ARTICLE. Keywords ACE-2. Akita mice. Angiotensinogen. Diabetes. Heterogeneous nuclear ribonucleoprotein F. Hypertension. Renal fibrosis.

Transcription

1 Dietologi (5) 58: DOI.7/s5-5-7-y ARTICLE Overexpression of heterogeneous nuler rionuleoprotein F stimultes renl Ae- gene expression nd prevents TGF-β-indued kidney injury in mouse model of dietes Cho-Sheng Lo & Yixun Shi & Shio-Ying Chng & Shn Ado & Iselle Chenier & Jnos G. Filep & Julie R. Ingelfinger & Sho-Ling Zhng & John S. D. Chn Reeived: 5 My 5 /Aepted: 6 June 5 /Pulished online: August 5 # The Author(s) 5. This rtile is pulished with open ess t Springerlink.om Astrt Aims/hypothesis We investigted whether heterogeneous nuler rionuleoprotein F () stimultes renl expression nd prevents TGF-β signlling, TGF-β inhiition of Ae- gene expression nd indution of tuulo-firosis in n Akit mouse model of type dietes. Methods Adult mle Akit trnsgeni (Tg) mie overexpressing speifilly in their renl proximl tuulr ells (s) were studied. Non-Akit littermtes nd Akit mie served s ontrols. Immortlised rt s stly trnsfeted with plsmid ontining either rt DNA or rt Ae- gene promoter were lso studied. Results Overexpression of ttenuted systemi hypertension, glomerulr filtrtion rte, lumin/retinine rtio, urinry ngiotensinogen (AGT) nd ngiotensin (Ang) II John S. D. Chn nd Sho-Ling Zhng re joint senior uthors Eletroni supplementry mteril The online version of this rtile (doi:.7/s5-5-7-y) ontins peer-reviewed ut unedited supplementry mteril, whih is ville to uthorised users. levels, renl firosis nd profiroti gene (Agt, Tgf-β, TGF-β reeptor II [Tgf-βrII]) expression, stimulted ntiprofiroti gene (Ae- nd Ang 7 reeptor [MsR]) expression, nd normlised urinry Ang 7 level in Akit - Tg mie s ompred with Akit mie. In vitro, overexpression stimulted Ae- gene promoter tivity, mrna nd protein expression, nd ttenuted Agt, Tgf-β nd Tgf-βrII gene expression. Furthermore, overexpression prevented TGF-β signllingnd TGF-β inhiition of Ae- gene expression. Conlusions/interprettion Thesedtdemonstrtetht hnrnpfstimultesae- gene trnsription, prevents TGF-β inhiition of Ae- gene trnsription nd indution of kidney injury in dietes. HnRNP F my e potentil trget for treting hypertension nd renl firosis in dietes. Keywords. Akit mie. Angiotensinogen. Dietes. Heterogeneous nuler rionuleoprotein F. Hypertension. Renl firosis. TGF-β Sho-Ling Zhng sho.ling.zhng@umontrel. John S. D. Chn john.hn@umontrel. Centre de reherhe, Centre hospitlier de l Université de Montrél (CRCHUM) Tour Viger Pvillon R, Université de Montrél, 9 Sint-Denis Street, Montrel, QC HX A9, Cnd Reserh Centre, Misonneuve-Rosemont Hospitl, Université de Montrél, Montrel, QC, Cnd Peditri Nephrology Unit, Msshusetts Generl Hospitl, Hrvrd Medil Shool, Boston, MA, USA Arevitions ACR AGT Ang BW DN EMSA ESRD KAP KW MsR Alumin/retinine rtio Angiotensinogen Angiotensin Body weight Dieti nephropthy Eletrophoreti moility shift ssy End-stge renl disese Heterogeneous nuler rionuleoprotein F Kidney-speifi ndrogen-regulted protein Kidney weight Angiotensin 7 reeptor

2 444 Dietologi (5) 58: RAS RE ROS RPTs s RT-qPCR SBP sirna STZ Tg TL (RII) WB WT Renin ngiotensin system Response element Retiveoxygenspeies Renl proximl tuules Renl proximl tuulr ells Rel-time-quntittive PCR Systoli BP Smll interfering RNA Streptozotoin Trnsgeni Tiil length TGF-β reeptor I(II) Western lotting Wild-type insulin-responsive element (IRE) in the Agt gene promoter [5 8], nd tht overexpression in s inhiits Agt gene expression nd kidney hypertrophy in Akit -Tg mie [9]. Here, we report tht overexpression of stimultes Ae- gene trnsription nd suppresses profiroti gene (Tgf-β, Tgf-βrII) expression in s of Akit -Tg mie. We hve onfirmed these hnges y in vitro studies in rt s. We lso show tht overexpression prevents TGF-β signlling nd inhiition of Ae- gene expression in s. Finlly, we identified the puttive DNA response elements (REs) in the Ae- gene promoter tht re responsive to nd TGF-β. Introdution Methods