White adipose tissue overproduces the lipid-mobilizing factor zinc a2-glycoprotein in chronic kidney disease

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1 si reserh & 13 Interntionl Soiety of Nephrology White dipose tissue overprodues the lipid-moilizing ftor zin -glyoprotein in hroni kidney disese Croline C. Pelletier 1,,3,5, Letiti Koppe 1,,3,5, Mrine L. Croze 1,, Emilie Klher 1,,3, Roxne E. Vell 1,, Fitsum Guere-Egziher 1,,3, Alin Géloën 1,, Lionel Bdet 1,4, Denis Fouque 1,,3 nd Christophe O. Soulge 1, 1 Université de Lyon, Lyon, Frne; INSERM U16, CrMeN, INSA-Lyon, Univ. Lyon-1, Villeurnne, Frne; 3 Hospies Civils de Lyon, Servie de Néphrologie, Hôpitl E Herriot Lyon, Lyon, Frne nd 4 Hospies Civils de Lyon, Servie d Urologie, Hôpitl E Herriot Lyon, Lyon, Frne Chroni kidney disese (CKD) is frequently ssoited with protein energy wsting, reognized strong preditive ftor of mortlity. Zin -glyoprotein () is new dipokine involved in ody weight ontrol through its lipid-moilizing tivity. Here we tested whether the uremi environment in CKD ould lter prodution y white dipose tissue nd ontriute to CKD-ssoited metoli disturnes. Compred with norml plsm, uremi plsm indued signifint inrese in synthesis (14%), ws ssoited with signifint inrese in sl lipolysis (31%), nd signifintly lunted lipogenesis ( 53%) in 3T3-L1 dipoytes in vitro. In 5/6 nephretomized rts nd mie in vivo, there ws signifint derese in white dipose tissue retion ( 44% nd 43%, respetively) nd signifintly higher white dipose tissue ontent of protein thn in shm-operted, pir-fed ontrol nimls (498% nd 16%, respetively). Suutneous white dipose tissue iopsies from ptients with end-stge renl disese exhiited higher ontent of (573%) thn ge-mthed ontrols. Thus, the ontent is inresed in white dipose tissue from ptients or niml models with CKD. Overprodution of in CKD ould e mjor ontriutor to metoli disturnes ssoited with CKD. Kidney Interntionl (13) 83, ; doi:1.138/ki.13.9; pulished online Ferury 13 KEYWORDS: hroni kidney disese; lipogenesis; lipolysis; white dipose tissue; ; zin--glyoprotein Correspondene: Christophe O. Soulge, CrMeN, Crdiovsulire, Métolisme, diétologie et Nutrition, Bâtiment IMBL, INSA-Lyon, venue Alert Einstein, Villeurnne edex 6961, Frne. E-mil: hristophe.soulge@ins-lyon.fr 5 These uthors ontriuted eqully to this work. Reeived 6 Jnury 1; revised 19 Novemer 1; epted 1 Novemer 1; pulished online Ferury 13 Chroni kidney disese (CKD) is hrterized y nonspeifi metoli disturnes s protein energy wsting, 1 insulin resistne, nd dyslipidemi.,3 Despite improved tretments nd nutritionl support, ll of these re still responsile for the high mortlity rte, mostly from rdiovsulr events. 4,5 Severl uses of CKD-ssoited dysmetolism hve een identified, inluding idosis, inflmmtion, nd oxidtive stress, 6 8 ut the pthogenesis remins unler. White dipose tissue (WAT) is n endorine orgn, s it releses lrge numer of iotive ompounds lled dipokines. Their endorine or prrine tivities ontriute to the regultion of energy lne nd inflmmtory response, nd ould ply key role in the etiology of uremi-ssoited insulin resistne nd hexi Zin -glyoprotein (therefter referred to s ) is 43-kD glyoprotein isolted from humn plsm. 1,13 is sereted in lrge mounts y WAT ut lso y epitheli ells from liver, rest, gstrointestinl trt, nd swet glnds. 14 hs ttrted extensive reserhes, s it ts s lipid-moilizing ftor. Clinilly, is overexpressed in some mlignnt tumors nd is strongly ssoited with the loss of dipose tissues in ner hexi In ontrst, expression of is rther low in oese rodents nd oese humn sujets nd is negtively orrelted with ody ft mss. 18,19 Russell et l. suessfully used injetions to redue ody ft nd ody weight in mie. Indeed, displys oth lipolyti nd ntilipogeni effets on WAT 19,1, nd promotes lipolysis through n intertion with 3-drenoreeptors.,1,3 Body ft loss ppers to e due to the lipolyti effets of, in omintion with n upregultion of UCP-1 (unoupling protein 1) in rown dipose tissue, inresing thermogenesis nd lipid utiliztion. CKD, nd espeilly end-stge renl disese, re ssoited with inresed plsm onentrtion of most dipokines. 4 These inreses re trditionlly thought to result from pssive umultion due to deresed renl lerne. 5 However, reent dt hllenged this view, s two independent studies demonstrted tht uremi plsm indues n upregulted 878 Kidney Interntionl (13) 83,

