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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 61:795-805 (2006)
© 2006 The Gerontological Society of America

Hypoxia and Expression of Hypoxia-Inducible Factor in the Aging Kidney

Tetsuhiro Tanaka, Hideki Kato, Ichiro Kojima, Takamoto Ohse, Daisuke Son, Takahisa Tawakami, Toshiya Yatagawa, Reiko Inagi, Toshiro Fujita and Masaomi Nangaku

Divisions of 1 Nephrology and Endocrinology, and 2 Pediatrics, University of Tokyo School of Medicine, Japan.

Address correspondence to Masaomi Nangaku, MD, PhD, Division of Nephrology and Endocrinology, University of Tokyo School of Medicine, 7-3-1, Hongo, Bunkyo-ku, Tokyo, Japan. E-mail: mnangaku-tky{at}umin.ac.jp


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Renal senescence is characterized by interstitial fibrosis and loss of peritubular capillaries. In this study, we provided evidence of tubulointerstitial hypoxia and the operation of hypoxia-inducible factor (HIF) in the aging kidney. Using two distinct methods, pimonidazole immunostaining and the expression of the "hypoxia-responsive" reporter of the transgenic rats, we identified the age-related expansion of hypoxia in all areas of the kidney. Expansion was most prominent in the cortex. Clusters of hypoxic tubules were observed in the superficial cortical zones, areas adjacent to the outer nephrons and expanded in the medullary rays. The degree of hypoxia was positively correlated with the age-related tubulointerstitial injury (R2 = 0.88, p <.01), which was associated with the upregulation of HIF-regulated genes, such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT1) (real-time polymerase chain reaction). These findings point to the involvement of hypoxia and highlight the pathological relevance of HIF and its target genes in the aging kidney.

AGING in the kidney has been linked to a number of physiological as well as structural derangements (1,2). Functionally, glomerular filtration rate (GFR) starts to decline at 30 years of age by approximately 8 mL/min per 1.73 m2 per decade (3,4). Renal blood flow (RBF) is also reduced, with redistribution of blood flow from the cortex to the medulla. As a result, renal cortex is likely to be exposed to an environment in which oxygen and nutrient supply is low. Structurally, various degrees of focal and segmental glomerular sclerosis develop with aging, as well as tubulointerstitial injury such as tubular basement membrane (TBM) thickening, tubular dilatation, cast formation, tubular atrophy, and interstitial fibrosis. In areas with severe tubulointerstitial injury, patchy loss of peritubular capillary networks has been documented (5).

These previous observations point to the potential involvement of hypoxia during the pathogenesis of age-related tubulointerstitial injury. In fact, tubulointerstitial hypoxia has been recognized not only as a hallmark of, but as a common mediator to, progressive glomerular diseases (6–8). It remains unclear, however, whether these scenarios apply in the process of physiological aging; still less is it certain whether the aging kidney is exposed to hypoxia, thus hampering the relevance of hypoxia to the age-related renal pathology.

At the cellular level, every intrinsic cell has an inborn mechanism to cope with hypoxia. On exposure to hypoxia, cells try to adapt to the environment by producing factors that promote for example, angiogenesis, erythropoiesis, and glycolysis. These adaptive mechanisms apply to all organs and tissues, including the kidney (9,10).

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that plays a central role in such cellular adaptation to hypoxia (11,12). It is composed of two basic helix–loop–helix proteins—HIF-{alpha} and HIF-ß—of the PAS (Per-Arnt-Sim) family. In hypoxia, the {alpha}/ß heterodimer binds to a core DNA motif in the hypoxia-responsive element (HRE) and transactivates its target genes, such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF). The transcriptional activity of HIF is regulated through post-transcriptional hydroxylation catalyzed by a set of oxygen-dependent enzymes that belong to the 2-oxoglutarate-dependent oxygenase superfamily (13). In the presence of molecular oxygen, the {alpha} subunit of HIF undergoes hydroxylation at specific prolyl residues in the oxygen-dependent degradation domain (ODD; prolyl hydroxylation) (14,15). Besides, the HIF transcriptional activity is modulated through asparaginyl hydroxylation. Oxygen promotes hydroxylation at the specific asparaginyl residue and blocks the binding of transcriptional coactivators such as p300 (16,17). Hence, the expression and the functional operation of HIF are under the tight regulation of hypoxia. On the basis of these previous observations, we postulated that the aging kidney might be exposed to hypoxia, investigated the activation of HIF, and characterized the expression of HIF-regulated genes in the aging kidney.