Dieti nephropthy (DN), leding use of endstge renl disese (ESRD), ounts for 5% of ll ESRD ses [, ]. While glomerulopthy is hllmrk of erly renl injury in DN [], tuulointerstitil firosis nd tuulr trophy re mjor fetures of lte-stge DN nd re losely ssoited with loss of renl funtion [4 7]. The mehnisms underlying tuulointerstitil firosis, however, re inompletely understood. TGF-β is onsidered to e the most potent induer of firogenesis [8]. Indeed, ptients nd niml models with type or dietes hve signifintly elevted serum nd urinry TGF-β levels [9 ] s well s heightened TGF-β mrna nd protein expression in glomeruli nd the tuulointerstitium [ 6]. We previously reported tht high gluose milieu enhnes expression of ngiotensinogen (AGT, the sole preursor of ll ngiotensins) through genertion of retive oxygen speies (ROS) in ultured rt renl proximl tuulr ells (s) [7, 8]. Rt AGT overexpression in s leds to hypertension, luminuri nd hypertrophy, nd enhnes TGF-β expression in dieti AGT-trnsgeni (Tg) mie [9, ]. Conversely, -seletive overexpression of tlse or phrmologil lokde of the renin ngiotensin system (RAS) ttenutes hypertension, ROS genertion, kidney injury nd normlised expression in mouse models of dietes [ 4]. Tken together, these oservtions indite tht oxidtive stress-indued upregultion of AGT expression nd downregultion of expression in s, resulting in higher ngiotensin (Ang)II/Ang 7 rtio, my e key determinnts of development of hypertension nd nephropthy in dietes. We reported tht insulin inhiits high gluose stimultion of rt renl Agt gene expression vi two nuler proteins heterogeneous nuler rionuleoproteins F nd K (, hnrnp K) tht intert with the Chemils nd onstruts Ative humn reominnt TGF-β ws otined from R&D Systems (Minnepolis, MN, USA). SB454 ( TGF-β reeptor I [RI] inhiitor) nd other hemils were purhsed from Sigm-Aldrih (Okville, ON, Cnd). The ntiodies used in the present study re listed in eletroni supplementry mteril (ESM) Tle. The pkap plsmid ontining the kidney-speifi ndrogen-regulted protein (KAP) promoter ws gift from C. D. Sigmund (University of Iow, Iow City, IA, USA) []. Full-length rt DNA fused with HA tg (enoding mino id residues 98 6 [YPYDVPDYA] of humn influenz virus hemgglutinin) ws inserted into pkap plsmid t the NotI site t oth 5 nd termini [5, 9]. pgl4. vetor ontining iferse reporter ws otined from Promeg (Sunnyvle, CA, USA). Rt Ae- gene promoter (N-,9/) ws loned from rt genomi DNA with speifi primers (ESM Tle ), s desried y Milsted et l [] nd then inserted into pgl4. plsmid t HindIII nd KpnI restrition sites. Srmled Silener Negtive Control no. smll interfering RNA (sirna) nd sirna were ought from Amion (Austin, TX, USA). QuikChnge II Site-Direted Mutgenesis Kit nd LightShift Chemiluminesent eletrophoreti moility shift ssy (EMSA) Kit were proured from Agilent Tehnologies (Snt Clr, CA, USA) nd Thermo Sientifi (Life Tehnologies, Burlington, ON, Cnd), respetively. The primer iotinlelling kit ws purhsed from Integrted DNATehnologies (Corlville, IA, USA). Physiologil studies Adult mle heterozygous Akit mie (Mus musulus) with mutted Ins gene (C57BL6-Ins Akit /J) were purhsed from Jkson Lortories (Br Hror, ME, USA: Akit Tg mie (C57Bl/6 kground) overexpressing rt -HA in s (line 97) were reted in our

3 Dietologi (5) 58: Tle Physiologil mesurements WT -Tg Akit Akit -Tg Blood gluose (mmol/l).8±.64.± ±.7 5.±.79 SBP (mmhg).7±.7.8±.67.4±.59.5±.5 KW (mg) 98.7± ±,96 55.± ±.97 BW (g) 8.±.4 4.9±. 6.4±.85 5.±.45 TL (mm).6±.6.7±..±.6 ±. KW/BW rtio.5±.57.±.8.7± ±.5 KW/TL rtio 7.6± ± ±.4 8.7±.5 GFR (μl min g ) 7.±.44 8.±.9 9.8±.6 6.±.85 Urinry ACR (mg/mmol).8±..8±.5.6±.5 5.8±.7 Urinry AGT/Cre rtio (pmol/μmol),48±4.4,49±7.5 4,5±75.6,84±4.7 Urinry Ang II/Cre rtio (pmol/μmol) 9.56± ± ±89.6.5±.68 Urinry Ang 7/Cre rtio (pmol/μmol) 7.97±.87 8.