Role of EP2 and EP4 receptor-selective agonists of prostaglandin E 2 in acute and chronic kidney failure

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1 & 26 Interntionl Society of Nephrology originl rticle Role of EP2 nd EP4 receptor-selective gonists of prostglndin E 2 in cute nd chronic kidney filure S Vukicevic 1, P Simic 1, F Borovecki 1, L Grgurevic 1, D Rogic 2, I Orlic 1, WA Grsser 3, DD Thompson 3 nd VM Prlkr 3 1 Lortory of Minerlized Tissues, Deprtment of Antomy, Zgre Medicl School, University of Zgre, Zgre, Croti; 2 Centrl Biochemicl Lortory, Clinicl Hospitl Center Rero, Zgre, Croti nd 3 Pfizer Glol Reserch nd Development, Groton Lortories, Groton, Connecticut, USA We tested the efficcy of three selective gonists of prostglndin E 2 (PGE 2 ) receptor, EP2 (CP-536,745-1), EP2/4 (CP-43,35-2), nd EP4 (CP-44,519-2), in two models of cute nd chronic kidney filure. In the nephrotoxic mercury chloride (HgCl 2 ) rt model of cute kidney filure systemiclly dministered EP4 gonist reduced the serum cretinine vlues nd incresed the survivl rte. Although the EP2 or the EP2/4 gonist did not chnge the serum cretinine vlues, the EP2 receptor gonist incresed the survivl rte. Histologicl evlution of kidneys from EP4-treted rts indicted less proximl tuulr necrosis nd less poptotic cells. In rt model of chronic renl filure, the three receptor gonists decresed the serum cretinine nd incresed the glomerulr filtrtion rte t 9 weeks following therpy. Kidneys treted with the EP4 gonist hd less glomerulr sclerosis, etter preservtion of proximl nd distl tuules nd lood vessels, incresed convoluted epithelium prolifertion nd less poptotic cells. Nephrectomy hd no influence on the expression of the EP4 receptor, wheres EP2 receptor expression ws reduced y 5% nd then corrected following tretment with EP2 nd EP2/4 receptor gonists. These findings suggest tht PGE 2 hs n importnt role in cute kidney filure vi the EP4 receptor, wheres in chronic kidney filure oth EP2 nd EP4 receptors re eqully importnt in preserving the progression of chronic kidney filure. Thus, gonism of EP2 nd EP4 receptors my provide sis for treting cute nd chronic kidney filure. Kidney Interntionl (26) 7, doi:1.138/sj.ki.51715; pulished online 26 July 26 KEYWORDS: prostglndin E 2 ; ARF; CRF; EP2 receptor; EP4 receptor Correspondence: S Vukicevic, Lortory of Minerlized Tissues, Deprtment of Antomy, Zgre Medicl School, University of Zgre, Slt 11, 1 Zgre, Croti. E-mil: vukicev@mef.hr Received 29 Septemer 25; revised 15 April 26; ccepted 9 My 26; pulished online 26 July 26 Acute nd chronic renl filures re directly responsile for high rte of mortlity, significnt moridity, nd high medicl costs. 1,2 Acute renl filure (ARF) is very morid nd costly disorder with severe proportion of ptients progressing to end-stge renl disese requiring dilysis. Despite the dvnces of supportive cre, the mortlity hs remined high (4 8%). 3 Although the dmged kidney is cple of complete repir nd regenertion fter cute injury, the successful tretment of ptients with ARF who require dilysis remins one of the gretest chllenges fcing nephrology tody. 4,5 The mechnisms controlling the cscde of cellulr migrtion, growth, nd prolifertion undoutedly comprise numer of utocrine nd prcrine growth fctors. 6,7 Severl novel compounds hve proved effective in the tretment of experimentl ARF, ut there hs een little success in humn clinicl trils, including synthetic tril ntriuretic peptide, recominnt humn insulin-like growth fctor-1, clcium chnnel ntgonists, thyroxine, nd endothelin receptor ntgonists. 8 Chronic renl filure (CRF) is nother entity, which progresses to end-stge renl filure, independently of the initil pthogenic mechnism. The erly stges of renl injury involve compenstory renl growth ssocited with cellulr hypertrophy nd hyperplsi. 9,1 The infiltrtion of pltelets, lymphocytes, nd monocytes into the glomeruli nd interstitium cuses the progression of renl scrring. 