Role of the EGF receptor in PPARγ-mediated sodium and water transport in human proximal tubule cells

Transcription

1 Dietologi (213) 56: DOI 1.17/s y ARTICLE Role of the EGF reeptor in PPARγ-meite soium n wter trnsport in humn proximl tuule ells S. S & J. Zhng & R. Yong & D. Yghoin & M. G. Wong & D. J. Kelly & X. M. Chen & C. A. Pollok Reeive: 1 Novemer 212 / Aepte: 4 Jnury 213 / Pulishe online: 31 Jnury 213 # Springer-Verlg Berlin Heielerg 213 Astrt Aim/hypothesis This stuy ime to etermine the intertion etween the EGF reeptor (EGFR) n peroxisome prolifertor-tivte reeptor γ (PPARγ) n the role of EGFR in soium n wter trnsport in the proximl tuule. Methos Primry humn proximl tuule ells (PTCs) were expose to high gluose in the presene n sene of pioglitzone. Totl n phospho-egfr levels n EGFR mrna expression were etermine y western lot n rel-time PCR, respetively. Soium hyrogen exhnger-3 (NHE3), PPARγ n quporin 1 (AQP1) levels were etermine y western lot. The role of EGFR ws eluite using the EGFR tyrosine kinse inhiitor, PKI166. The role of PPARγ in high-gluose onitions ws etermine using speifi PPARγ smll interfering (si)rna. P-EGFR, PPARγ, AQP1 n NHE3 proution in rt moel of ietes (streptozotoin-inue hypertensive Ren-2 trnsgeni [mren2]27 rts) n ontrols, with or without pioglitzone tretment, ws etermine y immunohistohemistry. The PPARγ n EGFR intertion ws etermine y hromtin immunopreipittion ssy, n the effet of pioglitzone on EGFR tivtion y luiferse ssy. Results PTCs expose to oth high gluose n pioglitzone inrese protein unne of P-EGFR, NHE3, AQP1 n PPARγ. Pioglitzone-inue upregultion of NHE3 n Eletroni supplementry mteril The online version of this rtile (oi:1.17/s y) ontins peer-reviewe ut uneite supplementry mteril, whih is ville to uthorise users. S. S () : J. Zhng : R. Yong : D. Yghoin : M. G. Wong : X. M. Chen : C. A. Pollok Renl Reserh Lortories, Kolling Institute of Meil Reserh, Royl North Shore Hospitl, Kolling Builing, St Leonrs, NSW 265, Austrli e-mil: sonis@me.usy.eu.u D. J. Kelly Deprtment of Meiine, St Vinent s Hospitl, University of Melourne, Melourne, Austrli AQP1 ws olishe y PKI166. High-gluose-inue inreses in P-EGFR, NHE3 n AQP1 were erese with PPARγ sirna. AQP1 n NHE3 ut not PPARγ were inrese in ieti rt moel n further inrese y pioglitzone tretment. Pioglitzone inue PPARγ ining to the EGFR promoter n susequent ownstrem tivtion. Conlusions/interprettion Our t suggest tht EGFR tivtion meites PPARγ-inue soium n wter resorption vi upregultion of NHE3 n AQP1 hnnels in the proximl tuule. EGFR inhiition my e therpeuti strtegy in the tretment of ieti nephropthy n in limiting slt n wter retention, whih urrently restrits the use of PPARγ gonists. Keywors Dieti nephropthy. EGF reeptor. PPARγ. Soium retention Arevitions AQP1 Aquporin-1 ChIP Chromtin immunopreipittion EGFR EGF reeptor NHE3 Soium hyrogen exhnger-3 P-EGFR Phospho-EGFR PPARγ Peroxisome prolifertor-tivte reeptor γ PTC Proximl tuulr ell Sgk1 Serum/gluoortioi kinse reeptor sirna Smll interfering RNA t-egfr Totl EGFR STZ Streptozotoin TZD Thizoliineione Introution Peroxisome prolifertor-tivte reeptor γ (PPARγ) is memer of the nuler hormone reeptor superfmily. Ativtion of the PPARγ pthwy requires heteroimeristion of

2 Dietologi (213) 56: lign-oun PPARγ with the retinoi X reeptor, whih ins speifi peroxisome prolifertor response elements in the promoter regions of trget genes [1]. Despite extensive literture on the enefiil effets of syntheti PPARγ gonists in limiting insulin resistne n proteting pnreti et ell funtion, n our own t suggesting protetive effet on nephropthy [2 5], slt n wter retention pose key limittion to the uptke of these gents in linil prtie. The proximl tuule meites 5 75% of totl tuulr soium n wter resorption, with the soium hyrogen exhnger-3 (NHE3) n quprorin-1 (AQP1) eing the key trnsporter n hnnel, respetively, meiting trnsellulr slt n wter resorption from the tuulr ultrfiltrte. We hve emonstrte tht PPARγ is proue in humn proximl tuulr ells (PTCs; HK2 ells) n is tivte y exposure to high gluose [6], s well s y the insulin-sensitising gents, thizoliineiones (TZDs) [7]. We hve lso emonstrte tht the serum/gluoortioi kinse reeptor gene, SGK1, is trget gene of PPARγ n meitor for PPARγ gonistinue NHE3 n AQP1 upregultion in humn PTCs [8]. High gluose hs