepts, Berkeley, CA) on an Apple Macintosh G4 computer (Apple Computer, Inc., Cupertino, CA). The correlation between VEGF and glomerulosclerosis was assessed non-parametrically using Spearman's rho. A P-value <0.05 was regarded as statistically significant.



   Results
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Renal functional and biochemical studies
Rats that underwent STNx surgery became hypertensive and developed heavy proteinuria, elevated serum creatinine and glomerulosclerosis. Each of these parameters was reduced with perindopril treatment (Table 1Go).


View this table:
[in this window]
[in a new window]
 
Table 1.  Clinical characteristics

 

RECA-1 expression
In sham rats there was intense localization of RECA-1 to the endothelial cells of glomerular capillary loops. RECA-1 immunolabelling was reduced to 19% of control levels in the glomeruli of STNx rats (P<0.01, Figures 1Go and 2Go). This decrease in RECA-1 immunolabelling was attenuated by perindopril to levels similar to that in sham animals (Figures 1Go and 2Go). Sections stained with normal IgG showed no staining (data not shown).



View larger version (124K):
[in this window]
[in a new window]
 
Fig. 1.  Representative photomicrographs of glomeruli labelled with RECA-1 antibody showing endothelial cell immunostaining in sham kidneys (A) and following subtotal nephrectomy in rats treated with either placebo (B) or perindopril (C). Sections show loss of microvascular endothelium in STNx kidneys that is attenuated by perindopril treatment. Magnification x380.

 


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.  Quantification of proportional endothelial cell density as the proportional area immunostained with RECA-1 antibody. Data are shown as mean±SEM. *P<0.01 STNx vs control; {dagger}P<0.01 perindopril-treated vs untreated STNx rats.

 

VEGF expression
In situ hybridization autoradiography revealed punctate cortical expression of VEGF mRNA consistent with localization of transcript to glomeruli (Figure 3Go). This pattern of distribution was not detected in autoradiographs from kidney sections of STNx animals but present in animals that were treated with perindopril following renal mass reduction. In emulsion-dipped in situ hybridization, sections showed that VEGF mRNA was localized to the glomerular visceral epithelial cells of sham and perindopril-treated STNx rats. While detectable and still confined to podocytes, the magnitude of its expression was substantially decreased in STNx rats (Figures 4Go and 5Go). Sections labelled with sense probe (negative control) showed no hybridization (data not shown).



View larger version (52K):
[in this window]
[in a new window]
 
Fig. 3.  In situ hybridization autoradiographs for VEGF mRNA in sham kidneys (A) and following subtotal nephrectomy in rats treated with either placebo (B) or perindopril (C). Punctate cortical expression in sham and perindopril-treated rats, consistent with glomerular expression. Magnification 8x.

 


View larger version (138K):
[in this window]
[in a new window]
 
Fig. 4.  Photomicrographs of glomeruli labelled in situ with antisense riboprobe to VEGF from sham kidneys (A) and following subtotal nephrectomy in rats treated with either placebo (B) or perindopril (C). Sections show abundant VEGF mRNA in glomerular podoctyes (arrow) from control and perindopril-treated STNx rats compared with untreated STNx. Magnification 570x.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 5.  Quantification of VEGF gene expression by film densitometry (upper panel) and by grain counting of glomerular transcript in emulsion-dipped sections (lower panel) from sham kidneys and following subtotal nephrectomy in untreated rats (STNx) and those treated with perindopril. Data are shown as mean±SEM of the relative optical density (OD) as arbitrary units (AU) and as proportional area occupied by autoradiographic grains. *P<0.01 STNx vs control, {dagger}P<0.01 perindopril-treated vs untreated STNx rats.

 
Densitometric analysis of autoradiographic images confirmed a significant reduction in renal VEGF expression in STNx rats, which was attenuated by perindopril (Figure 5Go). Similarly, quantification of emulsion-dipped sections also showed that treatment of STNx rats with perindopril was associated with restoration of renal VEGF levels, similar to those of sham animals (Figures 4Go and 5Go). A close inverse correlation between the degree of glomerulosclerosis and the magnitude of VEGF expression was noted (Figure 6Go).



View larger version (11K):
[in this window]
[in a new window]
 
Fig. 6.  Correlation between glomerular VEGF gene expression (as assessed in emulsion-dipped sections) and the extent of glomerulosclerosis. There is a significant inverse correlation between glomerular VEGF mRNA and the glomerulosclerotic index (rho=–0.48, P<0.01).

 



   Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In the present study of progressive renal disease, renal mass reduction was accompanied by a diminution in glomerular VEGF expression and a decrease in glomerular endothelial cell density. In the context of the known role of VEGF in the maintenance of capillary endothelium these findings suggest that reduction in this cytokine may contribute to the endothelial cell loss that develops in progressive renal disease.

