Formation of Renal Cysts and Tumors in -Deﬁcient Mice ... · renal cysts and tumors inVhl/Trp53...

Tumor and Stem Cell Biology

Formation of Renal Cysts and Tumors inVhl/Trp53-Deficient Mice Requires HIF1a andHIF2aD�esir�ee Sch€onenberger1, Sabine Harlander1,2, Michal Rajski1, Robert A. Jacobs1,2,3,Anne-Kristine Lundby1,2, Mojca Adlesic1, Tomas Hejhal1, Peter J.Wild4,Carsten Lundby1,2, and Ian J. Frew1,2

Abstract

The von Hippel–Lindau (VHL) tumor suppressor gene is inac-tivated in the majority of clear cell renal cell carcinomas (ccRCC),but genetic ablation of Vhl alone in mouse models is insufficientto recapitulate human tumorigenesis. One function of pVHL is toregulate the stability of the hypoxia-inducible factors (HIF),which become constitutively activated in the absence of pVHL.In established ccRCC,HIF1ahas been implicated as a renal tumorsuppressor, whereas HIF2a is considered an oncoprotein. In thisstudy, we investigated the contributions of HIF1a and HIF2a toccRCC initiation in the context of Vhl deficiency. We found thatdeleting Vhl plus Hif1a or Hif2a specifically in the renalepithelium did not induce tumor formation. However, HIF1aand HIF2a differentially regulated cell proliferation, mito-

chondrial abundance and oxidative capacity, glycogen accu-mulation, and acquisition of a clear cell phenotype in Vhl-deficient renal epithelial cells. HIF1a, but not HIF2a, inducedWarburg-like metabolism characterized by increased glycoly-sis, decreased oxygen consumption, and decreased ATP pro-duction in mouse embryonic fibroblasts, providing insightsinto the cellular changes potentially occurring in Vhl mutantrenal cells before ccRCC formation. Importantly, deletion ofeither Hif1a or Hif2a completely prevented the formation ofrenal cysts and tumors in Vhl/Trp53 mutant mice. These find-ings argue that both HIF1a and HIF2a exert protumorigenicfunctions during the earliest stages of cyst and tumor forma-tion in the kidney. Cancer Res; 76(7); 2025–36. �2016 AACR.

IntroductionClear cell renal cell carcinoma (ccRCC) is the most frequent

renal malignancy, and up to 92% of ccRCC tumors harborbiallelic inactivation of the von Hippel–Lindau (VHL) tumorsuppressor gene (1). VHL mutations occur at the earliest stageof tumor formation (2). The absence of pVHL function is clearlynecessary for the growth of fully transformed ccRCC cell lines asxenografts (3). However, the fact that the frequency of VHLmutant single epithelial cells vastly outweighs the number ofccRCC tumors in kidneys of familial VHL disease patients (4) andthe absence of tumors in a variety of renal epithelial cell–specificVhl knockout mice (reviewed in ref. 5) argues that ccRCC forma-tion requiresmutations in addition toVHL. There are currently no

autochthonous mouse models that fully reproduce all of thecharacteristic morphologic and invasive properties of humanccRCC; however, consistent with the hypothesis that multiplecooperating mutations are required for ccRCC development,kidney epithelial cell–specific codeletion of Vhl with Pten (6), orwith Kif3a to genetically ablate primary cilia (7), caused theformation of simple and atypical cystic lesions that are similarto the ccRCC precursor cystic lesions found in the kidneys ofpatients with inherited VHL disease. Deletion of Vhl together withhomozygous loss of Trp53 (8) or with heterozygous loss of Bap1(9) gave rise to very similar phenotypes, including simple andatypical cystic lesions as well as tumors containing cells thatdisplay cytoplasmic clearing and elevated mTORC1 activity, reca-pitulating many of the cellular and molecular changes that arecharacteristic of human ccRCC.

pVHL controls many biological activities, including regulationof the stability of the hypoxia-inducible transcription factors a(HIF1a, HIF2a, andHIF3a, collectively HIFa; ref. 10), regulationof NF-kB activity (11), maintenance of the primary cilium (12),activation of p53 (13), secretion of extracellular matrix compo-nents (14), promotion of DNA double-strand repair (15), regu-lationof theplaneof cellular division (16, 17), and suppression ofaneuploidy (16, 18). The combined loss of several or all of pVHL'sfunctions could contribute to tumor initiation and progression.Loss of function mutations of VHL occur in ccRCC mutuallyexclusively to rarer mutations in TCEB1, encoding Elongin C,and collectively these cause constitutive stabilization of HIFa inup to 95%of ccRCC tumors (1), implying thatHIFa activation is amajor oncogenic driving force in ccRCC.HIF1a andHIF2a appear

1Institute of Physiology, University of Zurich, Zurich, Switzerland.2Zurich Center for Integrative Human Physiology, University of Zurich,Zurich, Switzerland. 3Health and Physical Education, School of Teach-ing and Learning,Western Carolina University, Cullowhee, North Car-olina. 4Institute of Surgical Pathology, University Hospital Zurich,Zurich, Switzerland.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

D. Sch€onenberger, S. Harlander, and M. Rajski contributed equally to this article.

Corresponding Author: Ian J. Frew, University of Zurich, Winterthurerstrasse190, CH-8057 Zurich, Switzerland. Phone: 41-44-635-5004; Fax: 41-44-635-6814; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1859

�2016 American Association for Cancer Research.