11,12 This scrring process then leds to progressive dmge of glomerulr nd tuulr cells, nd eventully cuses glomerulr sclerosis nd tuulr trophy. In end-stge renl disese, renl cells re replced with firous tissue contriuting to the sclerotic chnges oserved in the glomeruli nd the interstitium. 11,12 When the glomerulr filtrtion rte (GFR) declines to less thn 1% of norml vlues (5 1 ml/min), the renl filure will rpidly progress to cuse deth unless the ptient receives renl replcement therpy (i.e. chronic hemodilysis, continuous peritonel dilysis, or kidney trnsplnttion) or lterntively therpy tht delys the progression of the CRF. At present, there is no specific therpy for chronic renl disese, nd tretment with growth fctors my meliorte the progression of the kidney disese. 13 A potentil tretment Kidney Interntionl (26) 7,

2 originl rticle S Vukicevic et l.: PGE 2 EP2 nd EP4 receptor gonist in renl filure for ARF nd CRF could e PGE 2 which hs een shown to hve multiple iologicl effects in mny tissues including kidney. PGE 2 plys mjor role in the norml renl physiology including production of renin, regultion of GFR, nd tuulr re-sorption of slt nd wter. 14 Similrly, in disese sttes such s dietic nephropthy, PGE 2 synthesis is elevted. PGE 2 plys n importnt role in mintining the lood flow especilly during conditions of enhnced vsoconstrictor ctivity. Furthermore, PGE 2 inhiits prolifertion of cultured mesngil cells in vitro, 15 nd given the diversity of PGE 2 ctions in the kidney it is ttrctive to speculte therpeutic role for PGE 2 in chronic nd/or cute kidney filure. However, owing to side effects tht include dirrhe, lethrgy, nd flushing, PGE 2 is n uncceptle therpeutic option. The phrmcologicl ctivity of PGE 2 is crried out vi four different cell surfce receptor sutypes: EP1, EP2, EP3, nd EP4. Of these four receptors, three re involved in modultion of cyclic denosine 5 -monophosphte (camp) levels (EP2 nd EP4 increse camp levels, EP3 reduces camp levels). The fourth receptor, EP1, is involved in regulting intrcellulr clcium levels. All four of the EP receptors hve een loclized in the kidney nd re responsile for distinct ctions of PGE 2. The EP1, EP3, nd EP4 receptors re present in the collecting duct. EP4 hs lso een shown to e expressed in the glomerulus nd EP2 mrna hs een loclized to the outer nd inner medull of the rt kidney. 16,17 Despite the well-known ctivities of PGE 2 in mintining the kidney function nd locliztion of vrious prostnoid receptors in the kidney, the precise role of their gonists or ntgonists s therpeutic options remins poorly understood. Recently, n EP1 selective ntgonist hs een demonstrted to e effective in prevention of dietic nephropthy in streptozotocin-induced rt model. 18 We initited discovery effort to identify the role of EP2 nd EP4 receptor-selective gonists in rt models of cute nd chronic kidney disese. In the present study, we hve used three different selective gonists of the PGE 2 receptors to ssess the ility of gonists of receptor sutypes to tret rts with cute nd chronic renl filure. Of the three receptor-specific gonists tested, CP- 43,35-2 ws selective for oth the EP2 nd EP4 receptors, CP-44,519-2 is n EP4-specific gonist, nd CP-536,745-1 is n EP2-specific gonist. In this report, we show tht CP- 44,519-2, highly selective nd potent functionl EP4 receptor gonist, is efficcious in rt model of cute renl filure, wheres oth EP2 nd EP4 receptor gonism prevents the progression of chronic renl filure. RESULTS EP2 nd EP4 receptor gonists We hypothesized tht specific PGE 2 receptor sutype gonists (Tle 1) would hve positive impct on renl dmge in the rt nd e devoid of severe side effects of PGE 2. To test our hypothesis, we sought EP2, EP4, nd EP2/4 receptorselective gonists. CP-43,35-2, CP-44,519-2, nd CP- Tle 1 PGE 2 receptor sutype gonists EP2 EC 5 (nm) EP4 EC 5 (nm) CP-536,745-1 (EP2) 1.2 CP-43,35-2 (EP2/4) CP-44,519-2 (EP4) ,745-1 ound with high ffinity nd were selective nd potent full gonists of the EP receptor, s mesured y their ility to increse