±.9.99± ±.8 All dt re expressed s mens±sem p<.5, p<. vs WT, p<.5, p<. vs Akit mie lortory (y J. S. D. Chn) [9]. Mle dult non-tg nd non-akit littermtes served s wild-type (WT) ontrols, nd were tested long with -Tg, Akit nd Akit -Tg mie. All nimls were housed individully in metoli ges for 4 h efore euthnsi t ge weeks. All nimls were fed stndrd mouse how nd wter d liitum. Animl re nd proedures were pproved y the CRCHUM Animl Cre Committee nd followed the Priniples of Lortory Animl Cre (NIH pulition no. 85-, revised 985: grnts.nih.gov/grnts/olw/referenes/phspol.htm). Blood gluose levels, following 4 5 h fsting, were determined with n Au-Chek Perform System (Rohe Dignostis, Lvl, QC, Cnd). Body weight (BW) ws reorded. Urine ws olleted nd ssyed for lumin/retinine rtio (ACR) y enzyme-linked immunosorent ssys (Aluwell nd Cretinine Compnion, Exoell, Phildelphi, PA, USA). GFR ws mesured s desried y Qi et l [] sreommended y the Animl Models of Dieti Complitions Consortium ( with fluoresein isothioynte inulin [, 8, ]. Kidneys were removed immeditely fter GFR mesurement, depsulted nd weighed. The left kidneys were proessed for histology nd immunostining, nd right renl orties were hrvested for renl proximl tuules (RPTs) isoltion y Peroll grdient entrifugtion [, 4, 8, 9]. Aliquots of freshly isolted RPTs from individul mie were immeditely proessed for totl RNA nd protein isoltion. Immunohistohemil stining Immunohistohemil stining ws performed y the stndrd vidin-iotin-peroxidse omplex method in four to five setions (4 μm thik) per kidney nd three mouse kidneys per group (ABC Stining System; Snt Cruz Biotehnology [Snt Cruz, CA, USA]) [, 4, 8, 9]. Stining ws nlysed under light mirosopy y two independent, linded oservers. The olleted imges were ssessed y Ntionl Institutes of Helth Imge J softwre ( [, 4, 8, 9]. Urinry AGT, Ang II nd Ang 7 mesurement Mouse urinry AGT, Ang II nd Ang 7 levels were nlysed y ELISA (Immuno-Biologil Lortories, IBL Ameri, Minnepolis, MN, USA) nd normlised y urinry retinine levels s desried [, 4, 8, 9, 4]. Cell ulture Immortlised rt s (pssges 8) [5] were ultured in 5 mmol/l D-gluose DMEM ontining 5% FBS until they rehed 6 7% onfluene. The medi were then hnged to serum-free DMEM, ensuring tht endogenously sereted TGF-β would not interfere in the ssy. After 45 min preinution, tive humn reominnt TGF-β [6] ( to ng/ml) ws dded (onsidered s time h) nd inuted for vrious time periods up to 4 h. In seprte experiments, s were inuted for 4 h in serum-free medium in the presene or sene of TGF-β± vrious onentrtions of SB454. Rel-time quntittive PCR, Ae, Ae-, MsR, Tgf-β, Tgf-βrI, Tgf-βrII, ollgen type IV, ollgen type I, fironetin nd β-tin mrna expression levels in RPTs were quntified y rel-time quntittive PCR (RT-qPCR) with forwrd nd reverse primers (ESM Tle ) [, 4, 8, 9]. Western lotting Western lotting (WB) ws performed s desried previously [, 4, 8, 9]. The reltive densities of, ACE,, Ang 7 reeptor (MsR), TGF-β,, I, fironetin, p-smd/, Smd/

4 446 Dietologi (5) 58: nd β-tin nds were quntified y omputerised lser densitometry (ImgeQunt softwre, version 5.; Moleulr Dynmis, Sunnyvle, CA, USA). Sttistil nlysis The dt re expressed s mens±sem. Sttistil nlysis ws performed y the Student s t test or one-wy nlysis of vrine nd the Bonferroni test s pproprite provided y Grphpd Softwre, Prism 5. ( A vlue of p.5 ws onsidered to e sttistilly signifint. Results Physiologil vriles in Akit nd Akit -Tg mie Tle douments signifintly higher lood gluose levels in WT -Tg Akit Akit/-Tg MsR d ACE e f g h F+HA mrna (46 KD),5,,5, 5 WT Akit WT Akit Akit Akit WT Akit Akit ( KD) Ae- mrna 5 5 Akit Akit WT Akit WT Akit WT Akit WT Akit Akit WT Akit Akit MsR (4 KD) MsR 5 5 WT Akit Akit i j k l Fig. overexpression upregultes nd MsR expression in mouse kidneys. Immunohistohemil stining of (), (), MsR () nd ACE (d) expression in kidney setions ( ); WB (e h) nd RT-qPCR (i l) of their respetive protein nd mrna MsR mrna WT Akit Akit ACE (8 KD) ACE ACE mrna Akit WT Akit Akit WT Akit Akit levels in freshly isolted RPTs from non-dieti WT ontrols, - Tg mie (), dieti Akit mie nd Akit -Tg mie (Akit F- Tg) t week. Vlues re mens+sem orreted to β-tin, n=6. p<.5; p<.; p<.

