J Am Soc Nephrol 15: 2569–2578, 2004 Divergent Expression Patterns for Hypoxia-Inducible Factor- 1 and Aryl Hydrocarbon Receptor Nuclear Transporter-2 in Developing Kidney
PAUL B. FREEBURG and DALE R. ABRAHAMSON Department of Anatomy and Cell Biology, University of Kansas Medical Center, Kansas City, Kansas
Abstract. The hypoxia-inducible factors (HIF) are ␣/ het- levels remained relatively constant. By immunohistochemical erodimeric transcription factors of the basic helix-loop-helix- analysis, widespread expression of HIF-1 was observed in Per-Arnt-Sim (bHLH-PAS) superfamily and are chiefly re- developing and mature kidneys. ARNT2/HIF-2 protein dis- sponsible for cellular adaptation to oxygen deprivation. HIF tribution was restricted to distal segments of developing function relies on the stabilization of the ␣ subunit. When nephrons and in mature kidney was confined specifically to oxygen tension falls, HIF-␣ subunits translocate to the nucleus thick ascending limb of Henle’s loop. The data presented here and, upon dimerization with HIF-, activate transcription of suggest that ARNT2/HIF-2 is required at high levels during target genes, including vascular endothelial growth factor, vas- nephrogenesis in distal tubules and later exclusively in thick cular endothelial growth factor receptor-1 and -2, and WT-1, ascending limb. Furthermore, Hypoxyprobe-1 and lotus lectin which are vital for kidney development. HIF- subunits are co-localization studies showed that developing proximal con- stable regardless of oxygen concentration and constitutively voluted tubules were the most severely hypoxic nephron seg- translocate to the nucleus. It was shown previously that HIF-1 ment in immature kidney. Because HIF-2 protein was not protein expression is nearly ubiquitous in newborn kidney and abundantly expressed in this segment, it may not be engaged in that HIF-1 dimerizes with either HIF-1␣ or -2␣. Here it is mediating responses to severe hypoxia. The differential distri- shown that aryl hydrocarbon receptor nuclear transporter-2 bution patterns for HIF-1 and -2 suggest divergent roles (ARNT2/HIF-2) also heterodimerized with HIF-1␣ and -2␣. during kidney development for these highly related bHLH- ARNT2/HIF-2 protein was highly expressed in newborn kid- PAS proteins. ney but decreased significantly with age, whereas HIF-1
The basic helix-loop-helix-Per-Arnt-Sim (bHLH-PAS) family the bHLH-PAS protein aryl hydrocarbon receptor (AHR) and of transcription factors functions as ␣/ heterodimeric DNA binds to the xenobiotic response element located in the en- binding complexes and regulates a remarkably diverse number hancer regions of the cytochrome P450 gene isoforms Cyp1a1, of biologic events. These include responses to oxygen depri- Cyp1a2, and Cyp1b1 (7). Expression of these proteins purges vation and exposure to toxins, control of circadian rhythms, the system of the toxin by way of their metabolic activity and regulation of hormone signaling (1). The bHLH-PAS toward benzo[a]pyrene. Ordinarily, AHR is held quiescent in heterodimers typically consist of one broadly expressed, the cytoplasm by binding heat shock protein 90 and AHR readily available member ( subunit) that heterodimerizes with interaction factor. AHR is released by this complex, combines a partner (␣ subunit) that has a tightly restricted expression with ARNT, and induces gene expression upon binding of the profile and is sensitive to environmental cues. Perhaps the most toxin (8). AHR therefore acts as the ␣ subunit of bHLH-PAS widely expressed and best understood protein in this family is heterodimer because its function is reliant on an environmental the aryl hydrocarbon receptor nuclear transporter (ARNT), insult, which in this case is the cellular toxin benzo[a]pyrene.  which acts as the subunit for transcriptional responses to both Another clearly defined role for ARNT as a  subunit of toxin exposure and hypoxic stimuli (2–6). To aid in metabo- bHLH-PAS heterodimers is in the hypoxia responsive path- lism of the toxin benzo[a]pyrene, ARNT heterodimerizes with 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- Received April 14, 2004. Accepted July 15, 2004. erodimeric transcription factor that stimulates Epo expression Correspondence to Dr. Dale R. Abrahamson, Department of Anatomy and Cell in response to low oxygen tension (9). This factor was purified Biology, University of Kansas Medical Center, MS 3038, 3901 Rainbow and found to contain ARNT (HIF-1) and a new member of Boulevard, Kansas City, KS 