2 CC Pelletier et l.: overprodution in uremi si reserh seretion of leptin y dipoytes. 6,7 At present, few dt re ville on metolism in CKD. Two studies reently showed n inrese of plsmti level in hemodilysis ptients, suggesting defet of its renl lerne. 8,9 In good greement, uremi ser from CKD stge 5 ptients stimulte sl lipolysis in ultured humn dipoytes. 3 We therefore hypothesized tht CKD ould e ssoited with n overprodution of in WAT tht ould, in turn, indue CKD-ssoited metoli disturnes nd prtiulrly loss of ody ft mss. The im of this study ws to explore the effet of CKD on ellulr ontent in WAT. To this end, 3T3-L1 ultured dipoytes were exposed to uremi plsm, nd ontent ws mesured y western lotting (WB). ontent ws lso mesured in WAT from CKD rodent models nd from CKD stge 5 ptients. RESULTS Ptient dt Clinil nd iologil hrteristis of helthy volunteers (HV),CKD,hemodilysis(HD),ndperitoneldilysis(PD) ptients re summrized in Tle 1. As expeted, plsm retinine nd renl funtion were the only signifint differenes etween the groups. ws quntified y enzyme immunossy in individul plsms. plsm onentrtion wstwofoldhigherinckdptientssompredwithhelthy volunteers ( þ 6%, n ¼ 8, Po.5; Tle 1). Pre nd postdilysis ptients lso exhiited further inreses in plsm onentrtion. The men plsm onentrtion ws 15±65 mg/ml efore the hemodilysis session nd 145±43 mg/ml fter the hemodilysis session ( þ 18%, Po.5). Uremi plsm pool inreses protein ontent in 3T3-L1 dipoytes 3T3-L1 dipoytes were inuted for 16 h with HV, CKD, HD, or PD humn plsm pools, nd ontent ws mesured using WB (Figure 1 nd ). Inution of 3T3-L1 dipoytes with CKD nd HV pools slightly deresed ell viility s mesured y MTT (3-(4,5-dimethylthizol--yl)-,5-diphenyltetrzolium romide) nd ltte dehydrogense ssys (Supplementry Tle S1 online). The presene of in ontrol 3T3-L1 ells ws lmost undetetle, wheres tretment with CKD pool indued signifint inrese in ontent s ompred with HV pool ( þ 14%, Po.1). Inution with HD pools yielded similr inrese of ontent ( þ 143%, Po.1) in dipoytes, wheres inution with PD pool hd no effet (Figure 1). As heprinized plsm tivtes lipoprotein lipse tht itself ould interfere with prodution, we ompred the effets on 3T3-L1 dipose ells of plsm nd serum from the sme CKD stge 5 ptients. It is noteworthy tht plsm nd ser pools yielded similr inrese in ontent, exluding suh n effet (Supplementry Figure S1 online). Ure t onentrtion hosen to mimi uremi (3 mmol/l) did not eliit ny inrese of in 3T3-L1 dipoytes. To get further insight into the moleulr effet of CKD plsm pool on ellulr ontent, plsm ws het intivted (het-shok, 65 1C for 3 min) or frtionted y ultrfiltrtion (Figure 1). Het denturtion of CKD did not signifintly lter prodution in dipoytes neither for HV nor CKD pools. Ultrfiltrtion of the CKD pool with 1 kd utoff dilysis memrne triggered overprodution in 3T3-L1 ells, wheres the ultrfiltrte (tht is, ompounds o1 kd) did not eliit ny overprodution. Tken together, these results suggest tht the stimulting ftor is not protein (s it resists to het denturtion) ut possess moleulr weight (MW) higher thn 1 kd or is tightly ound to plsm proteins. We therefore performed inutions with two prototypil representtives of the protein-ound uremi toxin fmily, p-resyl-sulfte nd indoxyl-sulfte. Both p-resyl-sulfte nd indoxyl-sulfte, t onentrtions hosen to mimi the levels enountered in Tle 1 Bseline hrteristis of helthy volunteers, CKD, hemodilysis, nd peritonel dilysis ptients Helthy volunteers CKD ptients Hemodilysis Peritonel dilysis P-vlue Sex, mle/femle 5M/7F 3M/5F M/3F M/F.81 Age, yers 56.± ±. 57.± ± Height, m 1.65± ± ± ±..156 Weight, kg 6.± ±9. 59.± ± BMI, kg/m.7±.6 4.±3. 4.3± ± Cretinine, mmol/l 71.3±.3 () 356.5±44.9 () 74.3±66.3 () 685.±187.5 () o.1 MDRD, ml/min per 1.73 m 1±3 () 18±8 () NA NA o.1 Birontes, mmol/l 5.6±.5.9±5. 3.± ± Gluose, mmol/l 5.±.6 5.1±1.3 7.± ± Triylglyerols, mmol/l 1.45± ± ± ±1.8.8 HDL holesterol, mmol/l.99± ±.4.96± ±.54.7 LDL holesterol, mmol/l.16±1.1.98±.68.63±1.9.57± CRP, mg/l 3.± ± ±4.6.9± Gluose-lowering tretment, % Lipid-lowering tretment, % RAA inhiitors, % Plsm, mg/ml 37.6± ± ±65.3 ND o.1 Arevitions: BMI, ody mss index; CKD, hroni kidney disese; CRP, C-retive protein; F, femle; HDL, high-density lipoprotein; LDL, low-density lipoprotein; M, mle; MDRD, Modifition of the Diet in Renl Disese; NA, not pplile; ND, not determined; RAA, renin ngiotensin ldosterone;, zin -glyoprotein. Different letters indite signifint differene t Po.5. Dt re expressed s mens±1 s.d. Mens were ompred with nlysis of vrine (ANOVA) one-wy test. Kidney Interntionl (13) 83,

