Angiogenin Mediates Cell-Autonomous Translational Control under Endoplasmic Reticulum...

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Angiogenin Mediates Cell-Autonomous TranslationalControl under Endoplasmic Reticulum Stress andAttenuates Kidney Injury

Iadh Mami,*† Nicolas Bouvier,‡ Khalil El Karoui,†§ Morgan Gallazzini,†§ Marion Rabant,†|

Pierre Laurent-Puig,*†¶ Shuping Li,** Pierre-Louis Tharaux,†† Philippe Beaune,*†¶

Eric Thervet,†‡‡ Eric Chevet,§§ Guo-Fu Hu,** and Nicolas Pallet*†¶‡‡

*Institut National de la Sante et de la Recherche Médicale (INSERM) U1147, Saints-Pères Research Center Paris,France; †Paris Descartes University Paris, France; ‡Nephrology Department Caen University Hospital, France; §INSERMU1151, Sick Childrens Necker Institute Paris, France; |Pathology Department, Necker Hospital Paris, France; ¶ClinicalChemistry and ‡‡Nephrology Departments, Georges Pompidou European Hospital Paris, France; **MolecularOncology Research Institute, Tufts Medical Center, Boston, Massachusetts; ††Paris Cardiovascular Research Centre,INSERM, Paris, France; and §§INSERM, UMR-U1053, Team Endoplasmic Reticulum Stress and Cancer, Bordeaux, France

ABSTRACTEndoplasmic reticulum (ER) stress is involved in the pathophysiology of kidney disease and aging, but themolecular bases underlying the biologic outcomes on the evolution of renal disease remain mostlyunknown. Angiogenin (ANG) is a ribonuclease that promotes cellular adaptation under stress but itscontribution to ER stress signaling remains elusive. In this study, we investigated the ANG-mediatedcontribution to the signaling and biologic outcomes of ER stress in kidney injury. ANG expression wassignificantly higher in samples from injured human kidneys than in samples from normal human kidneys,and in mouse and rat kidneys, ANG expression was specifically induced under ER stress. In human renalepithelial cells, ER stress induced ANG expression in a manner dependent on the activity of transcriptionfactor XBP1, and ANG promoted cellular adaptation to ER stress through induction of stress granules andinhibition of translation. Moreover, the severity of renal lesions induced by ER stress was dramaticallygreater in ANG knockout mice (Ang2/2) mice than in wild-type mice. These results indicate that ANG is acritical mediator of tissue adaptation to kidney injury and reveal a physiologically relevant ER stress-mediated adaptive translational control mechanism.

J Am Soc Nephrol 27: ccc–ccc, 2015. doi: 10.1681/ASN.2015020196

ESRD is a medical condition resulting from CKD,which affects millions of persons worldwide andconstitutes a major public health problem andeconomic challenge. AKI results in the destructionof the renal parenchyma and is amajor risk factor forthe development and progression of CKD and in-cident ESRD.1 Notably, the prognosis of injured kid-neys depends on the extent of the damage rather thanthe underlying disease.2 AKIs lead to profound adap-tive cellular reprogramming to maintain cellularhomeostasis, promote cell survival, and eliminatestressors. Although primarily protective, these eventsalso actively participate in tissue remodeling throughthe promotion of inflammation and fibrosis.3–5

Therefore, characterizing the molecular mechanismsunderlying the cellular response to acute stress andtheir structural and functional consequences at thetissue level is crucial for the development of preven-tive and therapeutic strategies in renal medicine.

Received February 21, 2015. Accepted June 10, 2015.

Published online ahead of print. Publication date available atwww.jasn.org.

Correspondence: Dr. Nicolas Pallet, INSERM U1147, CentreUniversitaire des Saints Pères, 45, rue des Saints-Pères, 75006Paris, France. Email: [email protected]

Copyright © 2015 by the American Society of Nephrology

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Figure 1. ANG is expressed in the kidney epithelium in association with ER stress. (A) Regression plots between ANG transcriptsexpression and acute Banff scores in 16 kidney transplant biopsies using quantitative RT-PCR. The ANG/RPL13A ratios were calculatedcompared with a normal kidney cDNA. Rho=Spearman’s rank correlation coefficient. (B) Representative photomicrographs of ANG

2 Journal of the American Society of Nephrology J Am Soc Nephrol 27: ccc–ccc, 2015


Many disturbances related to AKI, and including redoxregulation, aberrant calcium fluxes, glucose deprivation, viralinfections, altered glycosylation, or folding enzyme inhibition,interferewith the endoplasmic reticulum (ER) protein-foldingmachinery and subsequently lead to the accumulation ofmisfolded proteins in the ER lumen, a condition referred to asER stress.6 In the kidney, ER stress often occurs in podocytesand tubular cells under an injured microenvironment and hasthereby been implicated in the pathophysiology of variousrenal diseases.7–12 Upon ER stress, the unfolded protein re-sponse (UPR) is activated, engaging transcriptional, post-transcriptional and translational programs to reduce themisfolding burden in the ER by reducing the amount of proteinsentering this compartment, increasing its folding capacity andameliorating the clearance of accumulated proteins. The UPR issignaled through three ER stress sensors, including activatedtranscription factor 6 (ATF6), inositol-requiring enzyme 1a(IRE1a) and protein kinase RNA-like ER kinase (PERK). Inaddition, the UPR not only promotes the elimination of mis-folded proteins but also engages cell nonautonomous responses(angiogenesis, inflammation) to promote tissue-level adaptationand serve as a signal for the surrounding healthy tissue (i.e., thecell nonautonomous adaptive response).

