Epiphyseal growth plate growth hormone receptor signaling is decreased in chronic kidney disease related growth retardation

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1 & 213 Interntionl Soiety of Nephrology Epiphysel growth plte growth hormone reeptor signling is deresed in hroni kidney disese relted growth retrdtion Ariel Troi 1, Dniel Lndu 2, Leonid Khko 3, Rlph Rkin 4,5 nd Yel Segev 1 1 Shrg Segl Deprtment of Miroiology nd Immunology, Fulty of Helth Sienes, Ben Gurion University of the Negev, Beer Shev, Isrel; 2 Deprtment of Peditris, Sorok University Medil Center, Beer Shev, Isrel; 3 Deprtment of Pthology, Sorok University Medil Center, Beer Shev, Isrel; 4 Reserh Servie, Veterns Affirs Helth Cre Plo Alto, Stnford, Cliforni, USA nd 5 Mediine Deprtment/Renl Division, Stnford University, Stnford, Cliforni, USA Liner growth retrdtion in hildren with hroni kidney disese (CKD) hs een sried to insensitivity to growth hormone. This resistne stte hs een ttriuted to impired growth hormone signling through the JAK2/STAT5 pthwy in liver nd skeletl musle leding to redued insulin-like growth ftor-i (IGF-I). Here we determine whether systemi nd growth plte ltertions in growth hormone signling ontriute to CKD-indued liner growth retrdtion using prtilly nephretomized nd pir-fed ontrol 2-dy-old rts. Serum growth hormone did not hnge in rts with CKD, yet serum IGF-I levels were deresed nd growth retrded. The tiil growth plte hypertrophi zone ws wider nd vsulriztion t the primry ossifition enter ws redued in CKD. This ws ssoited with derese in growth plte vsulr endothelil growth ftor (VEGF) mrna nd immunostinle VEGF nd IGF-I levels. Growth plte growth hormone reeptor nd STAT5 protein levels were unhnged, while JAK2 ws redued. Despite omprle growth hormone nd growth hormone reeptor levels in CKD nd ontrol rts, reltive STAT5 phosphoryltion ws signifintly depressed in CKD. Of note, the mrna of SOCS2, n inhiitor of growth hormone signling, ws inresed. Thus, liner growth impirment in CKD n in prt e explined y impired long one growth plte growth hormone reeptor signling through the JAK2/STAT5 pthwy, n normlity tht my e used y n inrese in SOCS2 expression. Kidney Interntionl (213) 84, ; doi:1.138/ki ; pulished online 29 My 213 KEYWORDS: hroni kidney disese; growth hormone; growth plte; IGF-I; reeptor; somtotropin; STAT5 Correspondene: Yel Segev, Shrg Segl Deprtment of Miroiology nd Immunology, Fulty of Helth Sienes, Ben Gurion University of the Negev, PO Box 15, Beer Shev 8415, Isrel. E-mil: yelse@gu..il This work hs een presented s n strt t the nnul meeting of the Amerin Soiety of Nephrology, Sn Diego, Novemer 212. Reeived 2 August 212; revised 2 Mrh 213; epted 21 Mrh 213; pulished online 29 My 213 Liner growth retrdtion is mjor prolem in hildren with hroni kidney disese (CKD). Nerly 4% of North Amerin hildren with CKD suffer from short stture. Even though growth filure orreltes with the degree of renl impirment, hildren with mild redution of glomerulr filtrtion rte my lso exhiit short stture. In ddition, growth filure in CKD is ssoited with omoridity. 1 Severl ftors ontriute to this growth retrdtion, inluding the ge t onset nd severity of CKD, the existene of onomitnt idosis, hypovolemi nd hypontremi, seondry hyperprthyroidism, deresed ppetite, nd resistne to noli hormones suh s growth hormone (GH) nd insulin-like growth ftor-i (IGF-I). 2 GH stimultes liner growth, nd inreses musle strength nd one density. 3 After inding to its reeptor (growth hormone reeptor (GHR)), severl signl trnsdution pthwys n e tivted, inluding the Jnus kinse 2 (JAK2) signl trnsduer nd tivtor of trnsription (STAT) pthwy in whih GH-indued JAK2 tivtion phosphoryltes STAT5, whih dimerizes nd trnslotes to the nuleus, to ind response elements tht regulte trnsription of GH trget genes, mong them IGF-I. 4 Although STAT1, -3, -5, nd -5 n ll e tivted y GH, STAT5 is the mjor trget for growth promotion euse it regultes IGF-1 expression. 4 In hildren with CKD nd growth retrdtion, serum GH levels re norml or even elevted, refleting stte of quired GH resistne. 5 Liner growth is the produt of n elorte sde of events tht tkes ple in the rtilginous growth enter of the long ones, the epiphysel growth plte. Longitudinl growth is medited y endohondrl ossifition in the growth plte re. It egins with the prolifertion of erly hondroytes, followed y their lignment in olumns, nd finlly their mturtion into hypertrophi hondroytes. 6 The hypertrophi hondroytes then die, nd s they do so, the trnsverse sept of rtilge mtrix surrounding them re roken down, llowing entry of the invding ells of the ossifition front: lood vessels, osteolsts, nd preursors of osteolsts nd one mrrow ells. 6 Vsulr endothelil 94 Kidney Interntionl (213) 84,

