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Published ahead of print on July 11, 2007
J Am Soc Nephrol 18: 2320-2329, 2007
© 2007 American Society of Nephrology
doi: 10.1681/ASN.2006101093
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BASIC RESEARCH

Podocyte-Specific Expression of Angiopoietin-2 Causes Proteinuria and Apoptosis of Glomerular Endothelia

Belinda Davis*, Alessandra Dei Cas*, David A. Long{dagger}, Kathryn E. White{ddagger}, Anthea Hayward*, Ching-Hsin Ku*, Adrian S. Woolf{dagger}, Rudolf Bilous{ddagger}, Giancarlo Viberti* and Luigi Gnudi*

* Cardiovascular Division, King's College London School of Medicine, Guy's Hospital, King's College London, and {dagger} Nephro-Urology Unit, Institute of Child Health, University College London, and {ddagger} Department of Diabetes and Metabolism, School of Clinical Medical Sciences, University of Newcastle, Newcastle, United Kingdom

Correspondence: Dr. Luigi Gnudi, Unit for Metabolic Medicine, Department of Diabetes and Endocrinology, 5th floor Thomas Guy House, Guy's Hospital, London SE1 9RT, UK. Phone: +44-20-71881939; Fax: +44-20-71880146; E-mail: luigi.gnudi{at}kcl.ac.uk

Received for publication October 6, 2006. Accepted for publication April 27, 2007.


   Abstract
Top
 Abstract
Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
Angiopoietin-2 (Ang-2) modulates embryonic vascular differentiation primarily by inhibiting the antiapoptotic effects of Ang-1 on endothelia that express the Tie-2 receptor. Ang-2 is transiently expressed by developing glomeruli but is downregulated with normal maturation. Glomerular Ang-2 expression is, however, markedly upregulated in animal models of diabetic nephropathy and glomerulonephritis, both leading causes of human chronic renal disease, affecting 10% of the world population. It was hypothesized that Ang-2 might have significant roles in the pathobiology of glomerular disease. Mice with inducible podocyte-specific Ang-2 overexpression were generated. When the transgene was induced in adults for up to 10 wk, mice had significant increases in both albuminuria and glomerular endothelial apoptosis, with significant decreases of both vascular endothelial growth factor-A and nephrin proteins, critical for maintenance of glomerular endothelia and filtration barrier functional integrity, respectively. There was, however, no significant change of systemic BP, creatinine clearance, or markers of renal fibrosis, and podocytes appeared structurally intact. In kidneys of young animals in which Ang-2 had been upregulated during organogenesis, increased apoptosis occurred in just-formed glomeruli. In vitro, short-term exposure of isolated wild-type murine glomeruli to exogenous Ang-2 led to decreased levels of vascular endothelial growth factor-A protein. These novel results provide insight into molecular mechanisms underlying proteinuric disorders, highlight potentially complex interactions between subsets of glomerular cells, and emphasize how a vascular growth factor that has critical roles in normal development may be harmful when re-expressed in the context of adult disease.


   Introduction
Top
 Abstract
 Introduction
RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
Angiopoietins are growth factors involved in angiogenesis and vasculogenesis. Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are ligands for the Tie-2 receptor, found primarily on endothelial cells.1 Physiologic roles of Ang-1, the major physiologic ligand for Tie-2, include promotion of endothelial survival and restriction of endothelial permeability.2 Furthermore, Tie-2 activation stabilizes supporting perivascular cells, likely through Ang-1 paracrine-mediated mechanisms.3,4 Ang-2 is considered to be a natural antagonist of Ang-1 by virtue of its ability to competitively inhibit binding of Ang-1 to Tie-2, thereby reducing Tie-2 activation and signaling.5 Other work has suggested that Ang-1 and Ang-2, partly with the aid of integrins, could modulate cellular survival pathways in the absence of Tie-2.6 The actions of angiopoietins are critical for normal vascular development5,7,8 and for the maintenance and turnover of vessels in mature animals.7

The expression of Ang-2 in the kidney has been studied, although very little is known about its role(s) in health or disease. Ang-2 is expressed during mouse kidney development,9,10 whereas, postnatally, in the adult kidney, it is significantly downregulated. Mice that are null mutant for Ang-2 die soon after birth with chylous ascites,11 and neonates display dysmorphogenesis of cortical peritubular capillaries.12 In normal mature glomeruli, Ang-2 levels are low or undetectable,10,13 but they have been reported to be upregulated in certain disease models, including diabetic nephropathy14–17 and glomerulonephritis.13,18 In contrast, Ang-1 is normally expressed in the glomerular podocyte, and it has been suggested that a finely regulated Ang-1/Ang-2 level ratio (in favor of Ang-1) contributes toward the maintenance of the integrity of the glomerular filtration barrier.19

On the basis of these observations, we formulated the hypothesis that Ang-2 might have significant roles in the pathobiology of glomerular disease. We proceeded to explore this idea by generating mice in which Ang-2 could be overexpressed, in an inducible manner, in glomeruli.