Subtotal nephrectomy provides a well-characterized model of non-inflammatory proteinuric renal disease, frequently used to examine the pathogenesis of progressive kidney disease. Following renal mass reduction, endothelial cell loss due to apoptotic cell death occurs as an early and progressive feature, commensurate with the development of glomerulosclerosis [13]. Such endothelial cell loss has clear implications for the maintenance of blood flow within the glomerulus. However, in addition, the injured endothelium may also initiate platelet activation and the coagulation cascade, both also implicated in the pathogenesis of glomerulosclerosis [14]. In the present study, substantial glomerular capillary loss was noted in animals that had undergone subtotal nephrectomy. The cause of this endothelial cell loss cannot be directly determined from this study. However, given the important role of VEGF in endothelial cell survival and repair [3,15] these findings do infer that the demonstrated reduction in VEGF may be contributory.

The interaction between the renin–angiotensin system and VEGF expression has been a matter of controversy. For instance, in cultured vascular smooth muscle cells and in mesangial cells, angiotensin II induces VEGF expression [8,16]. Furthermore, renin gene transfer has also been shown to restore VEGF expression to skeletal muscle [17]. However, the opposite effect has been reported in tubular epithelial cells where angiotensin II leads to a diminution in VEGF expression [9]. Similarly, divergent effects have also been reported with blockade of the RAS in the in vivo setting. For instance, in the retina, ACE inhibition has been shown to reduce VEGF expression [18], contrasting the findings of the present study in which ACE inhibition was associated with an increase in VEGF mRNA. Together, these in vitro and in vivo studies, suggest that the effects of angiotensin II on VEGF expression are cell type-specific. A similar cell specificity may also apply to the relationship between blood pressure and VEGF where hypertension is associated with increased VEGF in the retina [19] contrasting the decrease in its renal expression demonstrated in the present study.

Like many other growth factors, VEGF is a multi-functional cytokine, initially noted for its permeability enhancing actions, leading to its implication in the pathogenesis of proteinuric renal disease [20]. However, in the present study, heavy proteinuria was instead accompanied by a reduction in VEGF expression suggesting that it is unlikely to have a major role in the pathogenesis of proteinuria in this model.

In addition to its angiogenic and permeability enhancing properties, VEGF also induces collagen synthesis in cultured mesangial cells [21], possibly via transforming growth factor-ß (TGF-ß)-dependent mechanisms [22]. However, in the present study, an inverse correlation between VEGF expression and the extent of glomerular matrix deposition was found. Furthermore, previous reports of increased TGF-ß following subtotal nephrectomy model and its attenuation with RAS blockade [4], suggest that VEGF is unlikely to directly contribute to the matrix accumulation and consequent sclerosis that develop in this animal model.

In summary, the present study indicates that progressive glomerulosclerosis in the remnant kidney model is associated with diminution in VEGF expression and reduced glomerular capillary endothelial cell density. These findings were both attenuated by treatment with the ACE inhibitor, perindopril. Whether these changes are due to a primary effect of ACE inhibition on VEGF expression, or whether they are a consequence of other renoprotective effects of this class of drug cannot be determined form the present study.



   Acknowledgments
 
This project was supported by a grant from the National Health and Medical Research Council of Australia. The authors thank Miss Mariana Pacheco for and Miss Giao Tran for technical assistance.

Conflict of interest statement. R.E.G. has received honoraria for speaking engagements and travel grants to attend scientific meeting from Servier Pharmaceuticals, the manufacturers of perindopril.



   Notes
 
Correspondence and offprint requests to: Dr Darren J. Kelly, Department of Medicine, University of Melbourne, St Vincent's Hospital, 29 Regent Street, Fitzroy, 3065 Victoria, Australia. Email: dkelly{at}medstv.unimelb.edu.au Back