The secreted ribonuclease angiogenin (ANG) was firstidentified as an angiogenic factor in tumor cell–conditionedmedium13 and is a component of stress response programs inyeast and mammalian cells.14,15 In mammalian cells, ANG pro-motes ribosomal RNA transcription and cell growth.16 Understressful conditions, such as heat shock or oxidative stress, cyto-plasmic ANGmay contribute to stress-induced translational re-pression through the promotion of tRNA cleavage.15,17–19

Therefore, ANG may act both intracellularly and extracellularlyto promote cell and tissue adaptation, respectively. WhetherANG signaling contributes to the UPR is currently unknown;however, this information would provide key insights into theregulatory and biologic functions of the UPR.

In the present study, we explored the molecular basisunderlying the regulation of ANG by the UPR and character-ized how this regulation promotes cellular adaptation duringER stress. Our results indicate that ANG, as a critical regulatorof the stress response regulated by the UPR, plays a critical rolein tissue adaptation in response to kidney injury.

RESULTS

ANG Expression is Induced in the Injured KidneyEpitheliumWe first determined whether ANG is expressed in injuredhumankidneys.To this end,weanalyzed the relative expression

ofANG transcripts in a series of 16 consecutive kidney allograftbiopsies, which are integrators of numerous injuries (toxic,immunologic and/or ischemic),20 systematically performed3 months after transplantation. The results showed that theexpression of ANG transcripts was significantly higher ininjured tissue samples assessed using acute Banff scores, inwhich higher numbers indicate extended injured areas21

(Figure 1A). To further document the link between ANG ex-pression and kidney injury, we performed an immunohisto-chemical analysis of kidney allografts. Whereas ANG was notexpressed in the normal kidneys, we observed the expressionof ANG in the tubular epithelium of allografts from injuredkidneys (Figure 1B). Together, these data indicate that ANG isexpressed in the acutely injured human kidney epithelium.

ER Stress Induces ANG Expression in Renal EpithelialCellsAccumulating evidence indicates that ER stress contributes tokidney disease, and constitutes a new progression factor.7,9 Inaddition, human renal epithelial cells express ANG during ERstress induced by tissue ischemia.22 Therefore, we investigatedthe biologic significance of ANG expression in injured kidneyepithelium, and we examined whether ER stress promotes theepithelial expression of ANG. Expression levels of bindingimmunoglobulin protein (BiP) (a surrogate marker of ERstress) and ANG transcripts were significantly higher in in-jured tissue samples assessed using Banff scores (SupplementalFigure 1, A and B). Consistently, immunohistochemistry re-vealed that ANG and BiP were coexpressed in the renal epi-thelium of injured kidney allografts (Figure 1, C and D). In thekidney tissues of mice treated with tunicamycin, a moleculethat inhibits N-linked glycosylation and promotes ER stressand AKI,23 ANG and BiP expression was induced in the renalepithelium (Figure 2, A and B, and Supplemental Figure 1C),with BiP and ANG being sometimes coexpressed in the sametubules (Supplemental Figure 1D). Similar findings were ob-served in rat kidneys subjected to cold ischemia. ANG and BiPwere also expressed during AKI associated with passive anti-glomerular basement membrane (GBM) nephritis (Figure 2,A and B) a condition associated with significant proteinuria,24

which is known to promote ER stress.25 Finally, BiP and ANGexpression was induced in rat kidneys with acute cyclosporinnephrotoxicity (Supplemental Figure 1C).

To confirm that ER stress controls ANG expression in thehuman renal epithelium, human renal epithelial cells (HREC)were exposed to thapsigargin (which inhibits Sarco/endoplas-mic reticulumCa21-ATPase pumps and disturbs calcium ho-meostasis) and tunicamycin. The topoisomerase inhibitorEtoposide, which promotes apoptosis without ER stress, wasused as a control. ANG mRNA expression was induced after 4

expression evaluated by immunohistochemistry using kidneys allografts from individuals with acute cyclosporine nephrotoxicity, acuteischemic damage (preimplantation biopsy), or without kidney injury (n=3 for each condition). Original magnification, 3200. (C, D) Rep-resentative photomicrographs of ANG and BiP coexpression evaluated by immunohistochemistry in kidneys allografts from individualswith AKI. Original magnification, 3100 (C); 3200 (D).

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Figure 2. ER stress induces ANG expression in renal epithelial cells. (A) Representative photomicrographs of ANG and BiP expression inkidneys of five mice treated with 1 mg/kg tunicamycin for 24 hours; or anti-GBM nephrotoxic serum; or five rats subjected to 24 hours ofcold ischemic injury; all being evaluated using immunohistochemistry. Original magnification, 3200. (B) Immunoblots representing



hours and peaked after 8 hours of treatment with the ER stres-sors but not Etoposide, and progressively declined thereafter(Figure 2C). The upregulation of ANG upon ER stress wasspecific, as among the various RNases typically expressed inhuman renal epithelial cells, only RNase4, which shares thesame promoter as ANG, was expressed (Supplemental Figure2). ER stress weakly induced ANG protein expression inHREC (Figure 2, D and E). Notably, ER stress also inducedANG expression in primary cultured renal epithelial cells re-covered from a human nephrectomy specimen26 (Supplemen-tal Figure 3).