   RESULTS
Top
 Abstract
 Introduction
 RESULTS
DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
Generation of Transgenic Mice
We obtained 10 founders for the pTRE bidirectional LacZ/Ang-2 construct (Figure 1); eight transmitted the transgene to the offspring. Mice that were derived from the F1 generation were bred with mice that were homozygous for podocin-rtTA expressing the reverse tetracycline-controlled transcriptional activator (rtTA) specifically in podocytes.20 First-generation offspring carrying both transgenes were studied for doxycycline-inducible transgene expression. The progeny from two pTRE bidirectional LacZ/Ang-2 founders showed inducible LacZ expression with no basal leaky expression of the transgene. We bred one parent that was homozygous for podocin-rtTA with another parent that was heterozygous for pTRE-LacZ/Ang-2 to generate two genotypes (abbreviated to Pod/Ang-2 and Pod/+), each to be administered either doxycycline or vehicle. Although the podocin-rtTA and pTRE-LacZ/Ang-2 lines were generated on different backgrounds (BL6/CBA and FVB/N, respectively), all experimental animals (littermates) in this study will have received one set of chromosomes from each transgenic parent and therefore would have a similar complements of "background alleles."


Figure 1
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  		Figure 1. Transgene constructs. Diagram of the two constructs used in the transgenic mouse lines for the generation of the reverse tetracycline–controlled transcriptional activator (rtTA) system (Tet-On System). Vector A is a 2.5-kb fragment of the NPHS2 (podocin) promoter-enhancer region and directs the expression of rtTA in podocytes. In the presence of tetracycline or a derivative such as doxycycline, rtTA binds to tetracycline-response operon promoter element (TRE) and initiates transcription from the cytomegalovirus (CMV) promoter of the LacZ/Ang-2 cDNA (Vector B).

 
Transgene Expression in Adult Mice
Eight-week-old adult mice were administered doxycycline or vehicle for up to 10 wk (both genders showed a similar phenotype and were analyzed together). X-gal staining revealed no signal in Pod/+ doxycycline- and vehicle-treated kidneys, confirming that the staining protocol did not detect endogenous galactosidase activity (data not shown). Furthermore, Pod/Ang-2 mice that were exposed to vehicle also failed to give a positive signal (Figure 2A), excluding "leakage" of LacZ expression in noninduced mice. In contrast, kidneys from adult Pod/Ang-2 mice that were exposed to doxycycline showed positive X-gal staining (Figure 2A), consistent with podocyte expression of the transgene, as described previously using the same podocin promoter.20 X-gal staining was analyzed at different time points after doxycycline administration (10 d and 5 to 10 wk), and the results were superimposable.


Figure 2
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  		Figure 2. Inducible expression of LacZ and angiopoietin-2 (Ang-2). (A) Nuclear X-gal staining of glomerular podocytes in Pod/Ang-2 mice after 10 wk of doxycycline administration. No staining was observed in vehicle-administered Pod/Ang-2 kidneys. Note that the "halo" around each podocyte results from the diffusion of high levels of color product from each positive cell. (B) Representative immunoblot and quantitative analysis of Ang-2 expression in kidney cortex lysates after 10 wk of vehicle or doxycycline administration to Pod/Ang-2 and Pod/+ mice: Each bar shows Ang-2 expression levels as a ratio factored for {alpha}-actin, a housekeeping protein (means ± SEM; n = 5 to 6 animals per group; *P < 0.01). g, glomerulus; t, tubuli.

 
Ang-2 protein expression was assessed by immunoblotting and immunogold electron microscopy (EM). Cortices of kidneys from Pod/Ang-2 mice that were exposed to doxycycline showed significantly (P < 0.01) increased Ang-2 levels (four- to five-fold) versus Pod/Ang-2 mice that were exposed to vehicle and versus Pod/+ mice that were exposed to either doxycycline or vehicle (Figure 2B). The Ang-2 overexpression in Pod/Ang-2 kidneys was detected at 10 d after the start of doxycycline exposure and was sustained for the whole study. These observations were supported by EM, which showed a significant increase in immunogold signal for Ang-2 in podocytes of Pod/Ang-2 doxycycline-treated mice: Negative control kidneys (i.e., those from vehicle Pod/Ang-2 and vehicle- or doxycycline-treated Pod/+ mice) showed significantly lower levels of Ang-2 expression in podocyte cell bodies and foot processes (Figure 3 and data not shown). During doxycycline administration, mice displayed neither changes in behaviors nor differences in body weight between groups (data not shown).