   MATERIALS AND METHODS
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Experimental Rats
Male, "hypoxia-sensing" transgenic rats of Wistar strain were used in this study (18). They are transgenic rats harboring a transgene composed of HRE (enhancer) and the FLAG-tagged luciferase reporter gene, allowing us to detect areas of hypoxia through HIF-mediated cellular hypoxic response. Rats of two age groups (young rats [YO; 4 months old, n = 8] and aging rats [AG; 20 months old, born in October 2003, n = 7]) were housed in a light- and temperature-controlled environment and were allowed free access to standard animal chow and water.

Study Protocol
One day prior to death, rats of both age groups were placed in metabolic cages to collect urine. Twenty-four hours later, they were anesthetized with ketamine (50 mg/kg i.p.), blood was taken via cardiac puncture, and the left kidneys were removed with midperitoneal incision. Removed kidneys were cut transversely and fixed with methyl Carnoy's or buffered formalin fixatives. A fraction of cortical tissues was set aside, snap-frozen in liquid nitrogen, and stored at –80°C for subsequent RNA and protein preparation. Proteinuria and serum creatinine levels were measured using a standard laboratory method (Bio-Rad, Hercules, CA and Wako, Osaka, Japan). One rat in the AG group was found later to have suffered from lung cancer with pleural invasion; the data on this rat were excluded from the study. All protocols conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the committee on ethical animal care at Tokyo University.

In another set of experiments, hypoxia in the aging kidney was corroborated with the use of pimonidazole, a 2-nitroimidazole (Hypoxyprobe-1 Kit; Chemicon, Temecula, CA). Both YO and AG rats received injections of pimonidazole at 60 mg/kg via tail vein, followed 2 hours later by removal of the kidneys for immunohistochemical detection of hypoxia.

Histological Evaluation
Paraffin-embedded sections (3 µm) were stained with periodic acid-Schiff, and tubulointerstitial injury was assessed semiquantitatively on the basis of morphological changes such as tubular dilatation, cast formation, sloughing of tubular epithelial cells, and thickening of the TBM, as follows: Grade 1, <10% of tubules involved; Grade 2, <25%; Grade 3, <50%; Grade 4, <75%; and Grade 5, ≥75%. Twenty consecutive fields in the cortex were examined at x400 magnification and averaged per slide. All quantification was made in a blinded manner.

Immunohistochemistry
Hypoxic tubular cells were identified using a modified indirect immunoperoxidase method, either on the basis of an increase in the hypoxia-responsive transgene expression or by the positive staining for pimonidazole. Methyl Carnoy's-fixed, paraffin-embedded sections were dewaxed and brought to water through graded ethanols. After quenching of endogenous peroxidase activity (0.3% H2O2 in methanol, 20 minutes), sections were probed with anti-FLAG (M2) antibody (1:350; Sigma, St. Louis, MO) or Hypoxyprobe-1Mb1 (1:200; Chemicon) followed by incubation with corresponding, biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA). Specific signals were detected with the tyramide signal amplification method (for FLAG; Perkin Elmer, Wellesley, MA) or the avidin-biotinylated peroxidase complex method (for Hypoxyprobe) method, followed by color development with H2O2 and diaminobenzidine.