intrcellulr camp levels to the sme levels s tretment with PGE 2 (Tle 1). Mercury chloride model of cute renl filure Serum cretinine (Cr) vlues of control rts incresed 7.5- fold on dy 1 nd 3 following HgCl 2 injection (Figure 1). In rts treted with 1 mg/kg of n EP4 (CP-44,519-2) receptor gonist, Cr vlues were significntly lower on dys 1 3 (Figure 1). A dose of 1 mg/kg EP4 receptor gonist ws less effective, showing sttisticl significnce only on dy 2 following mercury chloride (HgCl 2 ) injection. At 6 dys fter HgCl 2 ppliction, there were no differences etween tretment groups due to the low survivl rte of control nimls (Figure 1). Dynmic chnges of lood ure nitrogen showed similr trend s serum Cr vlue. Animls treted with 1 mg/kg of EP4 hd lower lood ure nitrogen on dys 1 nd 2 s compred to control nimls, wheres rts treted with 1 mg/kg hd lower vlues only on dy 1 (dt not shown). Therpy with the EP2 (CP-536,745-1) nd EP2/4 (CP-43,35-2) receptor gonist hd no effect on serum Cr vlues (Figure 1 nd c). At termintion on dy 6 following HgCl 2 dministrtion, 9 95% of rts treted with oth 1 nd 1 mg/kg EP4 survived, wheres 85% of the rts treted with EP2 survived (Figure 1). Aout 73% of rts survived in the group treted with 1 mg/kg of EP2/4 receptor gonist, wheres 1 mg/kg EP2/4 ws ineffective (Figure 1c). On the contrry, only 4% vehicle-treted control rts survived t dy 6 following HgCl 2 injection (Figure 1). Bsed on the functionl remnnt kidney prmeters, we next nlyzed the effect of the EP4 receptor gonist on the renl histopthology nd poptosis. The cellulr necrosis of the proximl tuules ws moderte in the EP4 receptor gonist-treted rts, wheres EP2 nd EP2/4 receptor gonists were less effective or hd no effect (Tle 2). However, no histopthologicl chnges were found in the glomeruli (Figure 2). There ws lower numer of poptotic cells in the kidneys treted with the EP4 receptor gonist t the termintion of the experiment, suggesting tht poptosis is n importnt event in the nephrotoxic kidney dmge nd tht prevention nd recovery re est pronounced in the EP4 receptor gonist-treted rts (Tle 3). These results suggest tht EP4 receptor gonism hd n effect on the iochemicl renl serum prmeters, correlted with significntly improved histopthologicl findings s compred to similr nlyses of kidney sections treted with EP2 nd EP2/4 receptor gonists (Tles 2 nd 3). 11 Kidney Interntionl (26) 7,

3 S Vukicevic et l.: PGE 2 EP2 nd EP4 receptor gonist in renl filure originl rticle c CP-44,519-2 (EP4) CP-53, (EP2) CP-43,35-2 (EP2/EP4) Survivl (%) mg/kg 1 mg/kg Survivl (%) mg/kg 1 mg/kg Survivl (%) mg/kg 1 mg/kg Figure 1 Therpeutic effect of () CP-44,519-2 (EP4), () CP-536,745-1 (EP2), nd (c) CP-43,35-2 (EP2/4) on serum Cr nd survivl in the HgCl 2 nephrotoxic rt model. EP4 receptor gonist decresed serum Cr nd incresed the survivl rte. EP2 nd EP2/4 receptor gonists hd no effect on serum Cr vlues; EP2 incresed the survivl rte; only the dose of 1 mg/kg EP2/4 incresed the survivl. Results re men7s.e.m. of 1 rts per tretment group. Po.5 with respect to HgCl 2 -control rts. Moriund rts were killed to prevent suffering (see Mterils nd Methods). Tle 2 Proximl tuulr necrosis in HgCl 2 control, CP-44,519-2 (EP4)-, CP-536,745-1 (EP2)-, nd CP-43, 35-2 (EP2/4)-treted rts Tretment groups Scores for the severity of necrosis in proximl tuules vehicle CP-44,519-2 (1 mg/kg) CP-44,519-2 (1 mg/kg) CP-536,745-1 (1 mg/kg) CP-536,745-1 (1 mg/kg) CP-43,35-2 (1 mg/kg) CP-43,35-2 (1 mg/kg) Results re men7s.e.m. of 1 kidneys (three sections ech) per tretment group. Po.5 with respect to HgCl 2 -control rts. Tle 3 Effect of CP-44,519-2 (EP4), CP-536,745-1 (EP2), nd CP-43,35-2 (EP2/4) on poptosis in rts with ARF Tretment groups Numer of poptotic cells per slice CP-44,519-2 (1 mg/kg) CP-44,519-2 (1 mg/kg) CP-536,745-1 (1 mg/kg) CP-536,745-1 (1 mg/kg) CP-43,35-2 (1 mg/kg) CP-43,35-2 (1 mg/kg) Results re men7s.e.m. of 1 kidneys (three sections ech) per tretment group. Po.5 with respect to HgCl 2 -control rts. Arevition: ARF, cute renl filure. Figure 2 Effect of CP-44,519-2 (EP4) on renl histology (periodic cid-schiff stin) in rts sujected to HgCl 2 injection. The cellulr necrosis of the proximl tuules ws decresed in the EP4 receptor gonist-treted rts nd no histopthologicl chnges were found in the glomeruli. Comprison of typicl histology from nimls treted with vehicle (, originl mgnifiction 25) or CP-44,519-2 (, originl mgnifiction 25) t dy 6 of therpy. 