een shown to trnstivte the EGF reeptor (EGFR), n the tivtion of EGFR through tyrosine phosphoryltion ours in the sene of extrellulr ligns in response to PPARγ gonists [9 11]. We hve emonstrte tht high gluose regultes NHE3 through n EGFR-meite pthwy [11]. This suggests tht exessive soium resorption in response to PPARγ tivtion my our through regulte ownstrem tivtion of EGFR in the proximl tuule. The role of the EGFR in PPARγmeite NHE3 n AQP1 proution in humn PTCs is not known n will e resse in this stuy. Methos Primry ulture of humn PTCs Segments of mrosopilly n histologilly norml renl ortex were otine uner septi onitions from ptients unergoing nephretomy for smll (<.6 m) tumours. Ptients were epte for inlusion in the stuy if there ws no history of renl or systemi isese known to e ssoite with tuulointerstitil pthology. Written informe onsent ws otine from eh ptient efore surgery, n ethis pprovl for the stuy ws otine from the Royl North Shore Hospitl Humn Reserh Ethis Committee. The methos for primry ulture of humn PTCs re esrie in etil elsewhere [12]. The ultrstruture, growth n immunohistohemistry of these ells hve een well hrterise in our lortory n shown to reprouily reflet the iology n physiology of their in vivo ounterprts [12 14]. Cell ulture Humn PTCs were grown in.1 m tissue ulture ishes (Beton Dikinson, North Rye, NSW, Austrli) n expose to 5 mmol/l D-gluose (ontrol meium), 25 mmol/l D-gluose, 5 mmol/l D-gluose+ 2 mmol/ll-gluose (osmoti ontrol) or 1 μmol/l pioglitzone in 5 mmol/l D-gluose for 24 h. L- n D-gluose (ICN Biomeil, Irvine, CA, USA) n pioglitzone (Cymn Chemil, Ann Aror, MI, USA) were use to etermine the speifi effets of PPARγ tivtion. Initil ose response experiments were unertken to etermine the onentrtion t whih pioglitzone mximlly stimulte PPARγ protein expression. On the sis of these stuies, we use 1 μmol/l pioglitzone in the experimentl protools s previously esrie [4, 6, 8]. As pioglitzone is issolve in DMSO,.13% DMSO ws use in the ontrol meium for ll the pioglitzone-relte experiments. The more seletive PPARγ gonist, L (Merk Biosienes, Drmstt, Germny), ws use in some experiments to etermine the speifi effets of PPARγ tivtion [8]. To etermine the role of EGFR in meiting the oserve hnges, the EGFR tyrosine kinse inhiitor of the pyrrolopyrimiine lss, PKI166 (4 μmol/l), ws use s previously esrie [11], in ells expose to either high gluose or pioglitzone. PKI166 ws synthesise in the lortories of NIBR (Novrtis Institutes for Biomeil Reserh, North Rye, NSW, Austrli) n ws provie y NIBR for the purpose of this stuy. PKI166 is onsiere highly speifi, with the inhiition onstnt (IC 5 ) for EGFR eing.7 nmol/l [15]. Western lotting Western lots were performe on Triton X- 1-solule frtions. PPARγ-speifi ntioy (Snt Cruz Biotehnology, Snt Cruz, CA, USA), AQP1 n NHE3 ntioies (Chemion Interntionl, Temeul, CA, USA), totl EGFR (t-egfr) ntioy (Cell Signling, Dnvers, MA, USA), phospho-egfr (P-EGFR) ntioy (py168; Invitrogen, Crls, CA, USA) or tin ntioy (Sigm- Alrih, St Louis, MO, USA) were use overnight followe y inution with nti-rit or nti-mouse ntioy (Amershm Phrmeutils, Rylmere, NSW, Austrli) for 1 h t room temperture. Dt were normlise to tin to ensure equl protein loing. The ns orresponing to AQP1 (28 kd), PPARγ (67 kd), NHE3 (85 kd), t-egfr or P-EGFR (17 kd) n tin (42 kd) were quntifie using NIH Imge softwre version 1.6. Rel-time RT-PCR RNA ws extrte using n RNesy Mini Kit (Qigen, Vleni, CA, USA) oring to the mnufturer s instrutions. DNA ws generte y reverse trnsriing 1 μg totl RNA in retion volume of 2 μl using VILO DNA Synthesis Kits (Invitrogen). Sequene-speifi primers for humn EGFR n β-tin re esrie in eletroni supplementry mteril (ESM) Tle 1. Primer speifiity in rel-time PCRs ws onfirme using RT-PCR. A 25 μl rel-time PCR inlue Brillint