5 Dietologi (5) 58: Akit ompred with WT mie nd -Tg mie. Overexpression of hd no effet on lood gluose levels in Akit -Tg mie. Systoli BP (SBP), kidney weight (KW)/BW nd KW/tiil length (TL) rtios, GFR nd ACR were ll elevted in Akit mie, ompred with oth WT ontrols nd -Tg mie. HnRNP F overexpression in s mrkedly ttenuted these hnges in dieti Akit - Tg mie. Furthermore, Akit mie exhiited elevted urinry AGTnd Ang II levels, prllel with deresed Ang 7levels, ompred with WT mie. HnRNP F overexpression prtilly redued urinry AGT nd Ang II levels, wheres it ompletely normlised urinry Ang 7 levels novel finding. Effet of overexpression on AGT, ACE, nd MsR expression in Akit -Tg mouse kidneys Immunostining reveled tht HnRNP F (Fig. ) ws overexpressed in s of -Tg nd Akit -Tg mie ompred with WT nd Akit mie, respetively. (Fig. ) nd MsR (Fig. ) expression ws deresed in Akit mie ompred with WT ontrols nd normlised in Akit -Tg mie. ACE (Fig. d) expression did not differ etween WT nd -Tg mie, wheres ACE expression ws signifintly higher in Akit mie thn in WT ontrols nd ws not normlised in Akit -Tg mie. WB nd RT-qPCR for,, MsR nd ACE protein nd their mrna levels (Fig. e l, respetively) onfirmed these oservtions. Effet of overexpression on TGF-β, TGF-β RII nd expression in Akit -Tg mouse kidneys Immunostining of TGF-β(Fig.)nd I (Fig. ), WB of TGF-β(Fig. d) nd I expression Fig. overexpression ttenutes TGF-β nd TGF-β RII expression in mouse kidneys. Immunohistohemil stining of TGF-β (), I () nd () expression in kidney setions ( ), WB (d f) nd RT-qPCR (g i) of their respetive protein nd mrna levels in freshly isolted RPTs from non-dieti WT ontrols, -Tg () mie, dieti Akit mie nd Akit -Tg mie (Akit ) t week. Vlues re mens+sem orreted to β-tin, n=6. p<.5; p<. TGF-β I WT -Tg Akit Akit/-Tg d e f TGF-β (5 KD) TGF-β Tgf-β mrna WT Akit Akit WT Akit Akit WT Akit Akit I (7~8 KD) I 5 5 WT Akit Akit WT Akit Akit g h i Tgf- β rii mrna WT Akit Akit (56 KD) Tgf- β ri mrna WT Akit Akit WT Akit Akit WT Akit Akit

6 448 Dietologi (5) 58: (Fig. e), nd RT-qPCR of Tgf-β (Fig. g) ndtgf-βrii (Fig. h) mrna expression showed signifintly higher TGF-β nd I expression in s of Akit mie thn in WT ontrols nd -Tg mie, nd they were ttenuted in Akit -Tg mie. In ontrst, expression ws similr in ll groups studied (Fig.,f,i). HnRNP F overexpression suppresses renl firosis in Akit -Tg mie Akit mie developed renl struturl dmge ompred with WT nd -Tg mie (ESM Fig., PAS stining), inluding tuulr luminl dilttion with umultion of ell deris, inresed extrellulr mtrix proteins in glomeruli nd tuules, nd proximl tuule ell trophy. HnRNP F overexpression mrkedly reversed ut never ompletely resolved these normlities in Akit mie. We deteted signifint inreses in Msson s trihrome stining (Fig. ) nd immunostining for ollgen type IV (Fig. ), fironetin expression (Fig. ) nd ollgen type I(Fig.d) in glomerulotuulr res in Akit mie ompred with WT ontrols nd -Tg mie. These hnges were redued in Akit -Tg mie. Quntifition of Msson s trihrome-stined (ESM Fig. ), immunostining of ollgen IV (Fig. e), fironetin (Fig. f) nd ollgen I (Fig. g), nd RT-qPCR quntifition of mrna levels (Fig. h j) onfirmed their expression. HnRNP F overexpression enhnes Ae- nd suppresses Agt, Tgf-β nd Tgf-βrII gene expression nd protein Fig. overexpression ttenutes renl firosis nd profiroti gene expression in mouse kidneys. Msson s trihrome stining (), immunostining of ollgen IV (Col IV) (), fironetin (FN) () ndollgeni(coli)(d) expression in kidney setions ( ); semiquntittive nlysis of immunostined ollgen IV (e), fironetin (f) nd ollgen I (g), nd RT-qPCR of ollgen IV (lso known s Col4) (h), Fn (i) nd ollgen I (lso known s Col) (j) mrna expression in freshly isolted RPTs from WT ontrol mie, -Tg mie (), Akit mie nd Akit -Tg mie (Akit ) t week. Vlues re men+sem orreted to β-tin, n=6. p<.5; p<. d Msson Col IV FN Col I WT -Tg Akit Akit/-Tg e f g Col IV immunostining glomerulo tuulr re (ritrry units) WT Akit Akit WT Akit Akit FN immunostining glomerulo tuulr re (ritrry units) WT Akit Akit h i j Col IV mrna Fn mrna WT Akit Akit Col I immunostining glomerulo tuulr re (ritrry units) Col I mrna WT Akit Akit WT Akit Akit