66160. Phone: 913-588-7000; Fax: 913-588- ␣ 2710; E-mail: [email protected] the bHLH-PAS family, named HIF-1 . Since the discovery of  ␣ 1046-6673/1519-2569 the HIF-1 /HIF-1 heterodimer (HIF-1), the biochemical be- Journal of the American Society of Nephrology havior of these proteins in response to oxygen deprivation has Copyright © 2004 by the American Society of Nephrology been studied extensively. Most evidence indicates that HIF-1 DOI: 10.1097/01.ASN.0000141464.02967.29 protein expression occurs in all cells and that the protein is 2570 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2569–2578, 2004 stable and is found abundantly in the nucleus regardless of whereas HIF-2 mRNA is expressed most prominently in the oxygen tension (10). HIF-1␣, conversely, is regulated directly brain and kidney at these stages (33). The in situ hybridizations by cellular oxygen availability (11). When intracellular oxygen in this earlier study demonstrated that HIF-2 mRNA is ex- tension is sufficient to satisfy metabolic demands (normoxia), pressed strongly in the outer cortex of developing kidney, HIF-1␣ protein undergoes an oxygen-dependent hydroxylation although cellular identification is not possible because of the on a highly conserved proline residue (12,13). This hy- low-magnification fields presented. Unlike HIF-1 mutants, droxyproline residue is vital, because it renders HIF-1␣ recog- HIF-2 knockout mice survive until birth and, with the excep- nizable by the von Hippel-Lindau ubiquitin ligase complex. tion of stunted hypothalamus formation, seem to develop nor- Hydroxylated HIF-1␣ becomes polyubiquinated and rapidly mally (34). Cultured neurons from HIF-2Ϫ/Ϫ mice show less destroyed by proteasomes. However, when oxygen tension induction of the hypoxia-inducible genes VEGF, Glut3, and falls below critical levels (hypoxia), HIF-1␣ escapes proline Pgk compared with HIF-2ϩ/ϩ neurons, and mobility shift hydroxylation, is not recognized by von Hippel-Lindau, and DNA-binding assays of nuclear extracts from hypoxic cultures therefore avoids proteasomal degradation. Stabilized HIF-1␣ of wild-type neurons show both HIF-1 and -2 binding DNA then translocates to the nucleus, heterodimerizes with the as a complex with HIF-1␣ (34). These observations therefore readily available HIF-1, and binds the hypoxia-responsive suggest that HIF-2 is capable of participating in hypoxic element (HRE) located in the promoter/enhancer of HIF target responses. genes. HIF-2␣, initially called endothelial PAS domain pro- Crosses between HIF-1ϩ/Ϫ and HIF-2ϩ/Ϫ mutants re- tein, has very similar structural and biochemical properties to vealed that progeny lacking any combination of at least two HIF-1␣ and also heterodimerizes with HIF-1 (14–16). wild-type alleles of either  subunit died by E8.5 (34). These Among the genes induced by HIF-1 and -2 are those highly experiments indicate that HIF-1 and HIF-2 have overlap- involved in kidney development, including vascular endothe- ping roles in early development and may compensate for loss lial growth factor (VEGF), VEGF receptor-1 (VEGFR-1) and of one another. After E8.5, however, HIF- subunit functions -2, angiopoietin-2, Tie-2, Epo, and WT-1 (17–23). begin to diverge, and each protein has a unique, indispensable Although much has been learned about the ␣ subunits of role in embryonic development, which has yet to be clearly bHLH-PAS heterodimers (AHR and HIF-1␣ and -2␣), rela- defined. This is consistent with the restricted expression pat- tively little is known about the HIF- counterparts. However, tern seen for HIF-2 during mouse development when com- recent studies have suggested that  bHLH-PAS proteins are pared with the ubiquitous expression pattern for HIF-1 (33). more complex than initially thought. For example, five differ- We previously immunolocalized HIF-1 protein expression in ent splice variants of HIF-1 have been identified in the rat newborn mouse kidney and found that every cell type seemed (24), and additional isoforms have been shown to contain to contain nuclear expression at that age (35). Although the deletions at exon 5, 6, or 11 or an insertion at exon 20. Other developing kidney is one of two sites of intense HIF-2 ex- HIF-1 mRNA isoforms contain a variable number of codons pression, this protein has not been studied in detail during in exon 16 encoding glutamine. Expression profiles and func- metanephrogenesis. Here, we examined HIF-2 protein ex- tions of each of the alternately spliced variants have yet to be pression