divergent expression patterns for hypoxia-inducible factor- 1 and

Divergent Expression Patterns for Hypoxia-Inducible Factor-1� and Aryl Hydrocarbon Receptor Nuclear Transporter-2 inDeveloping Kidney

PAUL B. FREEBURG and DALE R. ABRAHAMSONDepartment of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas

Abstract. The hypoxia-inducible factors (HIF) are �/� het-erodimeric transcription factors of the basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) superfamily and are chiefly re-sponsible for cellular adaptation to oxygen deprivation. HIFfunction relies on the stabilization of the � subunit. Whenoxygen tension falls, HIF-� subunits translocate to the nucleusand, upon dimerization with HIF-�, activate transcription oftarget genes, including vascular endothelial growth factor, vas-cular endothelial growth factor receptor-1 and -2, and WT-1,which are vital for kidney development. HIF-� subunits arestable regardless of oxygen concentration and constitutivelytranslocate to the nucleus. It was shown previously that HIF-1�protein expression is nearly ubiquitous in newborn kidney andthat HIF-1� dimerizes with either HIF-1� or -2�. Here it isshown that aryl hydrocarbon receptor nuclear transporter-2(ARNT2/HIF-2�) also heterodimerized with HIF-1� and -2�.ARNT2/HIF-2� protein was highly expressed in newborn kid-ney but decreased significantly with age, whereas HIF-1�

levels remained relatively constant. By immunohistochemicalanalysis, widespread expression of HIF-1� was observed indeveloping and mature kidneys. ARNT2/HIF-2� protein dis-tribution was restricted to distal segments of developingnephrons and in mature kidney was confined specifically tothick ascending limb of Henle’s loop. The data presented heresuggest that ARNT2/HIF-2� is required at high levels duringnephrogenesis in distal tubules and later exclusively in thickascending limb. Furthermore, Hypoxyprobe-1 and lotus lectinco-localization studies showed that developing proximal con-voluted tubules were the most severely hypoxic nephron seg-ment in immature kidney. Because HIF-2� protein was notabundantly expressed in this segment, it may not be engaged inmediating responses to severe hypoxia. The differential distri-bution patterns for HIF-1� and -2� suggest divergent rolesduring kidney development for these highly related bHLH-PAS proteins.

The basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) familyof transcription factors functions as �/� heterodimeric DNAbinding complexes and regulates a remarkably diverse numberof biologic events. These include responses to oxygen depri-vation and exposure to toxins, control of circadian rhythms,and regulation of hormone signaling (1). The bHLH-PASheterodimers typically consist of one broadly expressed,readily available member (� subunit) that heterodimerizes witha partner (� subunit) that has a tightly restricted expressionprofile and is sensitive to environmental cues. Perhaps the mostwidely expressed and best understood protein in this family isthe aryl hydrocarbon receptor nuclear transporter (ARNT),which acts as the � subunit for transcriptional responses to bothtoxin exposure and hypoxic stimuli (2–6). To aid in metabo-lism of the toxin benzo[a]pyrene, ARNT heterodimerizes with

the bHLH-PAS protein aryl hydrocarbon receptor (AHR) andbinds to the xenobiotic response element located in the en-hancer regions of the cytochrome P450 gene isoforms Cyp1a1,Cyp1a2, and Cyp1b1 (7). Expression of these proteins purgesthe system of the toxin by way of their metabolic activitytoward benzo[a]pyrene. Ordinarily, AHR is held quiescent inthe cytoplasm by binding heat shock protein 90 and AHRinteraction factor. AHR is released by this complex, combineswith ARNT, and induces gene expression upon binding of thetoxin (8). AHR therefore acts as the � subunit of bHLH-PASheterodimer because its function is reliant on an environmentalinsult, which in this case is the cellular toxin benzo[a]pyrene.

Another clearly defined role for ARNT as a � subunit ofbHLH-PAS heterodimers is in the hypoxia responsive path-way, where ARNT is now commonly known as hypoxia-inducible factor-1� (HIF-1�). Early studies of erythropoietin(Epo) gene expression led to the identification of a het-erodimeric transcription factor that stimulates Epo expressionin response to low oxygen tension (9). This factor was purifiedand found to contain ARNT (HIF-1�) and a new member ofthe bHLH-PAS family, named HIF-1�. Since the discovery ofthe HIF-1�/HIF-1� heterodimer (HIF-1), the biochemical be-havior of these proteins in response to oxygen deprivation hasbeen studied extensively. Most evidence indicates that HIF-1�protein expression occurs in all cells and that the protein is

Received April 14, 2004. Accepted July 15, 2004.Correspondence to Dr. Dale R. Abrahamson, Department of Anatomy and CellBiology, University of Kansas Medical Center, MS 3038, 3901 RainbowBoulevard, Kansas City, KS 66160. Phone: 913-588-7000; Fax: 913-588-2710; E-mail: [email protected]