7 Dietologi (5) 58: levels in rt s in vitro s stly trnsfeted with pdna./ (-pdna./) exhiited onsiderly higher levels of (Fig. 4,), lower mounts of AGT (Fig. 4,) nd higher mount of (Fig. 4,d) thn non-trnsfeted s or s stly trnsfeted with pdna. (-pdna.). In ontrst, TGF-β nd I protein levels were signifintly deresed in -pdna./ ompred with non-trnsfeted s or -pdna. (p<.) (Fig. 4e,f,g, respetively). protein level ws similr in non-trnsfeted s, -pdna. or -pdna./ (Fig. 4h). (46 KD) AGT d AGT (5 KD) (9 KD) / / / / e (46 KD) TGF-β (5 KD) I (7-8 KD) (56 KD) TGF-β I / / / / Fig. 4 overexpression inhiits AGT, TGF-β, I nd enhnes protein expression in s. Immunolotting () nd quntifition of (), AGT () nd (d) protein levels y densitometry in nive s, -pdna. or -pdna./ fter 4 h ulture. Immunolotting (e) nd quntifition of TGF-β (f), I (g) nd (h) protein levels in rt s. Vlues, orreted to β-tin protein levels, re men+sem, n=. The experiments were repeted twie. p<. f g h RT-qPCR of, Agt, Ae-, Tgf-β, Tgf-βrII nd Tgf-βrI mrna levels onfirmed these findings (ESM Fig. f). TGF-β signlling nd inhiition of Ae- gene expression in rt s TGF-β inhiited rt Ae- gene promoter tivity (Fig. 5), rt Ae- mrna expression (Fig. 5) nd rt protein level (Fig. 5) in onentrtion-dependent mnner, whih ws reversed y SB454 ( inhiitor) (Fig. 5d f, respetively). Furthermore, TGF-β stimulted Smd / phosphoryltion in onentrtion- nd timedependent mnner (Fig. 5g) nd reversed y SB454 (Fig. 5h). These dt demonstrte tht TGF-β inhiition of Ae- gene trnsription is medited, t lest in prt, vi Smd/ signlling. HnRNP F overexpression prevents TGF-β signlling, nd TGF-β inhiition of Ae- nd indution of firoti gene expression in s TGF-β hd no detetle effet on protein levels (Fig. 6,). Intriguingly, overexpression prevented TGF-β stimultion of Smd / phosphoryltion (Fig. 6,), I expression (Fig. 6, d) nd fironetin expression (Fig. 6,e). HnRNP F overexpression lso prevented TGF-β-indued downregultion of MsR (Fig. 6,f) ontent in s. Addition of TGF-β did not ffet expression in s (Fig. 6,g). Furthermore, overexpression of prevented the inhiitory effet of TGF-β on protein (Fig. 6,h) nd Ae- mrna (Fig. 6i) expression in -pdna./. Lolistion of - nd TGF-β (or SMAD)-RE in rt Ae- gene promoter To lolise the puttive DNA- RE(s) tht medite(s) the tion of or TGF-β on Ae- gene promoter tivity, plsmids ontining vrious lengths of the rt Ae- gene promoter were trnsiently trnsfeted into -pdna. or -pdna./. The tivity of pgl4.-ae- promoter (N-,9/+ 8) nd pgl 4.-Ae- promoter (N-499/) exhiited respetive fivefold nd -fold inrese s ompred with the ontrol plsmid, pgl 4. in -pdna. (Fig. 7). Further deletion of nuleotides N-499 to N-4 (pgl 4.- Ae- promoter [N-4/]) signifintly redued the rt Ae- promoter tivity. Moreover, the tivity of pgl4.- Ae- promoter (N-,9/) nd pgl4.-ae- promoter (N-499/) ws further inresed y.5.-fold, wheres the tivity of pgl4.-ae- promoter (N-4/) did not inrese in -pdna./ s ompred with -pdna. (Fig. 7). Interestingly, ddition of TGF-β inhiited the promoter tivity of pgl 4.-Ae- promoter (N-,9/) nd did not ffet the tivity of pgl 4.-Ae- promoter (N-499/) nd pgl 4.-Ae- promoter (N-4/) in -pdna. (Fig. 7).