3 1176 Dietologi (213) 56: SYBR Green QRT PCR Mster Mix oring to the mnufturer s instrutions (Strtgene, L Joll, CA, USA). Quntittive rel-time PCR ws performe using n ABI Prism 7,9 HT Sequene Detetion System (Applie Biosystems, Mulgrve, VIC, Austrli). Retions were performe in t lest triplite n nlyse y reltive quntifition using RQ Mnger softwre, version 1.2 (Applie Biosystems). All t re presente s fol hnge ompre with ontrol fter normlistion to the housekeeping gene, β-tin. PPARγ silening Smll interfering RNA (sirna) ws esigne to speifilly trget PPARγ mrna sites. PPARγ sirna trget is s follows: GCCTCATGAAGAGCCTTC- CAACTCCCTCA (Amion, Mulgrve, VIC, Austrli). PTCs were trnsfete with 8 nmol/l PPARγ sirna using Riojuie (Novgen, Mison, WI, USA) s per the mnufturer s instrutions. Protein levels were etermine using western lot. All sirna experiments inlue non-speifi ontrol (NSC sirnas; Amion). One PPARγ knokown ws onfirme, susequent experiments were onute to etermine the effet of exposure to high gluose (25 mmol/l) on AQP1, NHE3 n P-EGFR levels 24 h fter PPARγ knokown. Cell lystes were then ollete. Western lotting experiments were performe s esrie ove. In vivo experiments To etermine P-EGFR levels in n in vivo moel of ietes mellitus, we use the streptozotoininue hypertensive Ren-2 trnsgeni rt (mren2)27 moel. These nimls were onfirme to hve emonstrle hyperfiltrtion, proteinuri n pthologil signs of ieti nephropthy t the time of stuy. This moel hs een previously vlite for stuies in ieti nephropthy [16 2]. Briefly, 6-week-ol femle, heterozygous Ren-2 rts, weighing.125±5 kg, were rnomise to reeive either 55 mg/kg streptozotoin (STZ; Sigm) (ieti) or uffer lone (non-ieti) y til vein injetion fter n overnight fst. Dieti Ren-2 rts were susequently trete with 5 mg/kg pioglitzone (Cymn Chemil) fe y gvge or shm tretment for 12 weeks strting 7 ys fter STZ injetion. Experimentl proeures here to the guielines of the Ntionl Helth n Meil Reserh Counil of Austrli s Coe for the re n use of nimls for sientifi purposes n hve een pprove y St Vinent s Hospitl Animl Ethis Committee, Melourne, Austrli. Seril setions of 4 μmol/l thikness were fixe in 4% prformlehye, n prffin setions were prepre for immunohistohemistry. Levels of P-EGFR, PPARγ, NHE3 n AQP1 protein were etermine. Immunohistohemistry Prffin-emee setions were ewxe in xylene n rehyrte in gre onentrtions of ethnol. Enogenous peroxise tivity ws loke y immersion in 3% H 2 O 2 for 5 min. Epitope retrievl ws require for P-EGFR, AQP1 n NHE3, ut not PPARγ, stining. Het retrievl ws performe in.1 mol/l itrte uffer, ph6., using pressure ooker set t 121 C for 3 s. Non-speifi protein ining ws loke with protein lok (Dko, Troy, MI, USA). Inution with primry ntioies ws performe using Sequenz vertil overplte immunostining system (Thermofisher Sientifi, Soresy, VIC, Austrli) using.2 μg P-EGFR (Tyr1173) ntioy, 4 μg PPARγ ntioy (oth from Snt Cruz),.2 μg NHE3 ntioy (Novus Biologils, Littleton, CO, USA) or 4 μg AQP1 ntioy (Snt Cruz) overnight t 4 C. The primry ntioies were susequently lolise using iotinylte seonry nti-mouse or nti-rit IgG ntioies. Horserish peroxise-onjugte streptviin ws susequently use to visulise the tissue immune omplexes using the LSAB+etetion system (Dko). Antigen ntioy retions were visulise with the hromogen, iminoenziine, n ounterstining ws performe using Myer s Hemtoxylin followe y Sott s Blue stining (Fronine, Tren Point, NSW, Austrli). Control setions were lso prepre in whih the uthenti primry ntioies were reple with n irrelevnt isotype-mthe IgG. The tissue speimens were exmine y right fiel mirosopy using Lei photomirosope linke to DFC 48 igitl mer. Quntifition of histologil vriles In rief, 1 rnom non-overlpping fiels from three stine setions were pture n igitise using n AxioImger.A1 mirosope (Crl Zeiss AxioVision, Kirhorf, Germny) tthe to n AxioCm MR5 igitl mer (Crl Zeiss AxioVision). Ares of rown stining refleting P-EGFR, PPARγ, AQP1 or NHE3 proution were quntifie n lultion of the proportionl re stine rown ws then etermine using imge nlysis softwre AIS (Anlyti Imging Sttion version 6., Imging Reserh, St Ctherines, ON, Cn). Chromtin immunopreipittion ssy A hromtin immunopreipittion (ChIP) ssy ws performe using n EZ ChIP Kit (Millipore, Billeri, MA, USA) oring to the mnufturer s instrutions. In rief, ells were rosslinke in 1% formlehye fter ifferent ell ulture tretments. Cells were then lyse n sonite. Sonition ws optimise to hieve 2 to 1 p DNA frgments. Equl mounts of protein were inute with 4 μg/ml PPARγ ntioy (Snt Cruz Biotehnology). The rosslink of immunopreipitte protein DNA smples ws then reverse, n DNA smples were purifie using spin olumns. PPARγ-ining sites of the EGFR promoter were quntifie using rel-time PCR. Primers use for the promoter region re esrie in ESM Tle 2.