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to play opposite roles in determining the aggressiveness of estab-lished ccRCCs; HIF1a inhibits, whereas HIF2a promotes tumorformation in ccRCC xenografts (19–21), copy loss of the chro-mosomal locus harboring the HIF1A gene predicts poor patientoutcome (22), and sporadic ccRCCs expressingHIF1a andHIF2aexhibit lower proliferation rates than ccRCCs expressing onlyHIF2a (23). ccRCC cell lines frequently express only HIF2a anddo not express functional HIF1a due to biallelic alterations of theHIF1A locus (24). Knockdown of HIF1A in ccRCC cell lines thatexpress both HIF1a and HIF2a promotes xenograft tumor for-mation (24). These studies argue that whereas HIF2a activity istumor promoting, there is a selection against HIF1a expression oractivity during the progression of some cases of ccRCC. On theother hand, because HIF1a is strongly expressed in single andmulticellular clusters ofVHL-null cells and in cystic lesions inVHLpatients (4), and because transgenic overexpression of HIF1a inmouse proximal tubular epithelial cells causes a clear cell appear-ance, increased proliferation, and a disorganized tubular mor-phology (25), and because many human ccRCCs express HIF1aand HIF1a target genes (26), it may also be argued that HIF1aplays an important role in promoting ccRCC development. Thesestudies have largely focused on fully transformed, geneticallycomplex ccRCC cell lines derived from advanced tumors, and itremains unclear if and how HIF1a and HIF2a contribute to theearliest stages of initiation of ccRCC.

An early event following VHL mutation is likely to be aprofound alteration of cellular metabolic pathways. NumerousHIF1a and/or HIF2a-dependent metabolic changes occur inccRCC cell lines, including elevated glucose uptake and conver-sion to lactate with a concomitant reduction in mitochondrialoxidation of pyruvate, reduced mitochondrial biogenesis,reduced mitochondrial complex I activity, altered cytochrome coxidase activity, increased pentose phosphate pathway flux,decreased oxidative glutaminolysis, and increased lipogenesisthrough reductive glutamine metabolism (27–38). BecauseccRCC cell lines have complex genetic backgrounds that aroseduring tumor evolution, it is possible that some of these meta-bolic changes might be driven by late-occurring mutations thatfurther modify metabolic pathways and allow ccRCC cells toproliferate efficiently in the metabolic environment imposed byconstitutive HIFa activity. For example, ccRCCs can harbor acti-vating mutations in the PI3K–mTORC1 pathway, inactivatingTP53 mutations or can exhibit high levels of MYC expressionor decreased fructose-1,6-bisphospatase 1 (FBP1) expression(1, 38, 39), all of which are known to influence numerous cellularmetabolic pathways. The metabolic alterations that follow VHLmutation in otherwise genetically normal renal epithelial cells atthe start of the process of tumor formation remain unclear.

Given the importance of HIF1a andHIF2a in ccRCC, we askedwhether the loss or constitutive activation of HIF1a or HIF2aalters renal epithelial proliferative homeostasis, metabolism, andthe initiation of renal cysts and tumors by undertaking a system-atic approach of genetic deletion ofHif1a orHif2a alone, togetherwith Vhl and together with Vhl/Trp53 in renal epithelial cells inmice.

Materials and MethodsMouse strains

Hif1afl/fl (40) and Hif2afl/fl (41) mice were crossed with Ksp1.3-Cre;Vhlfl/fl (6),Ksp1.3-Cre;Vhlfl/fl;Trp53fl/fl (8), orKsp1.3-Cre/þ (42)

mice. Littermatemice that lacked the Cre transgene served aswild-type controls.

ImmunohistochemistryImmunohistochemistry and immunofluorescence of formalin-

fixed paraffin-embedded tissues were performed as previouslydescribed (6, 8) using antibodies listed in Supplementary Materi-als and Methods.

RespirometryBiopsies (1 mm3) of renal cortex andmedulla were assayed for

oxygen consumption following the addition of different respira-tory substrates and inhibitors using high-resolution respirometry(Oxygraph-2k, OROBOROS INSTRUMENTS Corp) according tothe protocol described in Supplementary Materials andMethods.

MicroarraysPrimary adult mouse renal epithelial cells were cultured for 3

days and infected with Adeno-GFP (Vector Biolabs, 1060) orAdeno-Cre (Vector Biolabs, 1700) as described (8). mRNAwas isolated 4 days after adenovirus infection, and Cy3- andCy5-labeled cDNA from GFP and Cre samples was competitivelyhybridized to Mouse GE 4 � 44 K v2 Microarrays (Agilent). Dyeswap experiments were used to control potential dye-specifichybridization effects. Analysis using R/Bioconductor packagelimma was based on the average fold expression level changes(Cre/GFP) of three independent biologic experiments.Microarraydata are accessible through GEO number GSE75745.

Histological stainsHematoxylin and eosin (H&E) and periodic acid-Schiff (PAS)

stains were conducted using standard protocols. For the visual-ization of lipids, frozen kidney sections were fixed with 10%formalin for 15minutes, washed in tapwater, equilibrated in 60%isopropanol, and stained with Oil Red O (3 mg/mL in 60%isopropanol) overnight at 4�C. After rinsing in 60% isopropanol,sections were washed with water, counterstained with hematox-ylin, and mounted in Mowiol.

Real-time PCRRNA isolation, cDNA preparation, and real-time PCR were

performed as described (6) using primers listed in SupplementaryMaterials and Methods.

Mouse embryo fibroblastsMouse embryo fibroblasts (MEF) were isolated from E13.5

embryos of relevant non–Cre-expressing floxed mouse strains orC57BL/6 embryos (for wild-type MEFs) and cultured as previ-ously described (8) at 5% O2 and 5% CO2. MEFs were infectedwith adenovirus expressing Cre recombinase and GFP (Ad-Cre-GFP; Vector Biolabs; #1700) or GFP only (Ad-CMV-GFP; VectorBiolabs; #1060). Detailed protocols for measuring ATP, extracel-lular acidification rate, oxygen consumption rate (OCR), lactateproduction, glucose utilization, and mitochondrial content aredescribed in Supplementary Materials and Methods.