3 /tuulin /tuulin /tuulin si reserh CC Pelletier et l.: overprodution in uremi end-stge renl disese ( did not eliit ny hnge in ontent, s ompred with potssium sulfte (K SO 4 ; dt not shown). Experiments with idosis (evluted with ulture medium djusted to ph 7.) yielded similr negtive result on ontent (dt not shown). As n elevtion of ontent my ffet lipolyti tivity of dipoytes, lipolysis ws mesured in sl onditions, s well s under isoproterenol stimultion (Figure ). Tretments with CKD nd HD plsm pools triggered n inrese of sl lipolysis ( þ 3%, Po.1) ompred with HV plsm pool. In ontrst, isoproterenol-indued lipolysis ws slightly redued y HV, CKD, nd HD pool tretments. Inution of Tuulin Control Ure HV CKD5 Control HV FCS HV HD PD HD PD 3T3-L1 ells with HV pool did not lter lipogenesis, wheres inution with CKD or HD pool signifintly deresed lipogenesis ( 5%, Po.5) ompred with the HV pool. WAT ontent is inresed in niml models of CKD To onfirm these in vitro results, WAT ontent ws mesured in rodent models of CKD indued y 5/6 nephretomy in rts nd mie. Nephretomized rts (Nx5/6) were ompred with shm-operted pir-fed rts (SO). The min fetures of renl funtion nd iometri dt of SO nd Nx5/6 rts re shown in Supplementry Tle S online. Nx5/6 rts exhiited n elevted plsm ure onentrtion ( þ 156%, P ¼.3), higher proteinuri ( þ 65%, P ¼.1), nd signifint derese in WAT retion ( 44%, P ¼.6) ompred with SO rts. As shown in Figure 3, WB nlysis of epididyml WAT evidened mrked inrese in ontent in the Nx5/6 rt WAT s ompred with SO rts ( þ 598%, Po.). This finding demonstrtes tht renl impirment inreses ontent in WAT. Using liner regression nlysis, we found positive orreltion etween tissue ontent nd proteinuri (Figure 3, P ¼.1) nd negtive orreltion with totl WAT weight (Figure 3, P ¼.6), suggesting tht, through its lipolyti nd ntilipogeni tions, ould ontriute to ft mss loss. Ft mss loss ws ssoited in Nx5/6 rts with etopi lipid redistriution in musle, s evidened y the mesurement of gstronemius totl lipid ontent (Figure 3d, P ¼.) nd liver (dt not shown). is rther low in WAT from oese rodents nd oese humn sujets. 18,19 The effets of nephretomy on tissue ontent ws therefore tested in mie fed high-ft diet to test the effets of inresed ft deposition. Biometri dt for these nimls re shown in Supplementry Tle S3 online. All nephretomized mie exhiited n elevted plsm ure onentrtion ( þ 191%, Po.1) ompred with SO mie. Regrdless of renl funtion, mie fed highft diet exhiited striking inrese in ft deposition when Tuulin Tuulin ,, HV pool HV pool HS CKD pool CKD pool HS Plsm > 1 kd Ultrfiltrte < 1 kd Figure 1 Uremi plsm inreses zin -glyoprotein () protein ontent in 3T3-L1 dipoytes. () Quntifition of protein ontent in 3T3-L1 ells inuted overnight with % (v/v) fetl lf serum (FCS; Control), % (v/v) fetl lf serum nd ure (3 mmol/l), % (v/v) helthy volunteers (HV) plsm pool, or % (v/v) hroni kidney disese (CKD5) plsm pool. Dt re mens±s.e.m. for n ¼ 6 7 in eh group. () Quntifition of protein ontent in 3T3-L1 ells inuted overnight with either % (v/v) fetl lf serum (FCS, Control), % (v/v) HV plsm pool, % (v/ v) hemodilysis (HD) plsm pool, or % (v/v) peritonel dilysis (PD) plsm pools. Dt re mens±s.e.m. for n ¼ 5 6 in eh group. () A high moleulr-weight non-protei ftor of uremi plsm indues prodution in 3T3-L1 dipoytes. Quntifition of protein ontent in ultured 3T3-L1 ells with either % (v/v) HV plsm pool, % (v/v) HV pool previously dentured y het shok (HS, 65 1C, 3 min), % (v/v) CKD plsm pool, % (v/v) CKD pool dentured y HS, % (v/v) CKD pool filtered in vitro on 1-kD utoff dilysis memrne (filter), or % (v/v) ultrfiltrte. Dt re mens±s.e.m. for n ¼ 4 5 in eh group. Quntifition of protein ws performed y western lotting nd normlized to tuulin. Different letters indite signifint differene t the Po.5 level. 88 Kidney Interntionl (13) 83,

4 [ 14 C]-ette inorported in TNL, nmol per mg protein Glyerol, μg/g /tuulin Musle lipid ontent, mg/g Glyerol, μg/g /tuulin /tuulin CC Pelletier et l.: overprodution in uremi si reserh P = Shm operted Nx5/6.5 r =.89 P = Proteinuri, mg 4 h. Tuulin r =.7 6 P=.6 r =.67 4 P = Totl WAT weight, g Totl WAT weight, g Figure 3 The zin -glyoprotein () ontent is inresed in white dipose tissue (WAT) from 5/6 nephretomized rt nd is ssoited with ft mss loss nd etopi lipid redistriution. () Quntifition of protein mounts ws performed y western lotting in epididyml white dipose tissues of shm-operted (SO) nd nephretomized (Nx5/6) rts. Quntifitions of ws normlized to tuulin nd expressed in ritrry units (AU). Dt re mens±s.e.m. for n ¼ 6 7 in eh group. () protein ontents in WAT is orrelted with proteinuri () ut negtively orrelted with totl WAT weight. (d) Intrmusulr tissue lipid ontent is negtively orrelted with totl WAT mss..5. Control HV Figure Effets of hroni kidney disese (CKD) plsm on lipolysis nd lipogenesis in 3T3-L1 dipoytes. () Bsl nd () isoproterenol-indued lipolysis in 3T3-L1 ell ultures inuted overnight with % (v/v) fetl lf serum (Control), % (v/v) helthy volunteers (HV) plsm pool, % (v/v) CKD plsm pool, or % (v/v) hemodilysis (HD) plsm pool. Lipolysis level ws expressed s the glyerol relese (mg) per grm (g) of ell protein in eh ulture well, in sl onditions or fter isoproterenol