loss of the tumor suppressor vhlh leads to upregulation of cxcr4 and rapidly progressive...
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Loss of the tumor suppressor Vhlh leads to upregulationof Cxcr4 and rapidly progressive glomerulonephritisin miceMei Ding1,2, Shiying Cui1, Chengjin Li1, Serge Jothy3, Volker Haase4, Brent M Steer5, Philip A Marsden5,Jeffrey Pippin6, Stuart Shankland6, Maria Pia Rastaldi7, Clemens D Cohen8, Matthias Kretzler8,9 &Susan E Quaggin1,2,5
Rapidly progressive glomerulonephritis (RPGN) is a clinical
syndrome characterized by loss of renal function within
days to weeks and by glomerular crescents on biopsy. The
pathogenesis of this disease is unclear, but circulating factors
are believed to have a major role1,2. Here, we show that
deletion of the Von Hippel–Lindau gene (Vhlh) from intrinsic
glomerular cells of mice is sufficient to initiate a necrotizing
crescentic glomerulonephritis and the clinical features that
accompany RPGN. Loss of Vhlh leads to stabilization of
hypoxia-inducible factor a subunits (HIFs). Using gene
expression profiling, we identified de novo expression of the
HIF target gene Cxcr4 (ref. 3) in glomeruli from both mice and
humans with RPGN. The course of RPGN is markedly improved
in mice treated with a blocking antibody to Cxcr4, whereas
overexpression of Cxcr4 alone in podocytes of transgenic mice
is sufficient to cause glomerular disease. Collectively, these
results indicate an alternative mechanism for the pathogenesis
of RPGN and glomerular disease in an animal model and
suggest novel molecular pathways for intervention in
this disease.
The renal glomerulus is a specialized vascular bed that functions as abarrier between the blood and urinary spaces. It permits the passage ofsmall molecules and water freely into the urine, while preventing theloss of large serum proteins such as albumin4. The barrier itself iscomposed of only two cell types, podocytes and fenestrated endo-thelial cells, separated by the glomerular basement membrane (GBM;Supplementary Fig. 1 online). Mesangial cells sit between the capillaryloops, providing support and producing extracellular matrix mole-cules and growth factors such as stromal-derived factor-1 (Sdf1),
whereas parietal epithelial cells surround the urinary space. Podocytesalso function as vasculature-supporting cells, producing basementmembrane components and a number of vascular growth factorsincluding Vegfa5.
Glomerular diseases account for 20% of all end-stage renal failure inNorth America. The most dramatic of these is RPGN, a potentiallyfatal disease and one of the few diagnostic emergencies that occur innephrology. If left untreated, it rapidly progresses to renal failure withindays to weeks. There are three major categories of RPGN based on thepresence and type of immune deposits. RPGN without immunedeposits is classified as pauci-immune and is characterized by necrotiz-ing glomerular vasculitis with prominent segmental fibrin deposits.
Currently, the pathogenesis of pauci-immune RPGN is incomple-tely understood. The identification of antineutrophil cytoplasmicantibodies (p-ANCA and c-ANCA) in a cohort of individuals withthis disease was recognized as a major breakthrough1,6, and much ofthe work in this area has focused on the role of this circulatingantibody and immune cells in the pathogenesis and progression ofthis disease2. What remains unexplained is why 20% of individualswith pauci-immune RPGN never have circulating ANCA-specificantibodies and, conversely, why 30% of individuals have persistentlevels of ANCA-specific antibodies upon resolution of RPGN7.
We hypothesized an alternative pathogenic mechanism for RPGN:that podocytes are required for maintenance of glomerular capillaryhealth and that an intrinsic defect within this cell population maytrigger glomerular vasculitis and RPGN. To test this model, we deletedthe product of the von Hippel–Lindau gene (Vhlh, encoding VHL)selectively from podocytes in mice. VHL is a component of the E3ubiquitin ligase that targets proteins for degradation in the protea-some. Loss of VHL leads to stabilization of HIF a subunits and
Received 22 March; accepted 5 July; published online 13 August 2006; doi:10.1038/nm1460
1Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada. 2Institute of MedicalScience, 7213 Medical Sciences Building, 1 King’s College Circle, University of Toronto, Toronto, Ontario M5S 1A8, Canada. 3Department of Laboratory Medicine andPathobiology, St. Michael’s Hospital, University of Toronto, 30 Bond Street, Toronto, Ontario M5B 1W8, Canada. 4Department of Medicine, Program in Cell Growth andCancer, University of Pennsylvania, 3451 Walnut Street, Philadelphia, Pennsylvania 19104, USA. 5Department of Medicine and Division of Nephrology, St. Michael’sHospital, University of Toronto, 30 Bond Street, Toronto, Ontario M5B 1W8, Canada. 6Division of Nephrology, University of Washington, 1959 NE Pacific Street, Box356521, Seattle, Washington 98195, USA. 7Renal Immunopathology Laboratory, Fondazione D’Amico per la Ricerca sulle Malattie Renali, c/o San Carlo Hospital, viaPio II, 3, Milan 20153, Italy. 8Nephrologisches Zentrum, Medizinische Poliklinik, Ludwig-Maximilians-Universitat Munchen, Pettenkoferstr. 8a, Munich 80336,Germany. 9Current address: Division of Nephrology, Department of Medicine, University of Michigan, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0676,USA.Correspondence should be addressed to S.E.Q. ([email protected]).