   References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

  1. Kang DH, Kanellis J, Hugo C et al. Role of the microvascular endothelium in progressive renal disease. J Am Soc Nephrol 2002; 13:806–816[Abstract/Free Full Text]
  2. Bohle A, von Gise H, Mackensen-Haen S, Stark-Jakob B. The obliteration of the postglomerular capillaries and its influence upon the function of both glomeruli and tubuli. Functional interpretation of morphologic findings. Klin Wochenschr 1981; 59:1043–1051[ISI][Medline]
  3. Ostendorf T, Kunter U, Eitner F et al. VEGF(165) mediates glomerular endothelial repair. J Clin Invest 1999; 104:913–923[Abstract/Free Full Text]
  4. Wu L, Cox A, Roe C, Dziadek M, Cooper ME, Gilbert RE. Transforming growth factor ß1 and renal injury following subtotal nephrectomy in the rat: role of the renin–angiotensin system. Kidney Int 1997; 51:1553–1567[ISI][Medline]
  5. Lewis EJ, Hunsicker LG, Clarke WR et al. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with nephropathy due to type 2 diabetes. N Engl J Med 2001; 345:851–860[Abstract/Free Full Text]
  6. Brenner BM. Hemodynamically mediated glomerular injury and the progressive nature of kidney disease. Kidney Int 1983; 23:647–655[ISI][Medline]
  7. Border WA, Noble NA. Interactions of transforming growth factor-beta and angiotensin Ii in renal fibrosis. Hypertension 1998; 31:181–188[Abstract/Free Full Text]
  8. Williams B, Baker AQ, Gallacher B, Lodwick D. Angiotensin II increases vascular permeability factor gene expression by human vascular smooth muscle cells. Hypertension 1995; 25:913–917[Abstract/Free Full Text]
  9. Kang DH, Joly AH, Oh SW et al. Impaired angiogenesis in the remnant kidney model: I. potential role of vascular endothelial growth factor and thrombospondin-1. J Am Soc Nephrol 2001; 12:1434–1447[Abstract/Free Full Text]
  10. Lehr HA, Mankoff DA, Corwin D, Santeusanio G, Gown AM. Application of photoshop-based image analysis to quantification of hormone receptor expression in breast cancer. J Histochem Cytochem 1997; 45:1559–1565[Abstract/Free Full Text]
  11. Gilbert RE, Wu LL, Kelly DJ et al. Pathological expression of renin and angiotensin II in the renal tubule after subtotal nephrectomy—implications for the pathogenesis of tubulointerstitial fibrosis. Am J Pathol 1999; 155:429–440[Abstract/Free Full Text]
  12. Baskin DG, Stahl WL. Fundamentals of quantitative autoradiography by computer densitometry for in situ hybridization, with emphasis on 33P. J Histochem Cytochem 1993; 41:1767–1776[Abstract/Free Full Text]
  13. Lee LK, Meyer TW, Pollock AS, Lovett DH. Endothelial cell injury initiates glomerular sclerosis in the rat remnant kidney. J Clin Invest 1995; 96:953–964[ISI][Medline]
  14. Schena FP, Gesualdo L, Grandaliano G, Montinaro V. Progression of renal damage in human glomerulonephritides: is there sleight of hand in winning the game? Kidney Int 1997; 52:1439–1457[ISI][Medline]
  15. Kang DH, Hughes J, Mazzali M, Schreiner GF, Johnson RJ. Impaired angiogenesis in the remnant kidney model: II. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function. J Am Soc Nephrol 2001; 12:1448–1457[Abstract/Free Full Text]
  16. Gruden G, Thomas S, Burt D et al. Interaction of angiotensin II and mechanical stretch on vascular endothelial growth factor production by human mesangial cells. J Am Soc Nephrol 1999; 10:730–737[Abstract/Free Full Text]
  17. Amaral SL, Roman RJ, Greene AS. Renin gene transfer restores angiogenesis and vascular endothelial growth factor expression in Dahl S rats. Hypertension 2001; 37:386–390[Abstract/Free Full Text]
  18. Moravski CJ, Kelly DJ, Cooper ME et al. Retinal neovascularization is prevented by blockade of the renin-angiotensin system. Hypertension 2000; 36:1099–1104[Abstract/Free Full Text]
  19. Suzuma I, Hata Y, Clermont A et al. Cyclic stretch and hypertension induce retinal expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2: potential mechanisms for exacerbation of diabetic retinopathy by hypertension. Diabetes 2001; 50:444–454[Abstract/Free Full Text]
  20. Brown LF, Berse B, Tognazzi K et al. Vascular permeability factor mRNA and protein expression in human kidney. Kidney Int 1992; 42:1457–1461[ISI][Medline]
  21. Amemiya T, Sasamura H, Mifune M et al. Vascular endothelial growth factor activates MAP kinase and enhances collagen synthesis in human mesangial cells. Kidney Int 1999; 56:2055–2063[CrossRef][ISI][Medline]
  22. Pertovaara L, Kaipainen A, Mustonen T et al. Vascular endothelial growth factor is induced in response to transforming growth factor-beta in fibroblastic and epithelial cells. J Biol Chem 1994; 269:6271–6274[Abstract/Free Full Text]
Received for publication: 19. 5.02
Accepted in revised form: 12. 2.03





This Article
Abstract
FREE Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (8)
Disclaimer
Request Permissions
Google Scholar
Articles by Kelly, D. J.
Articles by Gilbert, R. E.
PubMed
PubMed Citation
Articles by Kelly, D. J.
Articles by Gilbert, R. E.