beginning at the earliest stages of nephron develop- described. In addition, in some cell lines, HIF-1 protein is ment and continuing into maturity and compared expression apparently sensitive to oxygen concentration and accumulates patterns with those of HIF-1. Our findings show that HIF-2 in hypoxia, as observed in a human neuroblastoma cell culture is selectively expressed in distal segments of developing system (25). nephrons and becomes restricted to thick ascending limb HIF-1␣ and -2␣ both are required for proper development, (TAL) of loop of Henle. We also show that, like HIF-1, as demonstrated in mice with targeted deletions of these genes HIF-2 heterodimerizes with both HIF-1␣ and -2␣, and that (14,26,27). HIF-1␣ null embryos die at embryonic day 9.5 HIF-2 protein expression in organ-cultured metanephroi is (E9.5) from cardiovascular and neural tube defects, as well as not regulated by oxygen levels. widespread mesenchymal cell death. HIF-2␣ knockout mice on the C57Bl/6 or 129/Sv genetic background die at E13.5 to Materials and Methods E15.5 from bradycardia and lack of catecholamine synthesis. Western Blots and Immunoprecipitations Overall vascular development in these mice, at least at the Wild-type CD-1 mouse kidneys were dissected at 1 d, 7 d, and 6 wk gross anatomic level, seems normal (14). HIF-2␣ mutants on of age and disrupted in a Dounce homogenizer in RIPA buffer the ICR/129Sv background, however, show severe vascular supplemented with a protease inhibitor cocktail as described previ- defects and die by E13.5 (15). ously (35). After lysates were cleared by centrifugation at 4°C for 25 ϫ HIF-1 null embryos die at E11 from abnormal hematopoi- min at 15,000 g, total protein concentrations were determined by etic and vascular development (28). A second -class bHLH- the colorimetric Bio-Rad DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein (50 to 100 g) were sepa- PAS protein, ARNT2 (HIF-2), has been identified and found   rated by 5 to 15% SDS-PAGE and transferred to nitrocellulose. to share 63% homology with HIF-1 (29,30). HIF-2 has Standard immunoblot techniques were then applied with the following subsequently been shown to form functional heterodimers with antibodies: polyclonal HIF-1 (Novus-Biologicals, Littleton, CO; AHR, HIF-1␣, and HIF-2␣ (30–32). In situ hybridization 1:100) and polyclonal ARNT2 (HIF-2; sc-5581; Santa Cruz Bio- studies in developing mouse show that HIF-1 is nearly ubiq- technology, Santa Cruz, CA; 1:50). The ECL reagent and ECL Hy- uitously expressed at E11 through postnatal day 1.5 (P1.5), perfilm (Amersham Pharmacia Biotech, Piscataway, NJ) were used to J Am Soc Nephrol 15: 2569–2578, 2004 HIF-1 and ARNT2 (HIF-2) in Developing Kidney 2571 visualize the bands. For loading controls, membranes were also incu- as described above on the first serial section, and goat anti-THP bated with mouse monoclonal anti–smooth muscle actin antibody antibody (Cappel Laboratories, Durham, NC) was applied to the (1:100 dilution; Sigma-Aldrich, St. Louis, MO). second serial section for 30 min at 1:200. Rabbit anti-goat fluorescein For immunoprecipitation experiments, lysates from 1-d-old kid- was added as a secondary antibody to the anti-THP–labeled slides, neys were additionally cleared by incubation with protein A agarose which were also mounted with Prolong. Irrelevant IgG was incubated (1 l/10 g total protein) for 15 min and then centrifuged again at with sections and treated sequentially with fluorescein and rhodam- 15,000 ϫ g for 15 min. The clarified lysates then underwent immu- ine-conjugated secondary antibodies for controls. noprecipitation with HIF-2 antibodies, using procedures described For identifying hypoxic tissues, pimonidazole hydrochloride (Hy- previously (35). The immunoprecipitated proteins were analyzed on poxyprobe-1; Chemicon) was injected intraperitoneally into 6-d-old Western blots by standard protocols with monoclonal anti–HIF-1␣ mice (200 mg/kg), and fixation and labeling were carried out as before and -2␣ antibodies used at a dilution of 1:100. Purified rabbit IgG (35). In addition to the Hypoxyprobe-1 monoclonal antibody, sections (Sigma-Aldrich) was also used as a nonspecific control to demonstrate were incubated with Lotus lectin-fluorescein (1:200) for 30 min at the specificity of the ARNT2 antibodies, and no bands were detected room temperature. Sections from saline-injected mice were also im- on Western blots probed with anti–HIF-1␣ or -2␣. munolabeled for Hypoxyprobe-1 and served as controls.