1046-6673/1519-2569Journal of the American Society of NephrologyCopyright © 2004 by the American Society of Nephrology

DOI: 10.1097/01.ASN.0000141464.02967.29

J Am Soc Nephrol 15: 2569–2578, 2004

stable and is found abundantly in the nucleus regardless ofoxygen tension (10). HIF-1�, conversely, is regulated directlyby cellular oxygen availability (11). When intracellular oxygentension is sufficient to satisfy metabolic demands (normoxia),HIF-1� protein undergoes an oxygen-dependent hydroxylationon a highly conserved proline residue (12,13). This hy-droxyproline residue is vital, because it renders HIF-1� recog-nizable by the von Hippel-Lindau ubiquitin ligase complex.Hydroxylated HIF-1� becomes polyubiquinated and rapidlydestroyed by proteasomes. However, when oxygen tensionfalls below critical levels (hypoxia), HIF-1� escapes prolinehydroxylation, is not recognized by von Hippel-Lindau, andtherefore avoids proteasomal degradation. Stabilized HIF-1�then translocates to the nucleus, heterodimerizes with thereadily available HIF-1�, and binds the hypoxia-responsiveelement (HRE) located in the promoter/enhancer of HIF targetgenes. HIF-2�, initially called endothelial PAS domain pro-tein, has very similar structural and biochemical properties toHIF-1� and also heterodimerizes with HIF-1� (14–16).Among the genes induced by HIF-1 and -2 are those highlyinvolved in kidney development, including vascular endothe-lial growth factor (VEGF), VEGF receptor-1 (VEGFR-1) and-2, angiopoietin-2, Tie-2, Epo, and WT-1 (17–23).

Although much has been learned about the � subunits ofbHLH-PAS heterodimers (AHR and HIF-1� and -2�), rela-tively little is known about the HIF-� counterparts. However,recent studies have suggested that � bHLH-PAS proteins aremore complex than initially thought. For example, five differ-ent splice variants of HIF-1� have been identified in the rat(24), and additional isoforms have been shown to containdeletions at exon 5, 6, or 11 or an insertion at exon 20. OtherHIF-1� mRNA isoforms contain a variable number of codonsin exon 16 encoding glutamine. Expression profiles and func-tions of each of the alternately spliced variants have yet to bedescribed. In addition, in some cell lines, HIF-1� protein isapparently sensitive to oxygen concentration and accumulatesin hypoxia, as observed in a human neuroblastoma cell culturesystem (25).

HIF-1� and -2� both are required for proper development,as demonstrated in mice with targeted deletions of these genes(14,26,27). HIF-1� null embryos die at embryonic day 9.5(E9.5) from cardiovascular and neural tube defects, as well aswidespread mesenchymal cell death. HIF-2� knockout mice onthe C57Bl/6 or 129/Sv genetic background die at E13.5 toE15.5 from bradycardia and lack of catecholamine synthesis.Overall vascular development in these mice, at least at thegross anatomic level, seems normal (14). HIF-2� mutants onthe ICR/129Sv background, however, show severe vasculardefects and die by E13.5 (15).

HIF-1� null embryos die at E11 from abnormal hematopoi-etic and vascular development (28). A second �-class bHLH-PAS protein, ARNT2 (HIF-2�), has been identified and foundto share 63% homology with HIF-1� (29,30). HIF-2� hassubsequently been shown to form functional heterodimers withAHR, HIF-1�, and HIF-2� (30–32). In situ hybridizationstudies in developing mouse show that HIF-1� is nearly ubiq-uitously expressed at E11 through postnatal day 1.5 (P1.5),

whereas HIF-2� mRNA is expressed most prominently in thebrain and kidney at these stages (33). The in situ hybridizationsin this earlier study demonstrated that HIF-2� mRNA is ex-pressed strongly in the outer cortex of developing kidney,although cellular identification is not possible because of thelow-magnification fields presented. Unlike HIF-1� mutants,HIF-2� knockout mice survive until birth and, with the excep-tion of stunted hypothalamus formation, seem to develop nor-mally (34). Cultured neurons from HIF-2��/� mice show lessinduction of the hypoxia-inducible genes VEGF, Glut3, andPgk compared with HIF-2��/� neurons, and mobility shiftDNA-binding assays of nuclear extracts from hypoxic culturesof wild-type neurons show both HIF-1� and -2� binding DNAas a complex with HIF-1� (34). These observations thereforesuggest that HIF-2� is capable of participating in hypoxicresponses.