For the immunodetection of HIF-1{alpha} and HIF-2{alpha}, specific antibodies were used as follows: antihuman HIF-1{alpha} antibody (1:100; Novus Biologicals, Littleton, CO), antimouse HIF-2{alpha} antibody (1:3000, PM9; a gift from Dr. Michael S. Wiesener, University of Erlangen-Nuremberg, Germany). Sections were autoclaved (121°C, 20 minutes) in 10 mM citrate buffer (pH 6.0) for antigen retrieval. To reduce run-to-run variations in the staining intensity, all staining of the same antibody was performed in the same run for all YO and AG rat kidneys, as previously described (19).

Quantification of the Transgene and HIF-Regulated Gene Messenger RNA
Messenger RNA (mRNA) expression of the transgene (HRE-Luc), HIF-{alpha} isoforms (HIF-1{alpha} and HIF-2{alpha}), and several of known HIF-regulated genes was quantified by real-time polymerase chain reaction (PCR). RNA was isolated from the renal cortex by using ISOGEN (Nippon Gene, Tokyo, Japan) and 1 µg of template was reverse-transcribed (ImProm-II Reverse Transcription System; Promega, Madison, WI). One-twentieth (vol/vol) of the complementary DNA product was used as a template for subsequent quantification. Using iQ SYBR Green Supermix (Bio-Rad), PCRs were performed on an iCycler (Bio-Rad) under the following conditions: initial denaturation of the template at 94°C for 15 minutes, 40 cycles of amplification at 94°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. Data collection and analysis were performed using iCycler iQ Optical System software (version 3.0a; Bio-Rad), and the amount of mRNA of interest was corrected for that of ß-actin. The sets of primers used are as follows: (i) Luciferase: forward (fw): 5'-CGTTTCCAAAAAGGGGTTGC-3', reverse (rv): 5'-GAAGGACTCTGGCACAAAATCG-3' (accession No. U47295 [pGL3-Basic], expected PCR product length: 178 bp); (ii) HIF-1{alpha}: fw: 5'-GTTTACTAAAGGACAAGTCACC-3', rv: 5'-TTCTGTTTGTTGAAGGGAG-3' (accession No. NM 024359, expected PCR product length: 193 bp); (iii) HIF-2{alpha}: fw: 5'-GTCACCAGAACTTGTGC-3', rv: 5'-CAAAGATGCTGTTCATGG-3' (accession No. NM 023090, expected PCR product length: 249 bp); (iv) EPO: fw: 5'-TACGTAGCCTCACTTCACTGCTT-3', rv: 5'-GCAGAAAGTATCCGCTGTGAGTGTTC-3' (accession No. NM 017001, expected PCR product length: 113 bp); (v) VEGF (which recognizes all isoforms [VEGF120, VEGF144, VEGF164, VEGF188]): fw: 5'-TTACTGCTGTACCTCCAC -3', rv: 5'-ACAGGACGGCTTGAAGATA-3' (accession No. NM 031836, expected PCR product length: 189 bp); (vi) GLUT1: fw: 5'-CAGTTCGGCTATAACACCGGTGTC-3', rv: 5'-ATAGCGGTGGTTCCATGTTT-3' (accession No. NM 138827, expected PCR product length: 84 bp); (vii) heme oxygenase-1 (HO-1): fw: 5'-TCTATCGTGCTCGCATGAAC-3', rv: 5'-CAGCTCCTCAAACAGCTCAA-3' (accession No. NM 012580, expected PCR product length: 110 bp); and (viii) ß-actin: fw: 5'-CTTTCTACAATGAGCTGCGTG-3', rv: 5'-TCATGAGGTAGTCTGTCAGG-3' (accession No. NM 031144, expected PCR product length: 306 bp).