Figure 3
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  		Figure 3. Induced Ang-2 expression in podocytes. (Left) Representative transmission EM image of Ang-2 immunogold staining (black dots) in kidneys from Pod/Ang-2 mice that were treated with vehicle (VEH) or doxycycline (DOX). (Right) Quantitative analysis showing significant increase in Ang-2 particles in podocytes of Pod/Ang-2 doxycycline kidneys versus vehicle controls (means ± SEM particles/podocyte unit area; n = 5 animals per group; *P = 0.05). Note also the overtly normal podocyte foot processes and endothelial fenestrae in the Pod/Ang-2 doxycycline group. Pod, podocyte cell body; Pfp, podocyte foot process; BM, glomerular basement membrane; EC, endothelial cells. Bars = 0.2 µm.

 
Effects of Ang-2 Transgene Activation on Renal Function in Adult Mice
Eight-week-old mice of either Pod/+ or Pod/Ang-2 genotypes showed a similar, low level of albuminuria (approximately 20 µg/d; Figure 4), which was maintained at 5 and 10 wk after the start of the experiment in Pod/+ mice that were exposed to vehicle or doxycycline and in Pod/Ang-2 mice that were exposed to vehicle (Figure 4). At 5 wk after administration of doxycycline, Pod/Ang-2 mice showed a highly significant (P < 0.01) increased albumin excretion (approximately 60 µg/d), and a similar result was also noted at 10 wk (Figure 4). In accordance with the modest rise of albumin excretion, mice were not edematous. At 10 wk after the start of either vehicle or doxycycline administration, systolic and diastolic BP levels were not significantly different in the four groups of animals: Pod/+ vehicle 138.5 ± 1.6/112.5 ± 2 mmHg; Pod/+ doxycycline 136.5 ± 2.5/111 ± 1.6 mmHg; Pod/Ang-2 vehicle 132.7 ± 1.25/110 ± 1.4 mmHg; Pod/Ang-2 doxycycline 133.2 ± 1.5/108.8 ± 3.2 mmHg (n = 4 to 5 per group). Furthermore, no significant changes were observed in creatinine clearances (Pod/+ vehicle: 9.2 ± 2; Pod/+ doxycycline: 10.5 ± 2.5; Pod/Ang-2 vehicle: 11.4 ± 1.8; Pod/Ang-2 doxycycline: 10.9 ± 2.9 µl/min per g body weight; n = 8 to 10 per group).


Figure 4
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  		Figure 4. Albuminuria in Ang-2–overexpressing animals. Twenty-four-hour albumin excretion in urine of Pod/+ and Pod/Ang-2 animals, each genotype with either vehicle (VEH) or doxycycline (DOX) administration for 5 and 10 wk, expressed as geometric mean with 95% confidence intervals. Note significant increases in albuminuria in the Pod/Ang-2 doxycycline animals at 5 and 10 wk (n = 10 to 12/group; *P < 0.01). b, baseline.

 
Kidney Histology
Kidneys that were obtained from doxycycline-treated Pod/Ang-2 mice were morphologically grossly similar to those that were harvested from the negative control groups: They had smooth outlines and were neither swollen nor contracted. No differences in percentage of kidney/body weight ratio between groups Pod/+ and Pod/Ang-2 upon vehicle or doxycycline administration were observed. The proportion of glomeruli with apoptotic cells was approximately 2.5 times higher in Pod/Ang-2 doxycycline versus Pod/+ doxycycline and vehicle kidneys (Pod/+ vehicle 0.41 ± 0.4; Pod/+ doxycycline 1.15 ± 0.3; Pod/Ang-2 vehicle 1.33 ± 0.3; Pod/Ang-2 doxycycline 3.22 ± 0.5; Pod/Ang-2 doxycycline versus all other groups individually P < 0.05; n = 6 to 10 per group; Figure 5). EM demonstrated that glomerular apoptosis in Pod/Ang-2 doxycycline-administered mice was localized to the endothelium (Figure 6). EM (Figures 3 and 6) revealed no gross abnormalities in podocytes of kidneys from doxycycline-administered Pod/Ang-2 mice; in particular, neither foot process fusion/effacement nor nuclear morphology suggestive of apoptosis was observed. Furthermore, endothelial fenestrations and glomerular basement membranes appeared similar to those of controls (Figures 3 and 6 and data not shown). Quantitative analyses of Masson-Trichrome staining of glomeruli showed no significant differences among the four experimental groups (see Supplementary Figure 1). The tubules of doxycycline-exposed Pod/Ang-2 mice seemed similar to those of the other three experimental groups (data not shown).


Figure 5
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  		Figure 5. Glomerular apoptosis in Ang-2–overexpressing kidneys. Representative image of a positive apoptotic cell in a glomerulus (g): (i) terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining (FITC, green; note dull autofluorescence of other tissues, especially proximal tubules (t) and Bowman's capsule cells); (ii) propidium iodide staining (all nuclei are red); (iii) images merged with arrow indicating apoptotic nucleus; (iv) a 2.5-fold increase in glomerular apoptosis score in kidneys of Pod/Ang-2 animals administered doxycycline (DOX) versus vehicle (VEH) or Pod/+ DOX animals (means ± SEM; n = 8 to 10 animals per group; *P = 0.05).