Statistical analysesUnless otherwise stated, data are presented as mean � SD,

and statistical differences were assessed using the Student

Sch€onenberger et al.

Cancer Res; 76(7) April 1, 2016 Cancer Research2026



unpaired t test. �, ��, and �� denote P < 0.05; P < 0.01; andP < 0.001, respectively.

ResultsVhl deletion together with deletion of Hif1a or Hif2a does notcause renal tumors

Most cases of ccRCC appear to arise from the proximaltubule, although there is also evidence that some cases canarise from cells in other nephron segments (5). We employedthe Ksp1.3-Cre transgene that induces epithelial cell–specificgene deletion widely throughout different segments of thenephron, including in a significant number of proximal tubularcells, as well as in epithelia of several genital tract tissues (42).We generated Ksp1.3-Cre;Vhlfl/fl (VhlD/D; ref. 6), Ksp1.3-Cre;Hif1afl/fl (Hif1aD/D), Ksp1.3-Cre;Hif2afl/fl (Hif2aD/D), Ksp1.3-Cre;Vhlfl/fl;Hif1afl/fl (VhlD/DHif1aD/D), and Ksp1.3-Cre;Vhlfl/fl;Hif2afl/fl (VhlD/DHif2aD/D) mice to achieve renal epithelial–specific loss or constitutive stabilization of HIF1a and/or HIF2a(Fig. 1A and B). Immunohistochemical staining confirmed theexpected stabilization of either or both HIF1a and HIF2a in therelevant Vhlmutant genotypes (Fig. 1C and D). All of the above-described mutant mice were analyzed at 6, 12, and 18 months ofage. Neither Hif1a nor Hif2a deletion alone had any effect on themorphology of kidneys. Vhl/Hif1a double deletion, but not Vhl/Hif2a double deletion, fully rescued (data not shown) the previ-ously reported hydronephrosis phenotype caused by Vhl deletion(6). This phenotypic rescue will be described in detail elsewhere.

VhlD/D mice do not develop ccRCC precursor lesions or tumors(6). We reasoned that HIF1a might act as an antiproliferativefactor that prevents tumor development following Vhlmutation.However, none of the genotypes exhibited cysts, dysplasticlesions, or tumors in the cortex, medulla, or papilla even whenaged for 18 months. Tubular epithelial cells in VhlD/DHif2aD/D

mice frequently displayed a highly unusual "optically-clear"nucleus, characterized by a thin ring of chromatin surroundinga nonstained region in the center (Supplementary Fig. S1). Pap-illary thyroid carcinomas display optically clear nuclei, but thishistologic feature does not arise in ccRCCs. We conclude that thecombination of loss of the many putative tumor suppressorfunctions of pVHL plus constitutive expression of the putativeoncoprotein HIF2a plus the absence of the putative tumor sup-pressor activity of HIF1a does not cause cyst or tumor initiation.

Hif1a and Hif2a are both necessary for renal cyst and tumorformation caused by Vhl/Trp53 deletion

We next investigated potential requirements for Hif1a orHif2ain the initiation of renal cysts and tumors in the Vhl/Trp53 doublemutant background (8). We generated Ksp1.3-Cre;Vhlfl/fl;Trp53fl/fl;Hif1afl/fl (VhlD/DTrp53D/DHif1aD/D) and Ksp1.3-Cre;Vhlfl/fl;Trp53fl/fl;Hif2afl/fl (VhlD/DTrp53D/DHif2aD/D) mice (Fig. 2A and B) andconfirmed HIF1a and HIF2a stabilization in the relevant geno-types (Fig. 2C and D). PCRs specific for the recombined Vhl andTrp53 alleles demonstrated that these genes were deleted efficient-ly in these mice (Supplementary Fig. S2). We analyzed cohorts at6 and12months of age. Table 1 shows a summaryof the incidence

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Figure 1.Renal epithelial cell–specific deletion of Vhl, Hif1a, Hif2a, Vhl/Hif1a, and Vhl/Hif2a. H&E stainings of renal cortex (A) and medulla (B), and immunohistochemicalstainings for HIF1a (C), HIF2a (D), and GLUT1 (E) in the renal cortex.

HIF1a and HIF2a Are Renal Oncogenes

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Figure 2.Deletion of Vhl/Trp53, Vhl/Trp53/Hif1a, and Vhl/Trp53/Hif2a in renal epithelial cells. A and B, H&E stainings of renal cortex from mice aged 6 months (A)and 12 months (B). Arrowheads, disorganized tubules with clear cell cytoplasm; arrows, optically clear nuclei; Cy, cysts; Neo, neoplasm. C–E, immunohistochemicalstainings for HIF1a (C), HIF2a (D), and GLUT1 (E) in the renal cortex. F, H&E stainings of vesicular glands from mice aged 6 months. G and H, H&Estainings of epididymides from mice aged 12 months.





of different phenotypes in each genotype, and Fig. 2 showsexamples of these phenotypes. We previously demonstrated(8) that VhlD/DTrp53D/D mice display hydronephrosis, seminalvesicle developmental abnormalities (Fig. 2F), subfertility, andsubviability, and by 12months of age, these mice develop simpleand atypical kidney cysts (Fig. 2B), kidney tumors (Fig. 2B),disorganized and multilayered epididymal epithelial cell growth(Fig. 2G), epididymal squamous metaplasia (Fig. 2H), andbenign epididymal tumors at high penetrance. About one thirdof these mice also develop a variety of genital-urinary tractcarcinomas (8). All of these phenotypes were completely absentin VhlD/DTrp53D/DHif1aD/D mice. Six-month-old VhlD/DTrp53D/D

mice frequently displayed disorganized renal tubular epitheliawith a clear cell appearance, but this phenotypewas not present inVhlD/DTrp53D/DHif1aD/D mice (Fig. 2A). These mice developedonly a few micro-cysts at a frequency that was not higher thanlittermate control mice (data not shown), and importantlyno large renal cysts or tumors arose in mice aged 12 months(Fig. 2B, Table 1). Epididymides of VhlD/DTrp53D/DHif1aD/D micedisplayed nuclear atypia and mild epithelial disorganization(Fig. 2G and H), similar to the phenotypes observed in youngerVhlD/DTrp53D/Dmice (8), but did not develop epithelial dysplasiaor squamous metaplasia. No malignant tumors were observed inany other genital-urinary tract organs in these mice (Table 1).Thus, HIF1a activity is indispensible for the initiation of kidneycyst and tumor formation and for the formation of genital-urinarytract malignancies.

In contrast, VhlD/DTrp53D/DHif2aD/D mice displayed severalidentical phenotypes to VhlD/DTrp53D/D mice, including hydro-nephrosis (data not shown), seminal vesicle developmentalabnormalities (Fig. 2F), disorganized epididymal epithelialgrowth (Fig. 2G), and epididymal squamous metaplasia(Fig. 2H), although these epididymal phenotypes were presentin only half of the mice (Table 1) and were much smaller lesionsthan those seen in VhlD/DTrp53D/D mice. Similarly to VhlD/D

Trp53D/D mice, VhlD/DTrp53D/DHif2aD/D mice were also subfertileand subviable, with many mice dying at various ages, possiblydue to kidney failure caused by excessive hydronephrosis. While6-month-old VhlD/DTrp53D/DHif2aD/D mice displayed disorga-nized renal tubular epithelia with optically clear nuclei(Fig. 2A), no large renal cysts or tumors (Fig. 2B), nor genital-urinary tract carcinomas were observed in 12-month-old mice.Thus, many of the preneoplastic and all of the neoplastic phe-notypes resulting from combined deletion of Vhl and Trp53 arealso dependent on HIF2a activity.

HIF1a and HIF2a both contribute to increased cellularproliferation in Vhl and Vhl/Trp53 mutant mice

Because HIF1a and HIF2a have been implicated in regulatingthe proliferation of ccRCC cells, we analyzed the roles of HIF1a

and HIF2a in proliferative control in mouse renal epithelia. Ki67staining to label proliferating epithelial cells in the renal cortexrevealed that tubules in 6-month-old Vhl knockout mice dis-played an increased number of proliferating cells. This pheno-type was completely rescued by codeletion of Hif1a and partiallyrescued by codeletion of Hif2a (Fig. 3A and C), indicating amajor and minor role of HIF1a and HIF2a, respectively, incausing increased cellular proliferation. We next asked whetherthe rescue of cyst and tumor formation in the Vhl/Trp53 mutantbackground was due to a requirement for HIF1a or HIF2a incausing enhanced proliferation. In fact, Ki67 labeling revealedelevated numbers of proliferating epithelial cells in the renalcortices of 12-month-old VhlD/DTrp53D/D, VhlD/DTrp53D/DHif1aD/D,and VhlD/DTrp53D/DHif2aD/D mice in comparison with littermatecontrol mice (Fig. 3B and D), indicating that increased cellularproliferation is not the sole cause of cyst and tumor formation.Inter-genotype comparisons revealed that deletion of eitherHif1aor Hif2a lowered proliferation in the Vhl/Trp53 mutant back-ground with loss of Hif1a causing a stronger decrease in prolif-eration than loss of Hif2a. The absence of a complete rescue byHif1a deletion in the Vhl/Trp53 mutant background, in compar-ison with the complete rescue in the Vhl mutant background,argues that loss of Trp53 also contributes to increased prolifera-tion in the Vhl/Trp53 mutant background.

HIF1a regulates glycolysis, oxidative metabolism, andmitochondrial abundance in renal epithelial cells

To gain further molecular insights into HIF1a- and HIF2a-dependent processes in Vhl-null cells that might be necessary fortumor initiation, we developed a system to culture primary renalepithelial cells derived fromkidneys of adultVhlfl/fl,Vhlfl/flHif1afl/fl,Vhlfl/flHif2afl/fl, and Vhlfl/flHif1afl/flHif2afl/fl mice. Epithelial mor-phology (Supplementary Fig. S3A),Cdh1mRNA expression (Sup-plementary Fig. S3B), and E-Cadherin protein expression (Sup-plementary Fig. S3C) were maintained for at least 12 days ofculture, implying that the epithelial phenotype of these cells islargely preserved in this culture system. Cultures were infectedwith adenovirus expressing GFP as control or with adenovirusexpressing Cre (Fig. 4A), and real-time PCR confirmed the dele-tion of the floxed genes (Supplementary Fig. S3D). Microarraycomparisons of global mRNA expression 4 days after viral infec-tion revealed a transcriptional signature induced by loss of Vhlthat included 753 probes that were significantly (P < 0.05)upregulated more than 1.5-fold and 451 probes downregulatedmore than 1.5-fold. This transcriptional signature was used toprobe mRNA expression data derived from human normalkidney and ccRCC samples (GSE17895). Unsupervised clusteringusing the Vhl-deletion gene signature accurately segregatedhuman ccRCCs from normal kidney tissue (SupplementaryFig. S4), demonstrating that our culture system can identify

Table 1. Summary of phenotypes in VhlD/DTrp53D/D, VhlD/DTrp53D/DHif1aD/D, and VhlD/DTrp53D/DHif2aD/D mice aged 11 to 13 months

VhlD/DTrp53D/D VhlD/DTrp53D/DHif1aD/D VhlD/DTrp53D/DHif2aD/D

Number of renal cysts 399 (n ¼ 30 kidneys) 15 (n ¼ 30 kidneys) 10 (n ¼ 22 kidneys)Number of renal tumors 16 (n ¼ 30 kidneys) 0 (n ¼ 30 kidneys) 0 (n ¼ 22 kidneys)Number of genital-urinary tract carcinomas 6 (n ¼ 17 mice) 0 (n ¼ 15 mice) 0 (n ¼ 18 mice)Incidence of epididymal dysplasia/squamous metaplasia 100% (n ¼ 9 mice) 0% (n ¼ 7 mice) 50% (n ¼ 8 mice)Incidence of seminal vesicle abnormality 100% (n ¼ 9 mice) 0% (n ¼ 7 mice) 100% (n ¼ 8 mice)

NOTE: Numbers of renal cysts and renal tumors are derived from analysis of midline longitudinal sections of n kidneys of each genotype. Numbers of genital-urinarytract carcinomas represent the cumulative number of tumors arising in the epididymis, seminal vesicle, uterus, and urothelium of n mice of each genotype. Theincidence of epididymal and seminal vesicle phenotypes represents the percentage of n mice of each genotype that displays the listed phenotypes.





ccRCC-relevant gene expression changes. We identified genes thatwere upregulated by loss ofVhl and that were dependent solely onHif1aor solely onHif2aor that remained upregulatedwhen eitherHif1a or Hif2a were deleted but not when both were deleted,indicating that they are targets of both HIF1a andHIF2a (Fig. 4B,Supplementary Table S1). While theHif2a-dependent andHif1a/Hif2a-dependent gene sets were not strongly enriched for sets ofgenes that participate in particular biological processes, theHif1a-dependent target genes (Fig. 4C) encompassed numerous previ-ously identified hypoxia and HIFa targets (Bnip3, Bnip3l, Egln3,Loxl2, Ak4, Car9, Vegfa, P4ha2, Mif), including genes that regulatecellular metabolism (Supplementary Fig. S5) by promoting gly-colytic flux (Slc2a1, Gpi1, Pfkl, Pfkfb3, Aldoa, Aldoc, Tpi1, Gapdh,Pgk1, Pgam1, Eno1), promoting glycogenolysis (Pygl, Pgm2),diverting glucose-derived carbon from mitochondrial oxidationto lactate production and excretion (Pdk1, Ldha, Slc16a3), orregulating mitochondrial electron transport by inhibiting com-plex I (Ndufa4l2; ref. 36). Real-time PCR analyses of mRNAisolated from VhlD/D, VhlD/DHif1aD/D, and VhlD/DHif2aD/D kidneysconfirmed that several of these gene expression changes (Pgk1,Pdk1, Pfkfb3, Ldha, Ndufa4l2), as well as upregulation of Cox4-2,which alters the efficiencyof cytochrome coxidase (37), alsooccurupon Vhl deletion in vivo and demonstrated that, with theexception of Ndufa4l2, all were strictly dependent on Hif1aand independent ofHif2a (Fig. 4D). A similarHif1a-dependent,Hif2a-independent metabolic gene expression signature wasobserved in cultured primary renal epithelial cells (Supplemen-tary Fig. S6A and S6B) and kidneys (Supplementary Fig. S6C) inthe Vhl/Trp53 double mutant background, indicating that theinduction of expression of these genes is independent of p53function. Moreover, elevated GLUT1 protein expression was

detected in renal tubules in VhlD/D, VhlD/DTrp53D/D, VhlD/D

Hif2aD/D, and VhlD/DTrp53D/DHif2aD/D mice but not in VhlD/D

Hif1aD/D or VhlD/DTrp53D/DHif1aD/D mice (Figs. 1E and 2E).Because thisHif1a-activated transcriptional signature predicted

decreased flux of pyruvate into mitochondria and decreasedmitochondrial electron transport (Supplementary Fig. S5), wedeveloped a method to analyze mitochondrial respiration inbiopsies of the renal cortex and medulla. Oxygen consumptionresulting from various steps of mitochondrial oxidation wasassessed by sequential addition of the following metabolic inter-mediates or inhibitors: (i) malate, octanoyl-carnitine, and ADP todetermine medium chain fatty acid oxidation, (ii) pyruvate andglutamate to determine complex I–specific activity, (iii) succinateto determine total ATP synthase capacity, (iv) rotenone to inhibitcomplex I and determine complex II–specific activity, (v) anti-mycin A to block complex III, allowing the correction of residualO2 consumption, and (vi) N,N,N',N'-tetramethyl-p-phenylene-diamine (TMPD) and ascorbate to determine maximal cyto-chrome c oxidase activity (complex IV). Vhl deletion caused adecrease in all of the assays of respiratory capacity in the medullabut not cortex of these mice (Fig. 4E), consistent with the fact thatthemajority of tubules in themedulla are null for Vhl and the factthat proximal tubular cells, where gene deletion occurs in aminority of cells, are the most abundant cell type in the cortex.These effects were completely rescued in VhlD/DHif1aD/D but notVhlD/DHif2aD/D kidneys (Fig. 4E), consistent with theHif1a depen-dency of the gene expression changes. Because HIF1a has beenshown to decrease mitochondrial abundance via multiplemechanisms (32, 43), we analyzed mitochondrial abundancein AQP2 expressing collecting duct principal cells using immu-nofluorescence for the mitochondrial outer membrane protein

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Figure 3.Control of renal epithelial cellproliferation by HIF1a and HIF2a. A andB, Ki67 staining of renal cortices of6-month-old (A) or 12-month-old (B)mice. C and D, Ki67-positive tubularepithelial cells per field (0.36 mm2) in6-month-old (C) or 12-month-old (D)mice. Mean � SEM from five separateregions in 3 to 5 kidneys of eachgenotype.





TOM20. Reduced TOM20 staining intensity was observed inthese cells in VhlD/D and in VhlD/DHif2aD/D kidneys, whereasVhlD/DHif1aD/D kidneys exhibited increased TOM20 staining(Fig. 5A–H and U), consistent with the measures of cellularrespiration. Similar results were obtained in the Vhl/Trp53mutant background (Supplementary Fig. S7E–S7M). We con-clude that HIF1a but not HIF2a stabilization decreases mito-chondrial abundance and oxidative capacity independently ofp53 function in Vhl mutant renal tubular cells in vivo.

We next investigated whether the characteristic clear cell phe-notype of ccRCC, believed to be caused by the cytoplasmicaccumulation of lipids and glycogen, was also dependent onHif1a. Indeed, tubular epithelial cells in VhlD/D, VhlD/DTrp53D/D,VhlD/DHif2aD/D, and VhlD/DTrp53D/DHif2aD/D mice, but not inVhlD/DHif1aD/D and VhlD/DTrp53D/DHif1aD/D mice, frequentlydemonstrated clear cell morphology (Figs. 5I–L and 2A–D).

Tubules in Hif1aD/D and Hif2aD/D mice did not display morpho-logic alterations (Supplementary Fig. S1). Tubules in VhlD/D,VhlD/DTrp53D/D, VhlD/DHif1aD/D, VhlD/DTrp53D/DHif1aD/D, VhlD/D

Hif2aD/D, and VhlD/DTrp53D/DHif2aD/D kidneys all displayed strongaccumulation of cytoplasmic glycogen droplets as assessedby PAS staining (Fig. 5M–P, Supplementary Fig. S7A–S7D),and the optically clear nuclei in VhlD/DHif2aD/D and VhlD/D

Trp53D/DHif2aD/D mice also displayed strong glycogen accumu-lation (Fig. 5P, Supplementary Fig. S7D), whichmay be the causeof this phenotype. While several genes encoding enzymesof glycogenolysis and glycogen synthesis were upregulated inVhl-deficient kidneys, these gene expression changes were abol-ished by codeletion of eitherHif1a orHif2a (Fig. 5W), suggestingthat this is not the cause of glycogen accumulation. Surprisingly,no accumulation of lipids was observed in tubular cells in anygenotype, as assessed byOil RedO (Fig. 5Q–T) andNile red (data

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Figure 4.HIF1a stabilization inhibits mitochondrial oxidation in Vhl-deficient cells. A, workflow for microarray analysis of mRNA expression 4 days after deletion of Vhl aloneor together with Hif1a and/or Hif2a in primary renal epithelial cells. B, gene expression clustering of differentially expressed genes between Adeno-Cre andAdeno-GFP–treated cells. Rows represent mean log2 ratios values of three independent samples of each genotype. HIF1a-specific or HIF2a-specific target genes orgenes that are regulated by both HIF1a and HIF2 a are indicated. C, heat map of gene expression of HIF1a-specific genes. D, mRNA abundance of theindicated genes in kidneys deficient for Vhl, Vhl/Hif1a, and Vhl/Hif2a. Mean � SD (n ¼ 6 mice of each genotype) gene expression ratios between Ksp1.3-Cre andwild-type (WT) mice, normalized to expression of Rps12. E, respirometry of biopsies from cortex and medulla of wild-type (þ/þ, black bars) and knockout(Ksp1.3-Cre/þ, white bars) genotypes (n¼ 6 mice per genotype) in Vhlfl/fl (left column), Vhlfl/flHif1afl/fl (middle column), and Vhlfl/flHif2afl/fl (right column) kidneys.Graphs depict oxygen consumption attributable to total ATP synthase capacity, complex I, complex II, complex IV, and medium chain fatty acid oxidation.





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Figure 5.Cellular metabolic phenotypes in Vhl, Vhl/Hif1a, and Vhl/Hif2a-deficient mice. A–H, immunofluorescence stainings for AQP2, TOM20, and DAPI. A–D showoverlaid channels, E–H show TOM20 fluorescence only. Arrowheads, examples of collecting duct principal cells. I–L, H&E staining of renal cortex. Arrowheads,epithelial cells with clear cell cytoplasm; arrows, optically clear nuclei. M–P, PAS staining of the renal cortex. Arrows, optically clear nuclei that display strongaccumulation of glycogen. Q–T, Oil Red O staining in the renal cortex. U, average TOM20 intensity in AQP2-positive cells. A total of 43 to 64 tubules wereanalyzed in 7 to 11 separate regions per genotype. V, Oil Red O staining of perirenal fat. W, mRNA abundance of the indicated genes in kidneys deficient forVhl, Vhl/Hif1a, and Vhl/Hif2a (n ¼ 6 mice of each genotype) of the ratios of gene expression between Ksp1.3-Cre and wild-type (WT) mice, normalized toexpression of Rps12.





not shown) stainings. Perirenal fat in the same samples served aspositive control for these stainings (Fig. 5V). Thus, while HIF1a-dependent reductions inmitochondrial abundance and oxidativecapacity correlate with the presence of a clear cell cytoplasm,neither glycogennor lipid accumulation appears to be the cause ofthis phenotype.

HIF1a reduces cellular ATP levels in Vhl-deficient cellsindependently of p53

We next turned to a primary cell culture–based system to gainmore insight at the cellular level into the metabolic alterationsthat follow Vhl deletion. Because primary renal tubular epithelialcells rapidly dedifferentiate after trypsinization, they were notsuitable for these experiments. We instead utilized primary MEFsas a genetically tractable cell culture system and focused onalterations in glucose metabolism and oxygen consumption.We derived MEFs from Vhlfl/fl, Vhlfl/flHif1afl/fl, Vhlfl/flHif2afl/fl, andVhlfl/flHif1afl/flHif2afl/fl embryos and infected them with adeno-viruses expressing GFP (Adeno-GFP) or Cre (Adeno-Cre). Theefficient deletion of all genes in the relevant floxed genotypes wasconfirmed by real-time PCR (Supplementary Fig. S8). We inves-tigated the dependence on Hif1a andHif2a of the expression of aseries of the key HIF1a-dependent metabolic genes that weidentified in renal epithelial cells. The mRNA levels of Slc2a1,Pfkfb3, Pgk1, Ldha, Pdk1, NduFa4l2, and Cox 4-2 were elevated inVhl mutant cells in a Hif1a-dependent but Hif2a-independentmanner (Fig. 6A), identically to the results seen in primary renalepithelial cells and in knockout kidneys. Deletion of Vhl inducespremature senescence that can be rescued by codeletion of Trp53(8). Vhl/Trp53 double null MEFs also exhibited higher mRNAabundance of these genes (Fig. 6A), indicating that the geneexpression pattern is not a secondary consequence of senescence,nor is it dependent on p53. Because this gene expression pattern isexpected to promote the conversion of glucose-derived pyruvateinto lactate at the expense of entry of pyruvate into the mito-chondria for oxidative phosphorylation, we analyzed cellularreadouts of glycolytic flux in cells cultured at 20% O2. Modestincreases in glucose utilization (Fig. 6B) and extracellular acidi-fication rate (Fig. 6C), as well as increases in the amount of lactatesecreted by the cells (Fig. 6D),were observed inVhl andVhl/Trp53-null MEFs. In contrast to these relatively small increases, therewere large decreases in oxygen consumption (Fig. 6E) in Vhl andVhl/Trp53-null MEFs that were not attributable to reduced mito-chondrial mass, as assessed by NAO staining (Fig. 6F). Theseresults show that Vhl deletion causes a modest increase in glucoseuptake and conversion to lactate but a large decrease in the activityof the mitochondrial tricarboxylic acid cycle, reflected by a low-ered OCR. The reduced levels of oxidative phosphorylation thatare not compensated by a large elevation in glucose flux to lactatesuggested that cells may not be able to produce normal levels ofATP. Indeed, Vhl and Vhl/Trp53 deletion caused a decrease incellular ATP level (Fig. 6G) that is dependent on Hif1a butindependent ofHif2a (Fig. 6H), consistent with the transcription-al effects of these gene deletions on metabolic regulatory genes.

DiscussionThe molecular and cellular causes of ccRCC are incompletely

understood. Biallelic inactivation of VHL is an early event inmostccRCCs, but deletion of Vhl in renal epithelial cells in mice doesnot cause tumor formation (5). Correlative and functional genetic

studies of human ccRCC have demonstrated that HIF1a stabili-zation exerts a tumor suppressor–like activity, whereas HIF2abehaves oncogenically in the context of tumor cell proliferation(5). Here, we tested the hypothesis that abolishing HIF1a stabi-lization might allow hyperproliferation of Vhl mutant kidneyepithelial cells in vivo, potentially allowing cysts or ccRCC tumorsto form. In fact, Hif1a codeletion rescued the increased epithelialcell turnover caused by Vhl deletion, and VhlD/DHif1aD/D micedeveloped no renal proliferative abnormalities. Similar mousemodels in which Vhl and Hif1a were deleted in renal epithelialcells under the control of different nephron segment-specific Cretransgenes also did not lead to tumor formation (44, 45). OurVhlD/DHif2aD/D mice similarly did not develop renal cysts ortumors. Thus, despite the numerous putative tumor suppressorfunctions of Vhl, its deletion together with constitutive stabiliza-tion ofHIF1a andHIF2a alone or together is insufficient for renaltumor formation. Previous studies employing various strategiesto achieve HIF1a and/or HIF2a activation in Vhl wild-type renalepithelia also failed to induce tumor formation beyond the stageof simple cysts or small dysplastic lesions (25, 46–48). Vhl/Trp53deletion leads to the formation of simple and atypical renal cysts,renal tumors as well as carcinomas in other organs of the genitaltract (8). We anticipated that the tumor phenotype in these micemight be enhanced byHif1a codeletion. In fact, all phenotypes inthesemicewere completely rescued. Similarly, codeletionofHif2ain this genetic background also completely prevented the forma-tion of renal cysts, renal tumors, and malignancies of the genital-urinary tract. Insofar as the Vhl/Trp53 deletion model mimics theinitial stages of evolution of ccRCCs, these findings demonstratethatHIF1a andHIF2a stabilization both play essential oncogenicroles in cyst and tumor formation. It will be interesting in furthermouse genetic studies to determinewhetherHIF1a andHIF2a arealso both necessary for the formation of tumors induced by othermutations that cooperate with Vhl, such as Bap1 (9).

It seems likely that HIF1a andHIF2amight each alter multiplecellular processes that are separately or cooperatively necessary forcyst and tumor evolution. While our studies uncoupled increasedepithelial turnover from cyst and tumor formation, we identifiedseveralmetabolic alterations that precede tumor formation. InVhlmutant renal epithelial cells, HIF1a induces a transcriptionalprogram that promotes the glycolytic conversion of glucose tolactate and decreases mitochondrial oxidation of glucose- andfatty acid–derived carbons. HIF1a also decreased the abundanceof mitochondria in collecting duct epithelial cells, consistentwith previous findings in cultured human cells that HIF1adecreases mitochondrial biogenesis via MXI1-MYC-PGC1b andincreases mitophagy via BNIP3 (32, 43). Our microarray obser-vation that Bnip3 expression is upregulated in Vhl-deficientrenal epithelial cells (Fig. 4C) provides the basis for futurestudies to uncover how Vhl loss reduces mitochondrial abun-dance. In established ccRCC cell lines, increasing mitochondrialabundance by constitutive PGC-1a expression causes decreasedxenograft tumor growth (49), consistent with our correlative datathat VhlD/DTrp53D/DHif1aD/Dmice do not show decreases in mito-chondrial abundance and activity and do not develop cysts ortumors. These findings argue that a reduction in mitochondrialabundance promotes tumorigenesis. On the other hand, we showin MEFs that HIF1a induces Warburg-like metabolism associatedwith increased conversion of glucose to lactate, decreased mito-chondrial oxygen consumption, and a lowered cellular level ofATP, implying that reduced cellular ATP levels may be an early





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cellular consequence of VHL gene deletion. The role that thismode of metabolism might play in promoting or inhibitingccRCC formation remains to be determined, but it is noteworthyin human ccRCCs that high mRNA expression levels of several ofthe HIF1a-dependent metabolic genes that we identify in thisstudy (including PFKM, ALDOB, PGK1, PGM2, ENO1, LDHA,PDK1), as well as higher levels of phosphorylated AMPK, apredicted consequence of HIF-1a-mediated reduction in cellularATP levels, correlate with better patient outcome (39). Thisfinding argues that the HIF1ametabolic transcriptional signatureacts to restrain aggressive tumor behavior. Thus, it appears thatdifferent metabolic activities of HIF1amay contribute differentlyto different aspects of tumor formation and progression. Inaddition, it appears likely that mutations or alterations in theexpression of other metabolism-regulating genes, such asdecreased expression of FBP1 (38), may act together with HIF1ato further alter cellularmetabolic pathways to achieve a balance ofhigh rates of glycolytic and pentose phosphate pathway flux andsufficient oxidative phosphorylation to provide the necessarymetabolic intermediates and ATP to fuel efficient biosynthesis,cellular proliferation, and tumor growth.

VHL mutant "normal" tubular cells in VHL patient kidneysexhibit clear cell morphology (4), highlighting that this pheno-type precedes tumor formation. We demonstrate that HIF1astabilization, but not HIF2a stabilization, induces the clear cellphenotype, consistent with previous observations based on over-expression of stabilized mutants of HIF1a and HIF2a (25, 48).Somewhat surprisingly, while decreased mitochondrial activityand abundance correlated with the presence of clear cell cyto-plasm, the accumulation of glycogen deposits did not correlateand renal tubules did not accumulate lipids. These findingscontradict the widely held idea that the clear cell phenotyperesults from cytoplasmic lipid and glycogen accumulation.

Our studies encourage a modification of the idea that HIF2a isthe ccRCC oncogene and that HIF1a restrains tumor progression.We argue that while activation of HIF1a or HIF2a alone ortogether is insufficient for tumor formation following biallelicinactivation of VHL, their activities are each indispensible for theformation of cysts and tumors that arise as a consequence ofcooperating secondary mutations. The frequent selection in

human ccRCC for functional losses of the HIF1A gene (24) orfor other posttranscriptional alterations that impair HIF1a sta-bility and promote HIF2a activity (50) raises the intriguing ideathat HIF1a may switch during tumor evolution from being afactor that is initially necessary for tumor formation (oncogene)to one that later restrains tumor progression (tumor suppressor).Ourfindings also raise the idea that pharmacological inhibition ofeither HIF1a or HIF2a may be sufficient to prevent ccRCCformation in patients with inherited VHL mutations.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Rajski, C. Lundby, I.J. FrewDevelopment of methodology: M. RajskiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Rajski, R.A. Jacobs, A.-K. Lundby, M. Adlesic,T. Hejhal, P.J. Wild, C. Lundby, I.J. FrewAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Sch€onenberger, S. Harlander, M. Rajski,R.A. Jacobs, A.-K. Lundby, C. Lundby, I.J. FrewWriting, review, and/or revision of the manuscript: M. Rajski, R.A. Jacobs,I.J. FrewAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Adlesic, T. Hejhal, P.J. WildStudy supervision: I.J. Frew

AcknowledgmentsThe authors thank Strahil Georgiev and Wilhelm Krek for providing

Vhlfl/flHif1afl/fl andVhlfl/flHif2afl/flmice, and the Centre forMicroscopy and ImageAnalysis (UZH) and Tatiana Simka for assistance with SeaHorse assays.

Grant SupportThisworkwas supported by grants to I.J. Frew fromSNF F€orderungsprofessur

(PP00P3_128257), SNF NCCR Kidney. CH and ERC Starting Grant (260316).The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 9, 2015; revised January 4, 2016; accepted January 7, 2016;published OnlineFirst January 12, 2016.

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Formation of Renal Cysts and Tumors in -Deﬁcient Mice ... · renal cysts and tumors inVhl/Trp53...

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