Immunoblotting
The increase in the transgene, HIF-1{alpha}, VEGF, GLUT1, and HO-1 in the aging kidney was corroborated by western blotting. Whole cortical tissue was homogenized in 1% Nonidet P40 lysis buffer (150 mM sodium chloride, 1% Nonidet P40, 50 mM Tris–HCl, pH 8.0) containing leupeptin and pepstatin A (Sigma). Soluble lysates (50 µg) were loaded, resolved by 7.5% (HIF-1{alpha}), 10% (transgene and GLUT1), and 12% (VEGF and HO-1) sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions and transferred onto polyvinylidine difluoride (PVDF) membranes (Amersham, Piscataway, NJ). After blocking with 5% skim milk, membranes were probed with primary antibodies (above and antirat HO-1 antibody [1:1000; Stressgen, Victoria, BC, Canada]) followed by corresponding alkaline phosphatase-conjugated secondary antibodies (Promega). Specific bands were visualized with BCIP/NBT (Sigma). Coomassie brilliant blue (CBB) staining of the membrane confirmed equal loading and transfer.

Isolation of Inner Medullary Collecting Duct Cells
To test whether cells in the inner medulla activate HIF under physiological hypoxia, inner medullary collecting duct (IMCD) cells were isolated from HRE-Luc–transgenic rats and exposed to 2% O2, as previously described (20,21). In brief, renal medulla were excised, cut into small (1–2 mm3) pieces, and digested with 0.2% collagenase and 0.2% hyaluronidase (Sigma). After the enzymatic digestion, cells were isolated by three short low-speed centrifugations (175 g for 8 minutes, twice at 28 g for 2 minutes) and were seeded on six-well culture plates (Techno Plastic Products, Trasadingen, Switzerland) precoated with gelatin. With this method, more than 90% of obtained cells were collecting duct cells, as confirmed by positive immunostaining for aquaporin-2 (Chemicon). Then, cells were cultured with Dulbecco's modified Eagle medium containing 5% fetal calf serum (JRH Biosciences, Lenexa, KS) at 37°C under a humidified atmosphere of 5%CO2/95% air, grown to confluence, and exposed to hypoxia for 6 hours. Assuming the physiological medullary partial pressure of oxygen to be approximately 15 mmHg (22), we subjected IMCD cells to 2% oxygen (APM-30D multi-gas incubator; Astec, Fukuoka, Japan). The HIF activity was estimated by measuring the amount of the transgene (HRE-Luc) mRNA by real-time PCR, as described above. For practical reasons, only rats in the YO group were used in this study.

Statistical Analysis
Data were expressed as mean ± standard deviation or mean ± standard error of the mean, as appropriate. Analyses were carried out with StatView software (version 5.0; SAS Institute, Cary, NC). Differences among groups were compared by unpaired Student t tests with the Bonferroni correction or Dunn's method. Values of p below.05 were considered to be statistically significant.


   RESULTS
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 Materials and Methods
 Results
Discussion
 References
 
Animal Characteristics
Physical and biochemical characteristics of the YO and AG rats are summarized in Table 1. In the AG rats, there was a large individual difference in the serum creatinine level, which was yet significantly higher than in the YO rats (0.89 ± 0.14 mg/dL vs 0.69 ± 0.05 mg/dL, p <.01). In addition, the AG rats developed significant degrees of proteinuria as compared to the YO rats (85.9 ± 22.4 mg/day vs 13.0 ± 3.4 mg/day, p <.01), in keeping with the previous description on proteinuria in the aged laboratory rats (23).


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  		Table 1. Animal Characteristics.

 
Pathological Correlation of Hypoxia with Tubulointerstitial Injury
Periodic acid-Schiff staining of the AG rat kidneys revealed various degrees of glomerular and tubulointerstitial injury. Some glomeruli were small and globally sclerotic, whereas the thickening of the glomerular basement membrane (GBM) was more frequently observed. In the tubulointerstitium, TBMs appeared slightly thickened with positive immunohistochemical staining for fibronectin and collagen IV (not shown). In areas displaying moderate to severe tubulointerstitial injury, structural changes such as tubular dilatation, intratubular cast formation, tubular atrophy, and marked interstitial widening were observed (Figure 1A and B). Areas of fibrosis were also seen (Masson-Trichrome stain, not shown).