5/6 nephrectomy model of chronic renl filure Serum Cr concentrtions incresed 7% in Nx nimls throughout the durtion of the experiment (Figure 3). In contrst, nimls receiving EP4, EP2, nd EP2/4 receptor gonists once week hd lower serum Cr vlues t 9 nd 11 weeks following the eginning of therpy (Figure 3). In n nother experiment, Nx rts injected with EP4 receptor gonist three times week hd lower serum Cr vlues 8 nd 11 weeks following the eginning of therpy (Figure 3), nd did not show serious side effects tht include dirrhe, lethrgy, nd incresed ody temperture. Both, 1 nd/or 3 weekly dministrtions of EP4 receptor gonist were eqully effective in delying the progression of the disese (Figure 3). GFR ws 41% higher t 8 weeks following the eginning of therpy in rts receiving the EP4 receptor gonist three times week, wheres nimls dosed once weekly hd GFR incresed 45%, 11 weeks following tretment (Tle 4). EP4 receptor gonist-treted rts hd reduced glomerulr sclerosis, more vile glomeruli, less tuulointerstitil injury, nd etter preservtion of proximl nd distl tuule structures s compred to control Nx rts (Tle 5, Figure 4). Similr histopthologic prevention of the progression of kidney dmge ws detected in nimls treted with EP2 nd EP2/4 receptor gonists (dt not shown). The immunohistochemicl nlysis of perituulr cpillry-derived Kidney Interntionl (26) 7,

4 originl rticle S Vukicevic et l.: PGE 2 EP2 nd EP4 receptor gonist in renl filure /6 Nx 4 2/3 Nx CP-44,519-2 (EP4) 2 CP-53, (EP2) CP-43,35-2 (EP2/EP4) Weeks 2 Th 15 Th 1 5 5/6 Nx 2/3 Nx CP-44,519-2 (EP4) 3 week CP-44,519-2 (EP4) 1 week Weeks Figure 3 Therpeutic effect of 1 mg/kg of CP-44,519-2 (EP4), CP-536,745-1 (EP2), nd CP-43,35-2 (EP2/4) once week nd CP-44,519-2 (EP4) once nd three times week on serum Cr in 5/6 Nx rt model of chronic renl filure. () All three receptor gonists were effective in reducing serum Cr vlues of Nx rts 9 nd 11 weeks following Nx. () Both, 1 nd/or 3 weekly dministrtions of EP4 receptor gonist were eqully effective in delying the progression of the disese. Results re presented s men7s.e.m. of () 25 rts per tretment group in nd () 12 rts per tretment groups in. Po.5 vs control; Po.5 vs control. 2/3 Nx nd 5/6 Nx indictes surgicl nephrectomies s descried in Mterils nd Methods; Th indictes the eginning of therpy. Tle 5 Morphologicl lesions of Nx control nd CP-44, (EP4)-treted rts in CRF Group CP-44,519-2 CP-44,519-2 Vehicle 1 /week 3 /week Tuulointerstitium Tuulr dilttion/throphy Interstitil firosis Inflmmtory cells Glomeruli Glommerulr sclerosis Microneurysms Asence of vile glomeruli Morphologicl lesions were quntified s descried in the Mterils nd Methods section. Two independent oservers without significnt over- or underestimtion etween them nlyzed kidneys of 12 nimls per tretment group. The results re presented s men7s.e.m. of 1 kidneys (three sections ech) per tretment group. Po.5, Po.5 with respect to Nx-control rts. Arevition: Nx, nephrectomy. c d Tle 4 Effect of CP-44,519-2 (EP4) on GFR (ml/min/g) in rts with CRF GFR (ml/min/g) Weeks of therpy Therpy vehicle CP-44, (1 mg/kg) 3 /week CP-44,519-2 (1 mg/kg) 1 /week Results re men7s.e.m. of 1 rts per tretment group. Po.5 with respect to Nx-control rts. Arevitions: GFR, glomerulr filtrtion rte; Nx, nephrectomy. Figure 4 Effect of CP-44,519-2 (EP4) on renl histology (hemtoxylin nd eosin) in rts sujected to 5/6 Nx. EP4 receptor gonist-treted rts hd reduced glomerulr sclerosis, more vile glomeruli, less tuulointerstitil injury, nd etter preservtion of proximl nd distl tuule