4 Dietologi (213) 56: EGFR promoter tivity ssy The promoter tivity of EGFR ws etermine y the Dul-Luiferse Reporter Assy System (Promeg, Mison, WI, USA) s we hve esrie previously [21]. In rief, the DNA frgment (1,9 p) of the EGFR promoter ws mplifie using PfuUltr II HS Fusion DNA Polymerse (Agilent Tehnologies, Snt Clr, CA, USA). The DNA frgment of the EGFR promoter ontining the PPARγ-ining site ( 1271: AGGTCCTAGTGAA) ws sulone into pgl3 firefly luiferse vetor (Promeg). The plsmi, pgl3-egfr promoter, or the pgl3 sl plsmi ws introue into PTCs using Lipofetmine 2 (Invitrogen). prl-sv4 Renill vetor (Promeg) ws otrnsfete into ells, n its luiferse tivity ws use for normlistion of trnsfetion effiieny. Cells were then expose to pioglitzone or L for 24 h, n luiferse tivity ws etete y POLARstr (BMG Lteh, Offenurg, Germny). Sttistil nlysis Results re expresse s perentge or fol hnge ompre with the ontrol. Experiments were performe in t lest three ifferent ulture preprtions erive from eh ptient, n t lest three t points for eh experimentl onition were mesure in eh preprtion. Six nimls in eh group were use in the in vivo stuy. Results re expresse s men±sem, with n refleting the numer of ulture preprtions. Sttistil omprisons etween groups were me y ANOVA, with pirwise multiple omprisons me y Fisher s protete lest signifint ifferene test. Anlyses were performe using the softwre pkge, Sttview version 4.5 (Aus Conepts, Pistwy, NJ, USA). p vlues.5 were onsiere signifint. Results High gluose n pioglitzone inue PPARγ We hve previously emonstrte tht high gluose n pioglitzone inrese PPARγ protein expression in humn PTCs (HK2 ells) [6]. We hve onfirme tht PPARγ protein ontent ws signifintly inrese to 199±21.6% in humn primry PTCs when expose to 25 mmol/l gluose (HG) (p<.5). Exposure to the osmoti ontrol (OC) h no effet on PPARγ levels (19±23.4%; p=.7) (Fig. 1). Exposure to 1 μmol/l pioglitzone lso inrese PPARγ protein levels to 142±6.2% (p<.1) (Fig. 1). Comine effet of high gluose n pioglitzone on EGFR High gluose inrese t-egfr levels to 414± 15% of ontrol, most of whih (36±2%) ws phosphorylte (p<.5 vs ontrol) (Fig. 2, ). P-EGFR is proue t low levels in humn PTCs, s we hve previously esrie [11] n s is lso shown in Fig. 5. The omintion of high gluose n pioglitzone inrese t-egfr protein PPARγ protein level PPARγ Control HG OC ontent to 112±329% (p<.1), in ition to inresing EGFR phosphoryltion to 575±89% (p<.1) (Fig. 2, ). High gluose in the sene n presene of pioglitzone inrese EGFR mrna levels to 1.3±.3-fol (p<.1) n 1.7±.1-fol (p<.1), respetively (Fig. 2). Pioglitzone inreses NHE3 n AQP1 through n EGFR pthwy We hve onfirme tht NHE3 is signifintly inrese when PTCs re expose to pioglitzone, s we hve previously emonstrte [8], to 167±16.1% (p<.1). To etermine the role of EGFR in inrese NHE3 proution, we expose ells to the EGFR tyrosine kinse inhiitor, PKI166, in the presene of pioglitzone, n NHE3 protein ontent ws ssesse. The omintion of PKI166 with pioglitzone rogte the oserve inrese in NHE3 levels with the PPARγ gonist, pioglitzone, to 13±6.1% (Fig. 3). These results suggest tht the upregultion of NHE3 fter exposure to pioglitzone is EGFR meite. As we hve previously emonstrte [8], pioglitzone signifintly inrese AQP1 protein proution to 21± 23% of ontrol vlues (p<.1). The omintion of PKI166 with pioglitzone rogte the oserve inrese in AQP1 levels with the PPARγ gonist, pioglitzone, to 95± 21% (p=.7 vs ontrol) (Fig. 3). These results suggest tht the upregultion of AQP1 fter exposure to pioglitzone is lso meite through the EGFR. High-gluose-inue P-EGFR, NHE3 n AQP1 is PPARγ meite Sine pioglitzone hs PPARγ-meite n PPARγ-inepenent effets, to emonstrte the speifi role of PPARγ in the high-gluose-meite inrese in P- EGFR, we performe experiments in the presene of speifi PPARγ sirna. As lerly emonstrte, we were le PPARγ protein level Control Piog PPARγ Fig. 1 High gluose n pioglitzone inue PPARγ protein expression. PTCs were inute for 24 h with 5 mmol/l gluose meium (Control), 25 mmol/l gluose (HG), 5 mmol/l gluose meium with 2 mmol/l gluose (osmoti ontrol; OC) or 1 μmol/l pioglitzone (Piog) in ontrol meium. Representtive western lotting imges re shown for PPARγ n tin ns fter high-gluose () or pioglitzone () exposure. Normlise results re expresse s men±sem (n =3). p<.5 n p<.1 vs ontrol