Figure 01
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  		Figure 1. Progression of histological damage in the aging kidney. In the periodic acid-Schiff staining, various degrees of tubulointerstitial as well as glomerular injury were noted in the aging kidney. Whereas some kidneys showed almost unremarkable injury (A), others exhibited striking histological damage, such as tubular basement membrane thickening, tubular dilatation and atrophy, cast formation, brush border loss, and interstitial fibrosis (B). In the aging kidneys, a positive correlation was observed between the degree of hypoxia (x axis: transgene expression in the cortex, relative to the average of the young kidney counterparts) and the score of tubulointerstitial injury (y axis) (R2 = 0.88, p <.01) (C). Original magnification for A and B, x200

 
Pathological correlation between hypoxia and tubulointerstitial injury was obtained by measuring the transgene expression and the semiquantitative tubulointerstitial injury score in each individual AG rat (Figure 1C). A strong positive correlation was observed (R2 = 0.88, p <.01), suggesting the involvement of hypoxia in the development of age-related renal pathogenesis.

Identification of Hypoxic Tubules
Then, areas of hypoxic tubules were identified by pimonidazole, a chemical marker of hypoxia (Figure 2). In contrast to the YO kidney (A–C), the AG kidney (D–F) showed marked expansion of hypoxic areas in the cortex (A and D), medulla (B and E), and papilla (C and F), even in kidneys with mild to moderate tubulointerstitial injury. Overall, however, the age-related appearance of tubulointerstitial hypoxia was most prominent in the cortex. In the cortex with mild to moderate injury, areas of positive staining were broader in the medullary rays (D). On examination of the cortex with severe tubulointerstitial injury, areas of hypoxic tubules obviously extended from the medullary rays to the superficial cortex (G), with some tubules markedly dilated and distorted (H), whereas others appeared to reside in superficial cortical areas (I) and in those areas adjacent to glomeruli of outer nephrons (J).


Figure 02
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  		Figure 2. Hypoxia in the aging rat kidney. Hypoxic tubules in the aging rat kidney were identified by pimonidazole immunostaining. In the aging kidney, hypoxic areas were apparently larger in the medullary rays (D), outer medulla (E), and papilla (F), as compared to the young kidney (A–C). At higher magnifications of the cortex of the aging kidney, hypoxic areas were expanded from the medullary rays to the superficial cortex (G), consisting of tubules with moderate to severe thinning and dilatation (H). Spatially, hypoxic areas were also found in the superficial cortical regions (I) and in those regions adjacent to the glomeruli of outer nephrons (J, arrow). Note that E–G were captured from the aging kidney with only mild tubulointerstitial injury, to signify hypoxia beginning at the early stage. Original magnification: A–F, x40; G, x100; H–J, x200

 
Activation of HIF in the Aging Kidney
Identification of hypoxic tubules in the aging kidney led us to investigate whether there was any increase in HIF-activation (Figure 3). An age-related activation of HIF, as determined by the upregulation of the transgene, was observed in the cortex (A and B), outer medulla (C and D), and papilla (G and H), as could be envisaged from results of pimonidazole staining (Figure 2). Of interest, tubules in the inner medulla appeared to express the transgene to a similar degree in both groups, at levels higher than in the cortex of the young kidney (E and F). The inner medullary expression of the transgene was reminiscent of the borderline hypoxia in these areas, and suggested that HIF might be constitutively active in the physiologic conditions of the kidney, regardless of age.


Figure 03
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  		Figure 3. Expression of the "hypoxia-responsive" transgene. Tubular cell hypoxia was also identified by the upregulation of the hypoxia-responsive reporter of the transgenic rats. In the aging kidney, the expression was obviously higher in the cortex (B), outer medulla (D), and papilla (H), as compared to the young kidney (A, C, and G). In the inner medulla, however, the transgene appeared to be expressed at a higher level than in the cortex of the young kidney, regardless of age (E and F). Original magnification x200

 
To address this question, IMCD cells were isolated from the HRE-Luc–transgenic rats and exposed to 2% O2, assuming that the physiologic oxygenation in the deep medulla is approximately 15 mmHg (Figure 4). Immunostaining for aquaporin-2 confirmed that the isolated cells were collecting ducts (A). When these cells were exposed to hypoxia for 6 hours, the transgene expression became 2.1 ± 0.6-fold as compared to normoxia (p =.06) (B). These results further raise the possibility that HIF is constitutively active in the inner medulla, in the physiologic oxygenation status.


Figure 04
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  		Figure 4. Isolated inner medullary collecting duct (IMCD) cells activate hypoxia-inducible factor (HIF) in physiological regional hypoxia. IMCD cells were isolated from young rat kidneys and challenged to 2% O2, assuming the physiological oxygen gradient of the kidney. Immunostaining for aquaporin-2 clarified that more than 90% of isolated cells were those of collecting ducts (A). Stimulation with 2% O2 for 6 hours resulted in a 2.1 ± 0.6-fold increase in the transgene expression (p =.06) (B), raising the possibility that HIF is constitutively active at the physiological oxygen concentration in the inner medulla, even in young rats. The bar on the right (1% O2) serves as positive control ((**) p <.01). Original magnification (A) x400. Real-time polymerase chain reaction (B); n = 4

 
Expression of HIF-1{alpha} and HIF-2{alpha} Isoforms
To determine which of the major HIF-{alpha} isoforms was responsible for the tubular upregulation of HIF in the aging kidney, immunostaining for HIF-1{alpha} and HIF-2{alpha} was performed (Figure 5). In the cortex of the AG kidney, HIF-1{alpha} was expressed in the nuclei of tubules dominated by severe structural changes (B), whereas HIF-2{alpha} was restricted to the interstitial compartment (D). Both HIF-1{alpha} and HIF-2{alpha} were negative in the cortex of the YO group (A and C). Of note, the mRNA expression of HIF-1{alpha} and HIF-2{alpha} was similar between the YO and AG rat kidneys (Table 2), consistent with the notion that the activation of HIF by hypoxia is primarily determined at the post-translational level, namely, by the stabilization of HIF-{alpha} proteins (24). These observations indicate that HIF-1 rather than HIF-2 is responsible for the age-related activation of HIF in renal tubules.


Figure 05
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  		Figure 5. Immunostaining for hypoxia-inducible factor (HIF)-1{alpha} and HIF-2{alpha}. Immunostaining for HIF-{alpha} isoforms revealed the expression of HIF-1{alpha} in tubular epithelial cells (B) (aging) compared with (A) (young) and that of HIF-2{alpha} in the interstitial compartment (D) (aging) compared with (C) (young) in the aging kidney. Original magnification x400

 

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  		Table 2. Quantification of HIF-1{alpha} and HIF-2{alpha} mRNA.

 
Quantification of HIF-Regulated Genes
Activation of HIF in the aging kidney was corroborated by quantifying the transgene and several of the known HIF-regulated gene mRNAs by real-time PCR (Figure 6). As already stated, the transgene mRNA, although diverse among individuals, was significantly higher in the AG group (4.6 ± 3.3-fold, p =.03). The amount of EPO (25) (p <.01), VEGF (26) (p <.001), and GLUT1 (p <.01) mRNAs were all significantly larger in the AG group, whereas that of HO-1 (27) was comparable to the YO group, due to large individual variations (p =.08).