Embryonic Kidney Organ Culture and Western Blots Results Timed-pregnant CD-1 mice were killed and embryos were re- HIF- Protein Expression moved at E12. Metanephroi were dissected and cultured on PET Total mouse kidney protein was analyzed for HIF-1 and membrane cell culture inserts (0.4-m pore; BD Biosciences, San -2 by quantitative Western blots. Three stages of develop- Jose, CA). Organ culture media and growth conditions were as de- ment were examined: 1 d, 7 d, and 8 wk of age (Figure 1A). scribed previously (35). Culture oxygen concentrations were set at HIF-1 protein was readily detectable on blots at each age, either constant room air (~20% oxygen) or 5% oxygen for5dorat with a slight increase in expression at day 7. HIF-2 was 20% oxygen for 4 d and then reduced to 2% for the final 24 h. After culture periods, kidneys were homogenized in RIPA buffer, protein concentrations were determined, and Western blots were performed as described above. In addition to HIF-1 and -2, WT-1 (Santa Cruz Biotechnology; 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 Immunohistochemistry For immunohistochemical analysis, kidneys were dissected and frozen in OCT in isopentane in a dry-ice/acetone bath. Cryostat sections were cut at a thickness of 6 m and air-dried. Sections were fixed for 10 min in ice-cold methanol, washed in PBS, and placed in
3% H2O2 in methanol for 10 min. Blocking of nonspecific protein interactions was achieved by incubating sections with 10% goat serum in PBS. Slides were then incubated in primary antibodies: Polyclonal anti–HIF-1 and HIF-2 diluted in PBS (1:100 and 1:50, respec- tively) for1hatroom temperature in a humidified slide chamber. Sections were then washed with PBS, incubated sequentially with biotinylated secondary antibodies (15 g/ml) and streptavidin-HRP conjugates for 30 min each, and color-developed with 3,3'-diamino- benzidine tetrahydrochloride. Rabbit IgG diluted in PBS at the same concentration as the primary antibodies served as a negative control.
Immunofluorescence Analysis Figure 1. (A) Total renal hypoxia-inducible factor-2 (HIF-2) pro- Sections (6 m thick) of 3-d-old mouse kidney were fixed in 100% tein expression decreases with age. Quantitative Western blots for methanol on ice and incubated with polyclonal anti–HIF-2 (1:50) HIF-1 and -2 with kidney lysates from newborn (Nb), 7-d-old, and and either fluorescein-conjugated Lotus lectin, to label proximal con- 8-wk-old mouse kidneys revealed sustained expression of HIF-1 voluted tubule (Sigma Chemical Co., St. Louis, MO; 1:200), or throughout kidney maturation, with a peak at7dofage. By contrast, rhodamine-conjugated Dolichos Biflorus agglutinin (DBA), to label HIF-2 expression was most intense in newborn kidney, was signif- collecting duct (Vector Laboratories, Burlingame, CA; 1:10), for 30 icantly decreased by day 7, and was barely detectable at 8 wk. min at room temperature. After three washes in PBS, Alexa Fluor Anti–smooth muscle actin staining of blots serves as loading controls. 488–conjugated anti-rabbit antibody (Molecular Probes, Eugene, OR) (B) HIF-2 heterodimerizes with both HIF-1␣ and -2␣. Proteins was applied for 30 min at 1:200. Sections were again washed in PBS immunoprecipitated by HIF-2 IgG from 3-d-old mouse kidney were and permanently mounted with the fluorescence preserving reagent analyzed by Western blot with HIF-1␣– and -2␣–specific antibodies. Prolong (Molecular Probes). Serial sections of 6-d-old mouse kidney Both ␣ subunits were evident by Western blot, demonstrating that were fixed in the same way for co-localization of HIF-2 and Tamm- HIF-2 forms a complex with both ␣ subunits. NS, nonspecific bands Horsfall protein (THP). HIF-2 immunofluorescence was performed detected in both cases by secondary antibody. 2572 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2569–2578, 2004 strongly expressed in newborn kidney and decreased signifi- cantly at7dofage. In 8-wk-old kidney, HIF-2 protein was barely detectable by Western blot. To determine whether HIF-2 heterodimerizes with either HIF-1␣ or HIF-2␣, we conducted immunoprecipitation and Western blot experiments of newborn kidney lysates. Both HIF-1␣ and -2␣ proteins were observed on Western blots of the HIF- immune complexes, revealing that HIF-1 formed heterodimers with both HIF-1␣ and -2␣ in developing kidney (Figure 1B).