ANG Expression is Under the IRE1a/sX-Box BindingProtein 1 Axis in Response to ER StressEvidence suggests that ANG is induced in HREC subjected toER stress, so we determined whether and how the UPRregulates ANG expression using small interfering RNA(siRNA)-mediated silencing of PERK, ATF6 and IRE1a,respectively. Although PERK inhibition did not alter ANGexpression (Figure 3A), ATF6 and IRE1a expression was re-quired for ANG transcript expression upon ER stress (Figure3, B and C), with only IRE1a inhibition reaching a statisticallysignificant difference compared with controls. In line withthese findings, the inhibition of the ribonuclease activity ofIRE1a by the chemical compound 4m8C27 reduced the ex-pression levels of ANG under ER stress (Supplemental Figure4A). To understand the mechanism by which IRE1a regulatesANG expression, we then silenced the expression of X-boxbinding protein 1 (XBP1), the transcription factor activatedby the ribonuclease IRE1a, and which regulates the IRE1a-related transcriptional program.28 The siRNA-mediated XBP1silencing (Figure 3D, Supplemental Figure 4B) resulted in re-duced expression of ANG following ER stress (Figure 3D),consistent with the overexpression of sXBP1, the active formof XBP1, which induced the expression of ANG transcripts(Figure 3E). Notably, overexpression of ATF6 also led to aninduction of ANG expression, albeit not statistically signifi-cant. The sXBP1-mediated chromatin immunoprecipitationassay showed a significant enrichment of the ANG promoterregion (Figure 3F), consistent with the predicted presence ofsXBP1 binding sites in the ANG promoter (Supplemental Fig-ure 4C, Supplemental Table 1). These results indicate that theIRE1a/sXBP1 arm of the UPR controls ANG expression dur-ing ER stress.

ANG Participates in Translation Inhibition During ERStressProvided that ANGparticipates in cell stress responses throughintracellular pathways,18 we proposed that under ER stress,ANG could have intracellular properties. To examine the cellautonomous properties of ANG during ER stress, we focusedon the early period after ER stress induction (,4 h) to rule outany autocrine effect of secreted ANG, which is not yet secretedat this time (Supplemental Figure 5). We first analyzed theinteraction of ANG and the ribonuclease inhibitor ribonucle-ase/angiogenin inhibitor 1 (RNH1), an important regulator ofANG activity, whose dissociation under stress is required toallow ANG cellular activity.29 Co-immunoprecipitation anal-ysis revealed that the association of ANG and RNH1 was dis-rupted following 2 hours of exposure to tunicamycin (Figure4A), thus indicating that ER stress promotes the release ofANG from its inhibitor. Recent findings also indicate thatduring stress, ANG participates in the production of stressgranules (SG).30 SG are dense cytoplasmic aggregates (Sup-plemental Figure 6) containing messenger RNA stalled in theinitiation complex of translation, eventually promoting theadaptation and survival of stressed cells.31,32 Using elongationInitiation Factor 4E (eIF4E) and eIF3B as markers of SG,32

immunofluorescence analysis showed that siRNA-mediatedANG silencing (Supplemental Figure 7), was associated withthe decreased abundance of SG in the cytoplasm of HRECsearly after the induction of ER stress (Figure 4B), so suggestingthat ANG participates in SG formation. To examine whetherANG is involved in translation control upon ER stress, wemonitored protein synthesis using Click chemistry.33,34 Usingthe L-azidohomoalanine (AHA) + Alexa Fluor 488 alkyne re-action, or the inverse reaction, L-homopropargylglycine(HPG) and Alexa Fluor 488 azide, we observed that siRNA-mediated ANG silencing led to increased protein synthesis (byapproximately 20%) after 2 hours of ER stress (Figure 4, C andD), compared with control cells. This indicated that ANGcontributes to the reduction of protein synthesis during ERstress. Moreover, the analysis of small RNA in tunicamycin-treated HREC extracts resolved on denaturing gel and visual-ized using SYBR gold, revealed bands corresponding to RNAscentered at approximately 30–50 nucleotides (SupplementalFigure 8A). Northern blotting using cDNA probes comple-mentary to the 39 end of tRNAPro confirmed that thesestress-induced RNAs were tRNA fragments (tiRNA)

ANG, BiP, and actin protein expression in kidneys of five mice treated with 1 mg/kg tunicamycin for 24 hours, or five rats subjected to 24hours of cold ischemic injury, or five mice injected with anti-GBM nephrotoxic serum for 14 days. (C) Graph representing the relativeexpression (means6SEM) of BiP and ANG transcripts measured through quantitative RT-PCR during a time course experiment usingHREC incubated with 2 mg/ml tunicamycin, 0.25 mM thapsigargin, 100 mM etoposide or vehicle. The data were obtained from four in-dependent experiments. One-way ANOVA with Dunnett’s post test. ***P,0.001. (D) Immunoblot representing ANG and actin proteinexpression in HREC incubated with 2 mg/ml tunicamycin, 0.25 mM thapsigargin, 100 mM etoposide, or vehicle for 24 hours. The immu-noblot shown represents three independent experiments. Histogram shows densitometric analysis of three independent experiments.Mann–Whitney U test: *P,0.05 compared with control, with Dunnett’s post test for multiple comparisons to a single control. (E) Immu-nofluorescence analysis by confocal microscopy of ANG expression in HREC incubated 24 hours with 2 mg/ml tunicamycin, 0.25 mM

thapsigargin, 100 mM etoposide or vehicle. The bar represents 10 mm.



