structures s compred to control Nx rts. A typicl histology from nimls treted with vehicle (originl mgnifictions:, 12.5; c, 5) or CP-44,519-2 (, 12.5, d 5) t 11 weeks of therpy. smooth muscle cells reveled tht pproximtely fourfold more cells in EP4-treted kidneys expressed smooth muscle -ctin, suggesting tht EP4 receptor gonist supports the mintennce of vsculr smooth muscle cell phenotype (Figure 5 nd ). Cell prolifertion evluted y proliferting cell nucler ntigen (PCNA) stining indicted n increse in the proximl tuule cell prolifertion in EP4 receptor gonist-treted rts (Figure 5c nd d). EP4 lso ttenuted the expression of intrcellulr dhesion molecule-1, n importnt molecule mediting the progress nd intensity of renl necrosis nd susequent firosis (Figure 5e nd f). Mc CD 68 stining detected n ccumultion of mcrophges in vehicle-treted rts. In contrst, rts treted with EP4 receptor gonist hd 112 Kidney Interntionl (26) 7,

5 S Vukicevic et l.: PGE 2 EP2 nd EP4 receptor gonist in renl filure originl rticle SMA PCNA ICAM Mc CD-68 c e g Vehicle CP-44,519-2 (EP4) Figure 5 Immunohistochemicl stining of smooth muscle -ctin, proliferting cell nucler ntigen, intercellulr cell dhesion molecule, nd mcrophge-ssocited ntigen CD 68. Comprison of (, ) smooth muscle -ctin, (c, d) PCNA, (e, f) intrcellulr dhesion molecule nd (g, h) Mc stining of (, c, e, g) vehicle nd (, d, f, h) CP-44,519-2-treted kidneys t 11 weeks of therpy. Fourfold more cells in EP4-treted kidneys expressed smooth muscle -ctin, mintining vsculr smooth muscle cell phenotype. Cell prolifertion evluted y PCNA stining indicted n increse in proximl tuule cell prolifertion in EP4 receptor gonist-treted rts. EP4 lso ttenuted the expression of intrcellulr dhesion molecule -1, mediting renl firosis. Mc CD 68 stining detected ninefold less mcrophges ccumulted in the remining kidney prenchyme of rts treted with the EP4 receptor gonist. Originl mgnifiction pproximtely ninefold less mcrophges ccumulted in the remining kidney prenchyme (Figure 5g nd h). EP2 nd EP4 receptor expression The effect of EP4, EP2, nd EP2/4 receptor gonists on EP2 nd EP4 mrna ws evluted y rel-time polymerse chin rection in kidneys of 5/6 Nx control rts (Figure 6) nd in those treted with ll three PGE 2 receptor gonists. Following nephrectomy (Nx), the EP2 receptor expression ws reduced y 5% s compred to shm nimls, however, tretment with the EP2/4 receptor gonist rought the EP2 expression levels ck to shm vlues (Figure 6). EP4 expression level did not chnge following Nx nd tretment with EP2/4 hd slight effect on the EP4 gene expression (Figure 6). At the end of the therpy, there ws slight elevtion in EP2 nd EP d f h Fold chnge Fold chnge EP2 EP4 EP2 EP4 CP-44,519-2 (EP4) Weeks CP-536,745-1 (EP2) CP-43,35-22 (EP2/EP4) Figure 6 EP2 nd EP4 receptors expression during the therpy in the kidneys of 5/6 Nx rts treted with CP-43,35-2 (EP2/4) (), nd the end of the therpy in the kidneys of 5/6 Nx rts treted with CP-44,519-2 (EP4), CP-536,745-1 (EP2), nd CP-43,35-22 (EP2/4). Following Nx, the EP2 receptor expression ws reduced y 5% (.5-fold chnge) s compred to shm nimls (Po.5). Tretment with the EP2/4 receptor gonist rought the EP2 expression levels ck to shm vlues (). EP4 expression level did not chnge following Nx nd tretment with EP2/4 hd slight effect on the EP4 gene expression s compred to shm nimls (). At the end of the therpy, there ws slight elevtion in EP2 nd EP4 receptor expression in rts treted with ll three PGE 2 receptor gonists s compred to shm nimls (). All dt re represented s fold chnge to gene expression of shm nimls7s.e.m. (represented y 1. on the y-xis). 