Crosses between HIF-1��/� and HIF-2��/� mutants re-vealed that progeny lacking any combination of at least twowild-type alleles of either � subunit died by E8.5 (34). Theseexperiments indicate that HIF-1� and HIF-2� have overlap-ping roles in early development and may compensate for lossof one another. After E8.5, however, HIF-� subunit functionsbegin to diverge, and each protein has a unique, indispensablerole in embryonic development, which has yet to be clearlydefined. This is consistent with the restricted expression pat-tern seen for HIF-2� during mouse development when com-pared with the ubiquitous expression pattern for HIF-1� (33).We previously immunolocalized HIF-1� protein expression innewborn mouse kidney and found that every cell type seemedto contain nuclear expression at that age (35). Although thedeveloping kidney is one of two sites of intense HIF-2� ex-pression, this protein has not been studied in detail duringmetanephrogenesis. Here, we examined HIF-2� protein ex-pression beginning at the earliest stages of nephron develop-ment and continuing into maturity and compared expressionpatterns with those of HIF-1�. Our findings show that HIF-2�is selectively expressed in distal segments of developingnephrons and becomes restricted to thick ascending limb(TAL) of loop of Henle. We also show that, like HIF-1�,HIF-2� heterodimerizes with both HIF-1� and -2�, and thatHIF-2� protein expression in organ-cultured metanephroi isnot regulated by oxygen levels.

Materials and MethodsWestern Blots and Immunoprecipitations

Wild-type CD-1 mouse kidneys were dissected at 1 d, 7 d, and 6 wkof age and disrupted in a Dounce homogenizer in RIPA buffersupplemented with a protease inhibitor cocktail as described previ-ously (35). After lysates were cleared by centrifugation at 4°C for 25min at 15,000 � g, total protein concentrations were determined bythe colorimetric Bio-Rad DC Protein Assay (Bio-Rad Laboratories,Hercules, CA). Equal amounts of protein (50 to 100 �g) were sepa-rated by 5 to 15% SDS-PAGE and transferred to nitrocellulose.Standard immunoblot techniques were then applied with the followingantibodies: polyclonal HIF-1� (Novus-Biologicals, Littleton, CO;1:100) and polyclonal ARNT2 (HIF-2�; sc-5581; Santa Cruz Bio-technology, Santa Cruz, CA; 1:50). The ECL reagent and ECL Hy-perfilm (Amersham Pharmacia Biotech, Piscataway, NJ) were used to

2570 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2569–2578, 2004

visualize the bands. For loading controls, membranes were also incu-bated with mouse monoclonal anti–smooth muscle actin antibody(1:100 dilution; Sigma-Aldrich, St. Louis, MO).

For immunoprecipitation experiments, lysates from 1-d-old kid-neys were additionally cleared by incubation with protein A agarose(1 �l/10 �g total protein) for 15 min and then centrifuged again at15,000 � g for 15 min. The clarified lysates then underwent immu-noprecipitation with HIF-2� antibodies, using procedures describedpreviously (35). The immunoprecipitated proteins were analyzed onWestern blots by standard protocols with monoclonal anti–HIF-1�and -2� antibodies used at a dilution of 1:100. Purified rabbit IgG(Sigma-Aldrich) was also used as a nonspecific control to demonstratethe specificity of the ARNT2 antibodies, and no bands were detectedon Western blots probed with anti–HIF-1� or -2�.

Embryonic Kidney Organ Culture and Western BlotsTimed-pregnant CD-1 mice were killed and embryos were re-

moved at E12. Metanephroi were dissected and cultured on PETmembrane cell culture inserts (0.4-�m pore; BD Biosciences, SanJose, CA). Organ culture media and growth conditions were as de-scribed previously (35). Culture oxygen concentrations were set ateither constant room air (~20% oxygen) or 5% oxygen for 5 d or at20% oxygen for 4 d and then reduced to 2% for the final 24 h. Afterculture periods, kidneys were homogenized in RIPA buffer, proteinconcentrations were determined, and Western blots were performed asdescribed above. In addition to HIF-1� and -2�, WT-1 (Santa CruzBiotechnology; 1:50) and cyclo-oxygenase (Cox-2; Chemicon Inter-national, Temecula, CA; 1:200) antibodies were used to probe mem-branes. In some cases, explants were fixed and processed for immu-nohistochemistry as described below.

Peroxidase ImmunohistochemistryFor immunohistochemical analysis, kidneys were dissected and

frozen in OCT in isopentane in a dry-ice/acetone bath. Cryostatsections were cut at a thickness of 6 �m and air-dried. Sections werefixed for 10 min in ice-cold methanol, washed in PBS, and placed in3% H2O2 in methanol for 10 min. Blocking of nonspecific proteininteractions was achieved by incubating sections with 10% goat serumin PBS. Slides were then incubated in primary antibodies: Polyclonalanti–HIF-1� and HIF-2� diluted in PBS (1:100 and 1:50, respec-tively) for 1 h at room temperature in a humidified slide chamber.Sections were then washed with PBS, incubated sequentially withbiotinylated secondary antibodies (15 �g/ml) and streptavidin-HRPconjugates for 30 min each, and color-developed with 3,3'-diamino-benzidine tetrahydrochloride. Rabbit IgG diluted in PBS at the sameconcentration as the primary antibodies served as a negative control.