Figure 3. ANG synthesis during ER stress is mediated by IRE1a and sXBP1. (A) (Left) Immunoblot representing PERK and actin proteinexpression in HREC at 24 hours after transfection with siRNA targeting PERK mRNA. The immunoblot shown is representative of threeindependent experiments. (Right) Box and whiskers plots representing the relative expression levels of ANG transcripts in HREC



(Supplemental Figure 8B).15,18 To determine the role of ANGin the production of these tiRNA, wemonitored production ofsmall RNAs under ER stress in HREC extracts from cells si-lenced or not for ANG. These fragments were significantlyreduced in cells transfected with siANG, indicating that theirproduction might depend on ANG expression (Figure 4E).Notably, the activity of ANG on protein translation and SGformation was likely independent of eIF2a phosphorylation,as previously described during oxidative stress,15 because theinhibition of ANG expression did not alter ER stress-inducedeIF2a phosphorylation (Figure 4F). The inhibition of ANGexpression was not associated with degradation of total RNA,which could impact protein synthesis rate35,36 (Figure 4G). Inaddition, ANG silencing was associated with the increasedproduction of the spliced product of X-box binding protein1 (XBP1) mRNA, sXBP1, likely reflecting the exacerbation ofmisfolding burden on ER due to deficient inhibition of proteinsynthesis (Figure 4H). In line with an increased ER stress re-sponse, the expression of transcripts of UPR, including theproapoptotic factor CHOP, and the ER association degrada-tion pathway component HERP, were found to be significantlyincreased when ANG expression was inhibited (SupplementalFigure 8C). These results indicate that the cellular effects ofANG during ER stress are, at least in part, mediated through areduction in protein synthesis, potentially mediated throughtiRNA production.

ANG Deficiency Increases Susceptibility to ER Stress-Induced AKIWenext determinedwhether and howANG affords protectionunder ER stress. To this end, we inhibited ANG expressionusing siRNA-mediated RNA interference in tunicamycin-treated cells (Supplemental Figure 7), which results in theinhibition of both expression and secretion of ANG, andmon-itored cell death. ANG silencing increased signs of HREC ER

stress-induced cell death, possibly mediated by apoptosis andexemplified by increased PARP cleavage (Figure 5A). Thisindicated that ANG is cytoprotective upon ER stress. Accord-ingly, staining with the vital dyes Hoechst 33342 and propi-dium iodide,37 showed an increase in the number of dyingcells with fragmented and pycnotic nuclei and permeabilizedplasma membrane, compared with control cells (Figure 5, Band C). The incubation of HREC with monoclonal antibodiesthat block extracellular ANG reduced PARP cleavage under ERstress, as well as an increased number of apoptotic cells (Figure5D), suggesting that secreted ANGmay have deleterious auto-crine effects on HREC viability under ER stress. Together,these results indicate that the net effect of the activation ofANG during ER stress in vitro (both at the intracellular andextracellular levels) results in increased cell viability, an effectlikely supported by the intracellular adaptive properties ofANG.

We next examined a role for ANG in response to ER stress-induced kidney injury in vivo. To this end, we challenged micethat do not express ANG (Ang2/2 mice) with 1 mg/kg tunica-mycin. Ang2/2mice were born at the expected Mendelian ratioand exhibited no apparent renal phenotype under physiologicconditions (Supplemental Figures 9 and 10). Tunicamycin-treated wild-type mice developed severe renal lesions, includ-ing cell vacuolization, indicative of acute tubular necrosis(Figure 5E) and the frequency and severity of these lesionswere significantly increased in Ang2/2 mice (Figure 5F). Ofnote, 96 hours after tunicamycin injection, wild-type andAng2/2 mice displayed a similar frequency of tubular lesions,but the lesions were far more severe in Ang2/2mice (Figure 5E).The number of apoptotic cells, evaluated using terminaldeoxynucleotidyl transferase–mediated digoxigenin-deoxyuridinenick-end labeling staining, was higher in Ang2/2 mice chal-lenged with tunicamycin (Figure 5, G and H). These findingswere confirmed by an increased cleavage of caspase 3 in the