4 receptor expression in rts treted with ll three PGE 2 receptor gonists s compred to shm nimls (Figure 6). DISCUSSION PGE 2 is uiquitously present in nimls nd humns nd hs potent responses in numer of orgn systems. Dt in pre-clinicl niml models nd humns suggest tht PGE 2 plys crucil role in norml s well s pthologicl renl function The present study indictes tht CP-44, 519-2, selective gonist of the PGE 2 EP4 receptor, reduces nephrotoxic injury nd increses the survivl rte of nimls with ARF. Biochemicl serum prmeters indicte tht systemiclly dministered EP4 receptor gonist limits the extent of injury nd/or speeds up the recovery of injured tuulr cells. The sme trends of oth prmeters strongly indicte the eneficil effect of the EP4 receptor gonist in the HgCl 2 -cused kidney dmge. HgCl 2 exerts its toxic effects on kidney cells through vriety of mechnisms, with the principl trget eing the S3 segment of the proximl tuules. It interferes with respirtory chin nd oxidtive phosphoryltion enzymes nd cuses oxidtive injury with susequent lipid peroxidtion, DNA dmge, nd protein oxidtion. 29 The EP4 receptor gonist reduced the numer of poptotic cells in the kidney nd its cytoprotective ction ws responsile for the rpid regenertion of the renl Kidney Interntionl (26) 7,

6 originl rticle S Vukicevic et l.: PGE 2 EP2 nd EP4 receptor gonist in renl filure function. More thn 9% of rts survived in the EP4 receptor gonist-treted group, in comprison to 4% in the control group, indicting tht the EP4 receptor gonist reduced the severity of ARF nd, therefore, could potentilly e eneficil in treting ARF. As oth the EP2 nd EP2/4 receptor gonists hd no influence on serum iochemicl prmeters nd the EP2 receptor gonist did not improve the survivl of rts with ARF, we suggest tht the EP4 receptor is essentil for the prevention nd/or restortion of the kidney structure nd function in ARF. On the contrry, in model of CRF, ll three tested molecules were effective in preserving kidney function suggesting tht oth EP2 nd EP4 receptor gonism is cple of preventing progressive deteriortion of the remnnt glomerulr function in this rt model. CRF is ssocited with reduction of GFR, elevted serum Cr vlues, glomerulosclerosis, nd tuulointerstitil injury. These re reproduced in the rt remnnt kidney model of CRF, where the initil compenstory renl growth response to reduction of the prenchyme is followed in the long term y progressive renl scrring. Typiclly, the lte renl firotic chnges re ssocited with the progressive loss of glomerulr cells, tuulr trophy, nd their replcement y firous tissue. 3 EP receptors hve een shown to e expressed in vrious kidney cells including mesngil cells. 31 Recently, the role of the EP1 receptor in nephropthy using receptor-specific smll molecule inhiitors hs een demonstrted. 18 It ws suggested tht selective inhiition of the EP1 receptor inhiits glomerulr hypertrophy nd proteinuri, TGF-et nd fironectin trnscriptionl ctivtion, preventing the development of dietic renl injury in rts. 18 Here, we show in pre-clinicl models with impired renl function tht the expression of the EP2 nd the EP4 receptor is not significntly regulted t messge level. Interestingly, EP4 receptor gonist is more effective in ARF, wheres EP2, EP4, nd EP2/4 receptor gonism is oth effective in preventing the progression of the chronic renl filure. Thus, it is suggested tht oth EP2 nd EP4 receptors ply mjor role in regulting the kidney function. Following Nx, the expression of the EP4 receptor remins similr to shm rts, wheres the expression of the EP2 receptor is downregulted nd then upregulted t week 9 following therpy with ll three receptor gonists. Prostglndins nd in prticulr PGE 2 in the norml kidney physiology regulte vsculr smooth muscles, GFR, the relese of slt, wter re-sorption, nd the relese of renl hormones. 