PATTERNS & PHENOTYPES

Kidney Development and Gene Expression inthe HIF2� Knockout MouseBrooke M. Steenhard,1 Paul B. Freeburg,1 Kathryn Isom,1 Larysa Stroganova,1

Dorin-Bogdan Borza,2 Billy G. Hudson,2 Patricia L. St. John,1 Adrian Zelenchuk,1 andDale R. Abrahamson1*

The hypoxia-inducible transcription factor-2 (HIF2), a heterodimer composed of HIF2� and HIF1� subunits,drives expression of genes essential for vascularization, including vascular endothelial growth factor(VEGF) and VEGF receptor-2 (VEGFR-2, Flk-1). Here, we used a HIF2�/LacZ transgenic mouse to definepatterns of HIF2� transcription during kidney development and maturation. Our results from embryonicheterozygotes showed HIF2�/LacZ expression by apparently all renal endothelial cells. At 4 weeks of age,glomerular mesangial and vascular smooth muscle cells were also positive together with endothelial cells.These expression patterns were confirmed by electron microscopy using Bluo-gal as a �-galactosidasesubstrate. Small numbers of glomerular and tubular epithelial cells were also positive at all stagesexamined. Light and electron microscopic examination of kidneys from HIF2� null embryos showed nodefects in renal vascular development or nephrogenesis. Similarly, the same amounts of Flk-1 protein wereseen on Western blots of kidney extracts from homozygous and heterozygous HIF2� mutants. To examineresponsiveness of HIF2� null kidneys to hypoxia, embryonic day 13.5 metanephroi were cultured in roomair or in mild (5% O2) hypoxia. For both heterozygous and null samples, VEGF mRNA levels doubled whenmetanephroi were cultured in mild hypoxia. Anterior chamber grafts of embryonic HIF2� knockouts weremorphologically indistinguishable from heterozygous grafts. Endothelial markers, platelet endothelial celladhesion molecule and BsLB4, as well as glomerular epithelial markers, GLEPP1 and WT-1, were allexpressed appropriately. Finally, we undertook quantitative real-time polymerase chain reaction ofkidneys from HIF2� null embryos and wild-type siblings and found no compensatory up-regulation of HIF1�or -3�. Our results show that, although HIF2� was widely transcribed by kidney endothelium and vascularsmooth muscle, knockouts displayed no detectable deficits in vessel development or VEGF or Flk-1expression. Developmental Dynamics 236:1115–1125, 2007. © 2007 Wiley-Liss, Inc.

Key words: Flk-1; glomerulus; HIF; hypoxia; VEGF

Accepted 31 January 2007

INTRODUCTIONOne of the key molecules regulatingblood vessel development is vascularendothelial growth factor (VEGF; Ger-ber et al., 1999; Ferrara, 2001; Ros-sant and Howard, 2002). VEGF andtwo VEGF tyrosine kinase receptors,

Flk1 and Flt1, are highly expressedduring early kidney development(Robert and Abrahamson, 2001). Inthe developing glomerulus, VEGF sig-naling from immature podocytes isthought to recruit Flk1-expressing an-gioblasts to the vascular cleft (Robert

et al., 1998). Expression of VEGF bypodocytes and its receptors by glomer-ular endothelial cells continues inadult kidney (Simon et al., 1995; Rob-ert et al., 1998), perhaps to maintainthe highly specialized endothelium ofthe capillary loops. Mice with podo-

1Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas2Department of Medicine, Vanderbilt University, Nashville, TennesseeGrant sponsor: NIH; Grant number: DK052483; Grant number: DK065123.*Correspondence to: Dale R. Abrahamson, Ph.D., Department of Anatomy and Cell Biology, University of Kansas MedicalCenter, Mail Stop 3038, 3901 Rainbow Blvd., Kansas City, KS 66160. E-mail: dabrahamson@kumc.edu

DOI 10.1002/dvdy.21106Published online 2 March 2007 in Wiley InterScience (www.interscience.wiley.com).

DEVELOPMENTAL DYNAMICS 236:1115–1125, 2007

© 2007 Wiley-Liss, Inc.

cyte-specific deletions of VEGF, andthose overexpressing VEGF throughpodocyte-specific promoters, have se-vere defects in kidney function, lead-ing to early death (Eremina et al.,2003). VEGF is also expressed in de-veloping distal tubules (Kitamoto etal., 1997) and in developing and ma-ture collecting ducts (Simon et al.,1995). Some extraglomerular capil-lary endothelia also express VEGF re-ceptors during development and inmaturity (Robert et al., 1998). Injec-tion of anti-VEGF antibodies or solu-ble Flt1 causes glomerular endothelialcell detachment and hypertrophy,leading to proteinuria (Sugimoto etal., 2003). Mice lacking VEGF iso-forms 164 and 188 show defects inrenal arterial branching and glomeru-lar angiogenesis (Mattot et al., 2002),suggesting that VEGF signaling iscritical for renal vascular develop-ment and maintenance.