stimultion (1 mmol/l). Dt re mens±s.e.m. for n ¼ 8 1 for sl lipolysis nd n ¼ 6 for isoproterenol-indued lipolysis. () Lipogenesis ws mesured in 3T3-L1 ell ultures s the inorportion of [ 14 C]-ette into totl neutrl lipids (TNL). Dt re mens±s.e.m. for n ¼ 4 5 independent experiments in eh group. Different letters indite signifint differene t the Po.5 level. ompred with mie fed stndrd diet (Figure 4). Nephretomized mie, however, exhiited lunted ft retion when ompred with SO mie. dipose tissue ontent ws strikingly inresed in WAT from Nx5/6 mie fed stndrd diet, s well s in mie fed high-ft diet (Figure 4) ompred with their respetive ontrols. Thus, renl insuffiieny is le to trigger overprodution in dipose tissue of oth len nd oese nimls. ontent is inresed in suutneous WAT of CKD ptients Experimentl study in rts nd mie indited tht ontent ws inresed y renl impirment; therefore, we CKD HD imed to ssess ontent in WAT from CKD ptients. Seven CKD stge 5 ptients nd nine non-ckd ptients were reruited from n ongoing study to perform surgil suutneous WAT iopsies. To rule out potentil seletion ises tht my ontriute to distur WAT metolism, evidenes of inflmmtion, ner diseses, idosis, dietes, or oesity were onsidered s exlusion ftors. Bseline hrteristis of ptients re summrized in Tle. plsm onentrtion ws mesured y enzyme immunossy method. As expeted, higher plsm onentrtion of ws notied in CKD stge 5 ptients ompred with non- CKD ptients ( þ 71%, n ¼ 7 9, P ¼.4). CKD ptients exhiited higher ontent in dominl suutneous WAT thn the non-ckd ptients ( þ 573%, n ¼ 7 9, P ¼.3; Tle ; see Supplementry Figure S online for typil lots). Plsm onentrtion ws further ompred in non-ckd nd CKD overweighed/oese ptients (ody mss index 43 kg/m ) (Tle 3). A higher plsm onentrtion of ws notied in CKD stge 5 oese ptients ompred with non-ckd oese ptients ( þ 184%, n ¼ 7 8, Po.5). The protein WAT ontent ws nlyzed y WB in one oese CKD stge 5 ptient ompred with one non-ckd oese ptient (see Supplementry Figure S3 online). In good greement with niml studies, / tuulin rtio ws strongly inresed ( þ 633%) in WAT from the CKD oese ptient ompred with the non-ckd oese ptient (/tuulin rtio:.89 nd 6.53 for non-ckd nd CKD, respetively). Kidney Interntionl (13) 83,

5 /tuulin Totl WAT weight, mg si reserh CC Pelletier et l.: overprodution in uremi Shm Nx5/6 Shm Nx5/6 Chow diet High-ft diet Shm Nx5/6 Shm Nx5/6 Chow diet High-ft diet Tle Chrteristis of non-ckd ptients nd end-stge renl disese ptients Non-CKD CKD stge 5 P-vlue N 9 7 Age, yers 67.6± ±3.4.* Height, m 1.71±. 1.73±..38 Weight, kg 73.6± ± BMI, kg/m 5.3±.5 6.1±1..54 Cretinine, mm 89.7± ±39. o.1*** MDRD, ml/min per 1.73 m 8±5 13±1 o.1*** Birontes, mm 6.3±.9 4.±.7.6 Gluose, mm 5.4±.1 5.1±..18 Triylglyerols, mm 1.34±.14.1±.35.4* HDL holesterol, mm 1.1±.14.98±.9.5 LDL holesterol, mm.77± ±.33.1* CRP, mg/l.5± ±11.9. Zin -glyoprotein () Plsm onentrtion, mg/ml 75.6± ± * swat ontent.66± ±3.3.1* Arevitions: BMI, ody mss index, CKD, hroni kidney disese, CRP, C-retive protein, swat, suutneous white dipose tissue. Dt re expressed s mens ± 1s.d. Mens were ompred with Student s t-test, *Po.5, ***Po.5. tissue ontent ws nlyzed y western lotting s desried in methods nd normlized to tuulin ontent. Typil lots for non-ckd nd CKD ptients re shown in Supplementry Figure S4 online. Tuulin Figure 4 The zin -glyoprotein () ontent is inresed in white dipose tissue (WAT) from 5/6 nephretomized (Nx5/6) mie fed stndrd or high-ft diet. () WAT retion in shmoperted (SO) nd Nx5/6 mie fed low- or high-ft diet to inrese ft deposition. () Quntifition of protein mounts ws performed y western lotting in epididyml WAT of SO nd Nx5/6 mie. Quntifitions of in WAT ws normlized to tuulin, nd expressed in ritrry units (AU). Dt re mens±s.e.m. for n ¼ 6 7 in eh group. Different letters indite signifint differene t the Po.5 level. DISCUSSION We report in this study tht uremi plsm inreses protein prodution in 3T3-L1 dipose ells. The linil relevne of this finding is further onfirmed y the oservtion of inresed WAT ontent of in CKD niml models, s well s in suutneous WAT from CKD stge 5 ptients. These dt offer novel insight into the possile mehnisms of CKD-ssoited metoli disturnes suh s dyslipidemi nd protein energy wsting. In good greement with the study y Philipp et l. 8 in HD ptients, the CKD ptients inluded in the present investigtion exhiited mrked elevtion of plsm onentrtion (twofold, Po.5) tht ws lso oserved in HD ptients. Inution of 3T3-L1 dipose ells in the medium ontining plsm from the CKD ptients signifintly inresed intrellulr protein ontent ompred with tht found with plsm from HV. Tle 3 Chrteristis nd plsm onentrtion of oese non-ckd nd oese CKD ptients Non-CKD CKD stge 5 P-vlue N 1 8 Age, yers 61.6± ± Height, m 1.65± ±.1.34 Weight, kg 78.3± ± BMI, kg/m 3.