5 1178 Dietologi (213) 56: t-egfr protein level 2, 1,5 1, 5 t-egfr Control HG HG+Piog P-EGFR protein level P-EGFR Control HG HG+Piog EGFR mrna expression (fol inrese) Control HG HG+Piog Fig. 2 Effet of high gluose (HG) in the presene n sene of pioglitzone on EGFR. PTCs were inute for 24 h with 5 mmol/l gluose meium (Control) in the presene or sene of pioglitzone (Piog), n levels of P-EGFR were quntifie. Representtive western lotting imges for t-egfr () n P-EGFR () re shown. EGFR mrna expression is shown in (). Normlise results re expresse s men± SEM (n=3). p<.5, p<.1 n p<.1 vs ontrol n p<.5 vs HG to signifintly lok PPARγ protein expression using speifi PPARγ sirna to 27±4% of ontrol vlues (p<.1) (Fig. 4). Phosphoryltion of EGFR fter high gluose exposure ws onfirme using immunopreipittion. High gluose signifintly inue P-EGFR to 12.5±1.5% (p<.1). This inrese ws olishe in the presene of PPARγ sirna to 96.5±9.5% (Fig. 4). This suggests tht the effet of pioglitzone on P-EGFR is PPARγ meite. To onfirm tht the high-gluose effet on NHE3 n AQP1 is PPARγ meite, we repete experiments in the presene of PPARγ sirna. High-gluose-inue inreses in NHE3 n AQP1 levels were signifintly inhiite to sl levels in the presene of PPARγ sirna (to 91±17% n 78±12.8%, respetively) (Fig. 4, ). These t show tht the high-gluose inrese in NHE3 n AQP1 in humn PTCs is PPARγ meite. Effet of high gluose n pioglitzone on P-EGFR, AQP1, NHE-3 n PPARγ levels in vivo The expression of P-EGFR n the effet of high gluose n pioglitzone on P-EGFR expression in the proximl tuule were onfirme in n in vivo moel of ietes mellitus. P-EGFR levels were very low in the proximl tuule of non-ieti rts. However, its levels were inrese in the proximl tuule memrnes n ytoplsm of ieti rts n further inrese in ieti rts NHE3 protein level NHE Control Piog Control Piog Control Piog Control Piog PKI PKI AQP1 protein level AQP1 Fig. 3 PPARγ gonist-inue inrese in NHE3 n AQP1 is P- EGFR meite. PTCs were inute for 24 h with 5 mmol/l gluose meium (Control) or pioglitzone (Piog) with or without PKI166 (4 μmol/l), n levels of NHE3 () or AQP1 () were etermine y western lotting. Representtive imges for NHE3, AQP1 n tin ns re shown. Normlise results re expresse s men±sem (n= 4). p<.1 vs ontrol trete with pioglitzone (Fig. 5). As expete, nuler n ytoplsmi unne of PPARγ ws shown in ontrol rts (Fig. 6). However, this ws erese in the ieti rts. Interestingly only ytoplsmi stining of PPARγ ws shown in the ieti rts (Fig. 6). In ieti rts, ytoplsmi n nuler PPARγ levels were signifintly inrese in the presene of pioglitzone (Fig. 6), suggesting inrese PPARγ tivity. NHE3 is foun t low level in the tuules of ontrol rts (Fig. 7). Its levels were more onentrte in the luminl memrne of the tuule. This ws inrese in the ieti rts n further inrese in the presene of pioglitzone with inrese perinuler stining (Fig. 7, ). Similrly, inrese levels of AQP1 were emonstrte in the ieti rts omprewithontrols(fig.8, ). AQP1 is lerly proue in the luminl memrne of the tuules. Its ontent ws signifintly inrese in the presene of pioglitzone. Interestingly, PPARγ protein level NHE3 protein level NSC sirna PPARγ sirna PPARγ NHE3 Control HG Control HG NSC sirna PPARγ sirna P-EGFR protein level AQP1 protein level Control HG Control HG NSC sirna PPARγ sirna Control HG Control HG NSC sirna PPARγ sirna P-EGFR AQP1 Fig. 4 High-gluose-inue inrese in P-EGFR, NHE3 n AQP1 is PPARγ meite. Derese ontent of PPARγ ws onfirme in gene-silene PTCs using PPARγ sirna vs non-speifi sirna ontrol (NSC) (). PTCs were inute for 24 h with 5 mmol/l gluose meium (Control) or high gluose (HG) in the presene of PPARγ sirna or NSC sirna. P-EGFR (), NHE3 () or AQP1 () protein ontent ws etermine y western lotting. Representtive imges for P-EGFR, NHE3, AQP1 n tin ns re shown. Normlise results re expresse s men±sem (n=3). p<.5, p<.1 n p<.1 vs NSC sirna

6 Dietologi (213) 56: Proportionl re of P-EGFR pioglitzone lso inrese AQP1 levels in the pil rush orer s well s in the luminl memrne (Fig. 8). PPARγ regultes EGFR expression n tivity y ining to EGFR promoter, n thizoliineiones promote this ining Using motif sn progrm, we etermine tht the EGFR promoter ontins PPARγ-ining site t 1271: AGGTCCTAGTGAA. The ility of PPARγ to in the EGFR promoter ws teste using ChIP ssy. The t show tht pioglitzone signifintly inrese PPARγ ining to the EGFR promoter 1.66±.2-fol (p<.5) (Fig. 9). In ition, the more seletive PPARγ Control Dieti Dieti+Piog Fig. 5 P-EGFR levels in ieti rts with n without pioglitzone (Piog). Immunohistohemistry of P-EGFR in ontrol Ren-2 rts () n ieti () n ieti+pioglitzone-trete () nimls. Negtive IgG ws performe to onfirm stining speifiity (not shown). Mgnifition 4. Quntifition of P-EGFR immunostining is shown (). Vlues re represente s men±sem. p<.1 n p<.1 vs ontrol n p<.1 vs ieti Proportionl re of NHE3 gonist, L-85645, further inrese PPARγ EGFR promoter ining 8.8±2.6-fol (p<.5) (Fig. 9). This suggests tht PPARγ n in to the EGFR promoter to regulte its trnsriptionl tivity. Sine PPARγ n iretly in the EGFR promoter, n in orer to estlish if this ining inues EGFR tivtion, we etermine the promoter tivity of EGFR fter PPARγ tivtion y Dul-Luiferse Reporter Assy. Pioglitzone inrese luiferse tivity 1.5±.1-fol (p<.5), n L further