Hypoxia, and the hypoxia-inducibletranscription factors (HIFs), play keyroles in the expression of VEGF andVEGF receptors (Levy et al., 1995; Liuet al., 1995; Gerber et al., 1997; Kap-pel et al., 1999). HIFs are het-erodimeric transcription factors of thebasic helix–loop–helix–PAS (bHLH-PAS) superfamily that induce gene ex-pression in response to a wide varietyof environmental stimuli, includinghypoxia, xenobiotic exposure, and cir-cadian rhythms (Crews, 1998; Gu etal., 2000). The transcriptional activityof the alpha/beta heterodimeric HIFsare finely controlled by the stability ofthe alpha subunits in response to in-tracellular oxygen concentration.Three such HIF� subunits have beendescribed to date (Wang and Se-menza, 1995; Flamme et al., 1997;Tian et al., 1997; Gu et al., 1998; Haraet al., 2001), and all have similar bio-chemical properties (Jiang et al.,1996; O’Rourke et al., 1999; Maxwelland Ratcliffe, 2002). In the normoxicstate, HIF� proteins are hydroxylatedon a conserved proline residue, whichpromotes an interaction with the VHLubiquitin ligase complex (Ivan et al.,2001; Jaakkola et al., 2001). HIF�subunits are then polyubiquinatedand destroyed by the 26S proteasome.In hypoxia, proline hydroxylation andsubsequent proteasomal degradationare avoided, allowing HIF� proteinsto translocate to the nucleus, bind the

HIF� subunit, and induce transcrip-tion of target genes. An oxygen-depen-dent hydroxylation of HIFs also regu-lates gene transactivation properties.In hypoxia, hydroxylation of a con-served HIF� asparagine residue isblocked, which allows HIF to recruitp300/CBP to the transcriptional appa-ratus (Lando et al., 2002).

What role(s) HIFs play in kidneydevelopment has not yet been deter-mined (Haase, 2006). In developingkidney, where VEGF signaling is crit-ical for normal glomerular differenti-ation, we observed HIF1� and -2�mRNA expression in the nephrogeniczone of the outer cortex (Freeburg etal., 2003). Although originally thoughtto be confined to endothelial cells(Tian et al., 1997), HIF2� expressionhas also been localized to immaturepodocytes, suggesting that it may playa role in glomerular capillary develop-ment (Freeburg et al., 2003; Bern-hardt et al., 2006). In addition, HIF2�protein is stably expressed in avascu-lar embryonic kidney explants, and isup-regulated by hypoxia, which alsocauses an increase in VEGF mRNA(Freeburg et al., 2003).

Three different HIF2� knockoutmice have been generated, and allshow mid-gestational lethal pheno-types (Tian et al., 1998; Peng et al.,2000; Compernolle et al., 2002). In thefirst knockout analyzed, the authorsdescribed HIF2� expression in vascu-lar endothelium and also in the em-bryonic Organ of Zuckerkandl, whichis a site of catecholamine biosynthe-sis. Moreover, they documented se-vere bradycardia and decreased nor-adrenaline levels in embryonic day (E)12.5 null mutants and no viable em-bryos beyond E15.5. Supplementingpregnant dams with the catechol-amine precursor L-threo-3,4-dihy-droxyphenylserine (DOPS) allowed�40% of knockout mice to surviveonly until birth (Tian et al., 1998),implicating an essential role forHIF2� in catecholamine homeostasis.Whether deletion of HIF2� affectskidney development specifically hasnot been studied in detail previously.Here, we closely examined HIF2� ex-pression patterns in developing kid-ney in vivo after DOPS administra-tion, determined whether an absenceof HIF2� affected renal vascular de-velopment or expression of candidate

HIF target genes, and examined ef-fects of DOPS withdrawal on kidneydevelopment in metanephric organculture and by grafting mutant meta-nephroi into anterior eye chambers.

RESULTS AND DISCUSSION

Analysis of HIF2�/LacZExpression in Developingand Mature Kidney

Kidneys from E13.5 HIF2�/LacZ het-erozygotes were removed and sub-jected to whole-mount X-gal histo-chemistry. As shown in Figure 1A, aprominent network of HIF2�/LacZ-positive cells was observed, which re-sembled a vascular pattern. Sectionsof kidneys showed that, in outer corti-cal, nephrogenic zones, cells occupyingthe vascular clefts of comma- and S-shaped nephric figures were blue, aswere the newly formed capillaries inearly capillary loop stage glomeruli(Fig. 1B). Additionally, individualcells scattered in the metanephricmesenchyme not associated with glo-merular capillaries also expressed theLacZ transgene (Fig. 1B). After X-galhistochemistry, slides were also la-beled with the endothelial-specific lec-tin BsLB4-fluorescein (Fig. 1C). Therewas complete overlap of the blue andgreen signals (Fig. 1D), suggestingthat all HIF2�/LacZ-positive cells innephrogenic zones were endothelialcells or their progenitors. In additionto vascular endothelial cells, somepodocytes in immature and maturingglomeruli of newborn kidney also ex-pressed HIF2�/LacZ (Fig. 1E–G). By 4weeks of age, HIF2�/LacZ expressionwas widely expressed by most or allrenal endothelial cells, includingthose of glomerular and peritubularcapillaries (Fig. 1H). In addition to en-dothelium, vascular smooth musclecells in arteries and arterioles in kid-ney also expressed HIF2�/LacZ (Fig.1H,I). Rare expression of HIF2�/LacZby individual tubular epithelial cellswas also occasionally observed (Fig.1J).