Figure 06
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  		Figure 6. Quantification of hypoxia-inducible factor (HIF)-regulated genes. Messenger RNAs of the transgene and several of the known HIF-regulated genes were quantified by real-time polymerase chain reaction (PCR). In the aging kidney, the transgene expression was measured as 4.6 ± 3.3-fold over the young kidney (A). Overall, renal transcripts of erythropoietin (EPO) (B), vascular endothelial growth factor (VEGF) (C), glucose transporter-1 (GLUT1) (D), and heme oxygenase-1 (HO-1) (E) were higher in the aging kidney. The average amount of the target gene in the young kidneys was arbitrarily set at 1. The representative PCR amplification curves are also shown (F). Real-time PCR, n = 8 (for young rats; YO) and n = 6 (for aged rats; AG), respectively (unpaired t test, data expressed as mean ± standard deviation). Numbers in (F) are, respectively: 1, transgene-YO; 2, transgene-AG; 3, EPO-YO; 4, EPO-AG; 5. VEGF-YO; 6, VEGF-AG; 7, GLUT1-YO; 8, GLUT1-AG; 9, HO-1-YO; 10, HO-1-AG; 11, ß-actin-YO; and 12, ß-actin-AG

 
Quantitative changes in the transgene, HIF-1{alpha}, VEGF (p21 fragment), GLUT1, and HO-1 were analyzed at the protein level as well. Western blotting revealed the overall, coordinated upregulation of these HIF-associated genes in the AG group, although, once again, there were quantitative variations among aging individuals (Figure 7 and quantified in Table 3). Unfortunately, however, the age-related increase of HIF-1{alpha} and GLUT1 proteins was only marginal by this method, possibly due to technical difficulties and their limited expression at the basal level.


Figure 07
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  		Figure 7. Immunoblotting. The expression of hypoxia-inducible factor (HIF)-associated proteins was analyzed by western blotting. Cortical samples in the aged rat (AG) group contained larger amounts of the transgene, vascular endothelial growth factor (VEGF; p21 fragment), and heme oxygenase-1 (HO-1) proteins, than did those in the young rat (YO) group. Lanes 1–4: YO group; lanes 5–8: AG group

 

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  		Table 3. Quantification of the Transgene and HIF-Regulated Proteins.

 

   DISCUSSION
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 Materials and Methods
 Results
 Discussion
References
 
This study describes the age-related prevalence of hypoxia in the tubulointerstitium of the kidney. In the physiological, nonsenescent kidney, the medulla is exposed to borderline hypoxia to maintain its concentrating ability of the urine (22), whereas the cortex is exempt from it. With aging, the kidney was found to suffer more extensive degrees of hypoxia, which were most prominent in the cortex, but substantial in all areas.

Studies in the past have focused on describing the age-related structural derangements mainly in the cortex. Thickening of GBM and TBM (28), accumulation of the extracellular matrix and focal areas of tubular atrophy (5) have been characterized, and the loss of peritubular capillaries has been observed in the affected areas (29). These structural changes pointed to the potential relevance of hypoxia to the age-related tubulointerstitial injury. However, no direct proof has been provided so far.

We confirmed the prevalence of hypoxia in the aging kidney by using two distinct methods, namely: (i) positive staining for pimonidazole, and (ii) the upregulation of the "hypoxia-responsive" transgene. The former is based on the hypoxia (pO2 < 10 mmHg)-dependent binding of the 2-nitroimidazole to form adducts that serve as immunogens, and allows us to detect hypoxia immunohistochemically. Using this method, we identified areas of hypoxic tubules expanding in the medullary rays, superficial cortical areas and those adjacent to glomeruli of outer nephrons, in the aging kidney. Although how these areas are exposed to hypoxia is unclear, it seems plausible that renal hemodynamic changes play important roles. This may be envisaged by the anatomical characteristics that the medullary rays, areas most severely affected by hypoxia with aging, are partly vascularized by the ascending vasa recta draining the outer medulla until emptying into interlobular veins (30). In addition, the superficial area is known to be marginally supplied with blood flow for anatomical reasons. Furthermore, the perfusion of outer nephrons decreases more with age than does that of the corticomedullary nephrons (31). These previous observations are highly suggestive of the involvement of renal hemodynamics in the age-related development of hypoxia.