 

Figure 6
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  		Figure 6. Glomerular ultrastructural morphology in Ang-2–overexpressing kidneys. Glomerular electron microscopy (EM) in Pod/Ang-2 animals administered vehicle (i and iii) or doxycycline (ii and iv) for 10 wk. In i and ii, scanning EM images of the glomerular capillary lumen showing overtly normal endothelial fenestrae are shown, whereas in iii and iv, transmission EM images of glomerular capillaries are shown. In iv is shown condensed, irregular, and dark apoptotic nuclei (*) in an endothelial cell of doxycycline-treated Pod/Ang-2 animals. Bars = 2 µm.

 
Expression of Diverse Genes after Induction of Ang-2
The increased albuminuria that was observed after 10 wk activation of Ang-2 overexpression was accompanied by an approximately 50% reduction in both vascular endothelial growth factor-A (VEGF-A; P < 0.02) and nephrin (P < 0.01) expression (Figure 7), as assessed by immunoblotting of kidney cortices protein lysate; there was, however, no significant differences in levels of Ang-1 or Tie-2 proteins between Pod/+ and Pod/Ang-2 mice that were administered either vehicle or doxycycline (Supplementary Figure 2). Next, a set of "fibrotic genes" were analyzed, and we found no significant differences for levels of either TGF-beta1 (protein and mRNA) or collagen IV (mRNA) and fibronectin (mRNA) among the four groups, although there was a tendency for doxycycline administration to be associated with lower levels of TGF-beta1 and fibronectin transcripts (Supplementary Figure 1).


Figure 7
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  		Figure 7. Kidney cortex vascular endothelial growth factor-A (VEGF-A) and nephrin expression. Representative immunoblots and densitometric analysis of VEGF-A (A) and nephrin (B) protein expression in renal cortex of Pod/Ang-2 doxycycline (DOX)- or vehicle (VEH)-administered animals and their Pod/+ control. Graphs depict levels factored for {alpha}-actin (means ± SEM; n = 6 to 8 animals per group). Note the significant reductions in Pod/Ang-2 doxycycline versus vehicle and Pod/+ DOX kidneys for VEGF-A (*P < 0.02) and nephrin (**P < 0.01).

 
Angiopoietin Downregulates VEGF-A in Isolated Glomeruli In Vitro
Exposure of isolated adult rat glomeruli to Ang-2 induced a significant (approximately 30%; P = 0.01) downregulation of VEGF-A protein levels after 24 h of incubation. There was no effect on nephrin expression also at later time points up to 72 h of Ang-2 incubation. No changes in Ang-1 levels were observed in these same experiments (Figure 8).


Figure 8
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  		Figure 8. VEGF-A, nephrin, and Ang-1 expression in Ang-2–treated isolated glomeruli. VEGF-A, nephrin, and Ang-1 representative immunoblotting and densitometric protein expression analysis in isolated glomeruli incubated with Ang-2 (600 ng/ml) or vehicle (VEH) for 24 h. Data are means ± SEM percentage change over control (vehicle = 100%; vehicle versus Ang-2, *P = 0.01; n = 4, experiments conducted in duplicate).

 
LacZ/Ang-2 Transgene Expression in Maturing Kidneys
In other experiments, embryos and weaning mice were exposed to doxycycline by direct administration to mothers from the onset of pregnancy until 3 wk after birth. In these studies, litters contained animals that were heterozygous for both transgenes (Pod/Ang-2) and others that were heterozygous for podocin-rtTA as controls (Pod/+). At embryonic day 16, the first layer of glomeruli in the nephrogenic zone did express LacZ only in Pod/Ang-2 kidneys (Figure 9A). By 3 wk after birth, the nephrogenic zone had disappeared and LacZ expression was widespread in glomeruli, as for adult mice. Although it was not feasible to collect urine in the 3-wk-old mice, apoptosis was present in 7% of glomeruli from doxycycline-exposed Pod/+ mice, whereas the frequency was increased (P = 0.05) to approximately 20% in doxycycline-exposed Pod/Ang-2 mice (Figure 9B).


Figure 9
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  		Figure 9. Embryos and weaning mice that were exposed to doxycycline (DOX). (A) At embryonic day 16 (E16), Pod/+ organs that were exposed to doxycycline showed no LacZ expression, as assessed by X-gal staining; conversely, LacZ was expressed in nascent glomeruli of Pod/Ang-2 kidneys that were exposed to doxycycline. (B) terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining in 3-wk-old postnatal kidneys; on average, approximately 7.5% (7.57 ± 2.74) of glomeruli from doxycycline-exposed Pod/+ mice harbored an apoptotic cell, whereas the frequency was significantly increased (P = 0.05) to approximately 20% (20.36 ± 9.49) in doxycycline-exposed Pod/Ang-2 kidneys (n = 3 each group). nz, nephrogenic zone; pt, proximal tubule.