To examine the expression of HIF2�at the ultrastructural level, we pro-cessed kidney tissue from heterozy-gotes with an electron-dense LacZsubstrate, Bluo-gal, and carried outelectron microscopy. HIF2� expres-sion localized specifically to endothe-

1116 STEENHARD ET AL.

lial cells of capillary loop stage glo-meruli at E14.5 (Fig. 2A). At 4 weeksof age, both glomerular endothelialcells as well as mesangial cells ex-pressed HIF2�/LacZ (Fig. 2B).

Prior work in our lab with a now ob-solete antibody showed nuclear HIF2�

protein in developing glomerular cells,including podocytes, as well as in thevasculature (Freeburg et al., 2003). Incontrast, our findings presented hereshow that HIF2�/LacZ expression oc-curred predominantly in endothelialcells and vascular smooth muscle with

only rare reporter gene expression byrenal epithelial cells. Although LacZ isa convenient marker for promoter activ-ity, it certainly may not accurately re-flect stable HIF2� protein expression.Alternatively, the antibody used in ourprevious study may not have been

Fig. 1. Hypoxia-inducible transcription factor (HIF) 2� expression in kidney. A: Embryonic day (E) 13.5 HIF2��/� metanephros stained for�-galactosidase histochemistry shows a branching, vessel-like pattern. B: E14 HIF2��/� kidney showing �-galactosidase reaction product inglomerular endothelial cells migrating into the vascular cleft (VC), and in capillary loop stage glomeruli (G). Also, small arrows denote individual cellsin the metanephric mesenchyme containing �-galactosidase. C: The same slide in B was also labeled with BsLB4, an endothelial marker.D: Colocalization of �-galactosidase and BsLB4 shows complete overlap. E: Some visceral epithelial cells (developing podocytes) in early glomeruliexpress HIF2� (*). F: Intense vascular expression (arrowhead) of HIF2� is seen in newborn kidney, and within glomeruli (G). G: (Higher magnificationof boxed area in F): Some podocytes express HIF2� (arrows), whereas others do not (arrowheads). H: Frozen section of a 4-week HIF2��/� mouseshowing intense �-galactosidase product in an endothelial pattern, and in smooth muscle cells of arteries (arrowheads). I: Frozen section of a 4-weekHIF2��/� mouse showing a small artery. Reaction product is seen in both endothelial cells (arrow) and smooth muscle cells (arrowheads).J: Localization of HIF2�/LacZ to peritubular capillary endothelial cell (arrow) and (rarely) to a tubular epithelial cell (arrowhead).

HIF2� EXPRESSION IN DEVELOPING KIDNEY 1117

solely specific for HIF2�. Beyond thesecaveats, other factors may also explainthe different immunolocalization andreporter gene expression patterns. Forexample, HIF2� mRNA may only be ex-pressed transiently in podocytes, or theHIF2�/LacZ transcript may be highlyunstable (or degraded selectively) inpodocytes but not in endothelial andvascular smooth muscle cells. In sepa-rate studies, others have shown thatthe adult rat kidney is largely devoid ofHIF2� immunolabeling. However, ifrats are exposed to hypoxic conditions,abundant HIF2� labeling is seen in glo-meruli and in endothelial cells occupy-

ing the inner and outer medulla but notpapilla (Rosenberger et al., 2002).Therefore, perhaps the high levels ofHIF2�/LacZ reporter gene expressionwe observe abundantly in kidney endo-thelial cells helps ensure that appropri-ate amounts of HIF2� protein can bepromptly available in the event of hyp-oxia.

Normal Development ofHIF2�-Deficient Kidneys

In heterozygous matings and withoutDOPS supplementation, we observedan average litter size of 4.9 live pups/

litter. Genotype analysis of these new-borns revealed a non-Mendelian dis-tribution, with only two knockoutpups born (74/116 heterozygous,63.8%; 40/116 wild-type, 34.8%; and2/116 knockout, 1.7%). When preg-nant dams were supplemented dailywith DOPS beginning at E0.5 and em-bryos were allowed to develop toE13.5, the number of embryos aver-aged 7.6, and a Mendelian distribu-tion of genotypes was observed at thisgestational age (39/76 heterozygous,51.3%; 19/76 wild-type, 25.0%; 18/76knockout, 23.7%). At later gestationalstages (E17.5–E18.5), and despite thecontinued, daily administration ofDOPS, increased losses occurred innull embryos, with only �50% of theexpected ratio obtained. This lethalityis consistent with prior findings withthis knockout mouse (Tian et al.,1998).

HIF2� knockout embryos weregrossly indistinguishable from het-erozygous and wild-type counterpartsand none were edematous. Light mi-croscopic examination of kidneys fromall 11 HIF2� null embryos examined(2 at E12, 2 at E13, 1 at E14, 4 at E16,1 at E17, 1 at E18) showed no obviousdefects in either renal vascular devel-opment or in nephrogenesis. In earlynephric figures, condensing mesen-chyme was observed at the tips ofbranching ureteric buds (Fig. 3A).Comma- and S-shaped nephrons wereclearly visible and appeared morpho-logically normal, with endothelialcells migrating into the vascular cleftin the usual pattern (Fig. 3B, arrow).Red blood cells were seen within earlycapillary loop stage glomeruli (Fig.3C, arrow) as well as in more matureloops of glomeruli at E18.5 (Fig. 3D,arrow), indicating glomerular capil-lary continuity with the systemic vas-culature. Similarly, in frozen sectionsdeveloped for LacZ activity, no obvi-ous defects could be seen in renal vas-cular patterning: vascular clefts con-tained LacZ-positive endothelial cells(Fig. 3E, arrow), and capillary loopstage and maturing stage glomerulicontained endothelial cells in theusual pattern (Fig. 3F,G). Tubulemorphology also appeared entirelynormal, and there were no tubule di-latations or proteinaceous casts.