2 growth ftor (VEGF) is expressed in hypertrophi hondroytes nd is required for vsulr invsion to the primry ossifition enter. The sene of VEGF results in dely of vsulr invsion nd ossifition. 7 GH is n importnt regultor of longitudinl one growth. 8 Impired growth is seen in hildren with GH defiieny or geneti inility to respond to GH owing to muttions in its reeptor or omponents of its signling pthwy, inluding STAT5. 7 GH ffets one growth diretly, y inding to its reeptors in the growth plte, nd lso indiretly, vi IGF-I. GH stimultes mny ody tissues, espeilly the liver, ut lso the growth plte, to produe IGF-I, therey inresing irulting nd lol IGF-I levels. IGF-I serves s oth the min meditor of GH tion nd s GH-independent growth ftor. The min effet of GH on growth plte hondroytes is to stimulte their prolifertion 7,9 nd differentition (in sptildependent mnner 6 ) nd to protet ells from poptosis. Few studies hve previously speifilly investigted the one GHR signling pthwy in CKD. Rkin et l. exmined liver nd musle tissue of mture CKD rts nd showed tht CKD used postreeptor defet in GH signl trnsdution, whih is responsile for GH resistne, without ffeting GHR protein levels. 1,11 Our purpose ws to test the hypothesis tht there re hnges in the GH/IGF-I xis, oth in the growth plte nd systemilly, tht ould ontriute to the retrdtion of liner ody growth tht ours in CKD. To tht end, we hve studied young growing rts with surgilly indued CKD nd pir-fed shmoperted ontrols (C). RESULTS Kidney funtion ws modertely deresed in this sutotl nephretomy model, s refleted y 3-fold inrese in serum ure levels (Tle 1). Cretinine nd Ure lerne ws round 8-fold lower in the CKD group ompred with the C group (Tle 1). No hnges in serum phosphte or ironte levels were seen. Serum prthyroid hormone (PTH) ws mildly elevted nd plsm hemogloin ws mildly deresed in CKD (Tle 1). Serum GH did not Tle 1 Blood nd urine nlysis Prmeter Control CKD P-vlue Serum ure (mg/dl) 45.8± ±7 o.1 Serum retinine (mg/dl).36±.2.81±.4 o.1 Cretinine lerne (ml/min) 1658±12 19±23 o.1 Ure lerne (ml/min) 838±44 115±1 o.1 24-h urine volume (ml) 15.5±.7 2.5±.7 o.1 Plsm ironte (mmol/l) 24.7± ±1.5 NS Serum phosphte (mg/dl) 12.3± ±.6 NS TRP (%) 99.9± ±.14 o.5 Serum PTH (pg/ml) 195±35 385±38 o.5 Hemogloin (g/dl) 14.3± ±.4 o.1 Serum GH (ng/ml) 128±15 127±12 NS Serum IGF-I (ng/ml) 1138±83 662±57 o.1 Arevitions: CKD, hroni kidney disese; GH, growth hormone; IGF-I, insulin-like growth ftor-i; NS, not signifint; PTH, prthyroid hormone; TRP, totl resorption of phosphte. N ¼ 16 for eh group. hnge in CKD ompred with C, wheres serum IGF-I deresed signifintly in CKD vs. C (Tle 1). Similr endorine hnges were previously desried, 5 inluding reports in some humn studies. These results onfirm model of erly stge of CKD. Growth retrdtion in CKD Growth ws followed for 2 weeks sine the full indution of CKD. Body weight nd length, til length gin, nd tiil length ll deresed signifintly in the CKD group ompred with the C group (y 25±3, 42±4, 29±2 nd 15±1%, respetively). These hnges in growth prmeters were not the result of differenes in food onsumption, euse the nimls were pir-fed (Tle 2). Epiphysel growth plte width ws lrger in the CKD group ompred with the C group, ut there ws no hnge in the numer of ells per olumn etween groups (Tle 2). There were no hnges in prolifertive zone width etween the groups, wheres signifint inrese in the width of the hypertrophi zone ws seen (Tle 2). Aordingly, the rtios etween the widths of eh zone to the totl width of the growth plte showed derese in the frtion of the prolifertive zone nd n inrese in the frtion of the hypertrophi zone in CKD (Tle 2). In ddition, type X ollgen mrna, whih is undnt in the hypertrophi zone, ws inresed in CKD (Tle 2). Defets in growth plte GHR signl trnsdution Bovine GH ( mg/kg) or uffer ws injeted into the ven v 15 min efore killing the niml, reting four groups: ontrol þ uffer (C), ontrol þ GH (Cgh), CKD þ uffer (CKD), nd CKD þ GH (CKDgh). Tiil epiphysel growth plte GHR mrna levels were unhnged etween groups (1.2±.1-, 1.1±.3-, nd 1.3±.5-fold of C, in Cgh, CKD, nd CKDgh groups, respetively, p: NS) (Figure 1). Growth plte GHR protein levels were lso unhnged (88±11, Tle 2 Somti liner growth nd epiphysel growth plte prmeters C CKD P-vlue Somti growth Body weight gin (g) 8.9± ±2.3 o.1 Body length gin (m) 8.2±.3 4.8±.3 o.1 Til length gin (m) 5.1± ±.1 o.1 Tiil length (mm) 31.5± ±.4 o.1 Food onsumption/dy (g) 14.5± ±.4 NS EGP prmeters EGP width ± ±3.4 o.1 Prolifertive zone width ±3.5 15±4.2 NS Hypertrophi zone width ±2.2 14±6.2 o.1 Prolifertive/totl width rtio (%) 47.7± ±.9 o.1 Hypertrophi/totl width rtio (%) 45.5± ±1.1 o.1 Prolifertive/hypertrophi rtio (%) 14.8± ±1.1 o.1 Numer of ells/row 3.7± ±1.2 NS EGP type X ollgen mrna 1.±.1 3.7±.4 o.1 Arevitions: C, ontrol; CKD, hroni kidney disese; EGP, epiphysel growth plte; NS, not signifint. N ¼ 16 for eh group. Kidney Interntionl (213) 84,