± ±..91 Cretinine, mmol/l 84.4± ±19.7 o.1*** MDRD, ml/min per 1.73 m 84±4 13± o.1*** Ure, mmol/l 7.3± ±3.4 o.1*** Birontes, mmol/l 6.4±1. 3.3±1.4.9 Gluose, mmol/l 5.5±.8 5.8± Triylglyerols, mmol/l 1.73± ± HDL holesterol, mmol/l 1.6±.9 1.9± LDL holesterol, mmol/l.3±1.1.57± CRP, mg/l 4.6± ± Plsm, mg/ml 8.7± ±44.6.*** Arevitions: BMI, ody mss index; CKD, hroni kidney disese; CRP, C-retive protein; HDL, high-density lipoprotein; LDL, low-density lipoprotein; MDRD, Modifition of the Diet in Renl Disese;, zin -glyoprotein. Dt re expressed s mens±1 s.d. Mens were ompred with Student s t-test, ***Po.5. This inrese is minly due to neo-synthesis of protein, s it is olished y the use of protein synthesis inhiitor suh s yloheximide (dt not shown). This oservtion suggests tht inresed plsm onentrtion of reported in CKD ould, t lest in prt, e due to n inresed prodution nd seretion s opposed to the derese of its renl lerne. 8,31 To get n insight into the mehnism of uremi plsm-indued prodution, the plsm pool ws het intivted (3 min, 65 1C) or 88 Kidney Interntionl (13) 83,

6 CC Pelletier et l.: overprodution in uremi si reserh frtionted y ultrfiltrtion (utoff: 1 kd). Aminzdeh et l. 6 reported tht uremi plsm indues n exuernt seretion of leptin nd tht plsm tumor nerosis ftor- is involved in this proess. However, most ytokines re rpidly intivted during het shok (tht is, temperture C), exluding the ft tht the stimultory ftor of prodution is proinflmmtory ytokine. Supporting this view, overnight inution of 3T3-L1 dipose ells with two ommon inflmmtory ytokines, tumor nerosis ftor- nd interleukin-6, did not eliit ny hnge in ontent (Supplementry Figure S4 online). An in vitro ultrfiltrtion study indited tht the stimulting ftor hd high MW (41 kd) or ws tightly ound to plsm proteins. To test this hypothesis, 3T3-L1 ells were inuted with plsm extensively dilyzed through the ellulose memrne (MW utoff: 8 kda) to remove low-mw ompounds (see Supplementry Figure S5 online). Dilyzed plsm from CKD did not signifintly lter prodution ompred with HV plsm, suggesting tht stimulting ftor ould e smll protein-ound uremi toxin. In good greement, ultrfiltrte olleted from one CKD ptient undergoing ultrfiltrtion to tret fluid overlod lso filed to trigger prodution in 3T3-L1 dipose ells, onfirming tht the stimulting ftor ws either of high MW or ws tightly ound to plsm proteins (see Supplementry Figure S6 online). Tken together, these dt indite tht the stimulting ftor (s) elong(s) to the group of proteinound uremi toxin(s) with MW(s) o8 kd. Further studies nd frtiontion tehniques re, however, needed to deipher the ext moleulr nture of the stimulting ompound(s). Plsm for HD ptients eliited similr inrese in ontent in dipose ells, suggesting tht the stimulting ftor ws not removed y the HD proess. In ontrst, plsm from PD ptients did not eliit ny hnge in ontent (Figure 1). This oservtion deserves some omments. Beuse of their strong protein inding, proteinound toxins re poorly removed y lssil HD strtegies, even using lrge-pore dilysis memrnes. 3 In ontrst with HD (n intermittent dilysis strtegy), PD is hrterized y slow ut ontinuous removl of solutes. PD is lso ssoited with peritonel loss of lumin nd n improved preservtion of residul renl funtion. Although lerne of protein-ound solutes ws greter in HD ptients thn in those on PD, prdoxilly the plsm onentrtion of these protein-ound solutes ws lower in ptients on PD thn in those on HD. 33,34 As n exmple, Phm et l. 34 reported tht the totl onentrtion of p-resyl sulfte, the prototype memer of protein-ound uremi toxins, ws lower in nuri ptients on PD thn in those on HD. The disrepny etween the higher lood onentrtion nd higher lerne of protein-ound solutes in ptients on PD ompred with those on HD is urrently unexplined, ut differenes in intestinl genertion or metolism of these solutes ould e involved. 35 These oservtions support the ft tht the stimulting ftor(s) ould e protein-ound ompound(s). We nnot, however, exlude the ft tht physiologi nd/or metoli differenes in these ptients should underlie the sene of effet of PD plsm on 3T3-L1 ells. This issue will, however, require further reserh. We demonstrte for the first time tht protein ontent ws strikingly inresed in WAT from Nx5/6 rodents or stge 5 CKD ptients. We filed to find signifint orreltion etween dipose tissue protein level nd plsm onentrtion of in CKD stge 5 ptients (r ¼.3, P ¼.843). In good greement, the mesurement of rteriovenous differenes in humns showed tht there ws no signifint ontriution of WAT to the irulting levels of. 36 In this study, in oese sujets, seretion from WAT inresed signifintly, wheres serum levels remined unltered. is therefore relesed from humn WAT, ut this tissue does not ontriute signifintly to the irulting levels. ould therefore e regrded s lol ftor in dipose tissue, exerting its toli tion minly through utorine nd/or prrine tivity. displys oth lipolyti nd ntilipogeni effets on WAT. 19,1,,37 hs een shown to promote lipolysis through n intertion with 3-drenoreeptors 1,3 nd y upregultion of lipolyti enzymes. 19 On the other hnd, downregulted the expression of mjor lipogeni enzymes suh s ftty id synthse or etyl-oa roxylse in WAT 19 or liver. 37 Nephretomized rts exhiited signifint loss of totl WAT mss, nd we notied negtive orreltion etween tissue ontent nd WAT weight, suggesting tht, through lipolyti nd/or ntilipogeni tions, ould ontriute to ft mss loss. CKD results in mrked downregultion of lipoprotein lipse nd very-low-densitylipoprotein (VLDL) reeptor in dipose tissue nd skeletl musle The CKD-indued lipoprotein lipse nd VLDL reeptor defiienies signifintly ontriute to the redution of ft tissue mss y limiting the uptke of ftty ids from hylomirons nd VLDL nd their re-esterifition nd storge s triglyerides. Therefore, elevtion of ompounds the effets of lipoprotein lipse nd VLDL reeptor defiienies in CKD y promoting lipolysis nd inhiiting ftty id synthesis. Zho et l. 41,4 reported tht 1-month-old uninephretomized rts exhiit progressive loss of well-differentited WAT ssoited with etopi lipid deposition. Moreover, uninephretomized rts exhiited severl metoli disturnes suh s hyperlipidemi (elevtion of fsting serum totl holesterol, triglyeride, low-density lipoprotein holesterol, nd high-density lipoprotein holesterol), fsting hyperglyemi, nd insulin resistne. Blunted lipogenesis nd/or inresed lipolysis ould promote lipid redistriution in the ody, leding to lipotoxiity. Reently, Axelsson et l. 3 reported tht uremi serum stimulted sl lipolysis in humn predipoytes. Our study in 3T3-L1 yielded very similr results, suggesting tht inresed plsm onentrtion ould e entrl for the effet oserved on lipolysis. Supporting the notion tht ould lso e responsile for metoli disturnes, Philipp et l. 8 reported tht serum levels negtively orrelted with fsting insulin nd homeostsis model Kidney Interntionl (13) 83,

7 si reserh CC Pelletier et l.: overprodution in uremi UREMIC PLASMA Adipoyte Adipoyte 1) Protein-ound uremi toxin (s) Autorine effet LIPOLYSIS LIPOGENESIS FAT MASS LOSS DYSMETABOLISM ) Prrine effet ECTOPIC LIPID REDISTRIBUTION LIPOTOXICITY Figure 5 Shemti representtion of zin -glyoprotein () ontriution to dysmetolism ssoited with hroni kidney disese. Shemti representtion of zin -glyoprotein () ontriution to dysmetolism ssoited with hroni kidney disese. ssessment of insulin resistne. In preliminry report, Olofsson et l. 43 notied tht serum levels orrelted with serum levels of holesterol in helthy sujets nd during weight loss. Thus, s reported in ner ptients, 44 ould e mjor lipid-moilizing ftor nd my ontriute to the min CKD-ssoited metoli disturnes. In onlusion, s summrized in Figure 5, we demonstrte for the first time tht protein ontent is inresed in WAT from CKD ptients or CKD niml models. Our dt suggest tht uremi ser ontin protein-ound smll-mw ftor (or ftors) tht stimulte(s) prodution y dipoytes. Although our results re relevnt to understnd the metoli disturne in CKD ptients, we did not identify the ext moleule in uremi plsm tht medites overprodution in dipose tissue. Our results demonstrte the importne of overprodution in CKD, through inresed lipolysis nd deresed lipogenesis, tht ould e mjor ontriutor to dysmetolism ssoited with CKD. MATERIALS AND METHODS Humn sujets A totl of 1 helthy dult volunteers, 8 nondilyzed CKD ptients (renl disese: 3 nephrongioslerosis, dietes mellitus type, 1 polyysti disese, 1 glomerulr slerosis, nd 1 uropthy), 5 ptients mintined on HD for minimum of 1 yer, nd 4 PD ptients mthed for ge, gender, nd ody mss index were reruited t the University Hospitl in Lyon, Frne. HD (hemodifiltrtion) therpy ws performed three times weekly for 4 h. Men dilysis time ws 1.38±.75 h/week nd HD ptients exhiited n equilirted Kt/V of.7±.3. PD ptients exhiited totl Kt/V of.