Immunofluorescence AnalysisSections (6 �m thick) of 3-d-old mouse kidney were fixed in 100%

methanol on ice and incubated with polyclonal anti–HIF-2� (1:50)and either fluorescein-conjugated Lotus lectin, to label proximal con-voluted tubule (Sigma Chemical Co., St. Louis, MO; 1:200), orrhodamine-conjugated Dolichos Biflorus agglutinin (DBA), to labelcollecting duct (Vector Laboratories, Burlingame, CA; 1:10), for 30min at room temperature. After three washes in PBS, Alexa Fluor488–conjugated anti-rabbit antibody (Molecular Probes, Eugene, OR)was applied for 30 min at 1:200. Sections were again washed in PBSand permanently mounted with the fluorescence preserving reagentProlong (Molecular Probes). Serial sections of 6-d-old mouse kidneywere fixed in the same way for co-localization of HIF-2� and Tamm-Horsfall protein (THP). HIF-2� immunofluorescence was performed

as described above on the first serial section, and goat anti-THPantibody (Cappel Laboratories, Durham, NC) was applied to thesecond serial section for 30 min at 1:200. Rabbit anti-goat fluoresceinwas added as a secondary antibody to the anti-THP–labeled slides,which were also mounted with Prolong. Irrelevant IgG was incubatedwith sections and treated sequentially with fluorescein and rhodam-ine-conjugated secondary antibodies for controls.

For identifying hypoxic tissues, pimonidazole hydrochloride (Hy-poxyprobe-1; Chemicon) was injected intraperitoneally into 6-d-oldmice (200 mg/kg), and fixation and labeling were carried out as before(35). In addition to the Hypoxyprobe-1 monoclonal antibody, sectionswere incubated with Lotus lectin-fluorescein (1:200) for 30 min atroom temperature. Sections from saline-injected mice were also im-munolabeled for Hypoxyprobe-1 and served as controls.

ResultsHIF-� Protein Expression

Total mouse kidney protein was analyzed for HIF-1� and-2� by quantitative Western blots. Three stages of develop-ment were examined: 1 d, 7 d, and 8 wk of age (Figure 1A).HIF-1� protein was readily detectable on blots at each age,with a slight increase in expression at day 7. HIF-2� was

Figure 1. (A) Total renal hypoxia-inducible factor-2� (HIF-2�) pro-tein expression decreases with age. Quantitative Western blots forHIF-1� and -2� with kidney lysates from newborn (Nb), 7-d-old, and8-wk-old mouse kidneys revealed sustained expression of HIF-1�throughout kidney maturation, with a peak at 7 d of age. By contrast,HIF-2� expression was most intense in newborn kidney, was signif-icantly decreased by day 7, and was barely detectable at 8 wk.Anti–smooth muscle actin staining of blots serves as loading controls.(B) HIF-2� heterodimerizes with both HIF-1� and -2�. Proteinsimmunoprecipitated by HIF-2� IgG from 3-d-old mouse kidney wereanalyzed by Western blot with HIF-1�– and -2�–specific antibodies.Both � subunits were evident by Western blot, demonstrating thatHIF-2� forms a complex with both � subunits. NS, nonspecific bandsdetected in both cases by secondary antibody.

J Am Soc Nephrol 15: 2569–2578, 2004 HIF-1� and ARNT2 (HIF-2�) in Developing Kidney 2571

strongly expressed in newborn kidney and decreased signifi-cantly at 7 d of age. In 8-wk-old kidney, HIF-2� protein wasbarely detectable by Western blot.

To determine whether HIF-2� heterodimerizes with eitherHIF-1� or HIF-2�, we conducted immunoprecipitation andWestern blot experiments of newborn kidney lysates. BothHIF-1� and -2� proteins were observed on Western blots ofthe HIF-� immune complexes, revealing that HIF-1� formedheterodimers with both HIF-1� and -2� in developing kidney(Figure 1B).

Effect of Hypoxia on HIF-� Protein Expression inKidney Organ Culture

E12 kidney explants were cultured for 4 d at 20% oxygenfollowed by a 24-h exposure to 2% oxygen (Figure 2, left) orfor 5 d at 5% oxygen (Figure 2, right), followed by Westernblots for HIF1-� and -2�. In cultures that were exposed toacute or chronic hypoxia, no large changes in either HIF-1� or-2� protein expression were evident. However, the HIF-induc-ible genes WT-1 and Cox-2 both were induced by hypoxia inorgan culture, but WT-1 increased only modestly at 5% oxygen(Figure 2).