Effect of Hypoxia on HIF- Protein Expression in Kidney Organ Culture E12 kidney explants were cultured for 4 d at 20% oxygen followed by a 24-h exposure to 2% oxygen (Figure 2, left) or for5dat5%oxygen (Figure 2, right), followed by Western blots for HIF1- and -2. In cultures that were exposed to acute 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 in organ culture, but WT-1 increased only modestly at 5% oxygen (Figure 2).
HIF-1 and -2 Immunolocalization HIF-1 protein was found to be widely distributed in nuclei of apparently all cells in both developing and maturing kidney Figure 3. Immunolocalization of HIF-1 and -2 in E14 kidney. (A) at every stage examined. In E14 kidney, HIF-1 was expressed HIF-1 protein expression at E14 was nearly ubiquitous and uni- ubiquitously by mesenchymal cells, ureteric bud epithelium, formly intense. Strong nuclear staining was apparent in ureteric bud  and all cells in developing nephrons (Figure 3A). During later (UB), developing nephrons (N), and mesenchymal cells. (B) HIF-2 stages of kidney development and into maturity, widespread protein expression was much more restricted and variable at E14 compared with HIF-1. Faint nuclear labeling was found in ureteric HIF-1 protein expression persisted; nuclei within all tubular bud, uninduced mesenchyme in extreme outer cortex, but was partic- ularly prominent in developing nephrons. (C) Higher-power views show that HIF-2 protein was expressed strongly and specifically in the distal segment of developing nephrons (*), including cells that may form the macula densa. Weaker expression was also evident in the visceral (VE) and parietal epithelium (PE), which are destined to form the podocytes (Po) and Bowman’s capsule (BC), respectively (arrows). (D) Nonspecific rabbit IgG incubated with sections in place of primary antibodies served as controls, and no labeling was observed.
segments, interstitial cells, and cells within glomeruli all strongly labeled with anti–HIF-1 antibody (see A in Figures 4 through 7). Sections that were labeled with nonspecific rabbit IgG in place of HIF-1 antibodies were negative in all cases (Figure 3D). In contrast to HIF-1, the distribution of HIF-2 protein was Figure 2. HIF- and HIF target gene expression in embryonic day 12 highly restricted among the time points examined. At E14, (E12) organ cultures that were exposed to hypoxia. E12 mouse met- HIF-2 was seen in nuclei of ureteric bud epithelium and less anephroi were dissected and cultured for4din20%oxygen and then intensely in mesenchymal cells of the outer cortex (Figure 3B). shifted to a hypoxic environment (2% oxygen) for 1 d (left) or were In early nephrons, both the visceral and parietal epithelium cultured for5din5%oxygen (right). In both cases, HIF-1 and -2 were weakly positive, but developing distal segments showed protein levels remained constant. WT-1 and cyclo-oxengenase-2  (Cox-2), two potential HIF target genes during kidney development, particularly intense HIF-2 labeling (Figure 3, B and C).   however, showed increased protein levels in response to hypoxia in As an additional test for whether HIF-1 and/or -2 changes both cases, although upregulation of WT-1 at 5% oxygen seemed in abundance or expression pattern, organ cultures of E12 more modest. All blots are representative of two independent kidneys maintained for5dat20or5%oxygen were examined. experiments. As shown in Figure 4, no differences were observed. J Am Soc Nephrol 15: 2569–2578, 2004 HIF-1 and ARNT2 (HIF-2) in Developing Kidney 2573
Figure 4. Sections of E12 metanephroi maintained for5datroom 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 in either HIF-1 or -2 abundance or distribution. Note that a nephron segment that contained intense signal for HIF-2 (arrows) seemed similar to that observed in native, E14 kidney (Figure 3).