inrese luiferse tivity 5.5±1.5- fol (p<.5) (Fig. 9) Control Dieti Dieti+Piog Fig. 7 NHE3 levels in ieti rts with n without pioglitzone (Piog). Immunohistohemistry of NHE3 in ontrol Ren-2 rts () n ieti () n ieti+pioglitzone-trete () nimls. Negtive IgG ws performe to onfirm stining speifiity (not shown). Mgnifition 4. Quntifition of NHE3 immunostining is shown (). Vlues re represente s men±sem. p<.1 n p<.1 vs ontrol n p<.5 vs ieti Proportionl re of PPARγ Control Dieti Dieti+Piog Fig. 6 PPARγ levels in ieti rts with n without pioglitzone (Piog). Immunohistohemistry of PPARγ in ontrol Ren-2 rts () n ieti () n ieti+pioglitzone-trete () nimls. Negtive IgG ws performe to onfirm stining speifiity (not shown). Mgnifition 4. Quntifition of PPARγ immunostining is shown (). Vlues re represente s men±sem. p<.5 vs ontrol n p<.1 vs ieti Proportionl re of AQP Control Dieti Dieti+Piog Fig. 8 AQP1 expression in ieti rts with n without pioglitzone (Piog). Immunohistohemistry of AQP1 in ontrol Ren-2 rts () n ieti () n ieti+pioglitzone-trete () nimls. Negtive IgG ws performe to onfirm stining speifiity (not shown). Mgnifition 4. Quntifition of AQP1 immunostining is shown (). Vlues re represente s men±sem. p<.1 n p<.1 vs ontrol n p<.1 vs ieti

7 118 Dietologi (213) 56: PPARγ EGFR promoter ining (fol hnge) Disussion Control Piog L Cellulr soium n wter trnsport re ysregulte in ptients with ietes mellitus, whih re onsiere to e t lest in prt responsile for the high iniene of hypertension oserve in these ptients. EGFR is tivte/trnstivte y EGF, high gluose n ngiotensin II, ll ftors implite in the pthogenesis of ieti nephropthy. To te, only few stuies hve fouse on the role of EGFR in enhning tuulr flui resorption in the kiney proximl tuule. We hve emonstrte tht high gluose trnstivtes EGFR, whih then regultes the key trnsporter of proximl tuulr soium resorption, NHE3, through ownstrem regultion of Sgk1 [11]. In ition, we hve shown tht NHE3 is regulte through Sgk1 y TZDs [8]. The possile role of EGFR in the TZD-meite effets on soium n wter retention in the proximl tuule ws exmine in the present stuy. We hve onfirme tht PPARγ is proue in humn PTCs n its level is inrese fter exposure to high gluose n the linilly ville PPARγ lign (pioglitzone). This is onsistent with our previous finings using the HK2 primry humn PTC line [6]. We hve lso onfirme, using in vivo moels of STZinue ietes mellitus, tht PPARγ is proue in the proximl tuule n upregulte in ieti nimls in the presene of pioglitzone. Interestingly, ieti nimls h reue levels of nuler PPARγ. Our t show tht, in ieti nimls, PPARγ nuler trnslotion ws iminishe, suggesting reue PPARγ tivity, s hs een previously reporte [22]. In ition, we hve speifilly etermine tht omintion of high gluose with pioglitzone inrese totl EGFR mrna n protein expression n EGFR phosphoryltion. Although high gluose inrese P-EGFR levels, there ws no hnge in EGFR mrna levels. In vivo, ieti Ren-2 Luiferse tivity (fol hnge) Control Piog L Fig. 9 EGFR promoter ining tivity n Renill luiferse ssy. Cells were expose to ontrol meium, pioglitzone (Piog; 1 μmol/l) or the more seletive PPARγ gonist, L (8 μmol/l) for 24 h. () The ChIP ssy ws performe using the ChIP Kit oring to the mnufturer s instrutions. Rel-time PCR ws performe on purifie DNA smples whih were immunopreipitte with PPARγ ntioy. () Luiferse tivity ws mesure using the Renill Luiferse Assy System oring to the mnufturer s instrutions. Results re expresse s men±sem n shown s fol hnge ompre with ontrol. Three inepenent ell ulture preprtions were performe. p<.5 vs ontrol rts lso showe inrese P-EGFR expression, whih ws further potentite y tretment with pioglitzone. One of the most importnt funtions of the kiney is to mintin eletrolyte n metoli homeostsis. Our results show tht loke of EGFR phosphoryltion using PKI166, novel n highly speifi EGFR tyrosine kinse inhiitor of the pyrrolopyrimiine lss, ttenutes the inrese unne of NHE3 n AQP1 in PTCs in response to pioglitzone. This novel fining suggests role for EGFR in TZDmeite soium n wter trnsport in the proximl tuule. Drumm et l hve emonstrte tht losterone-inue NHE3 ell surfe expression n tivity in the proximl tuule is EGFR-epenent [23]. EGFR tivtion les to speifi erese in the levels of the tight juntion integrl protein, luin-2, whih results in moultion of prellulr soium trnsport in MDCK ells [24, 25]. Uner physiologil onitions, EGFR tivtion ppers to ply n importnt role in the regultion of renl hemoynmis n eletrolyte hnling y the kiney, wheres, in ifferent pthophysiologil sttes, EGFR tivtion my meite either enefiil or etrimentl effets on the kiney [26]. The interepenene of PPARγ n EGFR in the kiney hs not een previously shown. However, in other tissues suh s ner n enothelil ells, EGFR is moulte y PPARγ gonists in PPARγ-epenent