 

   DISCUSSION
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
CONCISE METHODS
 DISCLOSURES
 REFERENCES
 
As previously discussed, Ang-2 is downregulated in the normal mature glomeruli, but its expression is upregulated in experimental animal models of diabetic nephropathy and glomerulonephritis. We hypothesized that any disturbances in the Ang-2 expression levels might have significant roles in the pathobiology of glomerular disease. To explore this idea, we needed to experimentally upregulate its expression and activity while avoiding potentially confounding biologic effects that might accompany a generalized, systemic, and uncontrolled increase in levels of this vascular growth factor.21–23 Therefore, we generated mice in which Ang-2 could be overexpressed, in an inducible manner, specifically in glomeruli; furthermore, we elected to overexpress Ang-2 specifically in podocytes because in humans and murine species, they express Ang-1 and, at least in rats, the Tie-2 receptor.19 The novel observations arising from this experimental strategy support the contention that upregulation of Ang-2 that is observed in glomerular diseases not only is associated with glomerular injury as described13,14,16,17 but may also play a functional direct role in glomerular damage and exacerbate proteinuria.

Ang-2 Overexpression Leads to Enhanced Glomerular Endothelial Apoptosis
Increased glomerular expression of Ang-2 was paralleled by enhanced glomerular apoptosis, with transmission EM identifying these cells as endothelia. The biologic effects of Ang-2 are highly context dependent and, in vivo, depend on ambient levels of VEGF-A, such that vessel regression occurs when VEGF-A is lacking, whereas vessel destabilization is followed by angiogenesis when the local milieu is rich in VEGF-A.5,24 In this study, the increased levels of Ang-2 in the podocytes of Pod/Ang-2 adult mice that were exposed to doxycycline was paralleled by a constant level of Ang-1 and decreased VEGF-A. An increase of Ang-2 levels in the glomerulus would tend to antagonize Ang-1–induced Tie-2 activation, destabilizing capillaries, which would be further compromised by the parallel downregulation of VEGF-A, another endothelial survival factor. To an extent, these changes mimic those previously reported in experimental glomerulonephritis,18 whereby a reduction of glomerular VEGF-A expression is accompanied by an increased Ang-2/Ang-1 ratio; these changes might be expected to be antiangiogenic, and, indeed, they are accompanied by glomerular endothelial apoptosis.13

Of interest, Eremina et al.25 reported that a genetically mediated downregulation of VEGF-A in podocytes led to severe glomerular endothelial swelling ("endotheliosis") that progressed to endothelial necrosis rather than apoptosis; however, in that study, levels of Ang-1 and Ang-2 were not reported. Finally, in this study, it was found that, in young Pod/Ang-2 mice that had been exposed to doxycycline transplacentally and then via their mother's milk, the incidence of glomerular apoptosis was again increased versus Pod/+ mice, supporting further the proapoptotic role of excessive Ang-2 expression; notably, in the younger (3-wk-old) control mice, we found a higher incidence of glomerular apoptosis versus mature (18-wk-old) control mice, supporting a previous observation that immature glomeruli have a high incidence of endothelial cell programmed death.26

Ang-2 Overexpression in Glomeruli Leads to Enhanced Proteinuria
When Ang-2 was induced by doxycycline administration to adults for up to 10 wk, mice developed increased albuminuria. This was not accompanied by significant glomerulosclerosis or by upregulation of a panel of fibrotic genes. As assessed by electrophoresis analyses (data not shown), the urine of mice in which glomerular Ang-2 had been induced contained prominent bands for relatively high molecular weight proteins that were barely detectable in control mice. Although the levels of albuminuria were not in the "nephrotic range," we consider that the source of the enhanced proteinuria was most likely to be a leaky glomerular barrier. Podocytes and their slit diaphragms bridging the foot processes constitute the main barrier to protein filtration.27,28 In this context, we recorded reduced levels of nephrin protein in Pod/Ang-2 adult mice after doxycycline administration. The absence of major changes in the appearance of the glomerular basement membrane makes a structural cause for albuminuria less likely.29 Moreover, we did not observe fusion of podocyte foot processes, which is frequently observed in conditions leading to proteinuria of glomerular origin.30 This is not invariable, however31,32; for example, Liu et al.33 failed to find foot process fusion in the glomeruli of animals that were rendered proteinuric after administration of antibodies to Neph1 and nephrin proteins. Our data are consistent with the idea that (at least modest) protein leakage can occur through defective podocyte slit diaphragms via nephrin downregulation and that the podocyte structural alterations, frequently described in proteinuric diseases, are not always a prerequisite for the development of proteinuria and may be a later development.34