In 3-d-old kidney, developing nephrons of the extreme outer cortex showed the same overall expression pattern observed in E14 kidney with especially strong localization to distal seg- ments (Figure 5B). In addition, tubules projecting through the subcortical region also showed strong nuclear labeling at this age. The expression profile in 5-d-old kidney was indistin- guishable from that in 3-d-old kidney (data not shown). In    Figure 5. Expression of HIF-1 and -2 protein in 3-d-old (P3) mouse 1-wk-old kidney, HIF-2 protein distribution was restricted to kidney. (A) HIF-1 protein distribution was widespread at3dofage, certain tubular segments (Figure 6B), and podocyte nuclei of and most cells showed strong nuclear staining. (B) HIF-2, con- maturing stage glomeruli were also labeled (Figure 6B, inset). versely, remained tightly restricted at day 3. Developing nephrons (N) By 10 d of age, restricted tubular expression persisted in the again showed especially strong nuclear labeling. Portions of a specific cortex, but glomerular expression was now absent (Figure 7B). developing tubule segment projecting toward the medulla (arrows) The medulla of 10-d-old kidney showed intense HIF-2 ex- also displayed strong nuclear reaction product. pression (Figure 7C). Finally, in fully mature, 8-wk-old kidney, HIF-2 expression was diminished overall and found only sparsely in the cortex (Figure 8B). Medullary expression of controls, slides were incubated with nonspecific IgG and both HIF-2 was prominent (Figure 8B, inset upper right), but glomeruli showed little expression (Figure 8B, inset lower fluorescein- and rhodamine-conjugated secondary antibodies right). (Figure 9J).
Identification of Tubular Segment Expressing HIF-2 For identifying specifically which tubule segment expressed Identification of Hypoxic Nephron Segments HIF-2, co-localization studies with the tubule-specific mark- To identify nephron segments that contained hypoxic cells, ers Lotus lectin (specific for proximal convoluted tubule), we doubly labeled sections with Hypoxoyprobe-1 and tubule- DBA (specific for collecting duct), and THP (specific for TAL specific lectins. Nearly all proximal tubules (Lotus lectin pos- of Henle’s loop) were undertaken on sections of 6-d-old mouse itive) in 6-d-old kidney were positive for Hypoxyprobe-1 la- kidney. In serial sections that were merged digitally, co-local- beling as well (Figure 10). Because HIF-2 expression was ization with THP showed that HIF-2 protein was expressed in excluded from proximal tubules (Figure 9F), it is thereby also developing TAL of loop of Henle (Figure 9, A through C). excluded from the most severely hypoxic cells in developing Expression was restricted to TAL as no co-localization of kidney. In contrast to proximal tubules, medullary tissues of HIF-2 was apparent in the same section that dually labeled 6-d-old kidney were not labeled with Hypoxyprobe-1 (or Lotus with proximal tubular marker Lotus lectin (Figure 9, D through lectin; Figure 11) and therefore were not severely hypoxic at F) or collecting duct–specific DBA (Figure 9, G through I). For this time. 2574 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2569–2578, 2004
Figure 6. HIF-1 and -2 protein distribution in 7-d-old (P7) mouse Figure 8. Immunolocalization of HIF-1 and -2 in 8-wk-old mouse kidney. (A) HIF-1 protein localization at day 7 again showed ex- kidney. (A) HIF-1 protein expression persisted in all tubular seg- tensive nuclear staining throughout the kidney, including glomeruli ments and glomeruli (G), as seen throughout development. (B) (G). (B) At day 7, HIF-2 distribution was restricted to a specific HIF-2 protein expression in the cortex was limited, and few positive tubular segment. In addition, this was the first age point examined at tubular cells could be found (arrows). Glomeruli (G of inset) were which specific glomerular labeling was observed. The inset shows negative. The medulla was the site of the most intensive HIF-2 podocyte labeling for HIF-2 (P). labeling at this stage (inset top left).