NATURE MEDICINE VOLUME 12 [ NUMBER 9 [ SEPTEMBER 2006 1081
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subsequent upregulation of hypoxia-response downstream genes.Although VHL and HIFs have not been directly implicatedin RPGN, a number of VHL and HIF molecular targets are knownto be increased in RPGN, including tumor necrosis factor (TNF)-aand VEGFA8–10. Furthermore, a number of cases of RPGN havebeen reported in individuals with sporadic renal cell carcinoma, adisease associated with mutations in the VHL gene11,12. Here, we showthat loss of Vhlh selectively from podocytes causes a disease thatresembles pauci-immune RPGN in mice, and is characterized by theabsence of ANCA-specific antibodies. In contrast to the widelyaccepted paradigm that vascular injury in vasculitis and specificallyRPGN is the result of circulating factors, in our genetic model thisinjury is initiated by podocytes that sit on the ‘other side’ of theendothelium, away from the circulation and which reside in theurinary space.
To selectively delete Vhlh from renal podocytes, we bred a podo-cyte-specific Cre recombinase mouse line (Pod-Cre) with mice thatcarry a wild-type Vhlh allele flanked by loxP sites (SupplementaryFig. 2 online)13. Cre-mediated DNA excision generates a null Vhlhallele through deletion of the promoter and first exon13. Mice ofall genotypes were born at the expected mendelian frequencyand appeared healthy until 4 weeks of age. At this time, they developedthe explosive onset of renal disease with hematuria, proteinuriaand renal insufficiency (Fig. 1a,b). Mice rapidly succumbed to renalfailure by 7 weeks of age. Histologic examination of kidneys at4 weeks showed crescentic glomerulonephritis with prominentsegmental fibrin deposition and fibrinoid necrosis (Fig. 1a,c).Notably, immune deposits were not observed on immunofluo-rescent examination. Together, these are features characteristic ofpauci-immune RPGN.
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Figure 2 Podocyte proliferation initiates crescent formation. (a–f) Laser capture microdissection (LCM)
and lineage tagging show crescentic cells derive from podocytes. (a) A cellular crescent is shown by the
arrow. (b) Outline of crescent captured by LCM. (c) After capture, the cellular crescent has been removed
from the glomerulus. (d) Isolated cellular crescent. (e) PCR using genomic DNA isolated from cellular
crescents (Cr) shows a single 1-loxP band demonstrating that the origin of these cells is from podocytes. In contrast, tubular cells (T) exhibit only the
2-loxP band, whereas glomeruli (G) that contain podocyte and nonpodocyte cell types exhibit both bands. (f) Cre-mediated DNA excision occurs only within
podocytes leading to expression of GFP (brown cells). Vhlh heterozygotes (VhlhloxP/+ Cre/Z/EG) show expression in healthy-appearing podocytes; expression
in Vhlh knockouts (VhlhloxP/loxP Cre/Z/EG) is seen in cells that populate the glomerular crescent confirming that these cells originate from the podocyte cell
lineage. (g) At 3 weeks, cells in the glomeruli (white arrowheads) of all mutant (VhlhloxP/loxP Pod-Cre; –/–) mice are proliferating as shown by BrdU labeling
(red label). Unstained glomeruli are indicated by arrowheads in control (+/+) kidneys. Original magnification, �800–1,500. (h) Double immunostaining for a
podocyte-restricted marker, the zonula occludens protein (ZO-1, green label), and BrdU (red) confirm that podocytes are proliferating (yellow cells and white
arrowhead). Right panel shows merged images of ZO-1 and BrdU staining. Original magnification, �1,500.
Figure 1 Selective deletion of Vhlh from
podocytes leads to RPGN. (a) At 3 weeks of age,
glomeruli from VhlhloxP/loxP Pod-Cre (–/–) mice
have dilated capillary loops (arrows). By 4 weeks
of age, glomeruli (G) from mutant mice have
cellular crescents (Cr), fibrinoid necrosis (Ne) and
renal tubules (t) packed with pink proteinaceous
material. The glomerulus in the left bottom panel
also shows periglomerular monocytic infiltrate.
C, control. Original magnification, �250–1,500.
(b) SDS-PAGE gel. Urine was loaded in each of
the lanes from control (C) or mutant VhlhloxP/loxP
Pod-Cre (–/–) mice. There was a large amount of
albuminuria (66 kDa) in the mutant. MW,
molecular weight. Serum creatinine valuesincreased twofold by 4 weeks of age in mutant
mice compared to control littermates and indicate
renal failure. (c) Immunostaining for fibrin
demonstrates segmental staining in all glomeruli
from VhlhloxP/loxP Pod-Cre mice (mutant Vhlh)
that is absent from wild-type controls. Original
magnification, �1,000.