The question remains, however, as to the cause of the downregulation of nephrin and VEGF-A in adult mice overexpressing glomerular Ang-2. On the assumption that both VEGF-A and nephrin are exclusively expressed by podocytes within glomeruli, there are several possible explanations. The first is based on the assumption that glomerular endothelia are the only local target cells for Ang-1 and Ang-2. It was previously demonstrated that cultured human podocytes do not express Tie-2,19 whereas the receptor is expressed in cultured human glomerular endothelia.35 If these expression patterns can be taken as a paradigm for mouse glomeruli in vivo, then the observed downregulation of both VEGF-A and nephrin by podocytes in Ang-2–overexpressing glomeruli must be an indirect effect, secondary to perturbations in endothelial survival and/or paracrine signaling. An alternative explanation is that podocytes themselves express Tie-2 in addition to glomerular endothelia; that this may occur is suggested by the published observation of Satchell et al.19 that rat podocytes express Tie-2 in vivo and by our own unpublished observations that murine podocytes also express the same receptor in culture (data not shown). In addition, the biology of other cell types has been shown to be modulated by angiopoietins through integrin interactions given the absence of Tie-2 expression.6 In either case, it remains possible that podocytes are direct or indirect targets for Ang-1 and Ang-2, and this might explain the decreased levels of VEGF-A and nephrin.

Finally, the interpretation of our results is made more complex by the fact that experimental blockade of VEGF-A actions can itself be followed by reduced levels of nephrin and proteinuria.36 Furthermore, the use of VEGF inhibitors in neoplastic disease37 and the elevated circulating levels of soluble VEGF receptor in preeclampsia in humans38 have been associated with the development of proteinuria. Furthermore, in our in vitro experiments on isolated glomeruli, we observed that Ang-2 downregulates VEGF-A expression, which suggests that the reduction in nephrin expression observed in vivo could represent a delayed, secondary event, possibly related to VEGF-A downregulation as previously suggested.36

Our results provide insight into molecular mechanisms underlying proteinuric disorders, highlight potentially complex interactions between subsets of glomerular cells, and emphasize how a vascular growth factor that has critical roles in normal development may be harmful when reexpressed in adult life. In this work, the upregulation of Ang-2 (in vivo and in vitro) was accompanied by a downregulation of VEGF-A; this resembles the situation in experimental, immune-mediated glomerulonephritis.13,18 However, we also note that, in other experimental models characterized by proteinuria (e.g. diabetic nephropathy14–17), Ang-2 upregulation is accompanied by an increase of VEGF. Cleary, further experiments will be needed to unravel carefully the relationships between the observed changes in glomerular endothelial survival, enhanced albuminuria, and alterations in VEGF-A and nephrin expression that follow in vivo upregulation of Ang-2. Angiopoietins could represent novel pharmacologic targets for the treatment of glomerular diseases, and future studies will investigate the potential benefits of modulating the angiopoietin/Tie-2 receptor system in such disorders.


   CONCISE METHODS
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
DISCLOSURES
 REFERENCES
 
All chemicals were purchased from Sigma-Aldrich (Gillingham, Dorset, UK). Restriction endonucleases were obtained from Fermentas (St. Leon-Rot, Germany), and ligation kit was obtained from Roche (Lewes, Sussex, UK). Recombinant human Ang-2 was obtained from R&D Systems (Abingdon, UK). Mice were kept according to the Guidelines on the Use of Animals in Research. Mice were housed in a pathogen-free environment at 21°C, with 12-h light-dark cycle, all receiving a standard laboratory animal chow (Special Diet Services; Witham, Essex, UK) and water ad libitum.

Generation of Transgenic Mice
Mouse Ang-2 cDNA was obtained from the plasmid PEF–Ang-2 (gift of Dr. H.T. Yuan, Harvard University, Boston, MA)39 and cloned in the plasmid pBI-G Tet-Vector (GenBank #U89933; Clontech, Saint-Germain-en-Laye, France). The resulting plasmid, pTRE bidirectional LacZ/Ang-2, was used for generation of transgenic mice (Figure 1). Transgenic mice were generated in the King's College London Transgenic Unit by direct microinjection of the DNA construct into the pronuclei of BL6-CBA/F1 fertilized zygotes and subsequent transfer to pseudopregnant females. Transgene genomic integration was initially studied with Southern blotting technique. Subsequently, mice were genotyped by PCR using the following set of primers detecting the 3' end (SV40 polyA) of the transgene: Sense 5'-ACCTATAAAAATAGGCGTATCACGA-3' and antisense 5'-TGGCTGATTATGATCCTGCA-3' (amplicon size 281 bp). The genotyping for the podocin-rtTA mice (gift from Dr. J. Kopp, Bethesda, MD; Figure 1) was also studied with PCR, as described previously.20

Expression of Ang-2/LacZ was induced by administration of doxycycline (2 mg/ml) with drinking water. The dosage of doxycycline used in the study was obtained from previous work by Shigehara et al.20 Water was supplemented with sucrose (5% wt/vol) to enhance palatability, and doxycycline was replaced every third day and protected from light at all times. Control mice were treated with sucrose alone as vehicle.