The findings seen in the light micro-scope were supported by electron mi-

Fig. 2. High resolution view of two stages of glomerular development showing hypoxia-inducibletranscription factor (HIF) 2� expression in endothelial cells and mesangial cells. A: Photomicro-graph showing electron-dense Bluo-gal substrate reaction product (arrows) in endothelial cells ofan early capillary loop stage HIF2��/� glomerulus. B: Four-week-old glomerulus showing bothmesangial (Me) and endothelial (En) cells contain the Bluo-gal reaction product. Po, podocytes.

1118 STEENHARD ET AL.

croscopy of HIF2� null kidneys. Inmaturing glomeruli at E18, capillaryloops contained erythrocytes andshowed fenestrated endothelium,morphologically normal basementmembrane, and fully interdigitatedpodocyte foot processes with epithelialslit diaphragms spanning the filtra-tion slits (Fig. 3H), all of which wereindistinguishable from wild-type glo-meruli.

Amounts and Distribution ofFlk-1 Protein AppearUnaltered in HIF2�

Knockouts

Because the knockout had no promi-nent kidney phenotype, we next con-sidered possible downstream targetsof HIF2� transcriptional activity. Thereceptor tyrosine kinase Flk-1 is a

Fig. 3.

Fig. 4.

Fig. 3. Normal glomerular capillary develop-ment in hypoxia-inducible transcription factor(HIF) 2��/� mice. A–D: Toluidine blue stainingof thin sections from an embryonic day (E) 18.5HIF2��/� kidney. A: Ureteric bud (UB) andcondensing mesenchyme (CM) appear normal.B: Endothelial cells are migrating into the vas-cular cleft of S-shaped bodies (arrow). C,D: Redblood cells are seen in capillary loop stage glo-meruli (arrow in C), and open capillary loopswith red blood cells are readily apparent in ma-ture glomeruli (arrow in D). E–G: Frozen sec-tions from E14 HIF2��/� mutants show �-ga-lactosidase product in endothelial cellsmigrating into the vascular cleft (arrow) of com-ma-shaped nephric figures (E). F: Capillary loopstage glomeruli contain �-galactosidase-posi-tive endothelial cells. Afferent and efferent arte-rioles seen entering the glomerulus also containreaction product (arrowhead). G: The arrow-head depicts the open capillary loop within amature glomerulus. H: Ultrastructure of E18.5HIF2��/� capillary loop shows normal podo-cyte (Po) foot process interdigitation and slitdiaphragms, glomerular basement membrane,and fenestrated endothelium (arrow).

Fig. 4. Flk-1 and GLEPP1 expression appearnormal in hypoxia-inducible transcription factor(HIF) 2��/� mouse kidney. A: Western blot ofHIF2� wild-type (�/�) or knockout (�/�) kid-ney lysates at embryonic day (E) 13.5 wasprobed with anti–Flk-1, then stripped and re-probed with anti-HIF1�. B: Quantification ofFlk-1 from two separate litters of HIF2� miceshowing no difference in the levels of Flk-1 inwhole kidney lysates (n � 3 for each genotype).C: Double labeling with anti–Flk-1 (red) and an-ti-GLEPP1 (green) in a HIF2��/� mouse showspodocyte expression of GLEPP1 and endothe-lial labeling of Flk-1 as expected.D: Both GLEPP1 and Flk-1 expression appearnormal in the HIF2��/� glomeruli.

HIF2� EXPRESSION IN DEVELOPING KIDNEY 1119

likely candidate given its renal ex-pression restricted to endothelial cellsand the ability of HIF2� to activatethe Flk-1 promoter (Elvert et al.,2003). Whole kidney lysates from in-dividual E13.5 knockout or wild-typekidneys underwent sodium dodecylsulfate (SDS) -polyacrylamide gelelectrophoresis and then Westernblotting to detect Flk-1 protein. A highmolecular weight band representingFlk-1, at �200 kDa, was seen in boththe wild-type and knockout samples(Fig. 4A, upper panel). To verify equalloading, blots were stripped and re-probed with the ubiquitously ex-pressed HIF1� subunit (Fig. 4A, lowerpanel). Relative Flk-1 levels weremeasured by densitometry on blotsfrom three knockouts versus threewild-types, representing animals fromtwo separate E13.5 litters. As shownin Figure 4B, no significant differ-ences in levels of Flk-1 protein expres-sion were seen.

We also evaluated the tissue distri-bution of Flk-1 in HIF2� heterozy-gotes and knockouts by immunofluo-rescence microscopy. Frozen sectionswere labeled with antibodies to Flk-1(Fig. 4C,D, red) and the podocyte-spe-cific gene GLEPP1 (Fig. 4C,D, green).Glomeruli from heterozygous andknockout mice both expressed Flk-1and GLEPP1 in the endothelial andpodocyte compartments, respectively,and in the same distribution patternsand apparent intensities.