8 45 Dietologi (5) 58: Fig. 5 Humn reominnt TGF-β inhiits Ae- gene expression in rt s. TGF-β inhiits rt Ae- gene promoter tivity () (white rs, pgl4.; lk rs, pgl4.-rt Ae- promoter [N-,9/]), Ae- mrna () nd protein () expression in rt s in dose-dependent mnner. SB454 ( speifi inhiitor) reversed the suppressive effet of TGF-β on Ae- gene promoter tivity (d), Ae- mrna (e) nd protein (f) levels in rt s. TGF-β stimultedthe phosphoryltion of Smd/ in dose- nd time-dependent mnner (g) nd reversed it in the presene of SB454 (h). Rt Ae- gene promoter tivity ws mesured y luiferse tivity ssy. Vlues re men+sem, n=. Similr results were otined in three independent experiments. p<.5; p<., RLU, reltive light units RLU/(μg/μl), 8, 6, 4,, htgf-β (ng/ml).5 5 Ae- mrna (% of ontrol) htgf-β (ng/ml) htgf-β (ng/ml) htgf-β (ng/ml).5 5 (9 KD) d Ae- promoter tivity htgf-β (ng/ml) SB454 (mol/l) -6-6 e Ae- mrna (% of ontrol) htgf-β (ng/ml) SB454 (mol/l) -6-6 (9 KD) htgf-β (ng/ml) SB454 (mol/l) p-smd/ (6 KD) Smd/ (6 KD) p-smd/ (6 KD) Smd/ (6 KD) p-smd/ (6 KD) Smd/ (6 KD) htgf-β (ng/ml) DMSO (.%) SB454 (mol/l) p-smd/ (6 KD) Smd/ (6 KD) -6-6 htgf-β.5 5 (ng/ml) - + htgf-β (ng/ml) SB454 (mol/l) -6-6 g h f 5 min min 4 h However, TGF-β hd no inhiitory effet on the promoter tivity of these onstruts in -pdna./ (Fig. 7). In ontrst, trnsfetion of sirna signifintly inhiited the promoter tivity of pgl 4.-Ae- promoter (N-,9/) nd pgl 4.-Ae- promoter (N-499/) without ffeting the tivity of pgl 4.-Ae- promoter (N-4/) in -pdna. (Fig. 7d). Deletion of the nuleotides N-4 to N-9 (5 -ggggggg- ) in the Ae- gene promoter mrkedly ttenuted the promoter tivity of pgl 4.-Ae- promoter (N-,9/) nd pgl 4.-Ae- promoter (N-499/) in -pdna./ (Fig. 7e). Interestingly, deletion of the puttive proximl SMAD-RE (nuleotides N-5 to N-54 [5 -gg- ]) or distl puttive SMAD-RE (nuleotides N-789 to N-784 [5 - gg- ]) in the Ae- gene promoter prtilly ttenuted wheres deletion of oth REs (nuleotides N-5 to N-54 nd nuleotides N-789 to N-784) ompletely olished the inhiitory tion of TGF-β on pgl 4.-Ae- promoter (N-,9/) tivity in -pdna. (Fig. 7f). Furthermore, EMSA showed tht the doule strnd DNA frgments, nuleotides N-45 to N-87 (puttive -RE), nuleotides N-58 to N-497 (puttive proximl SMAD-RE)nd nuleotides N-797 to N-776 (puttive distl SMAD-RE)ind to the nuler proteins from s nd they ould e displed y the respetive WT DNA frgments, ut not y mutted DNA frgments (Fig. 7g,h, respetively). Importntly, ddition of nti- nd nti-smd / ntiody indued supershift of the respetive -RE nd SMAD- REs with the nuler proteins, respetively (Fig. 7g,h).