HIF-1� and -2� ImmunolocalizationHIF-1� protein was found to be widely distributed in nuclei

of apparently all cells in both developing and maturing kidneyat every stage examined. In E14 kidney, HIF-1� was expressedubiquitously by mesenchymal cells, ureteric bud epithelium,and all cells in developing nephrons (Figure 3A). During laterstages of kidney development and into maturity, widespreadHIF-1� protein expression persisted; nuclei within all tubular

segments, interstitial cells, and cells within glomeruli allstrongly labeled with anti–HIF-1� antibody (see A in Figures4 through 7). Sections that were labeled with nonspecific rabbitIgG in place of HIF-1� antibodies were negative in all cases(Figure 3D).

In contrast to HIF-1�, the distribution of HIF-2� protein washighly restricted among the time points examined. At E14,HIF-2� was seen in nuclei of ureteric bud epithelium and lessintensely in mesenchymal cells of the outer cortex (Figure 3B).In early nephrons, both the visceral and parietal epitheliumwere weakly positive, but developing distal segments showedparticularly intense HIF-2� labeling (Figure 3, B and C).

As an additional test for whether HIF-1� and/or -2� changesin abundance or expression pattern, organ cultures of E12kidneys maintained for 5 d at 20 or 5% oxygen were examined.As shown in Figure 4, no differences were observed.

Figure 2. HIF-� and HIF target gene expression in embryonic day 12(E12) organ cultures that were exposed to hypoxia. E12 mouse met-anephroi were dissected and cultured for 4 d in 20% oxygen and thenshifted to a hypoxic environment (2% oxygen) for 1 d (left) or werecultured for 5 d in 5% oxygen (right). In both cases, HIF-1� and -2�protein levels remained constant. WT-1 and cyclo-oxengenase-2(Cox-2), two potential HIF target genes during kidney development,however, showed increased protein levels in response to hypoxia inboth cases, although upregulation of WT-1 at 5% oxygen seemedmore modest. All blots are representative of two independentexperiments.

Figure 3. Immunolocalization of HIF-1� and -2� in E14 kidney. (A)HIF-1� protein expression at E14 was nearly ubiquitous and uni-formly intense. Strong nuclear staining was apparent in ureteric bud(UB), developing nephrons (N), and mesenchymal cells. (B) HIF-2�protein expression was much more restricted and variable at E14compared with HIF-1�. Faint nuclear labeling was found in uretericbud, uninduced mesenchyme in extreme outer cortex, but was partic-ularly prominent in developing nephrons. (C) Higher-power viewsshow that HIF-2� protein was expressed strongly and specifically inthe distal segment of developing nephrons (*), including cells thatmay form the macula densa. Weaker expression was also evident inthe visceral (VE) and parietal epithelium (PE), which are destined toform the podocytes (Po) and Bowman’s capsule (BC), respectively(arrows). (D) Nonspecific rabbit IgG incubated with sections in placeof primary antibodies served as controls, and no labeling wasobserved.


In 3-d-old kidney, developing nephrons of the extreme outercortex showed the same overall expression pattern observed inE14 kidney with especially strong localization to distal seg-ments (Figure 5B). In addition, tubules projecting through thesubcortical region also showed strong nuclear labeling at thisage. The expression profile in 5-d-old kidney was indistin-guishable from that in 3-d-old kidney (data not shown). In1-wk-old kidney, HIF-2� protein distribution was restricted tocertain tubular segments (Figure 6B), and podocyte nuclei ofmaturing stage glomeruli were also labeled (Figure 6B, inset).By 10 d of age, restricted tubular expression persisted in thecortex, but glomerular expression was now absent (Figure 7B).The medulla of 10-d-old kidney showed intense HIF-2� ex-pression (Figure 7C). Finally, in fully mature, 8-wk-old kidney,HIF-2� expression was diminished overall and found onlysparsely in the cortex (Figure 8B). Medullary expression ofHIF-2� was prominent (Figure 8B, inset upper right), butglomeruli showed little expression (Figure 8B, inset lowerright).

Identification of Tubular Segment Expressing HIF-2�For identifying specifically which tubule segment expressed

HIF-2�, co-localization studies with the tubule-specific mark-ers Lotus lectin (specific for proximal convoluted tubule),DBA (specific for collecting duct), and THP (specific for TALof Henle’s loop) were undertaken on sections of 6-d-old mousekidney. In serial sections that were merged digitally, co-local-ization with THP showed that HIF-2� protein was expressed indeveloping TAL of loop of Henle (Figure 9, A through C).Expression was restricted to TAL as no co-localization ofHIF-2� was apparent in the same section that dually labeledwith proximal tubular marker Lotus lectin (Figure 9, D throughF) or collecting duct–specific DBA (Figure 9, G through I). For

controls, slides were incubated with nonspecific IgG and bothfluorescein- and rhodamine-conjugated secondary antibodies(Figure 9J).