subunit of these transcriptional complexes, HIF-1 (〈RN⌻1), is expressed ubiquitously in newborn mouse kidney (35). A highly related  subunit, HIF-2 (ARNT2), is also expressed at very high levels in developing kidney and brain (33,36), but the expression patterns of HIF-2 had not yet been studied exten- sively. Our aims here were to define clearly the expression patterns 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 also sought 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 hypoxic cells, which we determined to be proximal tubule epithelium. By contrast, HIF-2 was expressed predominantly in distal Figure 7. HIF-1 and -2 protein expression in 10-d-old mouse segments of developing nephrons and became restricted to  kidney. (A) Similar to all other age points, HIF-1 protein distribution TAL of Henle’s loop. In addition, we found that neither HIF- at day 10 was nearly ubiquitous. Glomeruli (G) again were consis- subunit was prominently induced by hypoxia in metanephric tently positive for expression. (B) HIF-2 expression at day 10 organ cultures but that two potential HIF target genes that are showed relatively little protein in the outer cortex, and only a subset of tubular segments showed labeling. In contrast to day 7, glomeruli important for kidney development, WT-1 and Cox-2, were now showed little or no staining for HIF-2. (C) Certain medullary upregulated in low oxygen tension. tubules of 10-d-old kidney showed intense labeling for HIF-2. Northern blot analysis and in situ hybridization experiments have demonstrated intense mRNA expression of HIF-1 and -2 in developing kidney (36). These experiments, however, Discussion did not examine expression beyond P1.5 or specifically address During nephrogenesis, renal mesenchymal cells and ureteric protein expression. By quantitative Western blot, we showed bud epithelium differentiate into numerous, distinct cell types abundant HIF-1 protein expression in kidneys at birth, 7 d, in a spatially precise sequence. Transcriptional regulation of and 8 wk of age. Our data are consistent with the previous in this intricate process is poorly understood, but proteins that situ hybridization experiments (36) in that our Western blots belong to the bHLH-PAS family of transcription factors are showed very intense expression of HIF-2 in newborn kidney. highly expressed during kidney development, implicating them We extended the analysis to include maturing kidneys and as candidate regulators of nephron formation (33,35,36). One  showed that HIF-2 protein expression declined significantly J Am Soc Nephrol 15: 2569–2578, 2004 HIF-1 and ARNT2 (HIF-2) in Developing Kidney 2575
containing complexes has yet to be clearly defined. However, cultured neurons from HIF-2Ϫ/Ϫ mice show less induction of VEGF in response to hypoxia than HIF-2ϩ/ϩ neurons, sug- gesting that VEGF may be a target gene for heterodimers that contain HIF-2 (34). By applying immunoperoxidase techniques, we compared HIF-1 and -2 expression patterns at various stages of kidney development. At E14, HIF-2 protein was expressed specifi- cally in the ureteric bud and at particularly high levels in distal segments of developing nephrons. Visceral and parietal epithe- lia, which eventually form the podocytes and Bowman’s cap- sule, respectively, also showed positive staining, although at weaker levels. Mesenchymal cells of the outer cortex of E14 kidneys were also weakly positive. At3dofage, the expres- sion pattern in the early nephric figures of the extreme outer cortex persisted. In addition, a specific elongating tubular segment showed prominent nuclear labeling. This segment proved 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 pattern observed at day 7, where, in addition to TAL, glomerular Figure 9. Identification of HIF-2–positive tubular segment: Double podocytes were now positive for HIF-2. In mature, 8-wk-old labeling with tubule-specific markers. (A through C) Tamm-Hors- kidneys, expression was once again restricted to TAL, with no fall’s protein (THP) is expressed intensely in the thick ascending limb apparent glomerular labeling. In contrast to HIF-2, strong (TAL) of Henle’s loop. Serial cryostat sections from 6-d-old kidney nuclear HIF-1 labeling was observed at every stage examined were labeled for THP (A) and HIF-2 (B), and the resulting images were overlaid (C). THP and HIF-2 expression patterns overlap in the and in nearly every cell. These immunohistochemical expres- majority of cells, demonstrating that TAL is the primary site of sion patterns are consistent with the quantitative Western blots. HIF-2 protein expression. (D through F) The Lotus lectin specifi- HIF-1 expression was widespread and abundant at all ages cally binds carbohydrates found on proximal tubule epithelial mem- examined, whereas HIF-2 was highly expressed in develop- branes. Cryostat sections from 3-d-old mouse