32 Our results indicte tht role for PGE 2 signling vi the EP2 nd EP4 receptor in kidney my e more criticl thn hs een recognized to dte. The oserved cytoprotection ginst glomerulr degenertion, the preservtion of proximl nd distl tuule structures, nd the presence of newly formed nephrogenic structures seen in EP2 nd EP4 receptor gonist-treted kidneys suggest tht PGE 2 signling vi the EP2 nd EP4 receptor my provide morphogenic signl for the repir nd regenertion of postntl renl tissue in oth the cute nd chronic renl filure. PGE 2 is, therefore, not involved only in regulting renl functions ut it lso hs n importnt role in the repir of dmged renl tissue or in protecting renl tissue ginst insult nd injury. As the process of the chronic kidney filure in humns occurs over yers, delying the progression is criticl for the tretment of chronic kidney diseses. Selective gonists of the EP4 nd/or the EP2 receptor, thus, my e used to improve renl rchitecture during the course of renl disese nd recover the lost renl function. MATERIALS AND METHODS Screening for PGE 2 selective gonists Compounds were ctegorized sed on their ility to ind vrious prostnoid receptors nd ll three molecules were selective gonists when mesured ginst other PGE 2 receptors such s prostglndin I (IP) nd prostglndin D (DP) (Prlkr et l., 33 dt not shown). EP2 nd EP4 receptor gonism ws further defined y the ility of compounds to increse intrcellulr camp levels in humn emryonic kidney-293 cells overexpressing either the EP2 or the EP4 receptor. camp ws quntitted using rdioimmunossy kit ccording to the mnufcturer s instructions (DuPont/NEN Reserch Products, Boston, MA, USA). HgCl 2 model of ARF Wistr mle rts of g were mintined in cclimtized cges on 12 h light drk cycle nd hd free ccess to stndrd lortory chow nd tp wter. The nephrotoxin (mercury chloride, HgCl 2, 2.5 mg/kg) ws dministered sucutneously (s.c.) in olus upon sl serum iochemistry vlues nd nimls were rndomly ssigned to one of seven groups: (1) HgCl 2 control (n ¼ 1); (2) HgCl 2 þ CP-43,35-2 (1 mg/kg, n ¼ 1); (3) HgCl 2 þ CP-43, (1 mg/kg, n ¼ 1); (4) HgCl 2 þ CP-44,519-2 (1 mg/kg, n ¼ 1); (5) HgCl 2 þ CP-44,519-2 (1 mg/kg, n ¼ 1); (6) HgCl 2 þ CP-536,745-1 (1 mg/kg, n ¼ 1); nd (7) HgCl 2 þ CP- 536,745-1 (1 mg/kg, n ¼ 1). The compound ws dministered 1 min fter HgCl 2 injection nd continued t 24-h intervls until termintion. nimls received the sme volume of vehicle uffer (et-c-dextrn) t the sme time points. Animl models were pproved y the Institutionl Scientific Bord. 34 5/6 nephrectomy model of CRF Mle Wistr rts weighing pproximtely 4 g were fed stndrd rt chow d liitum nd were given free ccess to wter. A totl of 36 nimls underwent 5/6 nephrectomy (5/6 Nx). 35 The study followed the previously pulished protocol. 36 Animls were sujected to unilterl 2/3 nephrectomy (left kidney) under ketmin/dizepm nesthesi (1 or 2.5 mg/kg, respectively). After 2 weeks, the right kidney ws surgiclly removed under nesthesi. 13 In the first experiment, 5/6 Nx rts were rndomly ssigned into five groups: (1) Shm operted (n ¼ 5); (2) Nx-control (n ¼ 12); (3) Nx þ CP- 536,745-1 (1 mg/kg, 1 /week; n ¼ 25); (4) Nx þ CP-43,35-2 (1 mg/kg, 1 /week; n ¼ 25); nd (5) Nx þ CP-44,519-2 (1 mg/ kg, 1 /week; n ¼ 25). At 2 weeks following the second opertion, CP-536,745-1, CP-43,35-2, or CP-44,519-2 were dissolved in et-c-dextrn nd dministered vi the rt til vein once week throughout the period of 11 weeks. nimls received only et-c-dextrn s vehicle. Animls were killed 11 weeks following Nx y overdose of sodium pentoritl. In the second experiment, 5/6 Nx rts were rndomly ssigned into three groups: (1) Nx- 114 Kidney Interntionl (26) 7,

7 S Vukicevic et l.: PGE 2 EP2 nd EP4 receptor gonist in renl filure originl rticle control (n ¼ 12); (2) Nx þ CP-44,519-2 (1 mg/kg, 1 /week; n ¼ 12); nd (3) Nx þ CP-44,519-2 (1 mg/kg, 3 /week; n ¼ 12). Two weeks following the second opertion, CP-44,519-2 ws dissolved in et-c-dextrn dministered vi the rt til vein once or three times week throughout the period of 11 weeks. nimls received only et-c-dextrn s vehicle. Animls were killed 11 weeks following Nx y overdose of sodium pentoritl. RNA isoltion nd gene expression nlysis y rel-time polymerse chin rection Totl RNA ws isolted from kidneys of shm-operted rts nd rts receiving CP-536,745-1, CP-43,35-22, or CP-44,519-2 t, 2, 5, 7, nd 9 weeks following 5/6 Nx nd reltive quntifiction of gene expression ws crried out s previously descried. 