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Am J Physiol Renal Physiol 293: F1397-F1407, 2007. First published July 3, 2007; doi:10.1152/ajprenal.00133.2007
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Hypoxia and podocyte-specific Vhlh deletion confer risk of glomerular disease

Kirsten Brukamp,1 Belinda Jim,1,* Marcus J. Moeller,2 and Volker H. Haase1

1Renal-Electrolyte and Hypertension Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and 2Department of Internal Medicine, Rheinisch-Westfaelische Technische Hochschule, Aachen, Germany

Submitted 20 March 2007 ; accepted in final form 28 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Hypoxia is a potent regulator of a multitude of cellular processes, including metabolism and cell survival. The transcriptional response to oxygen deprivation is mainly mediated by hypoxia-inducible factors (HIFs), which are targeted for proteasomal degradation by the von Hippel-Lindau tumor suppressor protein (pVHL) under normoxia. Podocytes, as part of the glomerular filtration barrier, are prone to hypoxic injury during diseases affecting the glomerulus. VHL and HIF1 were functional in mature murine podocytes in vivo and in vitro, with HIF1 protein stabilization and target gene transcription under both hypoxia and VHL deficiency. Podocyte-specific Vhlh gene loss, mimicking podocyte hypoxia, in young mice of mixed background led to glomerulomegaly and occasional glomerulosclerosis, despite preserved glomerular development. In parallel, hypoxia effects on podocytes in cell culture included increased susceptibility to apoptosis, associated with nuclear translocation of apoptosis-inducing factor (AIF). Similarly, Vhlh gene inactivation in podocytes in vitro resulted in a significant survival disadvantage, particularly in conjunction with additional proapoptotic stimuli. Evaluation of the global transcriptional response to hypoxia in podocytes by microarray analysis revealed a typical upregulation of HIF target genes as well as the induction of genes relevant for stress response, cell-cell, and cell-extracellular matrix interaction. While the lack of a prominent phenotype in young mice with VHL-deficient podocytes is consistent with the absence of specific glomerular manifestations in human VHL disease, a low-oxygen environment of podocytes may contribute to the progression of glomerular disease by altering cellular metabolism and survival.

glomerulosclerosis; hypoxia-inducible factor; von Hippel-Lindau; apoptosis

PODOCYTE ALTERATIONS HAVE been implicated in the pathogenesis of various glomerulopathies, particularly those associated with nephrotic syndrome and glomerulosclerosis (37, 29). The pathophysiological contribution of podocyte hypoxia to glomerular diseases has not been elucidated in detail, although the role of hypoxia in tubulointerstitial injury is well established (26). Hypoxia-inducible transcription factors (HIFs) are key mediators of the cellular transcriptional response to hypoxia and belong to the basic-helix-loop-helix PER-ARNT-SIM (bHLH PAS) domain protein superfamily (25). Under hypoxia, the closely related and best studied members HIF1{alpha} and HIF2beta form heterodimers named HIF1 and HIF2 with the HIF1beta component, also termed aryl hydrocarbon receptor nuclear translocator (ARNT) (33). HIFs then bind hypoxia-responsive elements (HREs) on promoters of a variety of known HIF target genes, belonging to groups relevant for a multitude of biological processes, for example, energy metabolism, angiogenic signaling (such as the vascular endothelial growth factor Vegf), hormone regulation, growth, and apoptosis (such as the Wilms' tumor suppressor protein Wt1) (33). The heterodimers cannot associate after hydroxylation of prolyl residues by hydroxylases that are only active under normoxia and thereby act as oxygen sensors. After hydroxylation, the von Hippel-Lindau (VHL) tumor suppressor protein prevents the stabilization of HIFs by acting as the substrate recognition component of an E3 ubiquitin ligase complex that targets HIF{alpha} subunits for degradation in the 26S proteasome. This study was therefore designed to investigate the role of the HIF transcription factor system in podocytes to identify its physiological significance and its pathological contribution to glomerular disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of transgenic mice, genotyping, phenotypic assessment, and histopathological analysis. The generation of transgenic mouse strains (see Fig. 1 and RESULTS) used in this study has been previously described (13, 26, 15, 28). Mutant mice were of mixed genetic background based on BALB/c, 129Sv/J, and C57BL/6J strains. Tail DNA and cell DNA were isolated for genomic PCR to establish inheritance of transgenic constructs (21). A selection of primers is listed in Table 1. Proteinuria was evaluated by a protein assay kit according to the Bradford method (Bio-Rad). Histological sections were obtained after tissue freezing in OCT compound (Tissue-Tek), and staining was done with hematoxylin-eosin. Morphometric analysis was performed on three different histological sections from clinically healthy mice (4–8 wk; age not significantly different between Vhlh2lox/2lox and Vhlh1lox/1lox mice, which are labeled Vhlh+/+ and Vhlh–/– in the remainder of the article). All cortical glomeruli in a section were analyzed for a total number of >50 glomeruli/mouse. IP lab software (Scanalytics) was utilized for morphometric analysis and for tissue and cell images. All procedures involving mice were performed in accordance with the National Institutes of Health Guide for The Care and Use of Laboratory Animals and were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.