n -inepenent mnner. To etermine if the high-gluose effet on NHE3 n AQP1 is PPARγ meite, we use PPARγ sirna. Our t show tht P-EGFR is regulte ownstrem of PPARγ in response to high gluose. We hve shown tht high-gluoseinue P-EGFR is PPARγ meite, n the high-gluosemeite inrese in NHE3 n AQP1 is lso through PPARγ. In ition, we hve lerly emonstrte tht NHE3 n AQP1 re inrese in ieti rts n further inrese in the presene of pioglitzone n tht pioglitzone inrese AQP1 expression in the rush orer. Inrese levels of NHE3 n AQP1 represent inrese tivity, s previously esrie [8, 27]. This explins t lest in prt the enhne slt n wter retention oserve with pioglitzone use in ptients with ietes mellitus. Our t suggests ross-tlk etween PPARγ n EGFR in humn PTCs. PPARγ n EGFR signlling is known to interset in some ell types. For exmple, PPARγ regultion of urothelil ifferentition is moulte y ownstrem EGFR signlling [28]. Coorinte tivity etween PPARγ n EGFR hs een oumente in the inution of veolin-1 y rosiglitzone tretment in humn olon ner ells [29]. Interestingly, Lewis et l hve reently reporte tht ptients trete with pioglitzone hve n inrese risk of urinry ler ner if the rug is tken for more thn 2 yers n t high ose [3]. The risk of inrese ler ner is potentilly mitigte y the use of EGFR inhiitors. We hve uniquely emonstrte tht EGFR hs PPARγining site n tht TZD not only inrese PPARγ levels ut

8 Dietologi (213) 56: lso its ining to the EGFR promoter n EGFR tivtion. Our finings suggest tht high gluose n TZDs tivte/- trnstivte PPARγ, whih, through iret ining n ownstrem tivtion of EGFR, then inue NHE3 n AQP1 in the proximl tuule. Hene, the normlities in tuulr ell growth known to our in ieti nephropthy, ellulr soium n wter trnsport whih re ysregulte in ietes mellitus, n the inrese in soium resorption known to e responsile for the oserve flui retention with the use of TZDs re likely to our through n EGFR-epenent mehnism. These t lerly highlight the importne of EGFR in renl soium resorption n suggest tht PPARγ gonists in omintion with the linilly ville EGFR lokers my e enefiil in regulting the nephromegly n exessive soium resorption oserve in ieti nephropthy. In ft, EGFR tyrosine kinse inhiition meliortes the erly hnges tht our in ieti nephropthy suh s tuulr epithelil ell prolifertion, glomerulr enlrgement n hnges in kiney weight in ieti rts [31] n suppresses TGFβ-meite mtrix protein proution in rt kiney interstitil firolsts [32]. Dieti rts ministere n EGFR tyrosine kinse inhiitor showe ttenute kiney n glomerulr enlrgement n reution in luminuri, in ssoition with pooyte preservtion [31, 33]. These stuies, in ition to our t, suggest tht inhiition of the EGFR my provie n ttrtive therpeuti trget for the tretment of ieti nephropthy s well s limiting slt n wter retention, whih urrently restrits the use of PPARγ gonists. Aknowlegements We knowlege the support of the Deprtment of Urology of Royl North Shore Hospitl n Conor Hospitl for ssisting in prouring the kineys for primry ulture. We woul like to thnk S. Smith (Rymon Purves Bone n Joint Reserh Lortories, Kolling Institute for Meil Reserh, Austrli) for help with immunohistohemistry experiments n S. Kurukov (Funtionl Genomis, Kolling Institute of Meil Reserh, Austrli) for help with quntifition of histologil vriles. Funing This stuy ws prtly supporte y the Ntionl Helth n Meil Reserh Counil of Austrli n the Stff Speilist Reserh Grnt (Rmsy Helthre Fountion). Dulity of interest The uthors elre tht there is no ulity of interest ssoite with this mnusript. Contriution sttement SS ontriute to the stuy esign, t olletion, nlysis n interprettion of results n wrote the mnusript. JZ, RY n DY performe experiments, nlyse t n ontriute to the revision of the rtile. MGW provie tehnil support n ontriute to the quisition of t n revision of the rtile. XMC n DJK ontriute to the stuy esign, interprettion of results n mnusript revision. CAP ontriute to the stuy esign, interprettion of results, mnusript rfting n revision. All uthors pprove the finl version of the rtile. Referenes 1. Miyt KS, MCw SE, Mrus SL, Rhuinski RA, Cpone JP (1994) The peroxisome prolifertor-tivte reeptor interts with the retinoi X reeptor in vivo. Gene 148: Pnhpkesn U, Chen XM, Pollok CA (25) Drug insight: thizoliineiones n ieti nephropthy relevne to renoprotetion. Nt Clin Prt Nephrol 1: Pnhpkesn U, Sumul S, Pollok CA, Chen X (25) PPARgmm gonists exert ntifiroti effets in renl tuulr ells expose to high gluose. Am J Physiol Renl Physiol 289: F1153 F Zfiriou S, Stnners SR, Polhill TS, Poronnik P, Pollok CA (24) Pioglitzone inreses renl tuulr ell lumin uptke ut limits proinflmmtory n firoti responses. Kiney Int 65: Zfiriou S, Stnners SR, S S, Polhill TS, Poronnik P, Pollok CA (25) Pioglitzone inhiits ell growth n reues mtrix proution in humn kiney firolsts. J