VEGF mRNA Up-Regulationin HIF2� KnockoutMetanephric Organ Cultures

To evaluate responsiveness of HIF2�null kidneys to hypoxia, metanephroiwere dissected from E13.5 knockoutsand heterozygotes (obtained fromdams supplemented daily with DOPS)and maintained in organ culture atroom air (�20% oxygen) and mild hyp-oxia (5% oxygen) without DOPS for 5days. Total RNAs were isolated andVEGF mRNA levels quantified usingreal-time polymerase chain reaction(PCR), as described in the Experimen-tal Procedures section. As observedpreviously for mRNA obtained fromwild-type mice (Freeburg et al., 2003),VEGF mRNA levels from both het-erozygous and HIF2� knockouts weresignificantly greater in kidneys cul-

tured under hypoxic conditions (Fig.5), representing an approximatelytwofold increase over that seen whenkidneys were cultured in room air.

Anterior Chamber Grafts ofHIF2� KnockoutMetanephroi DevelopNormally

Because microvessel formation andglomerular endothelial differentiationfail to occur under typical metaneph-ric organ culture conditions, we trans-planted HIF2� knockout and het-erozygous metanephroi into anterioreye chambers of adult hosts, a sitethat normally supports the growthand development of endothelial cellsfrom graft-derived progenitors (Rob-ert et al., 1998). Hosts were not sup-plemented with DOPS. Five days aftertransplantation, grafts from knock-outs were morphologically indistin-guishable from heterozygotes and ro-bust HIF2�/LacZ was expressed in avascular pattern (Fig. 6A,B). As as-sessed by immunolabeling for the�3�4�5 chains of type IV collagen, theendothelial-specific markers plateletendothelial cell adhesion molecule(PECAM) and BsLB4, and the podo-cyte differentiation markers GLEPP-1and WT1, every aspect of glomerulardevelopment appeared normal ingrafts of HIF2� knockout kidneys(Fig. 6C–O).

Finally, in two rare instances, we

obtained female HIF2� knockout micethat survived birth. One died at 3weeks of age and was less than halfthe size of wild-type and heterozygouslittermates (4.26 g vs. 11.0 g), proba-bly reflecting a catecholamine deficit.However, light and electron micro-scopic analyses of kidneys from thismouse showed entirely normal tissuearchitecture (not shown). The secondmouse lived to 7 weeks of age and wasalso smaller than heterozygous litter-mates (10.9 g vs. 19.2 g). Urinalysisconducted at 6 weeks of age showed a24-hr albumin excretion rate of 19 �g,which was somewhat greater than aheterozygous sibling (11 �g), butwithin normal values for control, non-diabetic C57Bl/6 and 129Sv mice(Gurley et al., 2006).

Taken together, our findings indi-cate that there may be no develop-mental role for HIF2� in formation ofthe renal vasculature and glomeruli.Although DOPS administration is ef-ficacious to overcome deficits in cate-cholamine synthesis, the supplementdid not rescue every null mutant, and,as reported earlier, less than half theexpected number of knockout mice areborn despite DOPS supplementation(Tian et al., 1998). This finding sug-gests that HIF2� may be required fortranscriptional regulation in differentcell types. On a hybrid background,a small proportion of HIF2� knock-out mice are viable without DOPSsupplementation, but succumb to amultiorgan pathology stemming fromdysregulation of oxidative stress path-ways in mitochondria (Scortegagna etal., 2003a). These mice also had de-creases in all blood lineages, andtransplantation of wild-type bonemarrow into irradiated HIF2� knock-out hosts revealed a deficit in the bonemarrow microenvironment or a sys-temic effect on hematopoiesis (Scorte-gagna et al., 2003b). In another HIF2�knockout model, there was decreasedVEGF production by alveolar type IIpneumocytes, leading to impairedlung development and neonatal death(Compernolle et al., 2002).

The apparently normal vascular de-velopment observed in HIF2� knock-out kidney raised the likelihood ofcompensation by other HIF� familymembers. Indeed, HIF1� and -2�share 48% sequence identity and bothbind and activate transcription from

Fig. 5. Vascular endothelial growth factor(VEGF) mRNA increases under the low oxygencondition in cultured embryonic hypoxia-induc-ible transcription factor (HIF) 2��/� kidneys.Relative VEGF mRNA levels were measuredfrom embryonic day (E) 13.5 kidneys culturedfor 5 days in either room air (20% O2) or mildhypoxia (5% O2) from HIF2��/� (dark grey) orHIF2� �/� (light grey) mice (n � 3 for eachgenotype). Both genotypes show a twofold in-crease in the abundance of VEGF mRNA in mildhypoxia.

1120 STEENHARD ET AL.

Fig. 6. Hypoxia-inducible transcription factor (HIF) 2��/� kidney development in anterior chamber grafts proceeds normally. A: Frozen section of a graftedHIF2� �/� E13.5 kidney shows widespread vasculature development following �-galactosidase histochemistry. B,C: Serial frozen sections from a HIF2��/� graft were either reacted for �-galactosidase (B) or labeled (C) with the podocyte marker anti–WT-1 (green) and the endothelial marker anti- plateletendothelial cell adhesion molecule (PECAM, red). Both labels show normal distribution patterns. D–I: Double labeling of frozen sections with antibodies tothe basement membrane-specific type IV collagen (ColIV(�3, �4, �5), green) and the podocyte marker GLEPP1 (red). J–O: Double labeling of frozen sectionswith antibodies to the endothelial marker BsLB4 (green) and the podocyte marker WT-1 (red). The same expression pattern is seen for both heterozygousHIF2� grafts (D–F and J–L) and knockout HIF2� grafts (G–I and M–O). Merged images are shown in F, I, L, and O.