Figure 1
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  		Fig. 1. Generation of transgenic mice and phenotypic analysis. Von Hippel-Lindau gene (Vhlh) deletion in podocytes is associated with subtle glomerular changes at a young age and severe pathology in a minority of mice. A: transgenic constructs used in this study were the NPHS2(podocin)-Cre recombinase transgene, the Vhlh 2lox allele, the temperature-sensitive simian virus 40 large T antigen transgene, and the LacZ-enhanced green fluorescent protein (eGFP) transgene. LoxP sites are represented by triangles. Lines, boxes, and triangles are not drawn to relative scale. B and C: confirmation of Cre recombinase activation by visualization of eGFP in glomerular podocytes by epifluorescence microscopy (B; original magnification x40 and x400) and of Vhlh loop-out by immunohistochemistry for stabilized HIF1{alpha} (C; original magnification x400; brown on top of hematoxylin-eosin staining). Conventional, not confocal, epifluorescence microscopy of a whole mount kidney is shown in B. Signal does not represent individual cells, but numerous podocytes in several layers. D: histology in a mouse with a significant clinical and histological phenotype after podocyte-specific Vhlh deletion: severe glomerulosclerosis, tubular dilation, accumulation of proteinacious material in the tubules, interstitial fibrosis, and interstitial lymphocytic infiltration (hematoxylin-eosin staining; original magnification x400). E: glomerulomegaly as a typical glomerular finding in transgenic mice with podocyte-specific Vhlh deletion at 4–8 wk (hematoxylin-eosin staining; original magnification x100). F: increased areas of glomerular tuft and of glomerular size after podocyte-specific Vhlh deletion in young mice (4–8 wk), as evidenced by morphometric analysis of glomeruli (normalization to Vhlh+/+ glomerular size). **P < 0.01 and ***P < 0.001, respectively.

 

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  		Table 1. Primers

 
Cell culture, establishment of cell lines, cell-based assays, and flow cytometry. Podocytes were allowed to proliferate in the undifferentiated state under SV40 large T antigen activation by interferon-{gamma} (BD Biosciences) at 33°C. Cells were differentiated at nonpermissive conditions at 37°C under interferon depletion for 10–14 days. After kidneys were digested with collagenase type 4 (Worthington) and glomeruli isolated by density gradient centrifugation, novel cell lines were obtained from podocytes growing out of glomeruli under permissive conditions. Cell populations were subcloned to obtain homogenous cell lines. Expression analysis was performed between passages 10 and 30. Control MPC podocytes were a kind gift from Peter Mundel (27). Cell culture medium consisted of RPMI-1640 (Sigma) with or without D-glucose (Sigma), supplemented with fetal calf serum (Hyclone), penicillin, streptomycin, and L-glutamine (all GIBCO). LacZ and Cre recombinase expression adenoviruses (Children's Hospital of Philadelphia Vector Core Facility) were transduced at a magnitude of infection (MOI) of 100 virus particles/cell. X-Gal (Bethesda Research Labs) was utilized for beta-galactosidase staining. Neutral lipid content was assessed with oil red O (Sigma) staining after 10% neutral buffer formalin (Fisher Scientific) fixation. Proliferation was tested with a 5-Bromo-2'-deoxy-uridine (BrdU) Labeling and Detection Kit II (Roche). Cell viability was assessed by trypan blue (Sigma) exclusion with a hemocytometer and by metabolism of a tetrazolium compound to formazan (MTS assay; Promega). Live cell apoptosis staining was performed with combinations of Hoechst 33342 (Molecular Probes), propidium iodide (Molecular Probes), acridine orange (Sigma), ethidium bromide (Sigma), and 4',6-diamidino-2-phenylindole (DAPI). Defined O2 concentrations were generated in a sterile hypoxia workstation (Ruskinn Technologies) capable of achieving any desired O2 content from anoxia to room air. Cells were exposed to hydrogen peroxide (H2O2; Sigma) as a proapoptotic agent for some experiments. Cells were sorted for enhanced green fluorescent protein (eGFP) expression of novel podocyte cell lines with FACSVantage SE with DiVa Option (BD Biosciences, San Jose, CA), and flow cytometry was done by FACS Calibur (BD Biosciences) after propidium iodide staining.