9 Dietologi (5) 58: / htgf-β (ng/ml) (46 KD) p-smd/ (6 KD) Smd/ (6 KD) I (7-8 KD) (56 KD) FN ( KD) MsR (4 KD) (9 KD) p-smd//smd/ d I Disussion / / / FN MsR The present report identifies novel mehnism y whih prevents hypertension nd kidney injury in dieti Akit mie, i.e. stimultion of renl Ae- gene trnsription nd mitigtion of the inhiitory effet of TGF-β onae- gene trnsription. h i e f g Ae- mrna (% of ontrol) / / / / / Fig. 6 overexpression prevents TGF-β signlling, stimultion of profiroti gene nd inhiition of expression in rt s. () Immunolotting of, Smd/ phosphoryltion, I,, fironetin (FN), MsR nd ACE levels in nive s, -pdna. or -pdna./ in the presene or sene of TGF-β ( ng/ml) fter 4 h ulture. Quntifition of the level of (), Smd/ phosphoryltion (), I (d), fironetin (e), MsR (f), (g), (h) nd Ae- mrna (i). Vlues re men+sem, n=. Similr results were otined in three independent experiments. p<.5; p<. We reported previously tht overexpression of prevents systemi hypertension, nd inhiits renl Agt gene expression nd hypertrophy in dieti Akit - Tg mie [9]. The present pper provides new in vivo nd in vitro evidene tht stimultes Ae- gene trnsription vi inding to the DNA-RE of the Ae- gene promoter, whih is ritil for the formtion of renl Ang 7 nd susequent expression of its ntihypertensive nd renoprotetive tions in Akit mie [7]. HnRNP F, memer of the pre-mrna-inding protein fmily [8] regultes gene expression t oth the trnsriptionl nd post-trnsriptionl levels. Indeed, engges in lterntive spliing of vrious genes [9 4] nd ssoites with TATA-inding protein, RNA polymerse II, nuler p-inding protein omplex nd vrious trnsriptionl ftors.[4, 4] The Akit mouse is n utosoml-dominnt model of spontneous type dietes in whih the Ins gene is mutted. Akit mie develop hyperglyemi nd systemi hypertension, leding to rdi hypertrophy, left ventriulr distoli dysfuntion, glomeruloslerosis nd enhned oxidtive stress in RPTs, losely resemling those oserved in ptients with type dietes [44, 45]. Our study provides evidene for novel mehnism for lowering of SBP: inhiition of intrrenl Agt gene expression nd RAS tivtion, onomitnt with upregultion of the /Ang 7/MsR xis. Indeed, our results show tht overexpression inhiited renl AGT nd Agt mrna expression (ESM Fig. e), lowered urinry AGT nd Ang II levels nd normlised urinry Ang 7 levels. We onsistently oserved deresed renl expression in Akit mie s previously reported [, 4]. Deresed expression lso hs een reported in mle streptozotoin (STZ)-indued dieti mie [46], STZindued dieti rts [47, 48] nd humn type dieti kidneys [49, 5]. The preise mehnism y whih overexpression leds to upregultion of renl Ae- nd MsR gene expression in dietes remins unler. One possiility is tht inds to puttive -RE(s) in the Ae- nd MsR gene promoters, susequently enhning Ae- nd MsR gene trnsription. This possiility is supported y our findings tht onsiderly ugments the tivity of n Ae- gene promoter nd tht the sirna nd deletion of the puttive -RE mrkedly redued the rt Ae- gene promoter tivity in s. Furthermore, the iotinylted-lelled -RE speifilly ound to nuler proteins nd the ddition of nti- ntiody yielded supershift of iotinylted-lelled -RE inding with nuler proteins in EMSA. These dt demonstrte tht inds to the puttive -RE nd stimultes Ae- gene trnsription. Of note, is not speifi for

10 45 Dietologi (5) 58: Rt Ae- promoter tivity d Rt Ae- promoter tivity e GGGGA GA GG GGGGA GA GG 5 5 Rt Ae- promoter tivity f -9 CA GA GA CA GA GA CA -9 CA GA GA CA -9 GA GA CA Rt Ae- promoter tivity g Nuler extrt (5 μg) Biotinylted HnRNP F-RE HnRNP F-RE (WT) HnRNP F-RE (M) HnRNP F-RE (M) HnRNP F-RE (M) HnRNP F-RE (M4) Anti-hnRNPF ntiody Rit IgG 5 5 Rt Ae- promoter tivity -pdna./ nuler extrt - BSA X X X X h Rt Ae- promoter tivity -TGF-β nuler extrt Nuler extrt (5 μg) BSA+ +++ BSA Biotinylted SMAD-RE SMAD-RE (WT) SMAD-RE (M) -- -X SMAD-RE (M) X Biotinylted SMAD-RE SMAD-RE (WT) SMAD-RE (M) SMAD-RE (M) Anti-p-Smd/ ntiody Rit IgG X X SS SS Fig. 7 Identifition of -RE nd SMAD-RE in the Ae- gene promoter. () iferse tivity of the plsmid ontining vrious lengths of Ae- gene promoter in -pdna. (white rs) nd in -pdna./ (lk rs); () in -pdna.± TGF-β (white rs, without htgf-β; lk rs, with ng/ml htgf-β); nd () in -/±TGF-β (white rs, without htgf-β; lk rs, with ng/ml htgf-β); (d) in pdna.