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To determine the clinical course of the disease, mice and theirkidneys were examined at earlier timepoints. At 1 week of age, theglomeruli of VhlhloxP/loxP Pod-Cre mice were histologically normal. By3 weeks of age, proteinuria (1 g/L) was detected in the urine of allVhlhloxP/loxP Pod-Cre mice; but the mice were active and appearedhealthy, and blood measurements of renal function indicated that theywere not different from controls. On histologic examination, theglomeruli were normal, with the exception of dilated capillary loops(Fig. 1a). However, only 1 week later, 100% of VhlhloxP/loxP Pod-Cremice developed an acute onset of disease, similar to the presentation ofRPGN observed in humans. Given the interest in circulating factors indevelopment of this disease, we assessed mouse serum for circulatingANCA-specific antibodies at the height of their disease but did not findthem (n ¼ 3). Aside from vascular inflammation or glomerulitis,crescent formation is a notable and consistent finding in glomeruli ofhumans with RPGN and in our transgenic mice. It is currently acceptedthat crescentic cells are derived from parietal epithelium and influxinginflammatory cells such as macrophages (reviewed in Couser, B.Crescent formation. http://www.uptodate.com, 2004). More recently,lineage tagging showed that podocytes also contribute to crescent for-mation in an immune-mediated mouse model of anti-GBM RPGN14.
Given the experimental design, we speculated that the proliferatingcells that form the crescents in our model of RPGN originate from thepodocyte cell lineage. We used laser-capture microdissection to isolategenomic DNA from the cellular crescents (Fig. 2a–e). PCR analysisclearly showed excision of the floxed Vhlh allele from crescentic DNAbut not tubular DNA, confirming that these cells originate from
podocytes that express the Cre transgene. To confirm this finding, wealso tagged the podocyte cell lineage with a green fluorescent protein(GFP) reporter transgene15 that is activated only upon Cre-mediatedDNA excision; the majority of cells within the crescent express GFP,indicating that they originated from the podocyte lineage (Fig. 2f). Toexclude an environmental effect resulting from the ‘conventionalstatus’ of our mouse facility, we rederived the mice in a pathogen-free barrier facility and found no difference in phenotype. Together,our results show that an intrinsic defect in glomeruli is sufficient toinitiate RPGN.
It is widely accepted that terminally differentiated podocytes cannotproliferate and, to date, no genetic model exists in which podocytesare ‘switched back on’ to divide. To determine whether podocytesreenter the cell cycle and undergo proliferation to generate the cellularcrescents in our model, we pulsed the mice with bromodeoxyuridine(BrdU; Fig. 2g). Double immunostaining with the podocyte-restrictedmarker ZO-1 shows that in the early stages of disease (beforecrescent formation), podocytes are proliferating (Fig. 2h). Proliferat-ing cell nuclear antigen (PCNA) staining shows that glomerularepithelial cell proliferation continues within the crescent at 4 weeksand also includes parietal epithelial cells at this stage (SupplementaryFig. 3 online).
To characterize the molecular response in glomeruli of our trans-genic mice and to identify candidate targets for intervention in thisdisease, we performed gene expression profiling on glomeruli isolatedwith magnetic beads from mutant and wild-type littermates. The best-studied target(s) for VHL are the HIFa subunits. Loss of VHL
Blood
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Figure 3 Increased Cxcr4 is functionally important for the RPGN
phenotype. (a) At 4 weeks of age, Cxcr4 is upregulated in podocytes from
mutant (–/–) mice as shown by immunohistochemistry (black arrows).
Original magnification, �1,500. (b) LCM and real-time PCR was used to
quantify Cxcr4 and Sdf1 expression in glomeruli and tubules of Vhlh
knockout mice. Data shown represents the mean ± s.e.m. WT,
wild-type. (c) At 4 weeks of age, mutant mice treated with Cxcr4-specific
antibody had significantly lower proteinuria than PBS-treated littermates.
Similarly, the degree of hematuria was reduced in treated mice. Values
shown are averages for urinary dipstick values. Mutant, VhlhloxP/loxP Pod-Cre
(+) genotype; control, VhlhloxP/+ Pod-Cre littermates. *P o 0.025. (d) At
7.5 weeks of age, 100% of mutant mice (VhlhloxP/loxP Pod-Cre) treated withPBS vehicle showed global glomerulosclerosis (scarring of glomeruli) and
succumbed to renal failure. In contrast, all mutant littermates treated with
Cxcr4-specific antibody were alive and showed a range of glomerular phenotypes including glomeruli with no sclerosis or crescents. Low-magnification
images (top panels) show a marked difference between the degree of proteinuria and tubular dilation in treated versus untreated mice (arrowheads). Original
magnification, �250 (upper two panels) and �1,500 (lower four panels). (e) Model for RPGN in Vhlh mutant mice. Loss of VHL from the podocyte leads
to stabilization of HIFs and induction of downstream targets including CXCR4. This allows podocytes (Pod) to reenter the cell cycle, initiating crescent
formation. Concurrently, the adjacent endothelium (En) is activated (pink arrows) through upregulation of cytokines such as VEGFA and TNF-a.
Me, mesangial cell, Pa, parietal epithelial cell.
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stabilizes both the HIF1a and HIF2a subunits and leads to increasedexpression of hypoxia-response genes including VEGFA, CXCR4 andits ligand SDF1, and HIF1A itself16,17. Accordingly, both Hif1a andHif2a (encoded by Epas1) protein levels were increased in podocytesfrom VhlhloxP/loxP Pod-Cre mice (Supplementary Fig. 4 online and datanot shown), and microarray analysis confirmed that expected down-stream target genes of the VHL-HIF pathway as well as genes known tobe associated with RPGN were increased in glomeruli isolated fromVhlhloxP/loxP Pod-Cre compared to glomeruli from wild-type littermates(Supplementary Fig. 5 and Supplementary Table 1 online).
From the list of upregulated genes, we chose Cxcr4 as a candidate tounderlie the ‘phenotypic’ switch observed in podocytes from Vhlhmutants because it has been shown to have a role in the migratory andproliferative capacity of both cancer and hematopoietic cells3.Figure 3a demonstrates de novo expression of Cxcr4 within podocytesof mutant mice compared to absent expression in glomeruli of wild-type littermates. Laser-capture microdissection and real-time PCRconfirmed a 2.8-fold increase in glomerular Cxcr4 mRNA that wasabsent from tubules (Fig. 3b). CXCR4 is a seven-transmembrane,G-protein-coupled receptor that is activated through binding to itsonly known ligand, SDF1. Binding of SDF1 to CXCR4 leads toactivation of several signal transduction pathways that regulate moti-lity, chemotaxis, proliferation and survival of many cell types18. In the
developing glomerulus, Sdf1 is expressed in mesangial cells and in adynamic and segmental pattern in the adjacent podocyte-endothelialcompartment (Supplementary Fig. 6 online)19. Immunostaining andreal-time PCR confirmed that expression of Sdf1 persists and isincreased in glomeruli from adult mutant Vhlh mice compared tocontrol littermates (Fig. 3b and Supplementary Fig. 6).
To determine whether inhibition of Cxcr4 may be a therapeuticoption in RPGN, we injected mutant and control littermates at 19 d ofage with a blocking antibody to Cxcr4 (ref. 20). This timepoint waschosen because it precedes the onset of crescent formation and isthe earliest date that we could determine the genotype of the mice.The onset of disease was delayed in treated versus untreated mutantmice by 5–7 d, and the severity of glomerular disease was markedlydiminished, as determined by the degree of proteinuria (P o 0.025),hematuria (P o 0.02) and glomerular pathology (Fig. 3c,d). Mutantmice treated with PBS alone exhibited 100% mortality by 7 weeks ofage compared with 0% in the group receiving Cxcr4-specific antibodytherapy (Supplementary Fig. 7 online). Together, these results areconsistent with a model in which upregulation of Cxcr4 contributes tothe phenotypic switch in podocytes permitting them to proliferate andform the cellular crescents that surround the glomerular tuft (Fig. 3e).
In support of this hypothesis, differentiated immortalized podo-cytes that have been transfected with a constitutively active version of
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CXCR4PodControlFigure 4 De novo expression of CXCR4 in
mice and humans with glomerular disease.
(a) Podocytes infected with a constitutively
active Cxcr4 receptor show a higher portion of
cells in cell cycle compared with control cells
(Ki-67 staining) and an increase in cell number
during differentiation as shown by MTT. Western
blot analysis for EGFP and the Myc tag confirms
infection of podocytes and expression of Cxcr4.
(b) Immunostaining for Cxcr4 at 2 weeks of age
demonstrates podocyte-selective (po) expression
in transgenic (CXCR4Pod) mice (top row).
Glomeruli are markedly enlarged in CXCR4Pod
mice compared to wild-type littermates (shown
at same magnification) and some glomeruli showfocal crescents or crescent-like structures (arrow).
(c) Glomeruli from a Cxcr4Pod mouse (top) pulsed
with BrdU show numerous proliferating cells
including podocytes (arrows) compared to none in
controls (not shown). (d) Immunostaining for
CXCR4 shows a marked increase in expression in
representative glomeruli from two individuals with
pauci-immune RPGN compared to an individual
with a noncrescentic glomerulopathy (MePGN).
Expression of synaptopodin, a marker of
differentiated podocytes, is decreased in RPGN.
A few differentiated podocytes remain within
crescents (white arrows and inset panels) that
express both synaptopodin and CXCR4. A
glomerulus from an individual with lupus
nephritis (SLE) is shown as a positive control
where CXCR4 expression is upregulated
predominantly in glomerular endothelial cells.Light micrographs (LM) are shown from the same
individual for comparison. MePGN, mesangial
proliferative glomerulonephritis; SLE, Stage III
systemic lupus nephritis; Cr, crescent; Tri,
trichrome; H&E, hematoxylin and eosin.
Original magnification, �600.
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the Cxcr4 receptor that carries a conversion of Asn119 to a serineresidue21 show increased proliferation in vitro. A significantly higherproportion of Cxcr4-positive podocytes expressed Ki67 (Fig. 4a), amarker of entry into S phase of the cell cycle, at the onset of podocytedifferentiation (day 2). Cell number was also increased as measured bythe MTT assay (Fig. 4a).
We next generated transgenic mice that express Cxcr4 selectivelywithin their podocytes (CXCR4Pod). Our data show that de novoexpression of Cxcr4 alone was sufficient to cause glomerular diseaseand proliferation of podocytes in vivo. By 6 months of age, CXCR4Pod
mice were found to have blood (1+) and protein (1 g/L) in their urine,diagnostic of glomerular disease. Light microscopy showed that80–100% of glomeruli from CXCR4Pod mice were markedly enlargedand hypercellular (Fig. 4b; n ¼ 3) compared to control glomeruli.Mice were pulsed with BrdU; immunostaining confirmed the presenceof proliferating cells within glomeruli with positive staining inpodocytes (Fig. 4c). A portion of glomeruli had focal crescents orcrescent-like structures (Fig. 4b). These results suggest that Cxcr4 isboth required and sufficient for podocyte proliferation. Our data alsoindicate that other targets are required for full-blown RPGN asobserved in VhlhloxP/loxP Pod-Cre mice. Of note, SDF1, VCAM1 andICAM1 have previously been shown to enhance signaling through theSDF1-CXCR4 axis18 and were also increased in glomeruli fromVhlhloxP/loxP Pod-Cre mice (Supplementary Fig. 5 and SupplementaryTable 1) but not from CXCR4Pod mice (data not shown).Increased expression of Vegfa in podocytes may contribute to activa-tion of adjacent endothelium and vascular inflammation, but experi-ments in our lab showed that podocyte-selective upregulation of Vegfaalone (5–20-fold) is insufficient to cause glomerulitis or crescentictransformation in mice (S.E.Q. & V. Eremina, unpublished dataand ref. 5).
To determine whether stabilization of HIFs and upregulation ofdownstream targets also occur in glomeruli of individuals with pauci-immune RPGN, we performed real-time PCR analysis and immuno-staining (Supplementary Figs. 5 and 8 and Supplementary Noteonline). Notably, we found the same ‘expression fingerprint’ of HIFtarget genes, including a 7.2-fold increase (P o 0.05) in CXCR4(Supplementary Fig. 5). Immunostaining confirmed that CXCR4 ismarkedly increased in glomeruli from individuals with pauci-immuneRPGN compared to individuals with other renal diseases (Fig. 4d).Conversely, synaptopodin—a marker for differentiated podocytes—was decreased in RPGN; this loss of podocyte differentiation alsooccurs in Vhlh mutant mice (Supplementary Fig. 5). Currently, thereare no methods available to identify ‘de-differentiated’ podocytes increscents of humans and may explain why these cells have escapeddetection in biopsy specimens. Despite this, a few cells within thecrescents co-stain for both CXCR4 and synaptopodin (Fig. 4d),consistent with the lineage-tagging experiments in mice that showcrescentic cells originate from podocytes and express Cxcr4.
In summary, we have shown that Vhlh is required in the podocyteto maintain glomerular integrity. Loss of Vhlh permits terminallydifferentiated podocytes to reenter the cell cycle. Upregulation ofCxcr4 and rescue of the phenotype in VhlhloxP/loxP Pod-Cre transgenicmice with Cxcr4-blocking antibodies indicate that this pathway isfunctionally important in the disease pathogenesis. The absence of anysystemic or circulating perturbation in our model indicates thatintrinsic defects alone in the glomerulus are sufficient to initiatecrescent formation and renal vasculitis in mice. Together, our resultsprovide a new paradigm for the pathogenesis of small-vessel vasculitisand crescentic glomerular disease where the inciting endothelial injuryoccurs from the outside in.
METHODSGeneration of podocyte-specific Vhlh knockout and Cxcr4 transgenic mice.
We generated the podocin-Cre recombinase transgenic founder lines as pre-
viously described22. We crossed four individual founder lines with the Z/EG
reporter strain15 to determine the degree and timing of Cre-mediated DNA
excision in podocytes. We selected one founder line that gave 90% deletion of
the floxed b-geo cassette in podocytes. We bred the podocin-Cre mice with
homozygous floxed Vhlh mice (VhlhloxP/loxP) (strain Vhlhtm1Jae, Jackson Labs)13.
To generate VhlhloxP/loxP Pod-Cre recombinase mice, bitransgenic mice carrying
both the podocin-Cre transgene and one floxed Vhlh allele were bred to
homozygous floxed Vhlh mice (Supplementary Fig. 2).
We generated the Cxcr4 transgenic construct by inserting a sequence-
verified, full-length 2-kb coding cDNA for mouse Cxcr4 (Open Biosystems)
downstream of the 4.125-kb mouse Nphs1 (nephrin) promoter in the pNXPRS
vector, as previously described24 (Supplementary Fig. 2). We generated
multiple Cxcr4 transgenic founder lines. We identified three independent
founder lines and chose for further study the one with the most robust
expression of Cxcr4 protein in podocytes.
All animal experimentation was conducted in accordance with the
Canadian Guide for the Care and Use of Laboratory Animals, and protocols
were approved by the Animal Care Committee at the Samuel Lunenfeld
Research Institute.
Genotypic analysis. We isolated genomic DNA from tails of 3-week-old
transgenic mice and used them for genotypic analysis as described23. The
Cre transgene was detected by PCR using the primers 5¢-ATGTCCAATTTACT
GACCG-3¢ (forward) and 5¢-CGCCGCATAACCAGTGAAAC-3¢ (reverse),
which amplified a band of approximately 300 bp (Supplementary Fig. 2).
Conditions for the Cre PCR were as previously described5.
We detected the floxed Vhlh gene by PCR using the oligonucleotide primers
oIMR1555 (5¢-CTCAGGTCATCTTCTGCAACC-3¢) and oIMR1556 (5¢-TCTGTCTTGGCCTCCTGAGT-3¢), which generate a 945-bp fragment for
the floxed Vhlh allele and a 915-bp fragment for the wild-type Vhlh allele.
We separated bands on a 1.5% agarose gel (Supplementary Fig. 2).
We detected the Cxcr4 transgene by PCR analysis using a primer in the
Nphs1 promoter (5¢-AACAGAAAAGCAGGGCACAC-3¢) and a second primer
in the Cxcr4 cDNA (5¢-GTAGATGGTGGGCAGGAAGA-3¢).
Positive founders were identified by the presence of a 281-bp band
(Supplementary Fig. 2).
Injection of BrdU and Cxcr4-specific antibody. We injected 3-week old
VhlhloxP/loxP Pod-Cre or VhlhloxP/+ Pod-Cre mice with 100 mg/g body weight
BrdU (10 mg/ml; Sigma) in 0.9% NaCl solution. They received a second
injection 15 h later. Two hours after the second injection of BrdU, mice were
killed and kidneys were fixed in 4% paraformaldehyde overnight.
Each Cxcr4 treatment group (total of five treatment groups studied)
contained two VhlhloxP/loxP Pod-Cre and two VhlhloxP/+ Pod-Cre littermates.
At 19 d after birth and then daily, we administered rabbit anti-rat Cxcr4-
specific antibody (Torrey Pines Biolabs) to mice at a dose of 10 mg in 500 ml
PBS by a single daily intraperitoneal injection as previously described20. Within
each treatment group, one VhlhloxP/loxP Pod-Cre mouse and one control mouse
were given anti-rat Cxcr4-specific antibody, and the second VhlhloxP/loxP Pod-
Cre mouse and control mouse were given 500 ml PBS (placebo). We collected
urine from mice daily at the same time each morning. Blood was collected at
4 weeks and at the time of killing (7weeks). We treated a total of 20 mice.
Phenotypic analysis. We collected urine passively in an Eppendorf tube from
3-week-old mice. We used a urine dipstick (Chemstrip 5L; Roche Diagnostics
Corp.) to detect the presence or absence of protein and red blood cells in the
urine. We performed the standard colorimetric assay according to the manu-
facturer’s instructions. In addition, we placed 2 ml urine from transgenic or
control mice in 18 ml Laemmli buffer, boiled the mixture and loaded it on
a 12% SDS-PAGE gel. We loaded an SDS-PAGE low-range protein standard
(Bio-Rad Laboratories Inc.) in the first lane of the gel.
We took blood samples with a heparinized capillary tube by femoral vein
stab after warming. We collected a total of 120 ml of blood; we recorded
creatinine, urea and blood chemistry measurements using a Stat Profile M7
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(Nova Biomedical Corp.). We performed the complete blood count (CBC) on a
Coulter Counter (AcT diff; Beckman Coulter Canada).
Statistical analysis. Results are expressed as means. Student paired t-test was
used to analyze the difference between two groups. Values were regarded
significant at P o 0.05.
Histologic analysis. We dissected kidneys for histologic analysis, fixed them in
10% formalin in PBS and embedded them in paraffin. We cut 4-mm thick
sections and stained them with Periodic Acid–Schiff (PAS) stain or hematoxylin
and eosin (H&E), examined them and photographed them with a DC200 Leica
camera and Leica DMLB microscope (Leica Microsystems Inc.).
Laser-capture microdissection (LCM). We dissected one-half of a kidney from
VhlhloxP/loxP Cre mice and fixed it in 4% Paraformaldehyde overnight, cryo-
protected it in 30% sucrose overnight, embedded it in Tissue Tek OCT 4583
(Sakura Finetek USA Inc.) and snap froze it. We prepared 10-mm cryosections
on blood smear slides (Surgipath), stained them with toluidine blue and used
them for LCM. Pure cell populations from mouse glomeruli, renal tubules or
cellular crescents were obtained using the AutoPix Automated Laser Capture
Microdissection System according to the manufacturer’s instructions.
We extracted DNA from the LCM sample using the PicoPure DNA
Extraction Kit protocol. We stored digested DNA samples at –20 1C until we
used them for PCR amplification. We amplified DNA samples from LCM by
PCR using the following primers: VHL-FW5 primer 5¢-CTGGTACCCAC
GAAACTGTC-3¢ (upstream of 5¢ loxP); VHL-FWD primer 5¢-CTAGGCACC
GAGCTTAGAGGTTTG CG-3¢ (upstream of second loxP in intron 1); VHL-
RVS primer 5¢-CTGACTTCCACTGATGCTTGTCACAG-3¢ (downstream of
second loxP in intron 1). The Vhlh 2-loxP allele is represented by a 460-bp
band, the 1-loxP allele by a 350-bp band. The three primers together were used
to assess Cre-mediated DNA excision in 2-loxP homozygotes.
We subjected 5 ml of digested DNA to 42 cycles of PCR in a volume of 50 ml
containing 20 pmol VHL-FW5 primer, 20 pmol VHL-FWD primer, 40 pmol
VHL-RVS primer, 1 ml Taq DNA polymerase, 3 ml 25 mM MgCl2, 5 ml
10� PCR buffer, 1 ml 10 mM dNTP and 27 ml autoclaved ddH2O. PCR
conditions were 94 1C for 2 min 30 s, 94 1C for 50 s, 57 1C for 50 s, 72 1C for
1 min and last extension at 72 1C for 5 min. We separated PCR products by
electrophoresis on a 2% agarose gel.
We extracted RNA in a similar fashion from LCM samples and reverse-
transcribed cDNA from purified RNA using the Superscript First-Strand
Synthesis System for RT-PCR (Invitrogen, Life Technologies), and stored it at
–20 1C until using it for real-time PCR. We quantified cDNA samples from
LCM using the ABI 7900 (Applied Biosystems) according to the manufacturer’s
instructions, using the following mouse primers: Cxcr4 5¢-CAGAGGCCAAG
GAAACTGCT-3¢ (forward); Cxcr4 5¢-CTGACGTCGGCAAAGATGAA-3¢(reverse); 18S 5¢-AGGAATTGACGG AAGGGCAC-3¢ (forward); 18S 5¢-GGA
CATCTAAGGGCATCACA-3¢ (reverse), Cxcl12 (encoding SDF1) 5¢-CAAG
GTCGTCGCCGTGCTG-3¢ (forward); Cxcl12 5¢-CGTTGGCTC TGGCGAT
GTGG-3¢ (reverse).
We subjected 2 ml cDNA to real-time PCR on the ABI 7900 (Applied
Biosystems) in a volume of 10 ml containing 900 nM Cxcr4 forward, 900 nM
Cxcr4 reverse, 150 nM 18S forward and 150 nM 18-S primers or Cxcl2 primers.
We used SYBR Green Master Mix for all PCR reactions, and we followed
universal cycling conditions according to the ABI standard method as follows:
initial hold of 10 min at 95 1C followed by 40 cycles at 95 1C for 15 s and 60 1C
for 60 s. For quantitative analyses, we compared each VhlhloxP/loxP Cre cDNA
sample with a sample from a control littermate following the delta delta CT
(DD CT) method, and normalized all samples to 18S.
In situ hybridization and immunohistochemistry. We dissected kidneys from
mice on postnatal day 6 and at 3 weeks, 4 weeks or 7 weeks of age. We washed
kidneys briefly in RNase-free PBS and fixed them overnight in diethyl
pyrocarbonate (DEPC)-treated 4% paraformaldehyde. We then placed these
tissues in 30% sucrose for 12–24 h, embedded them in Tissue-Tek OCT and
snap froze them. We cut 10-mm tissue samples on a Leica Jung cryostat (model
CM3050; Leica Microsystems Inc.) and transferred them to Superfrost micro-
scope slides (Fisher Scientific Co.). We stored the slides at –20 1C until needed.
We prepared digoxigenin-labeled probes according to the Roche Molecular
Biochemicals protocol (Roche Molecular Biochemicals). Probes used for in situ
analysis were Nphs1 and Vegfa5. Details of the in situ analysis protocol may be
obtained upon request.
Primary antibodies used were: ZO-1 in a 1:10 dilution (gift of J. Miner,
St. Louis, Missouri), BrdU at a 1.5 U/ml dilution (Roche Diagnostics GmbH);
Cxcr4-specific monoclonal antibody at a 1:100 dilution (R&D Systems); GFP-
specific antibody at a 1:2,000 dilution for section immunostaining and at a
1:1,000 dilution for whole-mount immunostaining (Molecular Probes), Hif1a-
specific rabbit polyclonal antibody at a 1:100 dilution for both section (R & D
Systems; Affinity Bioreagents) and wholemount immunostaining (Affinity
Bioreagents); Sdf1 at a 1:50 dilution (SantaCruz) and fibrinogen at a 1:100
dilution (DakoCytomation).
Secondary antibodies used for anti-BrdU and Cxcr4 analyses were:
Cy3-conjugated AffiniPure donkey anti-mouse IgG at a 1:500 dilution (Jackson
ImmunoResearch laboratories Inc.); secondary for ZO-1 was FITC-conjugated
affiniPure goat anti-rat IgG at a 1:100 dilution (Jackson ImmunoResearch),
secondary for GFP and Hif1a for section immunostaining was anti-rabbit IgG
biotinylated antibody at a 1:200 dilution (ABC kit, Vector Laboratories);
secondary for GFP, Hif1a and Sdf1 whole-mount staining was FITC-conjugated
AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch), Cy3-conjugated
AffiniPure donkey anti-mouse IgG (Jackson ImmunoResearch) Cy3-conjugated
AffiniPure donkey anti-goat IgG (Jackson ImmunoResearch), respectively.
PCNA staining was performed with the ZYMED PCNA Staining Kit (ZYMED
Laboratories Inc.) according to the manufacturer’s instructions. Details of
whole-mount and section immunostaining are available upon request.
Human studies were performed on 6-mm cryosections taken from tissue
biopsies with primary antibodies to CXCR4, 1:50 dilution (Abcam), and
monoclonal mouse synaptopodin, prediluted (clone G1D4, Progen). Secondary
antibodies used for CXCR4-specific antibody were Alexa Fluor 546–labeled
anti-rabbit antibody (Molecular Probes, Invitrogen) and for synaptopodin-
specific antibody were Alexa Fluor 488–labeled goat anti-mouse antibody
(Molecular Probes, Invitrogen).