3 EGP GHR mrna EGP GHR/Tuulin. C Cgh CKD CKDgh GHR Tuulin C Cgh CKD CKDgh C CKD NC P H μm μm Figure 1 Growth hormone reeptor (GHR) expression. () Epiphysel growth plte GH reeptor (GHR) mrna. n ¼ 12 per group. Results re presented s fold of the ontrol (C) group. () Growth plte GHR/tuulin protein rtio. GHR ws deteted in 12 kd, determintion y western lotting, n ¼ 6 per group. () GHR distriution in the growth plte tested y immunohistohemistry. Br ¼ mm. Groups with ommon letters re similr. CKD, hroni kidney disese; EGP, epiphysel growth plte; H, hypertrophi zone; NC, negtive C without primry ntiody; P, prolifertive zone. 15±21, nd 11±22% of C in CKD, CKDgh, nd Cgh groups, respetively, p: NS) (Figure 1). Immunohistohemistry deteted GHR in the resting nd prolifertive zones of the growth plte (Figure 1). No hnges in the distriution or intensity of GHR etween C nd CKD were seen. Growth plte totl JAK2 protein ws signifintly deresed in CKD nd CKDgh ompred with C nd Cgh groups (53±11 nd 51±1 vs. ±1% nd 131±23% of ontrol, respetively, Po.5) (Figure 2d). Phosphorylted JAK2 ws not detetle in ny group. The levels of growth plte totl STAT5 protein were unhnged when CKD nd CKDgh groups were ompred with C nd Cgh groups (127±2 nd 122±23 vs. ±13% nd 98±19% of C respetively, p: NS) (Figure 2). Bsl (uffer treted) levels of phosphorylted STAT5 were signifintly lower in the CKD group vs. C group (6±18 vs. ±8% of verge ontrol vlue; Po.5). After olus of ovine GH, p-stat5 levels rose signifintly in the Cgh group (Po.5), ut it filed to hnge signifintly in the CKDgh group (16±27 vs. 79±18% of C vlue, Figure 2). Similrly, reltive STAT5 phosphoryltion (rtio etween p-stat5 nd STAT5), mesure of effiieny of phosphoryltion, showed signifint derese in the CKD nd CKDgh groups ompred with the C group (42±13 nd 74±2 in CKD nd CKDgh groups, respetively, vs. 99.3±1.2 nd 167±34% of ontrol in C nd Cgh groups, respetively, Po.5) (Figure 2). Tken together, solute nd reltive STAT5 phosphoryltion is depressed in the sl CKD stte, despite serum GH levels eing similr to C vlues (Tle 1), nd fter GH dministrtion STAT5 phosphoryltion inreses signifintly in the C group ut not in the CKDgh group, providing strong evidene for the presene of GH-resistnt stte. In ontrst to hnges in STAT5 phosphoryltion, the p-stat3/ STAT3 rtio ws unhnged in CKD nd CKDgh groups ompred with C nd Cgh groups (148±3 nd 13±32 vs. 97.7±18 nd 128±25.3% of ontrol, p: NS) (Figure 2e). Growth plte suppressor of ytokine signling 2 mrna levels Growth plte suppressor of ytokine signling 2 (SOCS2) mrna levels were inresed signifintly in CKD nd CKDgh groups ompred with C nd Cgh groups (1.38±.14- nd 1.36±.1- vs. 1±.1- nd 1±.1-fold of ontrol, respetively, Po.5) (Figure 2f). Over the 15-min period of study, there ws no hnge in SOCS2 mrna levels in response to GH in either treted group. IGF-I expression in the growth plte IGF-I protein expression in the growth plte, s determined y immunohistohemistry, ws lower in CKD ompred with C in ll of the growth plte lyers (resting, prolifertive, nd hypertrophi), s well s in the primry ossifition enter (Figure 3). The numer of IGF-I-positive hypertrophi hondroytes ws muh lower in the CKD group ompred with the C group (8.6±1.6% vs. 2±4.7%, respetively, Po.1). In ontrst to IGF-I protein expression, IGF-I mrna expression ws not downregulted in CKD nimls. Furthermore, IGF-I mrna ws influened y pir feeding, s seen when ompring ontrol d li fed nimls with ontrol pir-fed nimls nd CKD nimls (2.3±.3-, 1±.1-, nd 1.1±.2-fold of ontrol, respetively, Po.1) (Figure 3). IGF-inding protein 2 (IGFBP2) mrna, one of the min one IGFBPs, ws signifintly deresed in the CKD group 942 Kidney Interntionl (213) 84,

4 EGP STAT5 protein 1 STAT5 d EGP JAK2 protein JAK2 EGP p-stat5 protein 1 p-stat5 e EGP p-stat3/stat3 2 1 STAT3 p-stat3 EGP p-stat5/stat5 2 1 f EGP SOCS2 mrna. C Cgh CKD CKDgh C Cgh CKD CKDgh Figure 2 Defets in Jnus kinse 2/signl trnsduer nd tivtor of trnsription (JAK/STAT) signl trnsdution. () Epiphysel growth plte STAT5 deteted in 94 kd, determintion y western lotting, n ¼ 7 per group. () Growth plte p-stat5 deteted in 91 kd, determintion y western lotting, n ¼ 7 per group. () Growth plte p-stat5/stat5 rtio, n ¼ 7 per group. (d) Growth plte totl JAK2 deteted in 12 kd, determintion y western lotting, n ¼ 6 per group. (e) Growth plte p-stat3/stat3 rtio, deteted in 8 kd, determintion y western lotting, n ¼ 9 per group. (f) Growth plte SOCS2 mrna, n ¼ 12 per group. Results re presented s fold of ontrol (C). Different letters ove rs indite signifint differene etween groups (Po.5); groups with ommon letters re similr. CKD, hroni kidney disese; EGP, epiphysel growth plte ompred with the C group (.5±.1- vs. 1±.1-fold of ontrol, respetively, Po.1) (Figure 3). Growth plte IGF1R nd its downstrem signling moleules IGF1R mrna levels in the growth plte were signifintly inresed in the CKD group ompred with the C group (2.5±.2- vs. 1±.1-fold of ontrol, respetively, Po.1) (Figure 4). Similrly, growth plte IGF1R protein (determined y western lotting) ws lso inresed in the CKD group ompred with the C group (194±2.6 vs. ±9.1% of ontrol, respetively, Po.5). No differene ws seen etween the CKDgh group nd the Cgh group (12.6±23.6 vs ±15.7% of ontrol, respetively, p: NS) (Figure 4). Phosphorylted IGF1R