the hypoxia response element (HRE)found in the VEGF and erythropoietinpromoters (Ema et al., 1997; Tian etal., 1997). To test for possible up-reg-ulation of HIF1� or -3� in the HIF2�knockouts, we carried out quantita-tive, real-time PCR on RNAs obtainedfrom E13.5 kidneys (from two wild-type embryos and from two knock-outs), and from whole embryos (twowild-types, three knockouts). In kid-ney mRNAs, the wild-type:knockoutexpression ratios were closely similarfor both HIF1� (1:0.9) and HIF3� (1:0.9). Similarly, in HIF2� null em-bryos, there was no up-regulation ofHIF1� (1:0.7) or HIF3� (1:0.8), and nosignificant changes in expression ofFlk1 (1:0.9) or VEGF mRNAs (1:1)were observed. Much evidence sug-gests nonredundant roles for HIF1�and -2�. For example, glycolytic genesare not induced by hypoxia in a renalcell carcinoma cell line that only ex-presses HIF2�. However, glycolyticgenes are expressed in the RCC-4 line,which expresses both HIF1� and -2�(Hu et al., 2003). HIF1��/� embryonicstem cells lack induction of all glyco-lytic enzymes typical to a hypoxic re-sponse, suggesting that HIF2� cannotcompensate for loss of HIF1� in thisscenario (Iyer et al., 1998; Ryan et al.,1998). Despite stable expression,HIF2� is transcriptionally inactive inembryonic mouse fibroblast cell lines(Park et al., 2003). Furthermore, inA293 cell transient transfections, onlyHIF2� (but not -1�) is capable of acti-vating an Flk-1 promoter construct(Kappel et al., 1999). Thus, eventhough HIF1� and -2� are biochemi-cally similar, they most likely induceeither different genes and/or the samegenes in different cell types.

Despite biochemical evidence forHIF2� induction of Flk-1 (Kappel etal., 1999; Elvert et al., 2003), we didnot observe decreases in renal Flk-1levels in HIF2� knockouts, either byquantitative reverse transcriptase(RT) -PCR, Western blotting, or im-munofluorescence. Thus, regulation ofFlk-1 in developing kidney endothelialcells in vivo must occur independent ofHIF2�. Additionally, when E13.5HIF2� knockout kidneys were cul-tured in hypoxia, levels of VEGFmRNA increased significantly overthat seen in normoxia, and in parallelwith that seen in metanephric cul-

tures from heterozygotes. Again, thisfinding suggests that HIF2� alonemay not mediate the hypoxia-induc-ible response of the VEGF gene, atleast under the organ culture condi-tions used here. We, therefore, con-clude that, despite its widespread ex-pression throughout the developingkidney vasculature, deletion of HIF2�does not affect vascular patterning,the expression of VEGF or Flk1, ornephrogenesis. Nevertheless, we can-not exclude a role for HIF2� in renalvascular biology later in life and/orunder physiological stress. Futurestudies with HIF2� mutants may helpilluminate these possibilities.

EXPERIMENTALPROCEDURES

Transgenic Mice

Epas1tm1Rus mice were purchasedfrom the Jackson Laboratory (BarHarbor, ME), and the colony was es-tablished and maintained by inter-crossing heterozygotes (HIF2�LacZ/�;Tian et al., 1998). The following threeprimers were used to identify wild-typeHIF2��/�, heterozygous HIF2�LacZ/�,or homozygous null HIF2�LacZ/LacZ

mice by PCR of tail genomic DNA:5-GGAGGCTTTGTCCAGGTGGGA-GCTCACACTGTG-3, 5-GGTGCG-GGCCTCTTCGCTATTA-3, 5-GTGGG-TCACTACCGCGAGTGTGAATGG-3.To obtain homozygous mutants, thedrinking water of pregnant femaleswas supplemented with 3 mg/ml of thecatecholamine precursor DOPS(Sigma Chemical, St. Louis, MO) in 2mg/ml ascorbic acid. The solution wasmade fresh daily and administeredthroughout gestation.

�-GalactosidaseHistochemistry

Heterozygous or timed-bred pregnantmice were anesthetized with halo-thane and killed by cervical disloca-tion. For analysis of metanephroi, kid-neys were microdissected fromembryos and nonrenal tissues wereused for PCR genotyping. Dissectedkidneys were fixed overnight in 0.2%paraformaldehyde in 0.1 M PIPESbuffer (piperazine-N,N-bis[2-ethane-sulfonic acid]), pH 6.9, at 4°C. Tissueswere washed three times in 2 mM