Am So Nephrol 16: Pnhpkesn U, Pollok CA, Chen XM (24) The effet of high gluose n PPAR-gmm gonists on PPAR-gmm expression n funtion in HK-2 ells. Am J Physiol Renl Physiol 287: F528 F Izzeine H, Luny-Vher V, Buhesu I, Heurtier A, Bumelou A, Dery G (25) PPAR-gmm-gonists renl effets. Minerv Urol Nefrol 57: S S, Agpiou DJ, Chen XM, Stevens V, Pollok CA (29) The role of Sgk-1 in the upregultion of trnsport proteins y PPAR- > {gmm} gonists in humn proximl tuule ells. Nephrol Dil Trnsplnt 24: Grner OS, Dewr BJ, Erp HS, Smet JM, Grves LM (23) Depenene of peroxisome prolifertor-tivte reeptor ligninue mitogen-tivte protein kinse signling on epierml growth ftor reeptor trnstivtion. J Biol Chem 278: Slominy BL, Slominy A (24) Role of epierml growth ftor reeptor trnstivtion in PPAR gmm-epenent suppression of Helioter pylori interferene with gstri muin synthesis. Inflmmophrmology 12: S S, Stevens VA, Wssef L et l (25) High gluose trnstivtes the EGF reeptor n up-regultes serum gluoortioi kinse in the proximl tuule. Kiney Int 68: Johnson DWBB, Poronnik P, Cook DI, Fiel MJ, Pollok CA (1997) Trnsport hrteristis of humn proximl tuule ells in primry ulture. Nephrology 3: Johnson DW, Suners HJ, Brew BK et l (1997) Humn renl firolsts moulte proximl tuule ell growth n trnsport vi the IGF-I xis. Kiney Int 52: Qi W, Johnson DW, Vesey DA, Pollok CA, Chen X (27) Isoltion, propgtion n hrteriztion of primry tuule ell ulture from humn kiney. Nephrology (Crlton) 12: Bruns CJ, Solorzno CC, Hrison MT et l (2) Bloke of the epierml growth ftor reeptor signling y novel tyrosine kinse inhiitor les to poptosis of enothelil ells n therpy of humn pnreti rinom. Cner Res 6: Kelly DJ, Cox AJ, Tolos M, Cooper ME, Wilkinson-Berk JL, Gilert RE (22) Attenution of tuulr poptosis y loke of the renin-ngiotensin system in ieti Ren-2 rts. Kiney Int 61: Kelly DJ, Wilkinson-Berk JL, Gilert RE (27) Progressive ieti nephropthy in the Ren-2 rt. Am J Physiol Renl Physiol 292:F1662, uthor reply F Kelly DJ, Stein-Okley A, Zhng Y et l (24) Fs-inue poptosis is feture of progressive ieti nephropthy in

9 1182 Dietologi (213) 56: trnsgeni (mren-2)27 rts: ttenution with renin-ngiotensin loke. Nephrology (Crlton) 9: Wilkinson-Berk JL, Kelly DJ, Koerner SM et l (22) ALT-946 n minoguniine, inhiitors of vne glytion, improve severe nephropthy in the ieti trnsgeni (mren-2)27 rt. Dietes 51: Qi W, Chen X, Holin J, Tn CY, Kelly DJ, Pollok CA (29) Trnsription ftors Kruppel-like ftor 6 n peroxisome prolifertor-tivte reeptor-gluose-inue thioreoxininterting protein. Am J Pthol 175: Qi W, Chen X, Gilert RE et l (27) High gluose-inue thioreoxin-interting protein in renl proximl tuule ells is inepenent of trnsforming growth ftor-et1. Am J Pthol 171: Shiuy A, W K, Nkjim A et l (22) Nitrtion of PPARgmm inhiits lign-epenent trnslotion into the nuleus in mrophge-like ell line, RAW 264. FEBS Lett 525: Drumm K, Kress TR, Gssner B, Krug AW, Gekle M (26) Alosterone stimultes tivity n surfe expression of NHE3 in humn primry proximl tuule epithelil ells (RPTEC). Cell Physiol Biohem 17: Singh AB, Hrris RC (24) Epierml growth ftor reeptor tivtion ifferentilly regultes luin expression n enhnes trnsepithelil resistne in Min Dry nine kiney ells. J Biol Chem 279: Singh AB, Sugimoto K, Hrris RC (27) Juxtrine tivtion of epierml growth ftor (EGF) reeptor y memrne-nhore heprin-ining EGF-like growth ftor protets epithelil ells from noikis while mintining n epithelil phenotype. J Biol Chem 282: Zeng F, Singh AB, Hrris RC (29) The role of the EGF fmily of ligns n reeptors in renl evelopment, physiology n pthophysiology. Exp Cell Res 315: Stevens VA, S S, Poronnik P, Fenton-Lee CA, Polhill TS, Pollok CA (28) The role of SGK-1 in ngiotensin II-meite soium resorption in humn proximl tuulr ells. Nephrol Dil Trnsplnt 23: Vrley CL, Southgte J (28) Effets of PPAR gonists on prolifertion n ifferentition in humn urothelium. Exp Toxiol Pthol 6: Tener L, Burgermeister E, Eert MP, Lisovith M (28) Rosiglitzone inues veolin-1 y PPARgmm-epenent n PPRE-inepenent mehnisms: the role of EGF reeptor signling n its effet on ner ell rug resistne. Antiner Res 28: Lewis JD, Ferrr A, Peng T et l (211) Risk of ler ner mong ieti ptients trete with pioglitzone: interim report of longituinl ohort stuy. Dietes Cre 34: Wssef L, Kelly DJ, Gilert RE (24) Epierml growth ftor reeptor inhiition ttenutes erly kiney enlrgement in experimentl ietes. Kiney Int 66: Kng JH, Cho HJ, Lee IS, Kim M, Lee IK, Chng YC (29) Comprtive proteome nlysis of TGF-et1-inue firosis proesses in norml rt kiney interstitil firolst ells in response to sofurnone. Proteomis 9: Avni A, Wiggins KJ, Cox AJ, Zhng Y, Gilert RE, Kelly DJ (211) Inhiition of the epierml growth ftor reeptor preserves pooytes n ttenutes luminuri in experimentl ieti nephropthy. Nephrology (Crlton) 16: