Leptin induces TGF-b synthesis through functional leptin receptor expressed by human peritoneal mesothelial cell

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1 originl rticle & 2006 Interntionl Society of Nephrology Leptin induces TGF- synthesis through functionl leptin receptor expressed y humn peritonel mesothelil cell JCK Leung 1, LYY Chn 1, SCW Tng 1, KM Chu 2 nd KN Li 1 1 Deprtment of Medicine, Queen Mry Hospitl, The University of Hong Kong, Hong Kong; 2 Deprtment of Surgery, Queen Mry Hospitl, The University of Hong Kong, Hong Kong Mrked increse in leptin concentrtion in spent peritonel dilyste hs een reported following continuous multory peritonel dilysis tretment. The present study ws designed to determine whether functionl leptin receptor is expressed y humn peritonel mesothelil cells nd if so, the possile impliction in dilysis. Expression of leptin receptors in cultured mesothelil cells nd omentl tissue ws exmined. The effect of leptin on the production of trnsforming growth fctor- (TGF-) y mesothelil cells in the presence or sence of high glucose ws determined using in vitro culture model of humn peritonel mesothelil cells nd dipocytes. The signling mechnism involved in leptin-induced TGF- synthesis y mesothelil cells ws studied. Both mrna nd protein of the full-length leptin receptor re constitutively expressed in mesothelil cells. The leptin receptor expression in mesothelil cells ws upregulted y glucose ut not leptin. In dipocytes, glucose incresed the mrna expression nd synthesis of leptin. The Jnus kinse-signl trnsducers nd ctivtion (JAK-STAT) signl trnsduction pthwy in mesothelil cells ws ctivted y either exogenous or dipocytes-derived leptin. Exogenous leptin induced the relese of TGF- y mesothelil cells. The TGF- synthesis induced y leptin ws mplified y glucose through incresed leptin receptor expression. Our novel findings revel tht functionl leptin receptor is present on humn peritonel mesothelil cells. The leptin-induced TGF- synthesis in mesothelil cells is ssocited with the expression of leptin receptor nd the ctivtion of the JAK-STAT signl trnsduction pthwy. Kidney Interntionl (2006) 69, doi: /sj.ki ; pulished online 26 April 2006 KEYWORDS: leptin; leptin receptor; CAPD; mesothelil cell; TGF-; JAK-STAT Correspondence: KN Li, Deprtment of Medicine, Room 411, Professoril Block, Queen Mry Hospitl, The University of Hong Kong, Hong Kong. E-mil: knli@hku.hk Received 23 Septemer 2005; revised 8 Ferury 2006; ccepted 15 Ferury 2006; pulished online 26 April 2006 Continuous multory peritonel dilysis (CAPD) is n importnt tretment modlity of the renl replcement therpy. Unfortuntely, the peritonel memrne frequently exhiits structurl nd functionl chnges following longterm dilysis. 1 During CAPD, peritonel cells re repetedly exposed to nonphysiologicl hypertonic environment tht my led to peritonel firosis nd, ultimtely, ultrfiltrtion filure. 2 Humn peritonel dipocytes (HPAC) re uiquitously found in peritonel tissues. There is now compelling evidence suggesting dipocytes cn medite vrious physiologic processes through secretion of dipokines, including leptin, diponectin, resistin, tumour necrosis fctor-, interleukin-6, trnsforming growth fctor- (TGF-), tissue fctors, nd other growth fctors. 3,4 Adipocytes lso express receptors for leptin, insulin growth fctor-1, tumour necrosis fctor-, interleukin-6, TGF- tht orchestrte network of utocrine, prcrine, nd endocrine signls. 4 In prietl peritoneum, dipocytes lie deep underneth the mesothelium. During the fluid dwell in CAPD, solutes in peritonel dilysis fluid (PDF) re trnsported y pssive diffusion through the peritonel rrier nd come into contct with the dipocytes. In the omentum, dipocytes re in close contct with mesothelil cells. Ultrstructurl study revels tht portion of dipocytes re protruded from the mesothelil surfce, suggesting tht omentl dipocytes my e directly exposed to dilyste. 5 In ddition, dilyste cn lso rech the prietl dipose tissue when there is junctionl dmge or denudtion of the mesothelil monolyer. It is therefore logicl to postulte tht under repeted exposure to PDF nd the continuous chnges of the physiologic milieu of the peritonel cvity during CAPD, peritonel dipocytes will inevitly e ctivted. Yet, study on ny impct of CAPD on peritonel dipocytes is scrce. Leptin, n dipocytes-derived 16-kD hormone, exerts mny iologicl effects through leptin receptor. 4,6 Leptin receptors elong to the clss I cytokine fmily with six spliced isoforms (O-R to O-Rf). 4 Only the full-length isoform, O-R, contins the intrcellulr motifs essentil for the Jnus kinse-signl trnsducers nd ctivtion (JAK-STAT) 2078 Kidney Interntionl (2006) 69,

2 JCK Leung et l.: Leptin induces TGF- synthesis y mesothelil cell o r i g i n l r t i c l e signl trnsduction pthwy. 4,7 Accumulting evidence for systemic effects of leptin on specific tissues nd metolic pthwys indictes tht leptin opertes oth directly nd indirectly to orchestrte complex pthophysiologicl processes. 6,8 Leptin is clered principlly y the kidney nd the serum leptin concentrtion is incresed in ptients with chronic renl filure or undergoing dilysis. 9 Mrked increse in leptin concentrtion from serum nd spent dilyste hs een reported following CAPD tretment. 10 Leptin in peritonel dilyste is derived not only from plsm ut lso loclly from peritonel dipose tissue. In vitro experiments using dipocyte cell line 3T3-L1 showed glucose-sed PDFs induce higher leptin secretion. 11 In the present study, we determine whether functionl leptin receptors re expressed y humn peritonel mesothelil cells (HPMC) nd if so, whether these mesothelil leptin receptors could e triggered y leptin to modulte locl TGF- production. RESULTS Culture of HPMC nd HPAC Figure 1 shows the cultured cells used in our study. HPMC showed typicl colestone ppernce t confluence (Figure 1). For in vitro experiments, dipocyte differentition ws initited y culturing predipocytes (Figure 1) in dipogenic medium. After 15 dys of differentition, 90% of cells re positively stined with Oil Red O for neutrl lipid ccumultion (Figure 1C). Expression of O-R in omentl tissue nd cultured HPMC Expression of the O-R mrna (Figure 2) nd protein (Figure 2) ws demonstrted in three different lines of HPMC. Expression of O-R ws confirmed for peripherl lood mononucler cells nd HepG2 cells, which served s positive control for the reverse trnscriptse-polymerse chin rection (RT-PCR). 12,13 Expression of O-R ws detected on cell surfce of peritonel mesothelil cells nd dipocytes in omentum (Figure 3). However, HPMC do not produce leptin either constitutively or fter incution with glucose (dt not shown). c Figure 1 Culture of peritonel mesothelil cells nd dipocytes. Microgrphs showing () humn omentl mesothelil cells, () predipocytes, nd (c) differentited dipocytes in culture. The differentited dipocytes (HPAC) were positively stined with Oil Red O (originl mgnifiction 100). O-R O-R p 452 p upregultes O-R expression y HPMC Figure 4 shows the time course of O-R expression y HPMC cultured with glucose or mnnitol. Expression of O-R mrna (Figure 4) or protein (Figure 4) ws significntly incresed in HPMC y incution with glucose ut not with mnnitol fter 12 h nd plteued t 72 h (Po0.005). Similrly, glucose ut not mnnitol upregulted O-R mrna (Figure 5) nd protein expression (Figure 5) in HPMC in dose-dependent mnner. upregultes leptin mrna nd protein expression y HPAC Significnt increse in mrna expression of leptin ws detected in HPAC fter exposed to 40 mm glucose for h (Po0.005) (Figure 6). Then, the leptin expression fell t 72 h nd the level remined not different from the seline vlue. significntly incresed leptin relese from HPAC fter exposure for 48 h (Po0.005) (Figure 6). t concentrtion of 20 mm or greter significntly incresed the leptin mrna expression (Figure 7) nd M 125 kd 42 kd Figure 2 Expression of O-R in mesothelil cells. Expression of () full-length O-R mrna nd () protein in cultured humn mesothelil cells. Lnes 1 3 were HPMC cell lines. The liver cell line HepG2 (lne 4), humn peripherl mononucler cells (lne 5), nd omentl tissue (lne 6) were served s positive control for O-R expression. c Figure 3 Immunostining of O-R in omentl tissue. ( nd c) Representtive immunohistochemicl stining of O-R in humn omentl tissue. Omentl mesothelil cells (rrow hed) nd dipocytes (rrow) showed strong immunorectivity for O-R (rown). () No stining ws oserved for isotypic control (originl mgnifiction ( nd ) 200, (c) 1000). Kidney Interntionl (2006) 69,

3 o r i g i n l r t i c l e JCK Leung et l.: Leptin induces TGF- synthesis y mesothelil cell O-R protein (% of time zero) O-R/ rtio (h) (h) Mnnitol (h) (h) Mnnitol (h) Figure 4 Time-dependent upregultion of O-R expression in HPMC y glucose. Time response curve for () O-R mrna nd () protein expression in HPMC upon exposure to glucose. Tretment with glucose (40 mm; drk r) ut not mnnitol (40 mm; white r) led to time-dependent upregultion in O-R mrna nd protein expression in HPMC. Mesurement of O-R mrna t time intervls 12, 24, 48, nd 72 h; or O-R protein t time intervls 24, 48, nd 72 h differed significntly from vlues t time zero. Dt re men7s.d. of four individul experiments. *Po protein relese (Figure 7) (Po0.005 for oth). Similr upregultion ws not oserved with mnnitol. Expression of O-R HPAC ws not ffected y glucose (dt not shown). Leptin did not lter the expression of O-R ut incresed the TGF- relese y HPMC Incution with incresing dose of leptin (5 40 ng/ml) filed to upregulte the expression of OB-R in HPMC (Figure 8). However, leptin t concentrtion of 10 ng/ml or greter significntly incresed the TGF- mrna expression nd protein relese y HPMC (Po0.005) (Figure 8). Activtion of JAK-STAT signl trnsduction pthwy in HPMC y leptin Figure 9 shows the detection of phospho-jak2 nd phospho- STAT3 of the JAK-STAT pthwy, phospho-pn-protein kinse (PKC) or c-fos in lyste from HPMC following incution with medium contining glucose ( mm), medium contining leptin (0 40 ng/ml), control-hpac, or glucose-hpac medium. Incresing concentrtion of glucose ctivted the PKC ut not the JAK-STAT pthwy in stepwise fshion. In contrst, leptin ctivted the JAK2, STAT3, nd c-fos ut not PKC in HPMC. Activtion of the (h) O-R/ rtio O-R protein (% of 5.6 mm control) (mm) (mm) Mnnitol (mm) (mm) (mm) Mnnitol (mm) Figure 5 Dose-dependent upregultion of O-R expression in HPMC y glucose. Dose response curve for () O-R mrna nd () protein expression in HPMC upon exposure to glucose for 24 (mrna) or 48 h (protein). Tretment with incresing concentrtion of glucose (drk r) ut not mnnitol (white r) led to dosedependent upregultion in O-R mrna nd protein expression in HPMC. Mesurement of O-R mrna t high concentrtions of glucose (20, 40, nd 80 mm) differed significntly from the seline vlue t 5 mm. Dt re men7s.d. of four individul experiments. *Po JAK-STAT, c-fos, nd PKC pthwys were detected in HPMC incuted with glucose-hpac medium ut not with control- HPAC medium. Roles of PKC nd JAK-STAT signling pthwys To study the roles of PKC nd JAK-STAT signling pthwys in TGF- relese from HPMC induced y glucose or/nd leptin, HPMC ws cultured with medium contining 5.6 or 40 mm glucose, 40 mm glucose plus 20 ng/ml leptin, nd glucose-hpac medium in the sence or presence of neutrlizing ntiody to leptin receptor, inhiitor to JAK2 (AG490; 10 mm), or/nd PKC (clphostin; 100 nm). Leptin upregulted TGF- relese y HPMC (Figure 10). Addition of glucose further mplified this TGF- relese. There ws synergistic increse (960% of sl vlue) in TGF- synthesis y HPMC exposed to 40 mm glucose plus 20 ng/ml leptin. AG490, nti-leptin receptor or clphostin lone only reduced TGF- relese to 575, 637, or 340% of tht of the sl level. The TGF- relese in HPMC induced y simultneous exposure to glucose nd leptin ws completely olished only with the presence of oth AG490 nd clphostin. The 2080 Kidney Interntionl (2006) 69,

4 JCK Leung et l.: Leptin induces TGF- synthesis y mesothelil cell o r i g i n l r t i c l e Leptin/ rtio (h) (h) Mnnitol Leptin/ rtio (mm) (mm) Mnnitol Leptin (ng/10 5 cells) (h) (h) Figure 6 Time-dependent upregultion of leptin expression in HPAC y glucose. Time response curve for () leptin mrna nd () protein expression in HPAC upon exposure to glucose. Tretment with glucose (40 mm; drk r) ut not mnnitol (40 mm; white r) led to time-dependent upregultion in leptin gene nd protein expression in HPAC. Mesurement of leptin mrna t time intervls 12 h, 24, nd 48 h differed significntly from vlues t time zero. Significnce ws not oserved t 72 h when compred with seline vlues. Mesurement of leptin protein t 48 nd 72 h differed significntly from vlue t time zero. Dt re men7s.d. of four individul experiments. *Po Leptin (ng/10 5 cells) (mm) (mm) Figure 7 Dose-dependent upregultion of leptin expression in HPAC y glucose. Dose response curve for () leptin mrna nd () protein expression in HPAC upon exposure to glucose for 24 (mrna) or 48 h (protein). Tretment with incresing concentrtion of glucose (drk r) ut not mnnitol (white r) led to dosedependent upregultion in leptin gene nd leptin relese in HPAC. Mesurement of leptin mrna t high-dose concentrtions of glucose (20, 40, nd 80 mm) differed significntly from seline vlue t 5 mm. Dt re men7s.d. of four individul experiments. *Po concentrtions of glucose nd leptin in the conditioned medium were 3672 mm nd ng/ml, respectively. -HPAC medium significntly incresed TGF- synthesis y HPMC (803% of sl level). AG490, clphostin, nd ntileptin receptor reduced 39.7, 86.96, nd 33.1% of the incresed TGF- level, respectively. DISCUSSION To the est of our knowledge, this is the first study showing tht HPMC express functionl full-length leptin receptor, the O-R. We hve demonstrted tht O-R is constitutively expressed in cultured HPMC nd humn omentl tissue y RT-PCR, immunolotting, nd immunohistochemicl stining. The expression of O-R in HPMC ws upregulted following exposure to glucose. The synthesis of leptin y HPAC is upregulted y glucose ut not y equimolr mnnitol. These dt re in keeping with the recent finding tht glucose-sed PDF induces higher leptin secretion y murine dipocyte cell line 3T3-L1 compred with dilyste with lower glucose concentrtion or without glucose. 11 High glucose content in the dilyste is mjor fctor cusing the structurl nd functionl normlities in peritonel cells during CAPD. 14,15 Decresed expression of intercellulr junctionl proteins ZO-1 nd -ctenin is oserved in HPMC cultured with glucose. 16 upregultes TGF- nd fironectin synthesis y HPMC. 17,18 TGF- is involved in vrious pthophysiologicl events in peritonel iology. 19 Overexpressed TGF- in murine mesothelil cells results in peritonel memrne firosis nd loss of ultrfiltrtion. 20 In n niml model of peritonel dilysis, inhiition of TGF- function nd production reduces peritonel firosis. 21 -induced TGF- upregultion in kidney mesngil cells nd HPMC is medited y PKC. 18,22 High glucose induces de novo synthesis of dicylglycerol nd ctivte PKC in HPMC. 23 Activtion of PKC leds to trnscription of c-fos nd c-jun erly response gene. 24 The c-fos nd c-jun proteins cn form dimeric complexes with ech other tht ind to the ctivtor protein- 1 (AP-1) consensus sequence to ctivte the promoter ctivity. 25 It hs een shown tht the AP-1 ox B inding sites on the TGF- promoter medite the high glucose response in mesngil cells. 26 High glucose increses the TGF- promoter ctivity y enhncing the expression of c-fos, nd hence its inding to AP-1. The involvement of c-fos in regulting the TGF- promoter ctivity is well demonstrted y the oservtion tht heprin prevents TGF- ctivtion induced y glucose through reducing the c-fos expression, nd therey preventing the formtion of ctivted AP-1 complexes. 27 Kidney Interntionl (2006) 69,

5 o r i g i n l r t i c l e JCK Leung et l.: Leptin induces TGF- synthesis y mesothelil cell (ng/ml) (ng/ml) O-R mrna O-R protein O-R protein (% of control) TGF-β (pg/10 5 cells) Leptin (ng/ml) (ng/ml) TGF-β Leptin (ng/ml) Figure 8 Effect of exogenous leptin on O-R expression nd TGF- relese y HPMC. () Expression of O-R mrna (white r) or protein (drk r) ws not ltered in HPMC cultured with different concentrtion of leptin for 24 h (mrna) or for 48 h (protein). () Relese of TGF- in HPMC cultured with incresing doses of leptin for 48 h. The expression of TGF- mrna (white r) ws significntly incresed in HPMC incuted with leptin t 20 nd 40 ng/ml. The relese of TGF- (drk r) ws up-regulted in HPMC incuted with leptin t 10, 20, nd 40 ng/ml. Dt re men7s.d. of four individul experiments. *Po In the present study, we hve demonstrted tht leptin upregultes the gene expression nd protein relese of TGF- in HPMC. HPMC do not produce leptin constitutively or fter incution with high glucose, nd there could e prcrine effect of HPAC-derived leptin on HPMC expression of TGF-. It hd een shown tht leptin enhnced romtse expression in the rest cncer cell line vi AP We next exmined whether leptin-induced TGF- synthesis in HPMC is medited through signl pthwys involving PKC nd AP-1. Our dt showed tht PKC is not ctivted y leptin nd neither the leptin-induced TGF- synthesis is olished y PKC inhiitor. Hence, unlike TGF- synthesis in HPMC induced y high glucose, TGF- synthesis in HPMC induced y leptin is independent of the PKC pthwy. Leptin triggers O-R signling through the JAK-STAT signl trnsduction pthwy. 7 In vsculr smooth muscle cells, high glucose enhnces the ngiotensin II-induced cell prolifertion through the JAK-STAT pthwy. 29 In glomerulr mesngil cells, inhiition of the JAK-STAT pthwy prevents the glucose-induced TGF- nd fironectin synthesis. 30 Our O-R/ rtio TGF-β/ rtio (mm) (40 mm) Leptin (20 ng/ml) AG490 (10 μm) Clphostin (100 nm) Leptin (ng/ml) Phospho-pn-PKC Phospho-STAT3 Phospho-JAK2 c-fos immunolotting dt hve reveled tht leptin, ut not glucose, is le to ctivte JAK2 nd STAT3. Furthermore, the TGF- synthesis in HPMC induced y leptin ws olished y JAK2 specific inhiitor, AG490. Tken these oservtions together, TGF- synthesis in HPMC induced y leptin is triggered y O-R signling involving the JAK-STAT pthwy. It hs een shown tht ctivted STAT3 inds to c-fos promoter nd upregultes c-fos trnscription. 31 It is likely tht the downstrem event in leptin-induced TGF- synthesis y HPMC is similr to the mechnism of high glucose-medited TGF- synthesis, which involves n incresed c-fos expression nd TGF- promoter ctivity. Our dt show tht in the presence of 40 mm glucose or 20 ng/ml leptin, there ws 583 or 349% increse in TGF- synthesis y HPMC. However, glucose (40 mm) mplified the leptin-induced TGF- synthesis y HPMC (1113% (glucose þ leptin) versus 932% (glucose or leptin 583 þ 349 ¼ 932%). This mplifiction of TGF- synthesis is likely to e due to the incresed expression of O-R in HPMC y high glucose. Indeed, our inhiition studies show tht TGF- synthesis y HPMC in the presence of oth glucose nd leptin ws olished only y using oth PKC nd JAK2 inhiitors ut not with either one lone. Bsed on our present dt, hypotheticl model of leptin-induced TGF- synthesis in HPMC is proposed (Figure 11). TGF- synthesis y HPMC is upregulted y glucose through PKC-dependent mechnisms. HPMC exposed to high glucose will induce PKC ctivtion. PKC will then upregulte the trnscription fctors c-fos nd c-jun, which form complexes with the AP-1 Control-HPAC medium HPAC medium c-fos Figure 9 Detection of different phospho-specific signls in HPMC lyste following incution with glucose, leptin, glucose-hpac conditioned medium, or control-hpac conditioned medium. () Activtion of the PKC signl trnsduction pthwy ws detected in HPMC incuted with glucose or glucose-hpac medium ut not with leptin or control-hpac medium. () Activtion of the JAK2, STAT3, nd c-fos signl trnsduction pthwys ws detected in HPMC incuted with leptin or glucose-hpac conditioned medium ut not with glucose or control-hpac conditioned medium Kidney Interntionl (2006) 69,

6 JCK Leung et l.: Leptin induces TGF- synthesis y mesothelil cell o r i g i n l r t i c l e 1600 # 1400 TGF-β (pg/10 5 cells) # Φ Φ Ω# Φ mm glucose 40 mm glucose 20 ng/ml leptin 40 mm glucose -HPAC plus 20 ng/ml leptin medium Ω P < vs glucose plus leptin, without inhiitor # P < vs 40 mm glucose, without inhiitor P < vs 40 mm glucose, with AG490 ΦP < vs 40 mm glucose, with clphostin P < vs 40 mm glucose, with nti-leptin receptor A Figure 10 Blocking study of TGF- relese y HPMC. Concentrtion of TGF- in culture superntnt from HPMC incuted with medium contining 5.6 mm glucose, 40 mm glucose, 20 ng/ml leptin, 40 mm glucose plus 20 ng/ml leptin, or glucose-hpac conditioned medium in the sence (white r) presence of nti-leptin receptor ntiody (crossed r; lst r in ech group; 50mg/ml), inhiitor to JAK2 (AG490; 10 mm; dotted r; 2nd r in ech group), PKC (clphostin; 100 nm; gry r) or oth (10 mm AG490 plus 100 nm clphostin; drk r). Dt re men7s.d. of four individul experiments. P JAK2 AG490 P STAT3 Leptin O-R + c-fos c-jun c-fos AP HG + P PKC TGF-β trnscription HG Clphostin Figure 11 Hypotheticl model of leptin-induced TGF- synthesis in HPMC. HPMC exposed to high glucose will induce PKC ctivtion. PKC will then upregulte the trnscription of erly response genes, c-fos or/nd c-jun, which form complexes with the AP-1 inding site nd enhnce the inding of AP-1 proteins to the AP-1 ox B on the TGF- promoter. TGF- synthesis is upregulted y high glucose through this PKC-dependent mechnism. Although leptin cnnot ctivte the PKC pthwy directly, it does ctivte the JAK2 nd STAT3 through the functionl O-R expressed on HPMC. Activted STAT3 ccumultes on the c-fos promotor nd provides oost to c-fos trnscription. High glucose upregultes the expression of O-R y HPMC s well the PKC-dependent TGF- synthesis. In the presence of leptin nd incresed O-R expression y HPMC stimulted with high glucose, upregultion of TGF- synthesis will e further mplified through the JAK-STAT pthwy. inding site nd enhnce the inding of AP-1 proteins to the AP-1 ox B on the TGF- promoter. Although leptin cnnot ctivte the PKC pthwy directly, it does ctivte the JAK2 nd STAT3 through the functionl O-R expressed on HPMC. Activted STAT3 ccumultes on the c-fos promoter nd increses c-fos trnscription. Since high glucose lso upregultes the expression of O-R on HPMC, in the presence of leptin nd high glucose, upregultion of TGF- synthesis y leptin will further e mplified through O-R nd the JAK-STAT3-dependent pthwy. Pthwys other thn JAK-STAT could lso e involved in mediting leptin s ction. In peripherl lood mononucler cells, leptin ctivtes the MAPK nd phosphtidyl-inositol 3 0 -kinse pthwys, nd is implicted in regulting cell growth nd glucose metolism. 32 It hs een shown tht leptin directly suppressed peroxisome prolifertor-ctivted receptor-g expression in dipocytes. In kidney mesngil nd tuulr cells, peroxisome prolifertor-ctivted receptor-g gonist ttenuted high glucose-induced TGF- relese. It is not known whether HPMC expressed peroxisome prolifertor-ctivted receptorg nd if it is present, the effect of peroxisome prolifertorctivted receptor-g expression on leptin-induced TGF- relese y HPMC. Further exmintion of these dditionl pthwys in modulting the leptin-induced TGF- relese y HPMC is wrrnted. Leptin is elevted during cute infection, in response to proinflmmtory cytokines including interleukin-1 nd tumour necrosis fctor-. 9 In the kidney, leptin stimultes cell prolifertion, collgen IV nd TGF- synthesis in glomerulr endothelil cells, 33 nd increses the glucose trnsport, upregultes the expression of TGF- type II Kidney Interntionl (2006) 69,

7 o r i g i n l r t i c l e JCK Leung et l.: Leptin induces TGF- synthesis y mesothelil cell receptor nd collgen I synthesis through phosphtidylinositol-3-kinse relted pthwy in glomerulr mesngil cells. 34 These dt suggest tht leptin triggers prcrine interction etween glomerulr endothelil nd mesngil cells through upregultion of TGF- in glomerulr endothelil cells together with incresed TGF- receptor expression in mesngil cells. Whether such prcrine interction is operting etween peritonel dipocytes nd HPMC remins to e explored. Here, we present the first evidence tht dipocyte-derived leptin cn exert prcrine effect on HPMC through upregultion of O-R receptor nd ctivtion of the JAK-STAT pthwy. It must e emphsized tht dt otined from cultured cells model my not exctly reflect in vivo sitution. During CAPD, mesothelil cells nd dipocytes re exposed to unphysiologicl low ph nd high glucose PDF. PDF lso contins toxic sustnces including glucose degrdtion products. All these fctors work together with the cytokines nd dipokines networks from peritonel cells in ffecting the TGF- synthesis y mesothelil cells. For the present study, culture model ws employed to llow exmintion of specific vriles (high glucose, leptin nd O-R expression), nd this will give preliminry ide of wht could e hppened in vivo during peritonel dilysis. It is of importnt to exmine the in vivo expression of O-R nd relted signling pthwy in ptients efore nd under long-term CAPD nd future investigtion is wrrnted. In conclusion, our study provides novel dt tht leptin cn ctivte the JAK-STAT signl trnsduction pthwy nd increse the synthesis of TGF- y HPMC through triggering the functionl O-R receptor. In the presence of high glucose, leptin-induced TGF- synthesis y HPMC is enhnced. Our results implicte tht dipocyte-derived leptin my contriute to the peritonel dysfunction in dilysis through prcrine effect on mesothelil TGF- synthesis. MATERIALS AND METHODS Mterils All regents for tissue culture were otined from Invitogen Co (Crlsd, CA, USA). Monoclonl nti-o-r, neutrlizing nti-leptin receptor ntiody nd leptin protein were otined from R&D System (Minnepolis, MN, USA). Monoclonl mouse nti-ctin ws otined from L Vision (Fremont, CA, USA). Secondry ntiodies for immunohistochemicl stining nd immunolotting were otined from Dko (Crpinteri, CA, USA). Antiodies to phospho-jak2, phospho-stat3, pn-pkc nd c-fos were otined from Cell Signling Technology (Beverly, MA, USA). All other chemicls were otined from Sigm (St Louis, MO, USA). Culture of HPMC The study ws conducted in ccordnce with the Declrtion of Helsinki Principles nd ws pproved y the institutionl ethics committee for studies in humn. Full informed consent for removing smll pieces of omentl tissue for experimentl studies ws otined from ptients. Omentum tissue ws otined from five nonuremic, nondietic ptients (mle, ge 45 55) under elective dominl surgery for erly gstric cncer. The ptients hve not een previously exposed to peritonel dilysis tretment, nd were cliniclly not inflmed. The omentl smples collected were further exmined histologiclly to e free of other pthologicl conditions including inflmmtion, metstsis, nd endometriosis. Mesothelil cells were isolted nd chrcterized using procedures descried previously. 35 Once monolyer of HPMC reched confluence (HPMC of second or third pssge were used in ll experiments), the cells were growth-rrested with serum-free medium for 24 h prior to experiments. Under these conditions, the HPMC remined in nonprolifertive vile condition. Culture of peritonel dipocytes Omentl dipocyte precursors from the stroml vsculr frction were isolted y collgense digestion. 36 For in vitro experiments, dipocytes were differentited y culturing stroml vsculr frction in dipogenic medium (Dulecco s modified Egle s medium/hm s F12 medi supplemented with 10 mg/ml trnsferrin, 100 mm scoric cid 2-sodium, 0.85 mm insulin, 20 nm sodium selenite, 0.2 nm triiodothyronine, 1 mm dexmethsone, 100 mm isoutyl-methylxnthine, nd 1 mm rosiglitzone). The medium ws chnged 3 dys lter (dexmethsone nd isoutyl-methylxnthine were omitted). After 15 dys of differentition, 90% of cells re positively stined with Oil Red O for neutrl lipid ccumultion. These differentited dipocytes were cultured in serum-free M199 medium for 24 h prior to experiment. Immunohistochemicl stining The expression of O-R in 4 mm-thicked omentl prffin sections ws determined y immunohistochemicl stining using mouse nti-o-r (0.5 mg/ml). The ound ntiody ws visulised s rown colour using the Dko Envision Plus System (Dko). Preprtion of M199 medium contining mnnitol The sl glucose concentrtion in M199 medium is 5.6 mm, for ll experiment involving the use of mnnitol s osmolrity control, the concentrtion of mnnitol dded to the M199 medium re 0, 4.4, 14.4, 34.4, nd 74.4 mm (plus 5.6 mm glucose in the M199 medium) to generte different sugr concentrtion (5.6, 10, 20, 40, nd 80 mm). For simplicity, these 5.6 mm glucose þ mnnitol medium re nmed s 5.6, 10, 20, 40, or 80 mm mnnitol. Preprtion of conditioned medium from HPAC exposed to glucose HPAC were cultured with M199 medium contining glucose (40 mm) for 48 h. The conditioned medium (glucose-hpac medium) ws stored t 701C until used. The conditioned medi were centrifuged for 10 min t 2000 g efore experiment. Conditioned medium from HPAC cultured with 5.6 mm sl glucose level (control-hpac medium) ws used s control. Expression of O-R y HPMC exposed to glucose For time response experiment, HPMC ( cells) were exposed to 40 mm glucose or mnnitol for 0, 6, 12, 24, 48, nd 72 h. For dose response experiment, HPMC were cultured with 5.6, 10, 20, 40, nd 80 mm glucose or mnnitol for 24 h (mrna expression) or 48 h (protein relese). At ny prticulr time point or t the end of experiment, cells were hrvested for totl RNA or lyste preprtion for determintion of expression of O-R using RT- PCR or immunolotting Kidney Interntionl (2006) 69,

8 JCK Leung et l.: Leptin induces TGF- synthesis y mesothelil cell o r i g i n l r t i c l e Expression of leptin mrna nd protein y HPAC exposed to glucose For time response experiment, HPAC ( cells) were exposed to 40 mm glucose or mnnitol for 0, 6, 12, 24, 48, nd 72 h. For dose response experiment, HPAC were cultured with 5.6, 10, 20, 40, nd 80 mm glucose or mnnitol for 24 h (mrna expression) or 48 h (protein relese). At ny prticulr time point or t the end of experiment, culture superntnts were collected for ssy of leptin y ELISA nd cells were hrvested for totl RNA preprtion. Effect of leptin on the expression of O-R nd synthesis of TGF- y HPMC HPMC were exposed to 0, 5, 10, 20, nd 40 ng/ml leptin for 24 h (mrna) or 48 h (protein). At the end of experiment, culture superntnts were collected for ssy of TGF- y n ELISA nd cells were hrvested for totl RNA or cell lyste preprtions. O-R or TGF- mrna expression ws determined y RT-PCR nd O-R protein expression ws ssyed y immunolotting. Activtion of JAK-STAT nd PKC signl trnsduction pthwys HPMC/HPAC were exposed to glucose ( mm), leptin (0 40 ng/ml), glucose-hpac medium, or control medium for 30 min. The expression of phospho-jak2, phospho-stat3, nd phospho-pkc y HPMC ws determined using immunolotting. In nother sets of experiment, HPMC were cultured with M199 contining (i) 5.6 mm glucose, (ii) 40 mm glucose, (iii) 40 mm glucose plus 20 ng/ml leptin, or (iv) glucose-hpac medium in the sence or presence of inhiitor to JAK2 (AG490; 10 mm) nd/or PKC (clphostin; 100 nm); or nti-leptin receptor ntiody for 48 h (the inhiitors or ntiody were dded 1 h efore experiment strted). At the end of experiment, the TGF- concentrtion in the culture superntnt ws ssyed y ELISA. Gene expression study Cells were hrvested nd totl cellulr RT-PCR ws performed s previously descried. 37 The heptocellulr line HepG2 ws otined from Americn Type Culture Collection (ATCC; Rockville, MD, USA) nd the humn peripherl lood mononucler cells were isolted from helthy donors y Ficoll grdient seprtion s previously descried. 38 The RNAs otined from these cells were used s positive control for O-R expression. Primers for O-R, leptin, TGF-, nd glycerldehyde-3-phosphte dehydrogense were designed from GeneBnk sequences (O-R U43168; leptin D49487; TGF- X02812 nd glycerldehyde-3-phosphte dehydrogense AF261085). The sequences of primers (sense nd ntisense) re: (i) O-R, 5 0 -TCACCCAGTGATTACAAGCT nd 5 0 -CTGGAGAACTCT GATGTCCG; (ii) leptin, 5 0 -GCTGTGCCCATCCAAAAAGT nd ACTGCCAGTGTCTGGTCCAT; (iii) TGF-, 5 0 -GCCCTGGACAC CAACTATTGCT nd 5 0 -AGGCTCCAAATGTAGGGGCAGG; (iv) glycerldehyde-3-phosphte dehydrogense, 5 0 -TGAAGGTCGGAGT CAACGGATTTGGT nd 5 0 -CATGTGGGCCATGAGGTCCACCAC. The PCR products for O-R, leptin, or TGF- (1071, 179, or 161 p) nd control (glyurldehyde-3 phosphte dehydrogense; 452 p) were mixed nd seprted y 1.5% wt/vol grose gels. The results ws nlysed using the Gel Doc 1000 Gel Documenttion System (Bio-Rd Lortories Ltd., Hercules, CA, USA). Immunolotting Cells were lysed with uffer contining protese inhiitor cocktils (Sigm). The protein concentrtions in cell extrcts were mesured y modified Lowry method (DC protein ssy kit, BioRd). A totl of 10 mg of totl protein from the extrct ws electrophoresed nd then trnsferred onto polyvinylidine difluoride memrne. The memrne ws incuted with mouse nti-o-r ntiody (1:1000), nti-ctin ntiody (1:1000); rit nti-phospho JAK-2 (1:1000), nti-phospho STAT3 (1:1000) or nti-phospho pn-pkc ntiody (1:4000). The memrne ws incuted with peroxidseleled got nti-rit or nti-mouse immunogloulin (Dko) efore detection with enhnced chemiluminescence (Amershm Phrmci Biotech, Arlington, IL, USA). The immunolot imges were quntitted using ImgeQunt softwre (Moleculr Dynmic, Sunnyvle, CA, USA). Densitometry results were reported s % of control fter normliztion with the verge ritrry integrted vlues of the ctin signl. Mesurement of TGF- or leptin in culture superntnt The concentrtion of TGF- or leptin in superntnt ws mesured y ELISA (R&D System) with detection limit of 32 or 20 pg/ml nd coefficient of vrition of 8.5 or 7.6%, respectively. Sttisticl nlysis All dt were expressed s mens7s.d. Intergroup differences etween two vriles were ssessed y the unpired t-test. The dt from more thn two study groups were nlyzed using multivrite nlysis of vrince followed y Bonferroni correction. All P -vlues quoted re two-tiled nd the significnce is defined s Po ACKNOWLEDGMENTS The study ws supported y the Reserch Grnt Committee (Hong Kong SAR) [HKU7415/04] nd the Seed Funding for Bsic Reserch [No ] of the University of Hong Kong. JCK Leung is supported y the L&T Chritle Fund nd INDOCAFE. 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