±.7 per week (see Supplementry Tle S4 online for lerne results). After n overnight fst, lood smples were otined y venipunture, exept for dilysis lood smples tht were otined immeditely efore nd fter dilysis from mehnil loodstrem. Blood smples were entrifuged t 35g for 1 min to isolte plsm superntnt, snp-frozen in liquid nitrogen nd stored t 1C until use. Cretinine lerne ws lulted using the Modifition of the Diet in Renl Disese formul (MDRD). In seond experiment, seven CKD stge 5 ptients nd nine non-ckd ptients were reruited from n ongoing study to perform suutneous dipose tissue iopsies. Biopsies ( 3 g) were performed during eletive surgery (peritonel dilysis theter for CKD ptients nd rdil prosttetomy for non-ckd ptients), snp-frozen in liquid nitrogen, nd stored t 8 1C until use. In n dditionl experiment, 1 non-ckd nd 8 CKD stge 5 overweighed/oese ptients (ody mss index 43 kg/m ) were reruited from n ongoing study to perform plsm immunossy nd ptients to perform suutneous dipose tissue iopsies. The lol ethis ommittee (CPP Lyon Sud-Est IV, study MODAIR, ref D-9-17) pproved these studies, nd written informed onsent ws otined from eh sujet. Animl study Mle Wistr rts weighing 75 3 g were otined from HARLAN (Gnnt, Frne) nd housed in individul ges in temperturend humidity-ontrolled room with 1-h light/drk yle. The nimls were llowed free ess to wter nd stndrd lortory diet. Animls were rndomly divided into two groups: SO nd nephretomized (Nx5/6). Animl experiments were performed under the uthoriztion no (CrMeN L, Diretion Déprtementle des Servies Vétérinires du Rhône, Lyon, Frne). All experiments were rried out ording to the guidelines lid down y the Frenh Ministère de l Agriulture (no ) nd the E.U. Counil Diretive for the Cre nd Use of Lortory Animls of Novemer 1986 (86/69/EEC). After nesthesi with sodium pentoritl (35 mg/kg intrperitonelly (i.p.)) nd hlorpromzine (5 mg/kg i.p.), rts underwent surgil 5/6 renl mss redution in two stges or shm opertion. Animls were given morphine hlorhydrte (6 mg/kg i.p.) three times dy for dys to mnge postopertive pin. As uremi ould result in signifint hnges in food intke, SO rts were pir-fed with Nx5/6 nimls to prevent differene in feeding ehvior nd eliminte niml weight s vrile in this study. At 1 weeks fter surgery, rts were deeply nesthetized with sodium pentoritl (6 mg/kg i.p.). Blood ws withdrwn on heprinized syringe from punture of the ven v. Blood smples were entrifuged t 35g for 1 min to isolte the plsm superntnt nd snpfrozen in liquid nitrogen. Epididyml, retroperitonel, nd suutneous inguinl WAT were disseted out ording to ntomil lndmrks, weighed, nd snp-frozen in liquid nitrogen for immunodetetion. The musle nd hepti lipids were extrted using Chloroform Methnol (:1, v/v), nd totl lipid ontent ws estimted grvimetrilly. Cell ulture Mouse 3T3-L1 firolsts were otined from the Amerin Type Culture Colletion (CL-173; ATCC, LGC Stndrd SARL; Molsheim, 884 Kidney Interntionl (13) 83,

8 CC Pelletier et l.: overprodution in uremi si reserh Frne). Cells were grown in 1-well pltes in Duleo s modified Egle s medium ontining 5 mmol/l D-gluose, 4 mmol/l glutmine, 5 mmol/l Hepes uffer (GlutMAX, Invitrogen, Illkirh, Frne), nd supplemented with 1 IU/ml peniillin, 1 mg/ml streptomyin, nd 1% (v/v) het-intivted (3 min, 56 1C) fetl lf serum (Sigm-Aldrih, Sint Quentin-Fllvier, Frne). Cultures were mintined t 37 1C in wter-sturted tmosphere ontining 5% (v/v) CO. Adipoytes were studied 1 1 dys fter differentition, whih ws indued y insulin (.8 mm), IMBX (.5 mmol/l), dexmethsone (5 nmol/l), nd rosiglitzone (1 mmol/l). Plsm from HV, CKD, HD, PD ptients were pooled in equl mounts nd therefter referred to s Helthy Volunteer pool, CKD pool, HD pool, nd PD pool, respetively. Adipoytes were inuted for 16 h in ulture medium ontining % (v/v) fetl lf serum s ontrol onditions, % (v/v) fetl lf serum with 3 mmol/l ure, or % (v/v) of eh humn plsm pools. At the end of the inution period, the ulture medi were removed nd snp-frozen in liquid nitrogen. Cells were rinsed with ie-old phosphte-uffered sline, nd frozen nd stored t 1C until mesurements. Similr 16-h inutions of dipoyte ultures were performed with uremi toxins ording to the reommendtions of the Europen Uremi Toxin Work Group (EUTox, 3 (nmely 4 mg/ml of p-resyl-sulfte nd 6 mg/ml of indoxylsulfte) s ompred with K SO 4. The effet of idosis ws tested with ulture medium djusted to ph 7. with HCl. In further experiments, liquot frtions of HV nd CKD pool were het intivted (for 3 min t 65 1C) or frtionted y ultrfiltrtion using memrne with utoff of 1 kd, efore pplition on ells. Cells seeded in 96-well pltes were treted for 16 h under the sme onditions s mentioned ove. Viility ws determined with the MTT ssy (Cell Prolifertion Kit I; Rohe, Meyln, Frne) nd with the ltte dehydrogense ssy (Cytotoxiity Detetion kit; Rohe) ording to the mnufturer s reommendtions. Protein extrtion nd WB 3T3-L1 dipose ells were srped nd lysed in stndrd lysis uffer ( mmol/l Tris-HCL, 138 mmol/l NCl,.7 mmol/l KCl, 1 mmol/l MgCl, 5% Glyerol, nd 1% Nonidet P4) supplemented with protese nd phosphtse inhiitors (5 mmol/l EDTA, 1 mmol/l N 3 VO 4, mmol/l NF, 1 mmol/l DTT, Protein inhiitor oktil; Sigm-Aldrih). WAT ws homogenized using glss/teflon potter in stndrd lysis uffer, nd