MgCl2 in phosphate buffered saline(PBS) and then cryopreserved in 30%sucrose in 2 mM MgCl2 in PBS over-night at 4°C. Kidneys were thenplaced in optimal cutting temperaturecompound (OCT; Miles, Elkhart, IN)and frozen in isopentane in an ace-tone/dry ice bath. Cryostat sectionswere cut at a thickness of 8 �m, air-dried, and then fixed again in 0.2%paraformaldehyde in 0.1 M PIPES onice, pH 6.9, for 10 min. Slides werewashed three times in PBS with 2 mMMgCl2 and then incubated in deter-gent rinse (0.1 M phosphate buffer,pH 7.3, 2 mM MgCl2, 0.01% sodiumdeoxycholate, 0.02% Nonidet P-40) for10 min on ice. After detergent rinse,slides were placed in color develop-ment solution (detergent rinse con-taining 5 mM potassium ferricyanide,5 mM potassium ferrocyanide, 20 mMTris, pH 7.3, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-�-D-galactopyrano-side [X-gal; Sigma Chemical]) over-night at 37°C in the dark. Sectionswere then washed three times on icewith PBS with 2 mM MgCl2, post-fixed in 4% paraformaldehyde in PBS,dehydrated through ethanol gradi-ents, and permanently mounted inPermount (Fisher Scientific, Pitts-burgh, PA). Additionally, some sec-tions were post-fixed in 0.2% parafor-maldehyde after color developmentand labeled with Bandeiraea simplici-folia BS-L lectin (BsLB4; SigmaChemical) at a 1:50 dilution for 1 hr atroom temperature, followed by mount-ing with Prolong antifade mountingmedium (Molecular Probes, Eugene,OR).

Whole-Mount �-GalactosidaseHistochemistry

E13.5 kidneys were removed fromDOPS-supplemented females andfixed overnight in 0.2% paraformalde-hyde in 0.1 M PIPES buffer, pH 6.9, at4°C. Tissues were washed twice in 2mM MgCl2 in PBS, followed by deter-gent rinse for 10 min. Kidneys wereplaced in color development solutionovernight at 37°C in the dark. Kid-neys were rinsed twice in 2 mM MgCl2in PBS, and re-fixed with 4% parafor-maldehyde in PBS for 30 min on ice.

1122 STEENHARD ET AL.

Electron Microscopy

Kidney cortices were minced to 1-mmcubes, then fixed for 2 hr on ice with2% paraformaldehyde/0.4% glutaral-dehyde in PBS containing 2 mMMgCl2. Tissue was washed threetimes in PBS containing 2 mM MgCl2,and incubated at 37°C overnight withBluo-gal staining solution (20 mM po-tassium ferricyanide/ferrocyanide, 2mM MgCl2, 2 mM Bluo-gal [Invitro-gen, Carlsbad, CA], pH 7.3). Afterwashing three times with buffer, sam-ples were re-fixed with Karnovsky’sfixative for 30 min, washed threetimes with 0.1 M sodium-cacodylate in3.5% sucrose, pH 7.3, and then post-fixed for 1.5 hr with Palade’s OsO4.Tissues were dehydrated throughgraded ethanol and propylene oxideand embedded in Polybed 812 withovernight polymerization at 60°C. Ul-trathin sections were stained with 4%uranyl acetate and Reynold’s lead ci-trate. Other tissues were analyzedusing routine transmission electronmicroscopic methods as described pre-viously (Abrahamson and St. John,1992).

Western Blotting

Single metanephroi from E13.5 micewere dissected and immediately ho-mogenized in 75 �l of ice-cold RIPAbuffer (50 mM Tris, pH 8.0, 150 mMNaCl, 1.0% Triton X-100, 0.5% sodiumdeoxycholate, 0.1% SDS) supple-mented with a protease inhibitor cock-tail (Freeburg et al., 2003). Lysateswere incubated on ice for 10 min, fol-lowed by two freeze/thaw cycles. Cel-lular debris was cleared by centrifuga-tion (11,000 g, 15 min, 4°C). Totalprotein was quantified using the DCProtein Assay (Bio-Rad, Hercules,CA). Equal amounts of protein wereloaded onto precast gradient gels (Bio-Rad), and, after electrophoresis, pro-teins were transferred to nitrocellu-lose. Membranes were probed with a1:100 dilution of rabbit anti–Flk-1 an-tibody (C-20, Santa Cruz, Santa Cruz,CA) followed by donkey anti-rabbitimmunoglobulin G–horseradish per-oxidase (IgG-HRP) at 1:1,000 (Amer-sham). After ECL, membranes werestripped (Western Blot Recycling Kit,Alpha Diagnostic, San Antonio, TX)and re-probed with a 1:100 dilution ofgoat anti-HIF1� (C-19, Santa Cruz)

followed by rabbit anti-goat IgG-HRPat 1:10,000 (Sigma). Relative Flk-1intensity was calculated from densi-tometry values obtained using theBio-Rad ChemiDoc XRS gel documen-tation system.

Immunofluorescence

Kidneys were fixed in freshly pre-pared 0.2% paraformaldehyde (in PBScontaining 0.1 M PIPES, pH 6.9, 2mM MgCl2), cryoprotected in 30% su-crose, and frozen in Tissue-Tek OCTembedding compound. Frozen serialsections, 8 �m thick, were postfixed in0.2% paraformaldehyde, rinsed inPBS, then permeabilized in 0.5% Tri-ton X-100 in PBS for 5 min, followedby an additional rinse with PBS. Dou-ble labeling was carried out with a1:20 dilution of rat anti-Flk-1 (BDPharmingen, San Diego, CA) and 10�g/ml rabbit anti-GLEPP1 (a kind giftfrom Roger Wiggins, University ofMichigan [Wang et al., 2000]); a 1:50dilution of rabbit anti-WT1 (SantaCruz) and 50 �g/ml rat anti-PECAM(Serotec, Raleigh, NC); or with 10�g/ml rabbit anti-GLEPP1 and 100�g/ml mouse anti-collagen type IV(�3,�4,�5 chains; Heidet et al., 2003).The appropriate secondary antibodies(Alexa 488 and 594 conjugates, Molec-ular Probes) were applied after PBSrinses. In some cases, fluorescein-con-jugated BsLB4 lectin (Sigma) was ap-plied during the secondary antibodyincubation. Slides were mounted inProlong Gold plus DAPI (MolecularProbes). Fluorescent digital imageswere obtained using a Zeiss LSM510confocal microscope.