37,38 Betctin ws chosen s the most stle housekeeping gene for the CP- 43,35-2- nd CP-44,519-2-treted kidneys nd glycerldehyde- 3-phosphte dehydrogense for the CP-536,745-1 therpy. The following gene-specific primers were used: forwrd GTG CTG GTA ACG GAA CTG GT nd reverse CGT GGC CAG ACT AAA GAA GG for EP2; forwrd ACA CCA CCT CGC TGA GAA CT nd reverse GCT CCC ACT AAC CTC ATC CA for EP4; forwrd CAT CCT GAA CCG TCT GTG TG nd reverse TTT CCA CCA AGG ACC CAC TA for lumin; forwrd ATG ATT CTA CCC ACG GCA AG nd reverse CTG GAA GAT GGT GAT GGG TT for glycerldehyde-3-phosphte dehydrogense, nd forwrd GGG AAA TCG TGC GTG ACA TT nd reverse GCG GCA GTG GCC ATC TC for et-ctin. Renl function HgCl 2 model of ARF. Blood smples (.5 ml) were otined from the oritl venous plexus t, 24, 48, 72, nd 128 h fter HgCl 2 dministrtion. Serum cretinine ws mesured y the Jffe method (lkline picrte) nd lood ure nitrogen y enzymtic glutmte dehydrogense-uv procedure s previously descried in Vukicevic et l. 39 The cumultive survivl rte ws lso oserved nd recorded for oth control nd experimentl rts. Animls were killed 6 dys fter HgCl 2 tretment. 5/6 nephrectomy model of CRF. Blood smples nd 24-h urine collections in metolic cges were tken on weeks 2, 5, 8, nd 11. Serum nd urine cretinine ws mesured using the stndrd Jffè method. The GFR ws determined using serum cretinine over urine cretinine s djusted to ody weights. Histology HgCl 2 model of ARF. At termintion, kidneys were removed, cut longitudinlly nd one hlf of ech kidney ws fixed in 4% prformldehyde, emedded in prffin, nd 4 mm prffin sections were cut with microtome nd stined with periodic cid-schiff stin. Qulittive nd semiquntittive ssessments of the level of necrosis of proximl tuules nd other pthologicl chnges in kidney sections were mde lindly using light microscope. The level or severity of necrosis in proximl tuules ws rnked for ech niml on scle of 12, where represents no cellulr necrosis nd 12 represents level of necrosis tht involves virtully every proximl tuulr segment visile in the plne of section s previously descried. 4 5/6 nephrectomy model of CRF. Kidneys for histologicl exmintion were fixed in 2% prformldehide nd 7 mm prffin sections were cut nd stined with hemtoxylin nd eosin. Tuulointerstil injury, defined s tuulr dilttion nd/or trophy, interstitil firosis, nd inflmmtory cell infiltrte, s well s glomerulr dmge, were grded using semiquntittive scle from to 4 ccording to the following criteri: ¼ no chnges; 1 ¼ focl chnges involving 1 25% of the smple; 2 ¼ chnges ffecting 26 5% of the smple; 3 ¼ chnges involving 51 75% of the smple, nd 4 ¼ lesions ffecting more thn 75% of the smple. 41 Two independent oservers performed histologicl studies in linded fshion. Immunohistochemicl nlyses. Immunocytochemistry ws performed using the immunoperoxidse detection system (Zymed, Sn Frncisco, CA, USA). The following monoclonl ntiodies were used: PCNA (DAKO, Copenhgen, Denmrk), smooth muscle -ctin (DAKO), intrcellulr dhesion molecule (CD 54; DAKO), nd Mc (CD-68) (DAKO). A minimum of 3 cells ws counted per kidney section stined for PCNA nd smooth muscle -ctin, nd the numer of positive cells ws expressed s percentge of totl counted cells in sudivisions of cortex, nd/or the S3 zone. Apoptosis HgCl 2 model of ARF. Apoptotic cells were detected y TACS 2 TdT in situ poptosis system (Trevigen, Githersurg, MD, USA), which detects doule-strnd reks in genomic DNA y enzymtic incorportion of iotinylted nucleotide with terminl deoxynucleotidyl trnsferse followed y inding of streptvidin-horserdish peroxidse. The modifictions of the procedure include leling time of 9 min, coverslips in ll rections to prevent drying of smples, nd incution time of streptvidin HRP of 15 min. 42 Sttisticl nlysis All dt re presented s men7s.e.m. One-wy nlysis of vrince ws performed to determine the effect of tretment on iochemicl prmeters. Student t-test ws used to determine the effect of therpy on the gene expression level s compred to shm nimls. 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