levels, tested using speifi ntiody previously shown y us to urtely detet this frtion in rodent tissue, 12 were undetetle. Insulin reeptor sustrte-1 (IRS-1) levels were unhnged in CKD nd CKDgh groups ompred with the C nd Cgh groups (14±12% nd 149±21% vs. ±12% nd 13 ±25% of C respetively, p: NS) (Figure 4). AKT protein is inresed signifintly in CKD nd CKDgh groups ompred with C nd Cgh groups (152.8±2.7% nd 181.5±23.4% vs. ±2.8% nd 83.8 ±4.4% of C, respetively, Po.5), s is phosphoryltion of the protein (184.9±22.3% nd 185.8±26.3% vs. ±12% nd 75.5±15.2% of C, respetively, Po.5). However, the rtio of p-akt/akt showed no hnges in the CKD nd CKDgh groups ompred with C nd Cgh groups (115±8% nd 122±14% vs. 94±11% nd 11±12% of C, respetively, p: NS) (Figure 4d). Growth plte vsulriztion Growth plte stining with Movt s modified penthrome 13 showed redued lood vessels nd lood ells in the primry ossifition enter in the CKD group ompred with the C group (Figure 5). VEGF is expressed in hypertrophi hondroytes nd is required for vsulr invsion to the primry ossifition enter. The sene of VEGF results in dely of vsulr invsion nd ossifition. 8 In the tiil Kidney Interntionl (213) 84,

5 C CKD NC P H POC EGP IGF-I mrna 3 EGP IGFBP2 mrna C fed d-li C pir-fed CKD Figure 3 Insulin-like growth ftor-i (IGF-I) expression in the epiphysel growth plte. () Immunohistohemil stining for IGF-I expression in the growth plte. P, prolifertive zone; H, hypertrophi zone; POC, primry ossifition enter. Blk rrows point to IGF-I-positivestined hypertrophi hondroytes. NC, negtive ontrol without primry ntiody. Br ¼ mm. () Growth plte IGF-I mrna, n ¼ 16 per group. Results re presented s fold of the ontrol (C) group. C fed d li (gry r), C nimls tht were llowed free ess to food; C pir-fed, C nimls pir-fed with hroni kidney disese (CKD). Different letters ove rs indite signifint differene etween groups (Po.5). () Growth plte IGFBP2 mrna, n ¼ 16 per group. Results re presented s fold of the C group. *Po.5 vs. C. Different letters ove rs indite signifint differene etween groups (Po.5); groups with ommon letters re similr.. C * CKD EGP IGF1R mrna C * CKD EGP IRS-1 protein 1 IRS-1 C Cgh CKD CKDgh 2 d 1 1 IGF1R AKT p-akt C Cgh CKD CKDgh EGP IGF1R protein EGP p-akt/akt C Cgh CKD CKDgh Figure 4 Insulin-like growth ftor-1 reeptor (IGF1R) expression nd signling in the epiphysel growth plte. () Growth plte IGF1R mrna, n ¼ 16 per group. Results re presented s fold of ontrol (C) group. *Po.5 vs. C. () Growth plte IGF1R deteted in 97 kd, determintion y western lotting, n ¼ 8 per group. () Growth plte IRS-1 deteted in kd, determintion y western lotting, n ¼ 8 per group. (d) Growth plte p-akt/akt rtio, n ¼ 8 per group. Different letters ove rs indite signifint differene etween groups (Po.5); groups with ommon letters re similr. CKD, hroni kidney disese; EGP, epiphysel growth plte. growth plte, immunohistohemil stining for VEGF showed mrked derese in CKD ompred with C oth in the hypertrophi zone nd the primry ossifition enter (Figure 5). The numer of VEGF-positive hondroytes out of the totl hypertrophi hondroytes ws lower in the CKD group ompred with the C group (7.8±.9% vs. 27±2.4% respetively, Po.1). Tiil growth plte VEGF mrna ws lso deresed in the CKD group ompred with the C group (.78±.1- vs. 1.2±.4-fold of ontrol, respetively, Po.5) (Figure 5). 944 Kidney Interntionl (213) 84,

6 C CKD H 1.5 POC.5 C CKD NC EGP VEGF mrna 1. *. C CKD H POC H Figure 5 Vsulriztion distl to the epiphysel growth plte. () Movt s penthrome stining of tiil growth plte nd primry ossifition enter (POC). Blk rrow points to derese in vsulriztion in the POC. Br ¼ mm. () Immunohistohemil stining for vsulr endothelil growth ftor (VEGF) in the growth plte. Upper figures, mgnifition; lower figures, mgnifition; CKD, hroni kidney disese; H, hypertrophi zone; NC, negtive ontrol (C) without primry ntiody. Br ¼ mm. () Growth plte VEGF mrna, n ¼ 14 per group. Results re presented s fold of C. *Po.5 vs. C. EGP, epiphysel growth plte. DISCUSSION Anormlities of the GH/IGF-I xis re one of the mjor determinnts of growth retrdtion in hildren with hroni illnesses suh s CKD, inflmmtory owel diseses, nd juvenile idiopthi rthritis. 14 Growth impirment in CKD hildren hs een explined in the pst minly y redued IGF-I iovilility, in prt owing to impired GH-medited signl trnsdution, s shown in liver nd skeletl musle of rodents with CKD. 1,11,15 However, it is not known whether suh defet exists in the growth plte of long one, the site of liner growth. In this study, we desrie novel findings tht provide insight into the pthomehnisms operting t the level of long one growth plte tht proly ontriutes to the liner growth retrdtion of CKD. Our model shows n erly stge of nonidoti CKD in immture rts with n B2-fold inrese in serum retinine, mild hyperprthyroidism, impired liner ody growth, nd ttenuted GH-stimulted signling through the STAT5 pthwy (Tle 1 nd Figure 3). In the urrent study, growth impirment ws seen shortly (within 2 weeks) fter CKD indution (Tle 2), irrespetive of food onsumption, s ontrol rts were pir-fed ording to the CKD group level of food intke. Liner growth impirment ws ssoited with ounterintuitive widening of the growth plte, mostly t the level of the hypertrophi zone, s previously desried. 