Metanephric Kidney Culture

E13.5 kidneys were isolated fromembryos taken from DOPS-supple-mented HIF2��/� females andplaced into transwell culture with me-dium (DMEM with 10% fetal bovineserum, 1% Pen-Strep and 1% matrigel[BD Biosciences, Palo Alto, CA]).Genotyping was performed on embry-onic tail tissue. Paired kidneys wereplaced in separate dishes either at20% O2 (room air) or 5% O2 (mild hyp-oxia) for 5 days. Medium was ex-changed on the second day of culture.Total RNA was isolated from individ-ual kidneys using the RNAqueous-

Micro spin kit (Ambion, Austin, TX).Samples were diluted to 150 ng/�l andamplified using QuantiTect SYBRGreen RT-PCR kit (Qiagen, Valencia,CA) with the following primers: VEGFforward 5-GGAGATCCTTCGAGG-AGCACTT-3 and reverse 5-GGC-GATTTAGCAGCAGATATAAGAA-3;cyclophilin forward 5-CAGACGC-CACTGTCGCTTT-3 and reverse 5-TGTCTTTGGAACTTTGTCTGCAA-3(Shih et al., 2002). Real-time PCR wasperformed using an iCycler (Bio-Rad).The primer sets were validated forefficiency for the comparative Ctmethod using standard curve analysis(Livak and Schmittgen, 2001). PCRproducts were analyzed on agarosegels to confirm size, and melt curveanalysis was performed to reveal asingle amplified product.

Anterior Chamber Grafts

Adult CD1 hosts were anesthetizedwith a ketamine–xylazine combina-tion (100 mg and 15 mg, per kg bodyweight). Tropicamide was applied toone eye to dilate the iris, 0.5% tera-caine was supplied as corneal anes-thetic, and the cornea was piercedwith a 27-guage needle, and the inci-sion was extended �2 mm using Van-nas scissors. E13.5 kidneys (fromDOPS-supplemented embryos ob-tained from timed-bred HIF2� het-erozygous matings) were transferredthrough the corneal incision, and po-sitioned over the host iris as describedbefore (Robert et al., 1998). Ophthal-mic antibiotic ointment was applied tothe eye, and hosts were allowed torecover. Seven days later, hosts wereanesthetized by halothane and graftedkidneys were harvested. Grafts werefixed in cold 0.2% paraformaldehyde(in PBS containing 0.1 M PIPES, pH6.9, 2 mM MgCl2), cryoprotected in30% sucrose, and frozen in Tissue-TekOCT embedding compound. Serial8-micron sections were treated withthe various antibody combinations forimmunofluorescence and for �-galac-tosidase histochemistry as describedabove.

Quantitative Real-TimeRT-PCR

Total RNA was isolated from individ-ual kidneys or whole embryos using

HIF2� EXPRESSION IN DEVELOPING KIDNEY 1123

the RNeasy Micro Kit (Qiagen). Sam-ples were diluted to 10 ng/�l and am-plified with the following primers:VEGF forward 5-GGAGATCCTTC-GAGGAGCACTT-3 and reverse 5-GGCGATTTAGCAGCAGATATAA-GAA-3; cyclophilin forward 5-CAGACGCCACTGTCGCTTT-3 andreverse 5-TGTCTTTGGAACTTT-GTCTGCAA-3 (Shih et al., 2002), Su-perarray RT2 PCR Primer Sets formouse HIF1� (PPM03799A), mouseHIF3� (PPM05268A), and Mouse Kdr(Flk1; PPM03057A; SuperArray, Inc.,Bethesda, MD). Real time RT-PCRwas carried out on an iCycler (Bio-Rad) with a one-step RT-PCR kit (Qia-gen, catalog no. 204245), which usesthe double-stranded DNA-binding dyeSYBR Green. The primer sets werevalidated for efficacy using the com-parative Ct method and standardcurve analysis (Livak and Schmitt-gen, 2001). PCR products were ana-lyzed on agarose gels to confirm size,and melt curve analysis was per-formed to reveal a single amplifiedproduct. Normalization was per-formed by dividing each value calcu-lated for a specific gene by the value ofthe housekeeping gene (cyclophilin).

Urinalysis

Mice were weighed and maintained inmetabolic cages to collect 24-hr urines.Albumin excretion was measured usingthe Albuwell murine microalbuminuriaassay system, following manufacturer’sinstructions (Exocell, Inc., Philadel-phia, PA).

ACKNOWLEDGEMENTSThis manuscript is dedicated to thememory of our friend and colleague,Dr. Paul B. Freeburg. We thank Dr.Roger Wiggins, University of Michi-gan, for providing the GLEPP1 anti-body. Confocal images were acquiredat the KUMC Confocal Imaging Facil-ity, supported by the Kansas IDeANetwork of Biomedical Research Ex-cellence (grant no. RR016475), and wethank Dr. Elizabeth Petroske andEileen Roach for technical assistance.

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