Urinalyses and Creatinine Clearance Determination
Twenty-four-hour urine collections were made in adult mice, at baseline and after 5 and 10 wk of doxycycline or vehicle treatment. Urine volume was recorded, and aliquots were stored at –80°C for subsequent analysis. Albumin concentrations were measured in triplicate by ELISA using an anti-mouse albumin antibody (Bethyl Laboratories, Montgomery, TX) and expressed as µg/24 h. For measurement of creatinine clearance, blood samples were collected into heparinized tubes via cardiac puncture at the time of killing of the mice. Plasma and urinary creatinine was determined by isotope dilution electrospray mass spectrometry.40 Creatinine clearance (µl/min per g) was derived from the formula urinary creatinine x urine volume x 1440 min–1 x plasma creatinine–1 x body weight (g)–1.

Telemetry BP Determination
Mice were anesthetized (isoflurane), and a telemeter (DSI #TA11PA-C10 or C20; Data Sciences International, St. Paul, MN) was implanted via the left carotid artery as described previously.41 After complete recovery (2 to 3 wk), BP was recorded on two different days for 10 min repeated for three times within 1 h. The recording was performed in the morning in a room maintained at 21 to 22°C with the mice left undisturbed for at least 4 h before the BP determination. The average systolic and diastolic BP was then calculated for the two genotypes of adult mice exposed to either doxycycline or vehicle treatment for 10 wk.

Tissue Processing and X-gal (LacZ) Assays
Mice were either killed by cervical dislocation or were given terminal anesthesia with fentanyl/fluanisone (3 mg/kg) and midazolam (2 mg/kg) in saline (0.9% wt/vol). The left kidney was clamped and removed for further analysis. Mice were then were administered in vivo perfusion of phosphate-buffered 4% formalin and 1% glutaraldehyde solutions. Each kidney was then removed and stored in PBS 4% formalin at 4°C before electron microscopy studies. Tissue nuclear beta-galactosidase activity was studied as described previously.10

Quantification of Glomerulosclerosis with Masson-Trichrome Staining, TGF-beta1, Collagen IV, and Fibronectin mRNA
Paraffin-embedded tissue was stained with periodic Masson-Trichrome staining reagent, and the collagen deposition was determined in 50 glomerular profiles per kidney. The proportional area occupied by collagen fibers per glomerulus was calculated using a computer-assisted image analysis system (Soft Imaging System GmBH, Münster, Germany) as described previously.42 The calculated average of collagen fibers obtained from each animal was then used for statistical analysis. All quantitative assays were performed by two observers who were blinded to the experimental group of each sample. In addition, cDNA was prepared from the kidney cortex of Pod/+ and Pod/Ang-2 mice that were exposed to either vehicle or doxycycline, and quantitative real-time PCR was performed using the general methods previously described with the following primer sets for collagen IV, fibronectin, TGF-beta1, and hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a housekeeping gene: Collagen IV sense 5'-TTCCTTCGTGATGCACACCA-3' and antisense 5'-CCGTGGCACTCGATGAATG-3' (amplicon size 101 bp), fibronectin sense 5'-TGGCTGCCTTCAACTTCTCCT-3' and antisense 5'-TGTTTGATCTGGACTGGCAGTTT-3' (amplicon size 102 bp), TGF-beta1 sense 5'-AGGGCTACCATGCCAACTTCT-3' and antisense 5'-CCGGGTTGTGTTGGTTGTA-3' (amplicon size 102 bp), and HPRT sense 5'- AAGCTTGCTGGTGAAAAGGA-3' and antisense 5'-GCAAATCAAAAGTCTGGGGA-3' (amplicon size 354 bp).43,44

EM
For scanning EM analysis, pieces of cortical tissue were dehydrated in graded alcohol up to 70% and then freeze-cracked by immersion in liquid nitrogen. After cracking, dehydration was completed by immersion in 95% and then absolute ethanol. The tissue was critical point-dried, gold-coated, and examined under the scanning electron microscope. For transmission EM, kidney cortex specimens (1 mm3) were postfixed in osmium tetroxide, dehydrated in acetone, and embedded in epoxy resin. Ultrathin sections were stained with uranyl-acetate and lead citrate and examined.