Identification of Hypoxic Nephron SegmentsTo identify nephron segments that contained hypoxic cells,

we doubly labeled sections with Hypoxoyprobe-1 and tubule-specific lectins. Nearly all proximal tubules (Lotus lectin pos-itive) in 6-d-old kidney were positive for Hypoxyprobe-1 la-beling as well (Figure 10). Because HIF-2� expression wasexcluded from proximal tubules (Figure 9F), it is thereby alsoexcluded from the most severely hypoxic cells in developingkidney. In contrast to proximal tubules, medullary tissues of6-d-old kidney were not labeled with Hypoxyprobe-1 (or Lotuslectin; Figure 11) and therefore were not severely hypoxic atthis time.

Figure 4. Sections of E12 metanephroi maintained for 5 d at room air(~20% oxygen; A and C) or 5% oxygen (B and D) labeled for HIF-1�(A and B) and HIF-2� (C and D). Hypoxia did not induce changes ineither HIF-1� or -2� abundance or distribution. Note that a nephronsegment that contained intense signal for HIF-2� (arrows) seemedsimilar to that observed in native, E14 kidney (Figure 3).

Figure 5. Expression of HIF-1� and -2� protein in 3-d-old (P3) mousekidney. (A) HIF-1� protein distribution was widespread at 3 d of age,and most cells showed strong nuclear staining. (B) HIF-2�, con-versely, remained tightly restricted at day 3. Developing nephrons (N)again showed especially strong nuclear labeling. Portions of a specificdeveloping tubule segment projecting toward the medulla (arrows)also displayed strong nuclear reaction product.


DiscussionDuring nephrogenesis, renal mesenchymal cells and ureteric

bud epithelium differentiate into numerous, distinct cell typesin a spatially precise sequence. Transcriptional regulation ofthis intricate process is poorly understood, but proteins thatbelong to the bHLH-PAS family of transcription factors arehighly expressed during kidney development, implicating themas candidate regulators of nephron formation (33,35,36). One �

subunit of these transcriptional complexes, HIF-1� (�RN�1),is expressed ubiquitously in newborn mouse kidney (35). Ahighly related � subunit, HIF-2� (ARNT2), is also expressed atvery high levels in developing kidney and brain (33,36), but theexpression patterns of HIF-2� had not yet been studied exten-sively. Our aims here were to define clearly the expressionpatterns of HIF-2� in kidney development and compare di-rectly its distribution with HIF-1�. We found that HIF-1�protein was expressed ubiquitously at every age examined,whereas HIF-2� expression was tightly restricted. We alsosought to determine with which HIF-� proteins HIF-2� het-erodimerized and found both HIF-1� and -2� present in com-plexes immunoprecipitated with anti–HIF-2� antibodies. De-spite that HIF-2� heterodimerized with both HIF-� subunits,HIF-2� protein was not found in the most extremely hypoxiccells, which we determined to be proximal tubule epithelium.By contrast, HIF-2� was expressed predominantly in distalsegments of developing nephrons and became restricted toTAL of Henle’s loop. In addition, we found that neither HIF-�subunit was prominently induced by hypoxia in metanephricorgan cultures but that two potential HIF target genes that areimportant for kidney development, WT-1 and Cox-2, wereupregulated in low oxygen tension.

Northern blot analysis and in situ hybridization experimentshave demonstrated intense mRNA expression of HIF-1� and-2� in developing kidney (36). These experiments, however,did not examine expression beyond P1.5 or specifically addressprotein expression. By quantitative Western blot, we showedabundant HIF-1� protein expression in kidneys at birth, 7 d,and 8 wk of age. Our data are consistent with the previous insitu hybridization experiments (36) in that our Western blotsshowed very intense expression of HIF-2� in newborn kidney.We extended the analysis to include maturing kidneys andshowed that HIF-2� protein expression declined significantly

Figure 6. HIF-1� and -2� protein distribution in 7-d-old (P7) mousekidney. (A) HIF-1� protein localization at day 7 again showed ex-tensive nuclear staining throughout the kidney, including glomeruli(G). (B) At day 7, HIF-2� distribution was restricted to a specifictubular segment. In addition, this was the first age point examined atwhich specific glomerular labeling was observed. The inset showspodocyte labeling for HIF-2� (P).

Figure 7. HIF-1� and -2� protein expression in 10-d-old mousekidney. (A) Similar to all other age points, HIF-1� protein distributionat day 10 was nearly ubiquitous. Glomeruli (G) again were consis-tently positive for expression. (B) HIF-2� expression at day 10showed relatively little protein in the outer cortex, and only a subsetof tubular segments showed labeling. In contrast to day 7, glomerulinow showed little or no staining for HIF-2�. (C) Certain medullarytubules of 10-d-old kidney showed intense labeling for HIF-2�.