Age-related hypoxia, while undoubtedly most prominent in the cortex, was also evident in the medulla. This finding may be explained partly by the impaired oxygen diffusion, as can be envisaged by the accumulation of the extracellular matrix. In renal medulla, there is a significant increase in connective tissue with age, which is reportedly more striking than in the cortex (32). Once again, however, renal hemodynamics is also a candidate contributor, because the blood supply of the medulla is essentially postglomerular, except for a fraction of the "true medullary arterioles" originating directly from arcuate or interlobular arteries that can be seen after degeneration of the corresponding glomeruli and tubules.

Tubulointerstitial hypoxia was also evidenced by the activation of HIF. Based on the upregulation of the hypoxia-responsive transgene, the hypoxic response was calculated as 4.6 ± 3.3-fold in the aging rat kidneys, as compared to the young rat counterparts. Cortical immunodetection of HIF-{alpha} isoforms allowed us to identify HIF-1{alpha} in tubular epithelial cells and HIF-2{alpha} in the interstitial compartment; these identifications are consistent with previous findings in ischemic young rat kidneys (9) and suggest that HIF-1 plays a dominant role in the hypoxic response of the aging tubular cells. Furthermore, quantification of HIF-regulated genes, such as EPO, VEGF, GLUT1, and HO-1, revealed their coordinated upregulation in the cortex of the aging kidney, indicating the functional operation of the HIF-mediated cellular response to hypoxia. The pathological relevance of HIF and its target genes merits further study.

The expression of HIF-1{alpha} with aging has been reported in such systemic organs as brain, liver (33), and carotid body (34), but the background for the increase in HIF-1{alpha} protein remained largely unknown, except for one study reporting that the upregulation of HIF-1{alpha} in the aging liver was associated with an increase in the reactive oxygen species (35). However, an effect of reactive oxygen species on HIF generally remains elusive. Now that we identified hypoxic areas expanding in the aging kidney by pimonidazole staining, we propose in this study that the increase in HIF-{alpha} in the aging kidney (HIF-1{alpha} in tubular cells and HIF-2{alpha} in the interstitium) is associated with hypoxia.

Hypoxia in the aging kidney was found to have a positive correlation with the degree of tubulointerstitial injury. It has been documented from a human pathological study that the loss of peritubular capillaries is closely related to the degree of tubulointerstitial injury and the residual renal function (36). Considering that renal perfusion is a crucial component in determining regional oxygenation, it is tempting to propose that hypoxia in the tubulointerstitium serves as a hallmark of age-related renal injury as well.

Summary
This study demonstrated that the aging kidney is exposed to extensive degrees of hypoxia and that the intrinsic tubular cells respond to it by upregulating a number of HIF-regulated genes. The degree of hypoxia most likely serves as a hallmark of the age-related tubulointerstitial injury, as has been proposed and reported in glomerular diseases (6,18). Our future perspectives include the research for the expression of HIF and the transcriptional regulation of HIF target genes in aging individuals, in the context of additional ischemic insult. Although aging kidneys express HIF at a higher level than do young kidney counterparts, this finding does not necessarily mean that the former has enough potential to further upregulate HIF when exposed to additional hypoxic stimulation. This is potentially of therapeutic significance because maneuvers to activate HIF (37) have been suggested to work in a renoprotective manner in a number of acute (38,39) as well as chronic (40–42) renal diseases.


   Acknowledgments
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We acknowledge Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17390246).


   Footnotes
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Decision Editor: James R. Smith, PhD

Received September 30, 2005

Accepted February 16, 2006


   References
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 References
 

  1. Anderson S, Brenner BM. Effects of aging on the renal glomerulus. Am J Med. 1986;80:435-442.[Medline]
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