Immunogold
Pieces of cortical tissue (1 mm3) were fixed in 2% paraformaldehyde for 4 h, dehydrated in alcohol, and embedded in LR White resin. Ultrathin sections were taken and mounted on carbon-coated nickel grids. The grids were incubated overnight at 4°C with primary antibody (rabbit anti-mouse Ang-2; Alpha Diagnostics, San Antonio, TX; 1:100) in PBS/0.5% BSA. After washing, samples were incubated with 10-nm-diameter gold-conjugated goat anti-rabbit IgG (1:20) for 1 h at room temperature. Subsequently, the grids were stained with 2% aqueous uranyl-acetate and examined with transmission EM. Systematically sampled random micrographs were taken from two to three glomeruli per case. A grid of points was superimposed on each image, and the number of points hitting podocytes was counted along with the number of gold particles on podocytes. The density of labeling was expressed as the number of gold particles per unit area of podocyte (particles/µm2). The density of background staining was also estimated and subtracted from the density of specific labeling.45

Detection of Apoptosis
Detection of apoptosis was performed with terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling on paraffin sections (5 µm), as described previously13: Apoptotic nuclei were visualized using a labeling method (In Situ Cell-Death Detection kit; Roche). For each kidney, approximately 100 cross-sections of different glomeruli were scored and the percentage of glomeruli containing apoptotic cells was calculated. Glomeruli were predominantly negative for apoptosis; those that were positive always contained just one apoptotic cell nucleus. Apoptosis was also studied qualitatively using transmission EM. Cells were defined in a pre-/apoptotic status when condensed chromatin, dense cytoplasm, cytoplasmic vacuoles, and dense bodies were visualized.26

Western Immunoblotting
The following antibodies were used: Anti–Ang-1 and anti–Ang-2–specific antibodies (Alpha Diagnostic), anti–Tie-2 and anti–TGF-beta1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti–VEGF-A antibody (R&D Systems), and anti–{alpha}-actin antibody (Sigma-Aldrich). A rabbit polyclonal anti-nephrin antibody against the intracellular domain of rat nephrin was provided by Prof. H. Holthofer (Helsinki University, Helsinki, Finland).46,47 Renal cortex protein lysates were analyzed with PAGE standard procedures, and bands were visualized with chemiluminescence (Amersham Biosciences, Bucks, UK) and quantified using computerized densitometry (ImageJ; National Institutes of Health, Bethesda, MD).

Effect of Ang-2 on Isolated Murine Glomeruli
For these experiments we used rat glomeruli because they provide more material (per animal) than mouse ones. Glomeruli were isolated from adult (2 to 4 mo of age) male Wistar rats (Charles River, Margate, UK) using a sieving technique previously described.42 The isolated glomeruli were kept in culture for the whole duration of the experiment in RPMI 1640 with l-glutamine, 10% heat-inactivated FCS, normal glucose (5.5 mM), 5 µg/ml insulin-transferrin-selenite, 0.4 µg/ml hydrocortisone, 1 mM sodium pyruvate, 15 mM HEPES, 0.09% NaHCO3, 100 U/ml penicillin, and 100 µg/ml streptomycin and incubated with Ang-2 (600 ng/ml) or vehicle (PBS and 0.1% BSA), for 24 h. At the end of the incubation, glomeruli were collected and cell lysate was obtained. Nephrin, VEGF-A, and Ang-1 expression was analyzed by Western immunoblotting, and results were normalized by {alpha}-actin expression.

Statistical Analyses
All data are shown as means ± SEM unless otherwise specified. Data for albuminuria were log-transformed before analysis, and data are shown as geometric means with 95% confidence interval. Differences between groups were tested by t test. When more than two groups were compared, differences were tested by ANOVA followed by post hoc pair-wise comparisons using LSD (SPSS, Chicago, IL). Statistical significance was accepted at P < 0.05.


   DISCLOSURES
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
REFERENCES
 
None.


   Acknowledgments
 
This work was partly funded by a project grant from Biotechnology and Biological Sciences Research Council (S13745) and by European Foundation for the Study of Diabetes/Servier grant. B.D. is a recipient of a Biomedical (CJ Martin) Fellowship from the Australian National Health and Medical Research Council, visiting from the Baker Research Institute (Melbourne, Australia). A.S.W. and D.A.L. were recipients of a project grant from the Kids Kidney Appeal.

This work was partly presented at the annual meeting of the American Society of Nephrology; November 8 through 13, 2005, Philadelphia, PA; and the annual meeting of European Association for the Study of Diabetes; September 14 through 17, 2006, Copenhagen, Denmark.

We thank Dr. A. McGuigan (Transgenic Unit, King's College London) for the transgenic mice generation and the Biomedical Electron Microscopy Unit (Newcastle University) for the electron microscopy study. We are grateful to Dr. J. Kopp (NIH) for providing the podocin-rtTA mice, to Dr. N. Dalton and Dr. C. Turner (Guy's Hospital, London, UK) for the creatinine determination, and to Dr. Karen L. Price (University College London) for help with the quantitative PCR.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.

Supplemental information for this article is available online at http://www.jasn.org/.

See the related editorial, "Angiopoietin-2 and Glomerular Proteinuria," on pages 2217–2218.


   REFERENCES
Top
 Abstract
 Introduction
 RESULTS
 DISCUSSION
 CONCISE METHODS
 DISCLOSURES
 REFERENCES
 

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