transfectedwith siRNA, targetingPERK,or scrambled siRNAand incubatedwith2 mg/ml tunicamycin or vehicle for 16 hours. Thedatawereobtained from three independent experiments. (B) (Left) Immunoblot representing ATF6 and actin protein expression in HREC at 24 hoursafter transfection with siRNA targeting ATF6 mRNA. The immunoblot shown represents three independent experiments. (Right) Box andwhiskers plots representing the relative expression levels of ANG transcripts in HREC transfected with a siRNA targeting ATF6, ora scrambled siRNA, and incubated with 2 mg/ml tunicamycin or vehicle for 16 hours. The data were obtained from three independentexperiments. (C) (Left) Immunoblot representing IRE1a and actin protein expression in HREC at 24 hours after transfection of a siRNAtargeting IRE1a mRNA. The immunoblot shown is representative of three independent experiments. (Right) Box and whiskers plotsrepresenting the relative expression levels of ANG transcripts in HREC transfected with an siRNA targeting IRE1a, or a scrambled siRNA,and incubated with 2 mg/ml tunicamycin or vehicle for 16 hours. The data were obtained from three independent experiments. Mann–Whitney U test: *P,0.05. (D) (Left) Immunoblot representing unspliced XBP1, spliced BXP1 and actin protein expression in HREC at 24hours after transfection with siRNA targeting XBP1mRNA, or a scrambled siRNA, and after incubation with 2 mg/ml tunicamycin or vehiclefor 16 hours. The immunoblot shown is representative of three independent experiments. (Right) Box and whiskers plots representingANG transcripts expression in HREC transfected with an siRNA targeting XBP1, or a scrambled siRNA, and incubated with 2 mg/ml tu-nicamycin or vehicle for 16 hours, analyzed using quantitative RT-PCR. The data were obtained from three independent experiments.Mann–Whitney U test: *P,0.05. (E) Box and whiskers plots representing ANG transcripts relative expression in HREC transfected with anempty plasmid or a plasmid expressing ATF4, ATF6, or sXBP1 for 36 hours analyzed using quantitative RT-PCR. *P,0.05 compared withcontrol cells. In brackets are shown the expression levels of the transcripts of the respective transcription factors measured by quantitativePCR (F) Histograms representing the results of a chromatin immunoprecipitation assay of sXBP1, followed by amplification through thequantitative RT-PCR of ANG promoter using non-target primers or primers targeting two different regions of the promoter. HREC wereincubated with 2 mg/ml tunicamycin or vehicle for 2 hours. The graph is representative of two experiments.








Figure 4. ANG participates in translation inhibition during ER stress. (A) Blots representing a coimmunprecipitation assay of ANG,followed by RNH1 immunoblotting in HREC incubated with 2 mg/ml tunicamycin or vehicle for 2 hours. The immunoblot representsthree independent experiments. Histogram shows densitometric analysis of three independent experiments. Mann–Whitney U test:



idneys of Ang2/2 mice, which also displayed more severe ul-trastructural tubular lesions (Supplemental Figure 11). More-over, tunicamycin prompted acute renal failure, characterizedby an increase in plasma creatinine concentration, and Ang2/2

mouse kidneys experienced more severe dysfunction com-pared with wild-type littermates (Figure 5I). These resultsindicate that ANG affords protection against AKI during ERstress.

DISCUSSION

Understanding UPR signaling in injured tissues is of para-mount importance in medicine, and expanding the under-standing ofhowcells respond toER stresswill fuel thediscoveryof novel therapeutic targets and potential biomarkers relevantto tissue injury. In the present study, based on the character-ization of the cellular responses to kidney injuries, we providethefirst evidence that ANG is directly and specifically regulatedby the UPR through the IRE1a-activated transcription factorsXBP1. The discovery of a UPR-regulated original biologicpathway depending on ANG is of potential considerableimportance, as the biologic properties of ANG have been as-sociated with a wide range of pathophysiological processes,including tumorigenesis, tissue regeneration, inflammatorybowel disease, and inflammation.16,38–40

Based on our findings, we propose a working model inFigure 6. In the first few hours after ER stress arises, a basalpool of ANGdissociates from its inhibitor RNH1 and becomesactive, thereby cleaving tRNA, promoting SG formation andtranslation reduction. Themechanism bywhich ER stress pro-motes ANG–RNH1 dissociation is not known, but mightinvolve RNH1 nuclear translocation.29 In parallel, during ERstress ANG mRNA expression increases under the control ofthe IRE1a–XBP1 axis, as well as protein level, albeit to a lesserextent, maybe because the protein is in part secreted. Newlyproduced ANG is also involved in cytoprotection, and we can-not exclude that ANG has cytoprotective properties indepen-dent of tiRNA production. Therefore, the cytoprotectiveeffects of ANG that we observed in vitro are, at least in part,mediated by ANG-mediated translation reduction. Our

findings are consistent with those recently published indicat-ing that, in various models of tissue damage (ischemic reper-fusion, toxic injury and irradiation), the levels of circulatingtRNA derivatives, which correspond to the conformationalchange in tRNA structure that occurs before fragmentation,increased rapidly and may serve as a marker of early tissueinjury.41 Together, these and our results suggest that tRNAderivates are likely involved in renal cell adaptation understress and highlight the biologic relevance of tRNA catabolismin kidney injury.