16 Systemi GH IGF-I nlysis showed pttern of GH resistne in the CKD rts (norml serum GH ut deresed irulting totl IGF-I) (Tle 1). A similr serum pttern hs een previously desried in some (ut not ll) linil studies of hildren with end-stge renl disese. 5,15,17,18 Previous studies suggested derese in GHR undne in the growth plte in CKD rts. 19 We now show for the first time tht resistne to GH in growing one rises in CKD euse of growth plte postreeptor defet in signl trnsdution. In our study, immunoretive GHR ws deteted in the prolifertive zone, ut ws unhnged etween CKD nd C, oth t the mrna nd protein levels (Figures 1 ). Edmondson et l. 19 used immunohistohemistry nd found modest derese in GHR in CKD, onfined to the growth plte s prolifertive zone only. There re some differenes in the studies design (older nimls, femle vs. mle, lthough pir feeding ws performed in oth studies) tht my prtly explin the disrepny etween our results. In the present study, despite unhnged GHR levels, GH-stimulted tivtion of STAT5, s judged y the level of reltive STAT5 phosphoryltion (p-stat5/stat5), ws signifintly redued in CKD (Figure 2). Furthermore, totl JAK2 protein levels were signifintly lower (Figure 2d). STAT3 phosphoryltion ws unhnged (Figure 2e), whih suggests tht the impirment my e speifi to the JAK2/ STAT5 xis. In our study, growth plte p-stat5 ws upregulted in ontrol rts dministered GH, ut not so in CKD nimls (Figure 2). Moreover, s the mount of totl STAT5 protein ws unhnged in CKD (Figure 2) nd the sl serum GH ws similr etween the groups, the phosphoryltion effiieny in CKD nd CKDgh is muh lower. In ddition to the systemi redued IGF-I, lol protein expression of IGF-I in the growth plte ws redued, Kidney Interntionl (213) 84,

7 wheres IGF-I mrna ws unhnged (Figure 3 nd ). Tken together, our dt suggest derese in the tion of GH on one during CKD, whih is medited through the JAK2/STAT5 pthwy leding to deresed lol IGF-I. On refletion, our study is onsistent with the swith from the originl somtomedin hypothesis, impliting irulting GHinduing liver IGF-I to promote one growth to n lterntive hypothesis termed the dul effetor theory, 2 where GH ts diretly on germinl zone preursors of the growth plte nd stimultes the differentition of hondroytes nd the mplifition of lol IGF-I prodution. This lolly produed IGF-I, in turn, stimultes hondroyte lonl expnsion, hypertrophy, nd one growth in n utorine/prrine mnner. Although liverderived IGF-I is the min determinnt of systemi IGF-I levels, it ppers tht lolly derived IGF-I is proly more importnt for postntl growth. 21 Indeed, it is likely tht GH nd IGF-I hve oth dul nd overlpping funtions on hondroytes, s oth GHRndIGF-Imutntmieshowreduedgrowth,whihis more severe in doule GHR/IGF-I mutnts. 22 It is not ler whether GH medites ny IGF-I-independent effets on hondroytes, euse studies to dte hve lrgely een inonlusive. 23,24 Findings in experimentl nimls nd in humns hve shown tht the sene or muttions of the STAT5 gene re ssoited with diminished postntl growth, GH resistne, nd redued IGF-I synthesis. 8,25 Short stture in STAT5 knokout mie ws ssoited with defetive endohondrl ossifition proess. 26 STAT5 is the mjor meditor of growth y GH nd is essentil for GH-regulted IGF-1 gene expression. 27 Mny other ftors esides GH nd IGF-I ontriute to the rte of one growth through endohondrl ossifition, inluding the ontrol of hondroyte ell volume nd prolifertion rtes. These meditors inlude, mong others, the following: PTHrP, Ihh, Sox9, one morphogeneti proteins, nd firolst growth ftor. 28 Although uremi hyperprthyroidism with osteodystrophy is known seondry use of short stture in hildren with CKD nd ould oneivly ontriute to the GH-resistnt stte, our literture serh reveled no previous pulitions tht ddress the effets of hyperprthyroidism on GHR signling. When studying rts with CKD nd dvned seondry hyperprthyroidism, Snhez et l. 29 reported derese in growth plte PTH/ PTHrP reeptors, whih ould e normlized with dily GH injetions. However, in tht study, no growth retrdtion ws eliited, despite higher degrees of seondry hyperprthyroidism in omprison with our results (PTH levels 6.5- fold higher thn in ontrols vs. 2-fold in urrent study). Cortiosteroids tht inhiit liner growth fter long-term dministrtion, suh s in renl disese nd kidney trnsplnt mngement, do so y inhiiting IGF-I trnsription nd thus lol IGF-1 prodution in growth plte hondroytes nd osteolsts. 3 Nutrition lso hs n importnt influene on GH responsiveness. For exmple, potssium-defiient rts re GH-insensitive, nd the lk of response to GH dministrtion is ssoited with depressed growth plte IGF-I mrna expression. 