Figure 8. Immunolocalization of HIF-1� and -2� in 8-wk-old mousekidney. (A) HIF-1� protein expression persisted in all tubular seg-ments and glomeruli (G), as seen throughout development. (B)HIF-2� protein expression in the cortex was limited, and few positivetubular cells could be found (arrows). Glomeruli (G of inset) werenegative. The medulla was the site of the most intensive HIF-2�labeling at this stage (inset top left).


at P7 and was barely detectable on Western blots at 8 wk ofage. The relative abundance of HIF-2� in developing kidneyand decline in maturation therefore suggest a role for thisprotein in renal organogenesis.

HIF-1� and -2� are capable of binding a number of bHLH-PAS domain proteins, including HIF-1�, HIF-2�, and AHR(30–32). Of particular interest to kidney development are theheterodimers consisting of HIF-1� and either HIF-� subunit,because many of the genes that are known to be induced bythese complexes are crucial for kidney formation, includingVEGF, VEGFR-1 and -2, WT-1, angiopoietin-2, Tie-2, andEpo. We showed previously, by immunoprecipitation andWestern blotting, that HIF-1� forms heterodimers with bothHIF-1� and -2� in developing kidney (35). Here we show thatHIF-2�, in the same way, complexes with either � subunit in3-d-old mouse kidney. Which genes are targeted by HIF-2�–

containing complexes has yet to be clearly defined. However,cultured neurons from HIF-2��/� mice show less induction ofVEGF in response to hypoxia than HIF-2��/� neurons, sug-gesting that VEGF may be a target gene for heterodimers thatcontain HIF-2� (34).

By applying immunoperoxidase techniques, we comparedHIF-1� and -2� expression patterns at various stages of kidneydevelopment. At E14, HIF-2� protein was expressed specifi-cally in the ureteric bud and at particularly high levels in distalsegments of developing nephrons. Visceral and parietal epithe-lia, which eventually form the podocytes and Bowman’s cap-sule, respectively, also showed positive staining, although atweaker levels. Mesenchymal cells of the outer cortex of E14kidneys were also weakly positive. At 3 d of age, the expres-sion pattern in the early nephric figures of the extreme outercortex persisted. In addition, a specific elongating tubularsegment showed prominent nuclear labeling. This segmentproved to be developing TAL of Henle’s loop, by co-distribu-tion immunofluorescence analysis with THP on serial sections(37). There was a dramatic change in the expression patternobserved at day 7, where, in addition to TAL, glomerularpodocytes were now positive for HIF-2. In mature, 8-wk-oldkidneys, expression was once again restricted to TAL, with noapparent glomerular labeling. In contrast to HIF-2�, strongnuclear HIF-1� labeling was observed at every stage examinedand in nearly every cell. These immunohistochemical expres-sion patterns are consistent with the quantitative Western blots.HIF-1� expression was widespread and abundant at all agesexamined, whereas HIF-2� was highly expressed in develop-ing kidney in many cell types but with age became restricted toTAL, and, quantitatively, lower levels were detected onimmunoblots.

We have used the hypoxia marker Hypoxyprobe-1 and im-munofluorescence microscopy to identify extremely hypoxiccells within newborn mouse kidney (35). Here, we show in6-d-old kidney that the vast majority of hypoxic cells are Lotuslectin positive and therefore correspond to proximal tubularepithelium. Surprising, even though HIF-2� co-immunopre-cipitated the hypoxia responsive proteins HIF-1� and -2�,HIF-2� did not immunolocalize to proximal tubules. Thissuggests that HIF-2� may not participate in the hypoxic re-sponse pathway in the most intensely hypoxic cells. However,Hypoxyprobe-1 is optimally reactive only in extremely hy-poxic cells (�1% O2) (38). We therefore cannot rule out thatmild hypoxia (2 to 18% O2) may occur in glomeruli or TALand that HIF-2� could mediate hypoxia-induced gene expres-sion in these cells.

Whether HIF-1� gene expression is increased in hypoxia ornot is unclear. In most systems analyzed, HIF-1� protein levelsremain constant regardless of oxygen tension, whereas in cer-tain cell types, such as human neuroblastoma cells, HIF-1�protein accumulates under hypoxic stress (25,39). We showhere in E12 metanephric organ cultures that oxygen tensionhad no effect on either HIF-1� or -2� protein levels or theirexpression patterns. This evidence suggests that gene transac-tivation by HIF in developing kidney may be totally reliant on� subunit stabilization.

Figure 9. Identification of HIF-2�–positive tubular segment: Doublelabeling with tubule-specific markers. (A through C) Tamm-Hors-fall’s protein (THP) is expressed intensely in the thick ascending limb(TAL) of Henle’s loop. Serial cryostat sections from 6-d-old kidneywere labeled for THP (A) and HIF-2� (B), and the resulting imageswere overlaid (C). THP and HIF-2� expression patterns overlap in themajority of cells, demonstrating that TAL is the primary site ofHIF-2� protein expression. (D through F) The Lotus lectin specifi-cally binds carbohydrates found on proximal tubule epithelial mem-branes. Cryostat sections from 3-d-old mouse kidney double labeledfor fluorescein-conjugated Lotus lectin (D) and HIF-2� showed nooverlapping fluorescence, demonstrating that HIF-2� is not expressedby proximal tubules. (G through I) Dolichos Biflorus agglutinin(DBA) binds specifically to collecting duct epithelium. Double label-ing of DBA (G) and HIF-2� (H) on the same 3-d-old kidney sectionrevealed no co-localization (I), demonstrating that collecting ductepithelial cells are not sites of HIF-2� expression. (J) Controls withnonspecific IgG treated sequentially with fluorescein- and rhodamine-conjugated secondary antibodies were negative for each set ofexperiments.