We demonstrate herein that, under ER stress, ANG engage acell autonomous adaptive response, which implicates eIF2a-independent translation inhibition and SG formation. Thesefindings are consistent with the fact that ANG contributes totranslation repression and SG formation in response to oxi-dative stress.42 Mechanistically, ANG generates stress-inducedtiRNAs that contribute to the displacement of eIF4G/A fromcapped and uncappedmRNAand eIF4E/G/A (eIF4F) from them7G cap, thereby inhibiting translation and inducing SG as-sembly.18 In addition, ANG cleaves the conserved single-stranded 39-CCA termini of all tRNAs, thereby promotingthe deactivation of the aminoacyl-ends of tRNA and subse-quently inhibiting translation.43 Therefore, ANG participatesin translation attenuation in ER-stressed cells through an orig-inal process of RNA interference which therefore expandsUPR-induced mechanisms for the reduction of protein fluxinto the ER and comes in addition to the previously describedphosphorylation of eIF2a,44 regulated IRE1a-dependent de-cay of RNAs,36 and selective mRNA release from the ER.45

Our data also indicate that in the first hours after ER stressinduction, ANG-mediated translation repression correlateswith ANG dissociation from the ANG-inhibitor RNH1. It islikely that during early ER stress, the existing pool ANG ismobilized and activated, since adaptation occurs before ANGmRNA expression is induced by the IRE1a/XBP1 arm of theUPR. The mechanisms by which RNH1 dissociates from itsinhibitor are not clear. A model for RNH1–ANG dissociationhas been proposed whereby the RNH1–ANG complex is sen-sitive to oxidation, attributable to RNH1 cysteine residues.Indeed RNH1 contains 32 cysteines and loses activity in theabsence of reducing agents.46 Treatment of RNH1–ANG

*P,0.05 comparedwith control, with Dunnett’s post-test for multiple comparisons to a single control. (B) Immunofluorescence analysis byconfocalmicroscopy of eIF4E and eIF3b colocalization inHREC transfectedwith an siRNA targetingANGmRNAor a scrambled siRNA andincubated 4 hours with 2 mg/ml tunicamycin or vehicle. The bar represents 10 mm. Mann–Whitney U test: *P,0.05. (C, D) Histogramsshowing the incorporation rates of L- AHA and L- HPG in HREC transfected with siRNA targeting ANGmRNA or a scrambled siRNA after 2hours of incubation with 2 mg/ml tunicamycin. The data were obtained from three independent experiments. Mann–Whitney U test:*P,0.05. (E) PAGE of small RNA extracted from HREC transfected with siRNA targeting ANGmRNA or scrambled siRNA after 2 hours ofincubation with 2 mg/ml tunicamycin, followed by staining with SYBR gold. The data were obtained from three independent experiments.(F) Immunoblot representing phospho-eIF2a and eIF2a expression in HRECs transfected with an siRNA targeting ANG mRNA and in-cubated with 2 mg/ml tunicamycin for 2 hours. Immunoblot is representative of three independent experiments. Histogram shows den-sitometric analysis of three independent experiments. (G) Experion-based automated gel-based electrophoresis of total RNA extractedfrom HREC transfected with an siRNA targeting ANGmRNA and incubated with 2 mg/ml tunicamycin. (H) Agarose gel electrophoresis ofPCRproducts ofXBP1cDNAfromHREC transfectedwith siRNA targetingANGmRNAor scrambledsiRNAand incubated4 hourswith 2mg/mltunicamycin, or vehicle. Histogram shows densitometric analysis of three independent experiments. Mann–Whitney U test: *P,0.05.




Figure 5. ANG deficiency increases susceptibility to ER stress-induced kidney injury. (A) Immunoblot representing PARP and actin proteinexpression in HREC transfected with siRNA targeting ANG mRNA or scrambled siRNA and incubated 24 hours with 2mg/ml tunicamycin orvehicle. Immunoblot is representative of three independent experiments. (B, C) Representative epifluorescence photomicrograph of HREC



complexes with p-hydroxymercuribenzoate rapidly dissoci-ates the complex, releasing fully active RNase. Oxidation orderivatization of cysteine residues alters the structure ofRNH1,47 which may lead to dissociation of ANG from theRNH1–ANG complex.Whether thismechanism occurs underER stress, which can be associated with oxidative stress, re-mains to be established.

Inparallelwith this earlyANG-mediated stress response, ERstress also promotes the induction ofANGexpression. The factthat we observed a weak accumulation of ANG in whole celllysate may be related to a secretion process activated upon ERstress, which modalities remain to be determined. In additionto cell autonomous responses mediated by ANG to eliminatethe stress and promote cellular adaptation at the individualcellular level, one might propose that secreted ANG might aswell be involved in cell non-autonomous responses. Thosewould likely involve paracrine communication with neigh-boring cells not yet exposed to stress, and consequently, wouldact as warning signals.48 These cellular responses afford tissuelevel adaptations to a challenge, facilitating the preservation oftissue structure and function.

In conclusion, our results provide new insights into howANG modulates adaptation during kidney injury, and wedescribe an additional mechanism by which the UPR mightcontrol protein synthesis.We showedANGas a key regulator oftissue homeostasis during AKI associated with ER stress. Overtime, regulators of ANGactivity could be developed to increasetissue adaptation to stress.

CONCISE METHODS

Human StudiesRNA Isolation from Kidney Transplant BiopsiesSixteen surplus kidney allograft protocol biopsies, performed 3

months after transplantation, were retrospectively analyzed for BiP

and ANG mRNA expression. Detailed methods are available in the

Supplemental Material.

Immunohistochemistry of Human Kidney Biopsies with AcuteInjuryNine kidney allograft biopsies: three normal, three with acute

cyclosporine nephrotoxicity lesions, and three with ischemic damage

(preimplantation biopsy) were retrospectively analyzed for ANG

immunohistochemistry.

ApprovalsKidney biopsies were not performed for the purpose of this non-

interventional study, but only for patient care. Patients wrote in-

formed consent for the eventual use of their surplus biologic samples.