31 Clorie intke is lso importnt, euse in hroni norexi sttes or fter prolonged fsting or food restrition skeletl growth is impired, insensitivity to GH is pprent, nd in mie growth plte IGF-I mrna nd protein expression is ttenuted. 25 Indeed, in the urrent study, growth plte IGF-I mrna expression ws redued in the foodrestrited pir-fed norml ontrol rts ompred with d li fed norml rts (Figure 3). This indites tht the suppressed growth plte IGF-1 expression in CKD is in prt seondry to norexi nd redued nutrient intke. In CKD, mjor use of ttenuted JAK/STAT signling seems to e the inrese in tissue SOCS2 expression, whih is the hief negtive feedk inhiitor of GHR tion. GHtivted STAT5 inds the promoter of SOCS2 to indue its expression. In turn, SOCS2 inds t lest two phosphorylted tyrosines on the GHR to negtively regulte JAK2 nd STAT5 tivtion. 32 SOCS2 is known to hve role in liner growth: SOCS2( / ) mie show inresed longitudinl skeletl growth ssoited with deregulted GH/IGF-1 signling. 32,33 In our study, growth plte SOCS2 mrna levels were elevted in immture rts with growth retrdtion nd CKD (Figure 2f). Similr oservtions hve een reported for skeletl musle 1,34 nd liver 11 of hroni uremi rts nd musle of uremi mie. 35 There ould e severl mehnisms for suh upregultion of SOCS2 in CKD. One suh mehnism likely opertive in CKD is rised levels of inflmmtory ytokines, tht indue SOCS2 expression nd tht then in-turn, feedks nd negtively regultes the signling of GH 36 nd the very ytokines tht stimulte the SOCS2 expression. 25,33 As inflmmtion is ommon in CKD, this my ount in lrge prt for the inhiition of GH signling nd, onsequently, growth retrdtion. 25 Although the min fous of this study ws on the signling response to GH, we hve exmined some other key omponents of the GH/IGF-1 xis in the growth plte. Interestingly, lol expression of IGF1R mrna nd protein ws inresed in CKD (Figure 4 nd ), suggesting ompenstory mehnism for the low vilility of lol nd irulting IGF-I, whih ws redued in the CKD nimls. 4 Although IRS-1 protein levels were unhnged, AKT totl protein nd phosphoprotein levels were lso inresed signifintly (Figure 4d), perhps refleting retion to the ompenstory IGF1R response. As the reltive phosphoryltion (p-akt/akt rtio) is similr to the ontrol nd GHtreted ontrol vlues (Figure 4d), the inrese does not pper to e due to n inrese in phosphoryltion effiieny ut rther n inrese in the mount of sustrte, nmely AKT protein, ville to e phosphorylted. Previous studies showed defet in the IRS-1/AKT signling pthwy in skeletl musle of CKD rts with idosis, whih ws reversed with idosis orretion. 41 It is importnt to note tht our model ws designed to test only the GHR signling pthwy, y providing single olus of GH 15 min efore euthnsi, nd not IGF1R signling, s it tkes severl hours for IGF-1 protein levels to inrese fter GH tretment. 42 In CKD, the iotivity of IGFs is redued owing to exess mounts of irulting high-ffinity IGFBPs tht derese or 946 Kidney Interntionl (213) 84,

8 prevent IGF inding to its signling reeptor. 4 Even though systemi IGFBP levels were not tested, our results showed redued lol IGFBP2 mrna expression in CKD (Figure 3). Most studies indite tht IGFBP2 ts s negtive ftor in skeletl tissue, s IGFBP2 inhiits IGF-medited prolifertion nd mtrix synthesis in growth plte hondroytes nd osteolsts in vitro. Furthermore, systemi overexpression of IGFBP2 in trnsgeni mie redues postntl weight gin nd dult long one size. 43 On the other hnd, studies lso suggest tht IGFBP2 my t s n noli ftor, prtiulrly y inresing IGF-II iovilility. Indeed, systemi dministrtion of n IGFBP2/IGF-II omplex stimultes one formtion in rts. 43 Animls with CKD nd suffering from impired growth were expeted to hve nrrower growth plte, s seen in other models of growth retrdtion, suh s food restrition nd th-up growth. 6 However, we found tht the totl growth plte width nd, in prtiulr, the hypertrophi zone were wider in CKD ompred with ontrol nimls (Tle 2). These findings re onsistent with tht of others. 16,44 Furthermore, Snhez et l. 45, treting young helthy rts with rpmyin ( lssi mmmlin trget of rpmyin inhiitor) for 2 weeks, found tht the nimls were growthrrested nd their hypertrophi zone nd totl growth plte width were wider. In tht study, redution in the numer of proliferting hondroytes, lower rtilge resorption (refleted y deline in trtrte-resistnt id phosphte expression), nd dely in vsulr invsion were seen. 45 Interestingly, the mmmlin trget of rpmyin signling pthwy is lso tivted y IGF-1-reeptor signls. 