kidney double labeled ing kidney in many cell types but with age became restricted to for fluorescein-conjugated Lotus lectin (D) and HIF-2 showed no TAL, and, quantitatively, lower levels were detected on overlapping fluorescence, demonstrating that HIF-2 is not expressed immunoblots. by proximal tubules. (G through I) Dolichos Biflorus agglutinin We have used the hypoxia marker Hypoxyprobe-1 and im- (DBA) binds specifically to collecting duct epithelium. Double label- munofluorescence microscopy to identify extremely hypoxic  ing of DBA (G) and HIF-2 (H) on the same 3-d-old kidney section cells within newborn mouse kidney (35). Here, we show in revealed no co-localization (I), demonstrating that collecting duct 6-d-old kidney that the vast majority of hypoxic cells are Lotus epithelial cells are not sites of HIF-2 expression. (J) Controls with nonspecific IgG treated sequentially with fluorescein- and rhodamine- lectin positive and therefore correspond to proximal tubular  conjugated secondary antibodies were negative for each set of epithelium. Surprising, even though HIF-2 co-immunopre- experiments. cipitated the hypoxia responsive proteins HIF-1␣ and -2␣, HIF-2 did not immunolocalize to proximal tubules. This suggests that HIF-2 may not participate in the hypoxic re- at P7 and was barely detectable on Western blots at 8 wk of sponse pathway in the most intensely hypoxic cells. However, age. The relative abundance of HIF-2 in developing kidney Hypoxyprobe-1 is optimally reactive only in extremely hy- Ͻ and decline in maturation therefore suggest a role for this poxic cells ( 1% O2) (38). We therefore cannot rule out that protein in renal organogenesis. mild hypoxia (2 to 18% O2) may occur in glomeruli or TAL HIF-1 and -2 are capable of binding a number of bHLH- and that HIF-2 could mediate hypoxia-induced gene expres- PAS domain proteins, including HIF-1␣, HIF-2␣, and AHR sion in these cells. (30–32). Of particular interest to kidney development are the Whether HIF-1 gene expression is increased in hypoxia or heterodimers consisting of HIF-1 and either HIF-␣ subunit, not is unclear. In most systems analyzed, HIF-1 protein levels because many of the genes that are known to be induced by remain constant regardless of oxygen tension, whereas in cer- these complexes are crucial for kidney formation, including tain cell types, such as human neuroblastoma cells, HIF-1 VEGF, VEGFR-1 and -2, WT-1, angiopoietin-2, Tie-2, and protein accumulates under hypoxic stress (25,39). We show Epo. We showed previously, by immunoprecipitation and here in E12 metanephric organ cultures that oxygen tension Western blotting, that HIF-1 forms heterodimers with both had no effect on either HIF-1 or -2 protein levels or their HIF-1␣ and -2␣ in developing kidney (35). Here we show that expression patterns. This evidence suggests that gene transac- HIF-2, in the same way, complexes with either ␣ subunit in tivation by HIF in developing kidney may be totally reliant on 3-d-old mouse kidney. Which genes are targeted by HIF-2– ␣ subunit stabilization. 2576 Journal of the American Society of Nephrology J Am Soc Nephrol 15: 2569–2578, 2004
Figure 11. Section of medulla from 6-d-old mouse kidney showing only low levels of nonspecific background labeling for Hy- poxyprobe-1 (A) or Lotus lectin (B). Images are merged in C.
As described above, the VEGF gene is a potential target for HIF-2–containing heterodimers. What other genes relevant to kidney development might be induced by HIF-2 transcription factors? A recent study identified a HRE in the WT-1 gene promoter and shown that HIF-1 activates transcription of this gene in hypoxia (23). This study showed by DNA-binding electrophoretic mobility shift assays that the transcription fac- tor that induced the HRE in the WT-1 promoter contained HIF-1␣, but the  subunit was not identified. In early kidney
Figure 10. Identification of hypoxic cells in vivo. Using the hypoxia marker Hypoxyprobe-1, we identified severely hypoxic cells in 6-d- kidney (merged image C). Some proximal tubules were not positive old mouse kidney. By double labeling with the Hypoxyprobe-1 (A) for Hypoxyprobe-1 (arrow). Glomeruli did not show Hypoxyprobe- and Lotus lectin, a proximal convoluted tubule marker (B), we saw 1–specific fluorescence. (D) Control, saline-injected mice that were that proximal tubules were the only extremely hypoxic cells in 6-d-old treated with Hypoxyprobe-1 antibodies showed no fluorescence. J Am Soc Nephrol 15: 2569–2578, 2004 HIF-1 and ARNT2 (HIF-2) in Developing Kidney 2577 development, WT-1 is expressed in renal mesenchyme and References developing nephrons and then becomes localized specifically 1. Crews ST: Control of cell lineage-specific development and to podocytes (40). Our studies presented here show that transcription by bHLH-PAS proteins. 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