Analyses were performed anonymously. Paris Descartes University

ethics committee approved this observational study. The authors

adhere to the Declaration of Helsinki.

Experimental Animal ModelsIschemic InjuryAdultmale Sprague-Dawley rats (Charles River laboratories, L’Arbresle,

France) weighing 325–350 g were allowed free access to tap water. The

abdomen was then opened through a midline incision, and the aorta

was retrogradely cannulated below the renal arteries with an 18-gauge

needle. With the aorta occluded by ligation above the renal arteries,

and the renal vein opened by a small incision for outflow, the kidneys

were perfused with 20 mL of cold heparinized saline. Kidneys were

then washed with 10 mL IGL1 followed by incubation in IGL1 during

24 hours at 4°C.

Passive Anti-GBM NephritisPassive anti-GBM nephritis protocol has been induced as described

previously.49 Anti-GBM nephrotoxic serum was injected to C57Bl6/J

mice through the retro-orbital venous sinus at 6ml/g body wt for 3

days continuously. On day 14 animals were euthanized. Albuminuria

was significantly increased in the animals with anti-GBM nephritis.24

Ang knockout (Ang2/2) mice.Ang2/2 mice were generated by crossing Ang1 gene floxed mice with

EIIa-Cremice andwere backcrossed eight generations to obtainAng2/2

mice in pure C57BL/6 background. TheAng knockoutmice used in this

transfected with a siRNA targeting ANG mRNA or a scrambled siRNA and incubated 24 hours with 2 mg/ml tunicamycin or vehicle andstained with 1 mg/ml Hoechst 33342 (HO) and 5 mg/ml propidium iodide (PI), and quantification of the number of HOPI-positive cells perhigh-powered field. The bar represents 50 mm. The data represent three independent experiments. (D) (Left) Immunoblot representingPARP and actin inHREC incubatedwith 2 mg/ml tunicamycin or 0.25 mM thapsigargin for 24 hours andwith 30 mg/mlmonoclonal anti-ANGantibody (26-2F; Sigma-Aldrich) or mouse IgG. Immunoblot is representative of three independent experiments. (Right) Quantification ofthe number of HOPI-positive cells per high-powered field in HREC incubated with 2 mg/ml tunicamycin, 0.25 mM thapsigargin or vehiclefor 24 hours and with 30 mg/ml monoclonal anti-ANG antibody (26-2F; Sigma-Aldrich) or mouse IgG. Mann–Whitney U test: *P,0.05. (E)Photomicrographs (3400) of Ang2/2 or wild-type mouse kidneys stained with Masson’s trichrome 48 and 96 hours (black arrows) afterinjection with 1 mg/kg tunicamycin, or vehicle. (F) Quantification of the extent of tubular damage at 48 hours in tunicamycin- or vehicle-treated wild-type (n=6) and Ang2/2 mice (n=6), using a modified Shih scale.51 Mann–Whitney U test: *P,0.05. (G) Photomicrographs(3400) of Ang2/2 or wild-type mouse kidneys stained for terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridinenick-end labeling (TUNEL) 48 hours after injection with 1 mg/kg tunicamycin, or vehicle. (H) Quantification of the percentage of TUNEL-positive cells per high-powered field in kidneys from sixwild-type and sixAng2/2mice 48 hours after tunicamycin injection. The number ofTUNEL-positive nuclei per high-power field was reported to the number of DAPI positive nuclei (not shown) to calculate the percentage ofTUNEL-positive nuclei. Mann–Whitney U test: *P,0.05. (I) Plasma creatinine concentration from six wild-type and six Ang2/2 mice at 96hours after tunicamycin injection. The median and 95% CI are represented as box and whisker plots. Mann–Whitney U test: *P,0.05.




study were constitutive knockout. Eliia-Cre was used to delete floxed

Ang gene in the germline. All experiments were performed on 12-

week-old wild-type males and Ang2/2 littermates from Ang+/2 het-

erozygous crossings. Animals were fed ad libitum and housed at 25°C

in a 12-hour light cycle. Tunicamycin (1 mg/kg) or vehicle (DMSO)

was intraperitoneally injected at day 0, and mice were sacrificed 2 and

4 days post-injection. Kidneys were next processed for protein, RNA

extraction, and Masson’s trichrome staining. Detailed methods are

available in the Supplemental Material. Five to seven mice per group

were analyzed.

ApprovalsExperiments were conducted according to the French veterinary

guidelines and those formulated by the European Community for

experimental animal use (L358–86/609EEC), and were approved by

the Institut National de la Santé et de la RechercheMédicale (INSERM).

Animal procedures on Ang2 /2 mice were approved by the

Institutional Animal Care and Use Committee of Tufts University

(Protocol N°B2013–59).

Cell CultureNormal HREC of proximal origin (HK-2) were purchased from

ATCC/LGC Standards (lot number 60352186), and cultured accord-

ing to previously published methods.26 Detailed methods are avail-

able in the Supplemental Material.