Glomerular isolation and microarray analysis. We performed glomerular
isolation and microarray analysis as previously described24; using the 45K
mouse Affymetrix Chips (M0E430). All studies were performed at the micro-
array facility, The Centre for Applied Genomics, The Hospital for Sick
Children, Toronto. Briefly, we isolated glomeruli from 4-week-old wild-type
or VhlhloxP/loxP Cre mice. We isolated RNA and generated probes for microarray
hybridization using the Affymetrix two-cycle kit. We performed in vitro
transcription with the Affymetrix IVT kit, and miscanning with the Affymetrix
GeneChip Scanner 3000. Three independent experiments were performed.
Detailed protocols are available upon request.
Transfection of podocyte cell lines. We isolated a conditionally immortalized
mouse podocyte cell line from H-2Kb-tsA58 transgenic mice kidneys (Immor-
toMouse; Charles River Labs) and characterized it as previously described25.
We infected podocytes with murine stem cell virus puromycin
(pMSCVpuro) containing either enhanced green fluorescent protein (EGFP;
empty vector) or myc-Cxcr4 with an IRES (internal ribosomal entry site)-EGFP
(Clontech). Briefly, we transfected each vector into the Phoenix viral producing
cell line (Clontech). We applied supernatants containing shed virus to growth-
permissive podocytes for three infection cycles. We selected infected podocytes
for puromycin resistance at 3 mg/ml for 72 h and expanded resistant cells. We
replated infected podocytes under growth-restrictive conditions. We measured
proliferation by MTT assay (Promega) as per the manufacturer’s directions at
days 2, 4 and 6 of growth restriction. We then fixed infected podocytes in
methanol at the same time points and immunostained them with a mouse
monoclonal antibody to Ki-67 (BD Pharmingen) followed by a fluorescent
sheep anti-mouse secondary (Jackson ImmunoResearch). We quantified the
number of positive-staining nuclei as a percentage of the total nuclei. To
confirm infection of podocytes and expression of the Myc-tagged Cxcr4
protein, we separated 10 mg of whole-cell lysate under reduced conditions on
15% SDS-polyacrylamide gels and transferred this amount to PVDF mem-
branes (Immobilon-P; Millipore). We incubated membranes with a rabbit
antibody against GFP for infection efficiency or against c-Myc (Alpha Diag-
nostic International, Inc.) overnight at 4 1C, and then incubated them with an
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alkaline phosphatase–conjugated anti-rabbit IgG antibody (Promega) at 22 1C
for 60 min. Detection was performed using the chromagen 5-bromo-4-chloro-
3-indolyl phosphate/nitro blue tetrazolium (Sigma).
CXCR4 and VHL-HIF pathway target gene expression in human renal
biopsies. Human kidney biopsies, obtained in a multicenter study for renal
gene expression analysis (the European renal cDNA consortium, ERCB,
Supplementary Note online), were processed as described26. Informed
consent was obtained according to the respective local ethical committee
guidelines. Histologies were stratified by the reference pathologists of
the ERCB: IgA glomerulonephritis (n ¼ 15), pauci-immune RPGN (n ¼ 9)
and control biopsies from non-neoplastic parts of tumor nephrectomies
(n ¼ 5). We performed real-time RT-PCR as previously described26. We
used the following sequences of oligonucleotide primers (300 nM) and probes
(100 nM) for CXCR4: 5¢-GGCCGACCTCCTCTTTGTC-3¢ (sense), 5¢-CAAAG
TACCAGTTTGCCACGG-3¢ (antisense) and fluorescence-labeled probe
(FAM) 5¢-ACGCTTCCCTTCTGGGCAGTTGATC-3¢ (obtained from Applied
Biosystems). We used predeveloped TaqMan assay reagent for the internal
standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and HIF1A,
ITGB1 and TGFB1. Other primer sequences are available on request. Expres-
sion levels are shown as ratios to GAPDH and expressed as ratio to the mean
of controls. Quantification of the given templates was performed according
to the standard curve method. All measurements were performed
in duplicate; controls consisting of bi-distilled H2O were negative in
all runs.
Note: Supplementary information is available on the Nature Medicine website.
ACKNOWLEDGMENTSThe authors thank D. Vukasovic for secretarial assistance and the Centre forModelling Human Diseases for biochemical assays in the mice. We also thankB. Pressler (University of North Carolina at Chapel Hill) for performing ANCAassays, Y. Wang (Samuel Lunenfeld Research Institute) for help in LCM isolation,S. Peiper (Institute of Molecular Medicine and Genetics, Georgia) for providingthe constitutively active Cxcr4 constructs, V. Eremina for technical assistance,K. Kamel (St. Michael’s Hospital, Toronto) and D. Cattran (Toronto Hospital)for critically reviewing the manuscript. S.E.Q. is the recipient of a CanadaResearch Chair Tier II, and a Premier’s Research of Excellence Award. Thiswork was funded by Canadian Institute of Health Research grant MOP 77756,National Cancer Institute of Canada grant #16002 and Emerald Foundationgrant (to S.E.Q.).
COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturemedicine/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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