± sirna (white rs, treted with 5 nmol/l srmled sirna; lk rs, treted with 5 nmol/l sirna), ultured in norml gluose medi for 4 h. (e) Promoter tivity of the Ae- gene ±-RE in -pdna. (white rs) nd in -pdna./ (lk rs) or (f)±smad-res in -pdna. in the sene or presene of TGF-β (white rs, without htgf-β; lk rs, with ng/ml htgf-β). Vlues re men+sem, n=6. The experiments were repeted twie. p<.5; p<.. EMSA nd supershift EMSA of the puttive iotinylted -RE (g) nd iotinylted SMAD-REs (h) with nuler proteins±exess unlelled WT -RE or mutted -REs (M to M4 re mutnts of - RE with nuleotides mutted or deleted in the inding motif s shown in ESM Tle )orwt SMAD-RE or mutnt SMAD-REs (SMAD-RE [M nd M] nd SMAD-RE [M nd M] re mutnts of respetive SMAD- RE nd SMAD-RE with nuleotides mutted in the inding motif s shown in ESM Tle ). Rit IgG or rit nti- or nti- Smd/ ntiserum ws dded to the retion mixture nd inuted for min on ie efore inution with the iotinylted proe. Results re representtive of three independent experiments. SS, supershift nd

11 Dietologi (5) 58: Ae- gene expression ut lso ffets the expression of Agt [5] nd other genes [5, 5]. Currently, little is known out the mehnisms y whih TGF-β downregultes renl Ae- gene expression in dietes. Chou et l [5] reported tht SB454 inhiited high gluose nd TGF-β inhiition of Ae- mrna expression in ultured NRK-5 ells. Our findings onfirm these oservtions. Our present studies lso demonstrte tht TGF-β inhiits the tivity of pgl 4.-rt Ae- promoter (N-, 9/) nd tht deletion of puttive SMAD-REs in the Ae- gene promoter mitigtes the inhiitory effet of TGF-β on the Ae- gene promoter tivity. Furthermore, iotinylted-lelled SMAD-REs ound to nuler proteins nd the ddition of nti-smd/ ntiody yielded supershift of lelled DNA with nuler proteins. These dt demonstrte tht the inhiitory effet of TGF-β on Ae- gene trnsription is medited, t lest in prt, vi the SMAD-REs in the Ae- gene promoter. Intriguingly, overexpression prevented TGF-β signlling on Smd/ phosphoryltion nd on TGF-β inhiition of Ae- gene promoter tivity in s. At present, the underlying moleulr mehnism of how prevents TGF-β inhiition of Ae- gene trnsription is not yet defined. One possiility might e tht diretly inhiits Tgf-βrII gene expression s shown in our studies. The seond possiility is tht might interfere or prevent the intertion of Smd/ with other trnsriptionl ftor(s) to inhiit Ae- gene trnsription. Clerly, more studies re needed to define the moleulr intertion of with Smd/ on Ae- gene trnsription. In summry, the present study suggests mjor role for in ttenuting systemi hypertension nd renl firosis in experimentl dietes nd possily in dieti humn kidneys. Our oservtions rise the possiility tht seletive trgeting of this ntihypertensive nd nti-firoti protein my represent novel pproh for preventing or reversing the pthologil mnifesttions of DN, prtiulrly tuulr firosis. Aknowledgements This mnusript or ny signifint prt of it is not under onsidertion for pulition elsewhere. The dt, however, hve een presented in prt s free ommunition t the 45th Annul Meeting of the Amerin Soiety of Nephrology, Sn Diego, CA, USA, Otoer 4 Novemer (Free Communition, TH-OR5). Funding This work ws supported y grnts from the Cndin Institutes of Helth Reserh (MOP 846 nd MOP 6688 to JSDC, MOP to SLZ nd MOP-9774 to JGF), the Kidney Foundtion of Cnd (KFOC8 to JSDC), the Cndin Dietes Assoition (NOD_OG JC to JSDC), nd the Ntionl Institutes of Helth (NIH) of USA (HL to JRI). CSL is the reipient of fellowship from the Montrel Dietes Reserh Centre of the CRCHUM. Editoril ssistne ws provided y the CRCHUM Reserh Support Offie. Dulity of interest The uthors delre tht there is no dulity of interest ssoited with this mnusript. Contriution sttement JSDC is the prinipl investigtor nd ws responsile for the study oneption nd design. CSL drfted the mnusript nd ontriuted to the disussion. CSL, YS, SYC, SA, IC nd SLZ ontriuted to the in vivo nd in vitro experiments nd olletion of dt. JGF nd JRI ontriuted to the Disussion nd reviewed/edited the mnusript. All uthors were involved in nlysis nd interprettion of dt, nd ontriuted to the ritil revision of the mnusript. All uthors provided finl pprovl of the version to e pulished. JSDC is gurntor of this work nd, s suh, hd full ess to ll study dt, tking responsiility for dt integrity nd the ury of dt nlysis. Open Aess This rtile is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl Liense ( retiveommons.org/lienses/y/4./), whih permits unrestrited use, distriution, nd reprodution in ny medium, provided you give pproprite redit to the originl uthor(s) nd the soure, provide link to the Cretive Commons liense, nd indite if hnges were mde. Referenes. de Boer IH, Rue TC, Hll YN et l () Temporl trends in the prevlene of dieti kidney disese in the United Sttes. JAMA 5:5 59. 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