then insolule mterils were eliminted y entrifugtion (14,g, 15 min, 4 1C). Protein onentrtions in plsm nd 3T3-L1 ells were determined y the Brdford ssy (Bio-Rd, Mrnes-l-oquette, Frne). Proteins were oiled in Lemmli uffer, seprted y sodium dodeyl sulfte polyrylmide gel eletrophoresis, nd trnsferred onto nitroellulose memrne. After sturtion with skimmed milk (1% w/v), memrnes were proed with primry ntiodies, s speified. After inution with horserdish peroxidse oupled seondry ntiodies, memrnes were proessed for hemiluminesene (ECL plus, GE Helthre, Coutoeuf, Frne) nd quntified y densitometry using Quntity One softwre (Bio- Rd). Antiodies to were from Snt Cruz (Heidelerg, Germny, Ref. H-13) nd ntiodies to -tuulin were from Sigm-Aldrih (Ref. T674). Got nti-mouse IgGs were from Bio-Rd (Ref ). immunossy onentrtions in humn plsms were determined using humn enzyme immunossy purhsed from Ry Bioteh (Clinisienes, Montrouge, Frne) ording to the mnufturer s reommendtions. The detetion limit ws 1 pg/ml nd intrssy oeffiient of vrition ws.8%. Postdilysis onentrtions were orreted for hemoonentrtion using plsm protein onentrtions ssyed ording to the method of Brdford, with ovine serum lumin s stndrd. The orretion ws performed using the following eqution: [] Correted ¼ ([Proteins] pre-hd / [Proteins] post-hd ) [] post-hd with [Proteins] pre-hd, plsm protein onentrtion efore hemodilysis session, [Proteins] post-hd nd [] post-hd plsm protein nd plsm onentrtion fter hemodilysis session, respetively. Lipolysis mesurement Lipolysis ws mesured in seline nd stimulted (isoproterenol 1 mmol/l) onditions s the rte of glyerol relese over 1-h period. Medi were olleted from the ells, nd glyerol in ulture superntnts ws mesured y olorimetri method using glyerol ssy kits (R-iophrm, Sint-Didier, Frne). Lipogenesis mesurement 3T3-L1 dipoytes grown in six-well dishes were inuted for 16 h with plsm pools s desried ove. Lipogenesis ws then mesured s the inorportion of [ 14 C]-ette into totl neutrl lipids. 3T3-L1 ells were inuted in Duleo s modified Egle s medium ontining 1% ftty id free ovine serum lumin nd 1 mci of [ 14 C]-ette for 4 h t 37 1C. Totl neutrl lipids were extrted using Dole s extrtion fluid (isopropnol/heptne/h SO 4 1N, 4/1/1, v/v/v), nd [ 14 C] ws deteted y liquid sintilltion ounting. Lipogenesis dt were normlized y protein onentrtion estimted using the Brdford ssy. Sttistis All dt were nlyzed using the GrphPd Prism v5. softwre (Grphpd softwre, L Joll, CA) nd were expressed s mens± s.e.m or mens±s.d. Results were ompred y one-wy nlysis of vrine followed, when pproprite, y post ho Tukey s test. Simple omprisons were performed using Student s t-test. When pproprite, Welh s orretion for inhomogeneity of vrines ws pplied. Differenes were onsidered signifint t the Po.5 level. DISCLOSURE All the uthors delred no ompeting interests. ACKNOWLEDGMENTS CCP held grnt from Soiété Frn ise de Néphrologie, EK ws reipient of grnt from Adémie de Médeine nd LK held fellowship from Fondtion pour l Reherhe Médile. MLC nd REV were supported y grnts from the Frenh Ministère de l Reherhe et de l Tehnologie. This work ws supported y INSERM, INSA-Lyon, nd Fédértion Ntionle d Aide ux Insuffisnts Rénux (FNAIR). We grtefully knowledge ll ptients nd helthy volunteers for their prtiiption in this study. SUPPLEMENTARY MATERIAL Figure S1. Uremi plsms s well s uremi ser inrese protein ontent in 3T3-L1 dipoytes. Figure S. protein ontent is inresed in suutneous dominl white dipose tissue iopsies from CKD stge-5 ptients: typil lots. Figure S3. protein ontent is inresed in suutneous white dipose tissue iopsy from non-ckd nd CKD stge-5 oese ptients. Kidney Interntionl (13) 83,

9 si reserh CC Pelletier et l.: overprodution in uremi Figure S4. The inflmmtory ytokines TNF-lph (TNF-lph, 5 ng/ml) nd interleukin-6 (IL-6, 1 ng/ml) did not inrese protein ontent in 3T3-L1 dipoytes. Figure S5. Memrne dilyzed uremi plsm did not inrese protein ontent in 3T3-L1 dipoytes. Figure S6. Plsm ultrfiltrte from CKD ptient did not inrese protein ontent in 3T3-L1 dipoytes. Tle S1. Cell viility. Tle S. Chrteristis of shm-operted nd Nx5/6 rts. Tle S3. Biometri dt for shm-operted nd nephretomized mie. Tle S4. Clerne results for peritonel dilysis ptients. Tle S5. Bseline hrteristis of the ultrfiltrtion CKD ptient. Supplementry mteril is linked to the online version of the pper t REFERENCES 1. Fouque D, Klntr-Zdeh K, Kopple J et l. A proposed nomenlture nd dignosti riteri for protein-energy wsting in ute nd hroni kidney disese. Kidney Int 8; 73: Siew ED, Ikizler TA. Determinnts of insulin resistne nd its effets on protein metolism in ptients with dvned hroni kidney disese. 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