Small Interfering RNA TransfectionsThe transient inactivation of PERK, IRE1a, ATF6, ANG, and XBP1

was achieved using synthetic siRNAs designed and obtained from

Qiagen and transfected using HiPerFect (Qiagen) according to the

manufacturer’s protocol. The control siRNA we used is a scrambled

siRNA called AllStars Negative Control siRNA (Ref. 1027281; Qia-

gen). AllStars Negative Control siRNA has no homology to any

known mammalian gene. Validation has been performed using Affy-

metrix GeneChip arrays and a variety of cell-based assays to ensure

minimal nonspecific effects on gene expression and phenotype.

Chromatin Immunoprecipitation AssayChromatin immunoprecipitation (ChIP)was performed according to

the manufacturer’s protocol (EZ-Magna CHIP G; EMD Millipore).

Briefly, HREC were incubated with tunicamycin for 1 h. Subse-

quently, the cells were washed with PBS and crosslinked with 1%

formaldehyde at 37°C. After terminating the crosslinking reaction,

the cells were collected, washed, and resuspended in the SDS lysis

buffer containing protease inhibitor cocktail (Roche Diagnostics).

The lysates were sonicated five times followed by cooling on ice.

The cell debris was cleared, and the supernatant was diluted in a

ChIP dilution buffer. After a brief centrifugation, 1% of the total

supernatant was put aside and one-tenth of this material was used

as input control. Half of the remaining supernatant was incubated

with an anti-sXBP1 antibody (made from hybridoma, provided by

Dr. Eric Chevet,50 and the other half of the supernatant was incubated

with a nonimmune rabbit immunoglobulin G and protein G mag-

netic beads at 4°C overnight with rotation. The beads were washed

and pelleted using a magnetic separator. After elution, DNA frag-

ments were purified using a spin column. For PCR, 10% of the im-

munoprecipitated materials were used as the DNA template in 40

cycles of amplification using the following primer sets. The primers

sequences are listed in the Supplemental Table 2A. The results of the

ChIP analysis were calculated and recorded as fold-enrichment val-

ues. The default Input fraction was 1%, which represents a dilution

factor of 100 or 6.644 cycles (i.e., log2 of 100).

Coimmunoprecipitation AssayThe samples were immunoprecipitated using an anti-ANG antibody,

followed by immunoblotting for RHN1 and ANG. Detailed methods

are available in the Supplemental Material

Small RNAs Migration and Staining with SYBR GoldTotal RNAwas extracted using the miRNeasyMini Kit (Qiagen) RNA

(10mg per well) was analyzed using TBE-urea gels and stained with

Figure 6. Regulation of ANG activation and expression under ERstress. Under ER stress, ANG expression and activation are induced.ANGexpression is inducedby the transcription factor sXBP1, followingIRE1a activation. Part of ANG is secreted by HREC under ER stress. Inaddition, early ER stress promotes residual ANG activation by freeingit from its inhibitor RNH1, the exact mechanism being unknown, butwhich could involve post-translational modifications. One of themechanisms by which ANG promotes cell adaptation during ER stressimplicates its ribonuclease activity, which cleaves tRNA to producetiRNA that interfere with translation initiation. Consequently, nascentprotein synthesis and ER protein load are reduced, ER stress is miti-gated, and cell survival is prolonged. Importantly, this model does notexclude other mechanisms leading to cell adaptation mediated byANG under ER stress. Dashed lines represent hypothetical process.







SYBR gold to visualize stress-induced small RNAs. Detailed methods

are available in the Supplemental Material.

Click-It ChemistryNascent protein synthesis analysis was performed using the Click-iT

HPG/AHA Alexa Fluor 488 Protein Synthesis Assay Kits (Invitrogen)

according to the manufacturer’s instructions, with minor modifica-

tions. HREC cultured on 96-well plates in L-methionine-freemedium

supplemented with 200 mM L-cystine, 2 mM L-glutamine, and 10 mM

HEPES were fed with 50 mM L-HPG or L-AHA for 30 min. Subse-

quently, tunicamyin was added, and the cells were fixed after 2 hours

of incubation with 3.7% formaldehyde in PBS, followed by a perme-

abilization step using 0.5% Triton X-10. After incubation with

Click-iT reaction buffer containing Alexa Fluor 488 azide/alkyne,

the fluorescence was visualized using an imaging platformwith filters

appropriate for Alexa Fluor 488.

Statistical AnalysisDistribution of variables is represented using box-and-whiskers plots:

the bottomand top of the box are thefirst and third quartiles, the band

inside the box is themedian, and the ends of thewhiskers represent the

minimum and maximum of all the data. The proportions are

represented using histograms. We used the Mann–Whitney U test

for nonparametric data comparisons between two groups, and Stu-

dent’s t test for the comparison of parametric data.

One-way ANOVAwithDunnett’s post-test correction formultiple

comparisons to a single control was performed when necessary. Sta-

tistical analyses were performed using JMP.10 (SAS software) and

graphs were produced using Prism-GraphPad software. P values

,0.05 were considered significant.

ACKNOWLEDGMENTS

This work was funded by grants from the INSERM, la Fédération

Nationale pour l’Aide aux Insuffisants Rénaux (to N.P.), grants from

INSERM (to E.C.) and National Institutes of Health grants R01-

NS065237 and R01-CA105241 (to G.-F.H.). The authors thank the

HisIM facility (Cochin Institute), the Laboratoire de Biochimie

(Claude Bernard Institute), and Alain Schmitt (Electron Microscopy

facility, Cochin Institute).

DISCLOSURESNone.

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