As described above, the VEGF gene is a potential target forHIF-2�–containing heterodimers. What other genes relevant tokidney development might be induced by HIF-2� transcriptionfactors? A recent study identified a HRE in the WT-1 genepromoter and shown that HIF-1 activates transcription of thisgene in hypoxia (23). This study showed by DNA-bindingelectrophoretic mobility shift assays that the transcription fac-tor that induced the HRE in the WT-1 promoter containedHIF-1�, but the � subunit was not identified. In early kidney

Figure 10. Identification of hypoxic cells in vivo. Using the hypoxiamarker Hypoxyprobe-1, we identified severely hypoxic cells in 6-d-old mouse kidney. By double labeling with the Hypoxyprobe-1 (A)and Lotus lectin, a proximal convoluted tubule marker (B), we sawthat proximal tubules were the only extremely hypoxic cells in 6-d-old

Figure 11. Section of medulla from 6-d-old mouse kidney showingonly low levels of nonspecific background labeling for Hy-poxyprobe-1 (A) or Lotus lectin (B). Images are merged in C.

kidney (merged image C). Some proximal tubules were not positivefor Hypoxyprobe-1 (arrow). Glomeruli did not show Hypoxyprobe-1–specific fluorescence. (D) Control, saline-injected mice that weretreated with Hypoxyprobe-1 antibodies showed no fluorescence.


development, WT-1 is expressed in renal mesenchyme anddeveloping nephrons and then becomes localized specificallyto podocytes (40). Our studies presented here show thatHIF-2� was expressed in renal mesenchymal cells and earlynephrons as well as in podocytes at day 7. We thereforesuggest that the HIF complex driving WT-1 expression mayinclude either HIF-1� or -2�. Cox-2 displays an expressionpattern remarkably similar to that of HIF-2� in mouse kidney.Cox-2 is expressed exclusively in macula densa cells and TAL(41). Our studies also demonstrated that Cox-2 expression wasinduced in hypoxic kidney organ cultures, suggesting thatCox-2 might be an additional target of HIF-2�, but this spec-ulation demands further inquiry.

Studies of neuronal cell lines overexpressing HIF-2� withSIM-1, which is highly expressed in developing kidney, maygive more insight into which genes HIF-2� specifically targets(42,43). These earlier reports showed that Neuro-2a cells over-expressing HIF-2� showed strong induction of laminin �2,which is a component of the laminin-2 trimer (�2�1�1). Thislaminin isoform is expressed at low levels by Henle’s loop,distal tubule, Bowman’s capsule, and collecting duct epithe-lium (44). We observed HIF-2� expression in primitivenephrons in cells that eventually form distal tubule, Bowman’scapsule (parietal epithelium), and collecting duct (ureteric bud)as well as Henle’s loop. Perhaps HIF containing HIF-2� isdriving laminin �2 expression in these cells. Another potentialHIF-2� target identified in Neuro-2a cells is the janus kinase 2,which is partially responsible for expression of cytokine-stim-ulated inducible nitric oxide synthase in kidney epithelial cells(45). Again, these putative HIF-2� targets in developing kid-ney need further experimental confirmation.

We have hypothesized previously that a delicate balance ofHIF protein expression and stabilization occurs in various celltypes during nephrogenesis so that different HIF heterodimerspredominate in certain cell types and lead to selective tran-scription of different HIF target genes (46). The current studysupports this hypothesis by showing differential expressionpatterns for HIF-1� and -2� during kidney development:HIF-1� distribution was widespread, but HIF-2� was mostabundant initially in distal nephric segments and then becamerestricted to TAL. The cellular availability of � subunits andcompetition for binding HIF-� may determine which HIFtarget genes will be expressed and therefore has a vital role indetermining cellular phenotype. The events (e.g., microenvi-ronmental oxygen tension) that prompt formation of a partic-ular HIF heterodimer and the precise temporal and spatialoccurrence of these events need to be investigated thoroughlyin developing kidney to gain a better understanding of theHIF-mediated transcriptional regulation.

AcknowledgmentsThis study was funded by National Institutes of Health Grants

DK052483 and DK065123.We thank Kathryn Isom, Eileen Roach, and Pat St. John for

technical help.

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divergent expression patterns for hypoxia-inducible factor- 1 and

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