Immunostaining. For immunofluorescence, cultured podocytes were fixed and permeabilized with 10% neutral buffer formalin (Fisher Scientific) and 0.3% Triton X (Bio-Rad Laboratories). Antibodies included those against WT1 (rabbit polyclonal, dilution 1:400, Santa Cruz Biotechnology), synaptopodin (gift from P. Mundel; rabbit polyclonal), nephrin (guinea pig polyclonal, dilution 1:100, Progen), apoptosis-inducing factor (AIF; rabbit polyclonal, dilution 1:1,000, Chemicon), and respective Cy3-coupled donkey-derived secondary antibodies (dilution 1:600, Jackson ImmunoResearch). For tissue sections, immunohistochemistry was performed with an anti-HIF1 antibody (Novus), employing a horseradish peroxidase-conjugated ABC reagent (Vecta Elite Kit) and DAB Substrate Kit for Peroxidase (Vector Laboratories).

Protein isolation and Western blotting. Nuclear and cytoplasmic protein isolations were prepared under protease protection according to established protocols (5). Twenty grams of protein samples were separated on 3–8% SDS-PAGE (Invitrogen) and transferred to nitrocellulose membranes (Amersham Pharmacia Biotech). Equal loading was confirmed by Ponceau S staining (Sigma) before blocking. Incubation with primary antibodies at 4°C overnight was followed by labeling with horseradish peroxidase-conjugated secondary antibodies and analysis by chemiluminescence (Amersham Pharmacia Biotech). Primary antibodies used for Western blotting comprised HIF1{alpha} (Novus) and HIF2{alpha} (Invitrogen), and secondary antibodies for immunoblotting were goat anti-rabbit (Bio-Rad Laboratories) and sheep anti-mouse (Amersham Pharmacia Biotech).

RNA isolation, northern blotting, reverse transcription, semiquantitative PCR, quantitative PCR, and microarray analysis. RNA was prepared with TRIzol (Invitrogen) reagent and, for microarray analysis, with an RNeasy MidiKit (Qiagen) after initial TRIzol processing, according to the manufacturers' guidelines. Northern blotting was performed according to standard protocols (13). Reverse transcription was done with the SuperScript first-strand synthesis system (Invitrogen). Semiquantitative PCR was carried out after determination of the gene-specific linear phase of amplification. Quantitative real-time PCR was performed on the Prism 7700 Sequence Detection System with Sybr Green Master Mix for genes of interest and Taqman Universal PCR Master Mix (all from Applied Biosystems) for 18S rRNA expression, to which the cDNA levels were normalized. The quality of the RNA preparation for the microarray analysis was confirmed by the 18-to-28S rRNA ratio in an Agilent bioanalyzer. Microarray analysis was carried out with a cDNA microarray chip 6.0 (PancChip) with 13,059 murine transcripts expressed in the pancreas (16, 32). Microarray results were validated by quantitative real-time PCR for selected genes. Analysis was performed with Ingenuity Pathways Analysis 3.1 Software (Ingenuity Systems).

Statistical analysis. Data are reported as means ± SD. An unpaired t-test was used to compare mean values. A P value of <0.05 was regarded as statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Phenotypic analysis of mice with podocyte-specific Vhlh deletion. Conditional knockout mice with podocyte-specific Vhlh gene deletion via the Cre/loxP-mediated recombination technology were created to investigate the role of constitutively active HIF signaling in vivo. Mice bearing an NPHS2-Cre recombinase transgene (26) were mated to those with a conditional Vhlh 2lox allele (13) (Fig. 1A). To establish Cre recombinase activity and for the derivation of cell lines, mice were bred with those carrying the temperature-sensitive simian virus 40 large T antigen (15) and a conditional allele for enhanced GFP (eGFP) expression (28) (Fig. 1A). The latter allele mediates bacterial beta-galactosidase expression at baseline; after Cre recombinase activation, eGFP is transcribed. Recombination efficiency in mice was therefore confirmed by visualization of eGFP expression (Fig. 1B), loop-out of genomic DNA in freshly isolated glomeruli (1lox band greatly favored over 2lox band; data not shown; compare configuration in Fig. 2B), as well as immunohistochemistry for HIF1{alpha}, which is stabilized after Vhlh gene deletion (Fig. 1C).


Figure 2
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  		Fig. 2. Establishment of conditionally Vhlh-deficient podocyte cell lines (CLL). A: transducibility of cells in culture with adenovirus (AV) is highly efficient as assessed by beta-galactosidase expression on LacZ-AV treatment vs. none after Cre recombinase-AV transduction, with a transduction efficiency estimated as 98% at a magnitude of infection (MOI) of 100:1. B: exemplary loop-out in the Vhlh gene by genomic PCR in a Vhlhwild-type/2lox mouse (right), resulting in a Vhlhwild-type/1lox configuration (left). C: molecular confirmation of podocyte origin (mRNA expression profiling). D: morphological confirmation of podocyte origin (immunofluorescence). Top, WT1; bottom, nephrin; red: podocyte-typical proteins, blue: DAPI counterstain; middle, synaptopodin black-and-white photography without application of false colors. Original magnification x200–x600).

 
Eleven mice with podocyte-specific Vhlh gene deletion were compared with four littermate control mice possessing Vhlh 2lox, but not Cre recombinase, alleles. While the majority of Vhlh–/– mice appeared clinically healthy (n = 6 at 4–8 wk and n = 3 at 6 mo of age), one mouse developed end-stage renal disease and succumbed to early death at 8 wk. Extensive histological changes consisted of severe glomerulosclerosis, tubular dilatation, accumulation of proteinacious material in the tubules, interstitial fibrosis, and interstitial lymphocytic infiltration characteristic of advanced renal failure (Fig. 1D). Two other mice exhibited less renal destruction with dilated, protein-containing tubules at 8 wk and lymphocytic infiltration at 25 wk, respectively. Nondiseased mice at an age of 4–8 wk also exhibited subtle glomerular changes characterized by glomerulomegaly (Fig. 1E). Both the glomerular tuft size and the overall glomerular area were significantly increased for mice with VHL-deficient podocytes (n = 6) compared with controls (n = 3) (Fig. 1F). Proteinuria was determined at several time points for the same animal between 4 and 8 wk in three healthy mice each for the Vhlh+/+ and Vhlh–/– genotype, and the assay revealed neither remarkable proteinuria nor a significant difference between the mouse groups.

To investigate the role of HIF signaling in podocytes further, Vhlh 2lox-conditional podocyte cell lines (CLL) were derived from 3- to 8-wk-old mice, with differentiation characteristics similar to previously established podocyte cell lines (27). Adenovirus-mediated delivery of Cre recombinase was efficient (Fig. 2A) and generated Vhlh-deficient cells as demonstrated by genomic DNA analysis (Fig. 2B for three bands of Vhlhwild-type/2lox loop-out to Vhlhwild-type/1lox configuration; data not shown for 2 bands of Vhlh2lox/2lox loop-out to Vhlh1lox/1lox configuration). CLL exhibited WT1, synaptopodin, and nephrin staining (Fig. 2D) and expressed podocyte-typical and -specific markers comparable to previously described MPC podocytes (27) (Fig. 2C), demonstrating that CLL possessed the morphological and molecular characteristics of podocytes.

Hypoxia results in typical HIF1 target gene induction in podocytes. HIF1{alpha} protein was stabilized in the nucleus under hypoxia (O2 of 0.5% for 8 h) in MPC in vitro (Fig. 3A), while HIF2{alpha} protein did not significantly change under these conditions. Similarly, stabilized HIF1{alpha} protein was found in Vhlh–/– podocytes compared with Vhlh+/+ cells (data not shown). Classic HIF1 target genes, such as phosphoglycerate kinase 1 (Pgk), lactate dehydrogenase A (Ldha), and glucose transporter 1 (Glut1), were upregulated under low-oxygen conditions of 0.5%: hypoxia induced PGK in the undifferentiated and the differentiated state in a time-dependent fashion, and Vhlh–/– podocytes exhibited an even higher level of PGK transcripts (Fig. 3C). Ldha induction was similar under hypoxia and VHL deficiency in CLL, whereas Glut1 was more strongly induced under acutely low-oxygen concentrations compared with permanent VHL loss (Fig. 3B).


Figure 3
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  		Fig. 3. Hypoxia-inducible factor 1 (HIF1) is functional in podocytes. Metabolism-related HIF1 target genes are upregulated. A: HIF1{alpha} protein is stabilized in the nuclear compartment during hypoxia, while HIF2{alpha} remains without significant change acutely (MPC; 0.5% O2 for 8 h; Western blotting). B: lactate dehydrogenase (Ldha) and glucose transporter 1 (Glut1) upregulation (CLL: Vhlh+/+ and Vhlh–/–; normoxia vs. 0.5% O2 for 24 h; Northern blotting). C: phosphoglycerate kinase (Pgk) upregulation compared with beta-actin control (CLL: Vhlh+/– and Vhlh–/–; normoxia vs. 0.5% O2 for up to 9 h; semiquantitative PCR).

 
On the transcriptional level, both short and prolonged exposure to hypoxia led to an upregulation of podocyte-typical genes that have previously been described as direct HIF1 target genes. By quantitative real time PCR, Vegf was induced 2- to 2.5-fold in MPC after exposure to 0.5% O2 for 4–24 h, and a progressive upregulation was also seen at the more moderate hypoxia level of 1.5% O2 for 12–48 h (Fig. 4A). Wt1 was induced by hypoxia as well (Fig. 4B). Regarding podocyte-typical genes without prior data on HIF regulation, nephrin transcripts remained the same, whereas synaptopodin exhibited a downregulation after prolonged exposure to hypoxia (Fig. 4C). Specific hypoxic regulation was not found for ZO1, Lmx1b, P-cadherin, podoplanin, and Cd2ap (data not shown).


Figure 4
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  		Fig. 4. Hypoxia induces Vegf and Wt1 as podocyte-typical markers, but not nephrin and synaptopodin, on the transcriptional level (quantitative real-time PCR). A: Vegf upregulation (MPC; 0.5 and 1.5% O2 for up to 24 and 48 h, respectively). B: Wt1 upregulation (MPC; 0.5 and 1.5% O2 for up to 8 and 48 h, respectively). C and D: stable nephrin and decreasing synaptopodin transcripts (MPC; 0.5 and 1.5% O2 for up to 24 and 48 h, respectively). *P < 0.05, **P < 0.01 and ***P < 0.001.

 
Both hypoxia and VHL deficiency lead to increased podocyte apoptosis. When undifferentiated CLL cells were transduced with Cre recombinase adenovirus, compared with LacZ adenovirus control transduction, a decrease in viable cells was seen after 1 wk for Vhlh–/– podocytes, whereas Vhlh+/+ ones continued to expand in culture (Fig. 5A). To rule out an immediate unspecific posttransduction effect underlying this phenomenon, Vhlh+/+ and Vhlh–/– podocytes were reseeded at equal densities. The cell number was again significantly different after 3 days in culture, and the slope of increase in cell numbers was greatly reduced (Fig. 5B). Transduction of differentiated, nonproliferating CLL podocytes resulted in decreased survival of Vhlh–/– cells after 10 days compared with stable numbers of Vhlh+/+ podocytes (Fig. 5C). A difference between Vhlh+/+ and Vhlh–/– cells after 2 days could be detected by exposure to supplementary proapoptotic stimuli, such as withdrawal of growth factors in cell culture serum and addition of hydrogen peroxide (Fig. 5D).


Figure 5
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  		Fig. 5. Hypoxia and HIF stabilization through VHL loss result in increased apoptosis after cell cycle dysregulation. A: cell death of undifferentiated Vhlh–/– CLL podocytes (after Cre recombinase-AV transduction; cell numbers as fold-increase over baseline at day 0). B: decreased cell expansion of undifferentiated Vhlh–/– CLL podocytes (after Cre recombinase-AV transduction and reseeding; cell numbers as fold-increase over baseline at day 0). C: cell loss of differentiated Vhlh–/– CLL podocytes over time in culture (square denotes Vhlh+/+ CLL, circle denotes Vhlh–/– CLL; MTS assay; numbers as percentage of baseline at day 0). D: cell loss of differentiated Vhlh–/– CLL podocytes within 2 days in conjunction with withdrawal of growth factors present in the culture serum and supplementation of hydrogen peroxide (H2O2) at concentrations of 10 and 100 mM as an additional proapoptotic stimulus (MTS assay; numbers as percentage of baseline at day 0). E: cell cycle analysis for undifferentiated MPC and CLL Vhlh+/+ podocytes under hypoxia (0.5% O2 for 2 days; flow cytometry with propidium iodide). *P < 0.05, **P < 0.01 and ***P < 0.001.

 
Live cell apoptosis staining revealed typical correlations of nuclear morphology with expected color staining patterns for live, apoptotic, and dead cells (representative control images of live cell apoptosis staining with acridine orange for green viable cells and ethidium bromide for red apoptotic cells; Fig. 6A). MPC were more susceptible to apoptosis under hypoxia (staining with Hoechst 33342 for blue apoptotic cells, with a faint background staining of live cells, and propidium iodide for red dead cells; Fig. 6B), but were protected by the presence of glucose in the cell culture medium (numbers compared with normoxia with glucose category as control; Fig. 6D). Overall, hypoxia and glucose depletion were additive in their proapoptotic effects, leading to decreased live cell numbers after 2 days (Fig. 6D). The quantification for apoptotic cells (Fig. 6C) demonstrates a significant increase in apoptosis under hypoxia in the presence of glucose; for the hypoxic, glucose-depleted setting, the measurement likely underestimates the considerable apoptotic effect because programmed cell death occurs rapidly and thereby foregoes direct observation after 2 days, particularly in this category. Under hypoxia, fixed podocytes with hallmark features of apoptosis, such as chromatin condensation and pyknotic nuclei, showed translocation of apoptosis-inducing factor to the nucleus (Fig. 6E). This association was also observed in apoptotic nuclei under normoxia; however, the degree of apoptosis was marginal compared with hypoxic conditions.


Figure 6
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  		Fig. 6. Podocyte hypoxia leads to apoptosis, associated with nuclear translocation of apoptosis-inducing factor (AIF). A: control images demonstrate good correlations between fluorescent signal and nuclear morphology in apoptotic cells [live cell apoptosis staining with acridine orange for viable cells (green), and ethidium bromide for apoptotic cells with nuclear condensation and fragmentation (red); original magnification x600]. B–D: increased apoptosis and decreased cell viability of differentiated MPC podocytes under hypoxia, especially under simultaneous glucose withdrawal (normoxia vs. 0.5% O2 for 2 days; 200 vs. 0 mg/dl glucose). B: live cell apoptosis staining with propidium iodide for dead cells (red) and with Hoechst 33342 for differential staining between apoptotic cells with condensed chromatin (intense blue) and viable cells (faint background blue); original magnification x400]. C and D: quantification of apoptotic podocytes (C) and remaining live cell number (D) numbers compared with normoxia with glucose category as control). E: podocyte apoptosis under hypoxia (0.5% O2 for 2 days) is associated with translocation of AIF into the nucleus (immunofluorescence; original magnification x400). F: undifferentiated podocytes have a delayed onset of proliferation under hypoxia [5-bromo-2'-deoxy-uridine (BrdU) incorporation; 0.5% O2 for 2 days). G: moderate prolonged hypoxia (0.5% O2 for 7 days) leads to fat accumulation in differentiated podocytes (oil red O staining; original magnifications x400 and x600 for top and bottom, respectively). Note also the decreased cell density for hypoxia compared with normoxia samples. H: quantification of podocyte number with neutral fat accumulation (see G). *P < 0.05, **P < 0.01 and ***P < 0.001.

 
Cell cycle analysis by flow cytometry was performed for undifferentiated, proliferating podocytes after exposure to hypoxia for 2 days and revealed a prominent increase in the percentage of MPC podocytes in the G2 phase and of CLL podocytes in the S phase, while both MPC and CLL cells showed a modest drop in G0/G1 phase (Fig. 5E). MPC podocytes did not reveal any evidence for an increased proliferative ability under hypoxia, but rather an initial decrease that was indicated by lower BrdU incorporation after 1 day, which became indistinguishable from normoxic controls at 2 days (Fig. 6F).

Hypoxia regulates stress genes and cell adhesion in podocytes. A microarray analysis of podocytes exposed to hypoxia aided in evaluating the global transcriptional response to low-oxygen conditions (0.5% O2 for 24 h; Table 2). The Significance Analysis of Microarrays (SAM) testing revealed 1,174 differentially regulated genes with a false discovery rate (FDR) of 0.66%. Three hundred and forty-three genes exhibited a change of >1.4-fold. Known HIF target genes were upregulated, such as those related to glucose metabolism, hypoxia, iron metabolism, and Bnip3 (Table 2). Conversely, genes implicated in the ubiquitin pathway as well as in transcription and translation were suppressed (Table 2).


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  		Table 2. Microarray analysis

 
The microarray assay also revealed groups of genes that may carry a previously unknown significance of podocyte response to hypoxic stress. For example, gene transcripts for migration inhibitory factor, metallothionein 2, and immediate early response genes were increased (Table 2). Factors that regulate cell-cell adhesion and stabilization of extracellular matrix were upregulated as well, such as desmoplakin, lysyl oxidase-like 2 gene, and procollagen lysine dioxygenase. Among these genes, migration inhibitory factor, metallothionein 2, lysyl oxidase-like 2, and procollagen lysine dioxygenase were confirmed as hypoxia-regulated genes by quantitative real-time PCR (data not shown). A further interesting phenomenon was the differential regulation of enzymes and proteins relevant for lipid metabolism (Table 2). Overall, genes were only regulated modestly on the transcription level, e.g., steaoryl-coenzyme A desaturase, sterol element binding protein (SREBP), acyl-coenzyme A synthetase, and apolipoprotein E (1.5- to 3.5-fold; confirmed by quantitative real-time PCR; data not shown), while adipose differentiation-related protein (Adrp, also known as Adfp or ADPH) did not exhibit any significant change in transcription (data not shown). However, the significance of lipid metabolism regulation by hypoxia is clearly demonstrated by the accumulation of neutral fat in differentiated podocytes after 1 wk of exposure to 0.5% O2 (Fig. 6, G and H).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Prolonged exposure to low oxygen has detrimental effects on podocyte survival. Programmed cell death could be observed more frequently both in hypoxic and in VHL-deficient podocytes with the common feature of HIF1 stabilization, which implies that apoptosis under hypoxia may be mediated by HIF1. In parallel, VHL deficiency in podocytes of in vivo mouse models of constitutive HIF stabilization resulted in glomerulomegaly at a young age and occasional glomerulosclerosis. Glomerular tuft increase may have resulted from hyperfiltration as a compensatory mechanism in some glomeruli to balance nephron loss due to damage of other glomeruli, and potential secondary capillary proliferation in glomeruli could also be explainable through endothelial cell regulation across the glomerular basement membrane by podocytes with HIF stabilization. Overall, podocytes responded to hypoxia not only by induction of known HIF target genes such as Vegf and Wt1, but also of genes related to stress response, antitoxic and potentially cytoprotective genes, and genes known for establishing cell-cell contacts and increased extracellular matrix.

During glomerular development, HIF1 and HIF2 are abundantly expressed in podocytes, and VEGF is as well (2, 12). The lack of immediate nuclear stabilization of HIF2 observed here in MPC does not rule out relevance of HIF2 in vivo. The importance of apoptosis-inducing factor (AIF) in podocytes has previously been demonstrated for puromycin aminonucleoside-induced apoptosis (38). Gene expression changes under hypoxia seen in the microarray analysis could be classified into several groups with concerted effects. Upregulation of lysyl oxidase-like 2 and procollagen lysine dioxygenase may act synergistically to enhance cell-cell interaction and extracellular matrix stability. Lysyl oxidase has recently been identified as a crucial factor of HIF-dependent metastatic growth because it increases cancer cell motility and invasion as well as cell-matrix adhesions (9). Notably, migration inhibitory factor (MIF), which is directly regulated by HIF via an HRE (1), is expressed in podocytes and mesangial cells during lipid-induced renal injury (14), and MIF transgene overexpression in podocytes results in progressive mesangial sclerosis and end-stage renal disease in mice (31).

In this study, both hypoxic and VHL-deficient podocytes had a propensity to undergo apoptosis, and undifferentiated MPC and CLL did not show an appropriate increase in cell number during proliferation and exhibited changes in the cell cycling profile under hypoxia. While parallel effects of hypoxia and VHL deficiency in podocytes propose mediation through HIF stabilization as the final common pathway, a definitive study with podocyte-specific HIF1{alpha}, HIF2{alpha}, and HIF1beta, in addition to VHL, deletion is still under investigation in the senior author's laboratory. Reconciliation of the survival, apoptosis, and cell cycle studies are suggestive of cell cycle arrest of podocytes under hypoxia and an exit to apoptosis potentially in the G1 phase. Abortive cell cycle reentry is frequently observed as a mechanism of apoptosis (8). Obviously, the data obtained do not allow direct conclusions about cell cycle regulation in differentiated podocytes in vivo.

The proliferative and apoptotic features of VHL loss may depend on the cell type, as VHL-negative, HIF1-expressing fibrosarcomas and murine embryonic fibroblasts exhibit a proliferation defect without an increase in apoptosis (23, 24), whereas VHL loss leads to apoptosis in thymocytes via a caspase 8-dependent mechanism (3). The balance of pro- and antiapoptotic factors through differential gene regulation under hypoxia may be tilted in favor of apoptosis in a complex manner consistent with the rheostat hypothesis (20). Given the observation of delayed apoptosis, some effects of HIF1 stabilization may actually be beneficial for podocytes in the early stage of hypoxia and then wane later on, allowing cell death. For example, induction of Vegf, Wt1, metallothionein 2, Mxi1 (26), pyruvate dehydrogenase kinase 1 (Pdk) (17), and genes of stress response may confer a cytoprotective effect. Metallothionein 2 expression is an anticytotoxic mechanism that likens podocytes to astrocytes, and it may be relevant for xenobiotic defense in the vicinity of the capillary wall (4).

The lack of a prominent disease pattern in vivo is consistent with the absence of relevant glomerular manifestations in human VHL disease. VHL disease is a familial tumor syndrome that can present with cyst and tumor formation, which solely originates from distinct cell types, such as hemangiomas, pancreatic cysts, and renal cell cancer derived from renal tubular epithelial cells (22). Affected patients inherit one mutated Vhlh allele, and according to the two-hit hypothesis frequently explaining malignant degeneration (19), another insult to the remaining allele may mediate tumor formation. It is conceivable that all cell types of VHL patients are equally exposed to these secondary insults. Therefore, the particular pattern of tumor formation indicates that only specific cell types are susceptible to the effects of functional VHL loss as a precancerous and preproliferative event. Obviously, evaluation of VHL and HIF biology in cell types not usually affected by VHL-associated tumors is scientifically relevant as well. Glomerular disease is not a manifestation of human VHL disease, and podocytes may be inert to the loss of this tumor suppressor gene because of their proposed terminally differentiated status and probably less active metabolism compared with renal tubular epithelial cells under normal conditions.

Two other groups (34, 7) have examined the role of podocyte-specific Vhlh deletion via the same transgenic approach. Some of the histological manifestations in one group (34) are similar to those seen in our diseased mice, although the preliminary description is most comparable to a human manifestation of immune complex disease. The other laboratory (7) mainly observed podocyte-derived crescent formation with nephrotic-range proteinuria and severely decreased life expectancy with the death of the majority of mice within 3 mo, despite initially normal glomerular development. Rapidly progressive glomerulonephritis (RPGN) would liken the phenotype to proproliferative lesions of VHL disease. Although this finding may provide insight into the role of some HIF target genes in glomerular disease pathogenesis, RPGN due to Vhlh mutations in murine podocytes does not constitute a disease model for a parallel phenomenon in human VHL patients, and as such its significance for human pathophysiology is difficult to assess. The differences in phenotypes may be the result of environmental conditions and the murine strain background, two factors that have already been demonstrated to modulate glomerular disease pathogenesis (30, 10). Disease patterns may be ameliorated by compensatory mechanisms or redundant pathways in healthy mice, which may counteract the effect of a mutation in one single gene. Mouse exposure to systemic hypoxia (35) leads to glomerulomegaly and VEGF upregulation, results that are consistent with our data.

In summary, hypoxia may contribute to progression of human glomerular disease by altering podocyte metabolism and survival. Podocytes are exposed to low-oxygen conditions in diseases affecting primarily the vascular compartment of the glomerulus. This applies to acute ischemia as well as vasculitis, endocapillary proliferation, thrombotic microangiopathy, and capillary loss due to glomerulosclerosis and mesangial proliferation. Although constitutive HIF1 stabilization in podocytes does not prevent glomerular development and maintenance per se, it appears to confer an increased risk of glomerular defects, namely, glomerulomegaly and glomerulosclerosis. This phenomenon may play a role in human glomerular disease progression, as podocyte hypoxia can result from systemic and vascular disorders and from local effects of diseased endothelial and mesangial cells. Additionally, steady podocyte loss under hypoxia offers novel hypotheses about the pathophysiological explanation for human glomerular disease related to obstructive sleep apnea, which is characterized by proteinuria, glomerular hypertrophy, and focal-segmental glomerulosclerosis (11, 18).


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health (NIH) Grant R01-CA-100787 and seed money from the Department of Medicine to V. H. Haase and Morphology Core Center Grant P30-DK-50306. K. Brukamp was the recipient of a National Kidney Foundation Postdoctoral Research Fellowship from July 2005 to June 2006.


   ACKNOWLEDGMENTS
 
We thank Erinn B. Rankin, Mangatt P. Biju, Gary Swain, Tim Cash, Peter White, John Tobias, the Morphology Core Facility staff, and the Flow Cytometry and Cell Sorting Facility staff for valuable technical advice; Jana Havranova and Jennifer Rha for technical assistance; John E. Tomaszewski for review of mouse histopathology; Lawrence B. Holzman, Peter Mundel, and Russ Carstens for sharing reagents; and M. Celeste Simon, Brian Keith, Michael P. Madaio, and Vicente E. Torres for helpful discussions.

V. H. Haase created the Vhlh 2lox allele transgenic mice, and M. J. Moeller and Lawrence B. Holzman contributed the NPHS2-Cre recombinase transgenic mice. V. H. Haase performed the breeding and allocation of transgenic mice for experiments, and B. Jim and V. H. Haase conducted the in vivo phenotype analysis, except for the morphometric analysis. K. Brukamp carried out the majority of the remaining experiments independently under V. H. Haase's supervision and direction.

Parts of this study have been presented at the American Society of Nephrology meetings in St. Louis, MO, in 2004, in Philadelphia, PA, in 2005, in San Diego, CA, in 2006, as well as at the National Kidney Foundation meeting in Chicago, IL, in 2006 and the Experimental Biology meeting in Washington, DC, 2007.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Brukamp, Massachusetts General Hospital, Harvard Medical School, Dept. of Medicine, Nephrology Div., 149 13th St., 8th floor, Charlestown/Boston, MA 02129

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* Present address of B. Jim: Jacobi Medical Center, Albert Einstein College of Medicine, Bronx, NY. Back


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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