12 Our study ws not designed to explore the lol effets of GH suh s promotion of hondroyte prolifertion nd mturtion nd stimultion of IGF-1 synthesis, s for these tions prolonged exposure to GH is required. In this regrd, Mk nd Pk 46 studying epiphysel hondroytes isolted from rts with hroni renl filure desried resistne to oth GH nd IGF-I. Nevertheless, it ppers tht when GH is dministered in high doses in vivo, resistne to GH n e overridden nd growth n e improved. In ddition, niml in vivo studies 19 reported on the enefiil roles of exogenous high-dose GH for the improvement of CKD-relted growth retrdtion. The oservtion in our study of inresed hypertrophi zone width without hnge in the growth plte ell numer suggests the possiility of n rrest in the hondroyte mturtion proess distl to tht stge, whih requires the reruitment of osteolsts through vsulr invsion. VEGF is minly expressed in hypertrophi hondroytes 7 nd promotes this vsulriztion proess. In our model, some evidene for deresed vsulriztion ould e seen from the Movt s penthrome stin (Figure 5). In ddition, growth plte VEGF mrna nd protein expression deresed in CKD (Figures 5 nd ). Interestingly, onnetion etween GH signling nd vsulriztion hs een previously reported in retin development, dieti retinopthy nd nephropthy, rheumtoid rthritis, nd tumor development. 46,47 We therefore offer for onsidertion the possiility tht the widened growth plte together with dereses in VEGF expression, growth plte vsulriztion, nd IGF-I expression re n interrelted onsequene of impired GH-medited signling through the JAK/STAT5 pthwy. However, to prove this postulte, further studies re required. In onlusion, in this study, we hve provided evidene tht supports our thesis tht hnges in the GH/IGF-I xis oth in the growth plte nd systemilly ontriute to the impired liner ody growth tht ours in CKD. In prtiulr, we hve unovered lol defet in the GH-stimulted JAK/STAT5 signling pthwy in the growth plte tht my led to the ttenuted growth of CKD. As the lol expression of SOCS2 ws elevted, we suggest tht this my provide one mehnism ounting for the signling defet. Although extrpoltion from nimls to humns must e rried out with ution, suh defet my well ontriute to the impired growth of hildren with CKD. MATERIALS AND METHODS Animl experimenttion Twenty-dy-old mle Sprgue Dwley rts were used in the experiment (Hrln Lortories, Jeruslem, Isrel). Animl reeding omplied with the NIH Guide for the Cre nd Use of Lortory Animls. The lol institutionl review ommittee pproved the study protool. Animls were housed in stndrd lortory ges provided with norml rt how (ontining.8% phosphte, 3.1 kl/g) (Hrln Lortories) with free ess to n unlimited mount of tp wter. The nimls where 5/6 nephretomized in two stges. 48 Rts were nesthetized using 8 mg/kg of ketmine nd 16 mg/kg of xylzine. First, uninephretomy ws performed on the right kidney of CKD rts, nd shm opertion ws performed in the ontrol group. Two dys lter, 2/3 of the remining left kidney ws removed from the CKD group or shm opertion ws performed in the ontrol group. Three dys lter, pir feeding egn, providing the ontrol group the sme mount of food onsumed y CKD nimls. An dditionl ontrol group ws fed d li, nd it is presented in Figure 3. All other results mentioning the ontrol group (C) refer only to pir-fed nimls. Weight nd food onsumption mesurements were reorded dily. Urine ws olleted for 24 h efore euthnsi, using metoli ges. There ws 3-dy differene etween urine olletion nd euthnsi. Euthnsi ws performed in the fed stte 2 weeks fter strting pir-feeding onditions. Under ketmine/xylzine nesthesi, n injetion of ovine GH ( mg/kg) (Monsnto, St Louis, MO), dissolved in uffer ironte or uffer lone, ws dministered vi the inferior ven v 15 min efore euthnsi s follows: ontrol þ uffer (C), ontrol þ GH (Cgh), CKD þ uffer (CKD), nd CKD þ GH (CKDgh). The dose of GH used in this experiment is sed on preliminry dose response studies, where doses etween 25 nd 1 mg/kg were used nd liver p-stat5/stat5 levels were heked. Aording to those experiments, mg/kg/dose ws found to exert lredy mximl STAT5 phosphoryltion, nd higher doses did not inrese this effet. This dose is muh lower thn the 5-mg/kg/dy dose used to promote growth. At the time of killing, nose-to-til tip (ody length), nus-to-til tip (til length), nd knee-to-heel (tiil length) distnes were mesured in nesthetized nimls under omplete musle relxtion. Blood ws drwn from the ven v t the time of killing. Serum ws seprted nd frozen t 8 1C. Kidney Interntionl (213) 84,

9 The right tiil growth plte ws removed nd frozen t 8 1C for western lot nlysis or rel-time PCR, wheres the left tii length ws mesured nd then fixed in 4% formlin for histologil nlysis. Immunossys Blood nd urine smples were olleted nd frozen t 8 1C until nlysis. Serum IGF-I nd GH onentrtions were mesured using enzyme-linked immunosorent ssy kits (DSL-1-29 nd -1-72, respetively, Dignosti Systems L., Bre, CA) ording to the mnufturer s instrution. Serum nd urine iohemistries were nlyzed y the Biohemistry lortory of Sorok Medil Center (Beer Shev, Isrel). Totl resorption of phosphte ws lulted s follows: 1 (urine phosphte serum retinine)/ urine retinine serum phosphte). Histology Five-miron-thik longitudinl setions were ut nd olleted on superfrost-plus slides for histology using modifition of Movt s Penthrome stining (lue to lk ¼ nulei, red ¼ ytoplsm, red to lil ¼ elsti fiers, yellow ¼ ollgen nd minerlized one mtrix, lue green ¼ osteoid or minerlized rtilge, nd light lue ¼ rtilge) 13 for morphometry nlysis. Totl growth plte width ws mesured s the distne from the one mtrix in the reserve zone to one mtrix in etween the zone of lifying rtilge nd developing treule of the metphysis. Prolifertive zone width ws tken s the distne from the one mtrix in the reserve zone to the distl order of the lst line of prolifertive ells efore ler hypertrophi zone hnge. Hypertrophi zone width ws tken s the distne from this line to the mture edge of the growth plte. The widths of totl growth plte, prolifertive nd hypertrophi zones, nd growth plte numer of ells per olumn were mesured. Two setions per niml t five different fields per setion were nlyzed. This mesurement ws reorded y two different oservers, nd the results re shown s the verge of mesurements. Immunohistohemistry The following ntiodies were used in this study: mouse nti-rt VEGF (Snt Cruz Biotehnology, Dlls, TX), mouse nti-rt GHR (Am, Cmridge, UK), nd rit nti-rt IGF-I (GroPep Bioregents, Therton, SA, Austrli). For VEGF, GHR, nd IGF-I immunohistohemistry, deprffinized, rehydrted setions were treted with 3% H 2 O 2 for 15 min t room temperture to lok endogenous peroxidse tivity. The setions were wshed with PBS efore loking them with 2.5% norml horse serum (Vetor Lortories, Burlingme, CA) for 1 h, followed y overnight inution in primry ntiody diluted in the loking serum VEGF nd GHR 1: nd IGF-I 1:75. After inution in primry ntiody, the setions were wshed in PBS. Next, they were inuted in ppropritely diluted iotinylted seondry ntiody for 1 min, wshed with PBS, followed y inution in streptvidin peroxidse for 1 min, nd then gin wshed in PBS. Next, the setions were inuted with uffered sustrte solution (ph 7.5) ontining hydrogen peroxide nd 3,3-diminoenzidine hromogen solution (Vetor Lortories). The setions were then dehydrted nd mounted with permount nd exmined y light mirosopy. For imge proessing, the Cellsense Entry softwre (MATIMOP, Tel Aviv, Isrel) ws used. For IGF-I- nd VEGF-positive ell mesurement in the hypertrophi zone, the numer of positive hondroytes, Tle 3 Primers used for rel-time PCR Primer Forwrd sequene Reverse sequene GHR 5 -ATCTTTGGCGGGTGTTCTTA-3 5 -TAGCTGGTGTAGCCCCACTT-3 SOCS2 5 -TTAAAAGAGGCGCCAGAAGGA-3 5 -GCCCGGCTGATGTCTTAACA-3 VEGF 5 -CAGAAACACGACAAACCCATCC-3 5 -TAAGCCACTCACACACACAGCC-3 Type X ollgen 5 -CCCAGGGTTACCAGGACCAA-3 5 -GTTCACCTCTTGGACCTGCC-3 IGFBP2 5 -AAGGCGCATGGTGGAGAT-3 5 -CCCTAAGTCAGGCATGAAGG-3 IGF1R 5 -ATTGCCTCGGAGTTGGGAGAA-3 5 -AGCTGCTGCAAGTTCTGGTT-3 -Atin 5 -CCCGCGAGTACAACCTTCT-3 5 -CGTCATCCATGGCGAACT-3 Arevitions: GHR, growth hormone reeptor; IGFBP2, IGF-inding protein 2; IGF1R, insulin-like growth ftor-1 reeptor; SOCS2, suppressor of ytokine signling 2; VEGF, vsulr endothelil growth ftor. indited y drk-rown peroxidse retion, were ounted y linded oserver. One-hundred ells were ounted in four different mirosopi fields per growth plte. Dt were expressed s the perentge of ells tht were IGF-I nd VEGF positive per totl numer of ells ounted. All the experiments nd oservtions were repeted t lest four times. Western immunolot nlysis The following ntiodies were used for evlution of the growth plte extrts: JAK2 nd p-jak2 (Tyr 7/Tyr 8) (Merk- Millipore, Drmstdt, Germny); STAT5, GHR, IRS-1, IGF1R, p-akt(thr 38), nd tuulin (Snt Cruz Biotehnology); p-igf1r (Tyr1131) p-stat5 (Tyr 694), p-stat3 (Tyr 75), nd STAT3 (Cell signling Tehnology, Dnvers, MA); nd -tin (MP Biomedil, Solon, OH). The full protool is previously desried in our previous pulition. 12 RNA extrtion nd rel-time PCR Totl RNA ws extrted from growth plte using the PerfetPure RNA Tissue kit (Gentr Systems, Minnepolis, MN), nd DNAs were synthesized using high-pity DNA reverse trnsription kit (Applied Biosystems, Foster City, CA) s previously desried. 12 Quntittive rel-time PCR ssys were performed with power SYBR green PCR mster mix (Applied Biosystems) s previously desried 12 using the ABI Prism 73 Sequene Detetion System (Applied Biosystems). Primers for quntifition of GHR, IGF-I, SOCS2, VEGF, type X, IGFBP2, IGF1R, ollgen, nd -tin (Sigm-Aldrih, Rehovot, Isrel) re summrized in Tle 3. Eh smple ws nlyzed in triplite in individul ssys. The speifiity of the retion is given y the detetion of the melting tempertures (Tms) of the mplifition produts immeditely fter the lst retion yle. The trget gene expression vlue ws lulted y the DDt method fter normliztion with housekeeping gene (-tin). Sttistil nlysis Two-tiled unpired Student s t-tests were pplied for omprison of two normlly distriuted groups. Comprisons etween more thn two normlly distriuted groups were mde y one-wy nlysis of vrine. P-vlue o.5 ws onsidered signifint. DISCLOSURE All the uthors delred no ompeting interests. ACKNOWLEDGMENTS This study ws supported y Grnt-in-id of the US-Isrel Bintionl Siene Foundtion (no. 767) to DL, RR, nd YS, the Isreli Ministry of Helth Chief Sientist (no ) to YS nd DL, nd Reserh Servie, Veterns Affirs Helth Cre Plo Alto to RR. 948 Kidney Interntionl (213) 84,

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