Rho kinase inhibition protects kidneys from diabetic nephropathy without reducing blood pressure

Transcription

1 originl rticle & 211 Interntionl Society of Nephrology Rho kinse inhiition protects kidneys from dietic nephropthy without reducing lood pressure Rdko Komers 1, Terry T. Oym 1, Dougls R. Berd 2, Chris Tikellis 3, Bei Xu 1, Dniel F. Lotspeich 1 nd Shron Anderson 1,2 1 Division of Nephrology nd Hypertension, Oregon Helth & Science University, Portlnd, Oregon, USA; 2 Portlnd VA Medicl Center, Portlnd, Oregon, USA nd 3 Juvenile Dietes Reserch Foundtion/Dnielle Alerti Memoril Centre for Dietes Complictions, Bker Medicl Reserch Institute, Melourne, Austrli Rho-ssocited kinses (ROCK) re ctivted in the kidney s well s in cultured cells of dietic models nd hve een implicted in renl pthophysiology. To explore whether inhiition of ROCK is protective, we studied its role in model of ccelerted dietic nephropthy where uninephrectomized rts were mde dietic y streptozotocin. After estlishing dietes, rts were treted with the ROCK inhiitor fsudil continuously or for the finl 6 weeks of n 18-week experimentl period. The results were compred to similr rts given losrtn, n estlished tretment of clinicl nd experimentl dietic nephropthy, or comintion of oth gents. Vehicle-treted dietic nd non-dietic uninephrectomized rts served s controls. Dietes resulted in rpid development of luminuri, higher glomerulosclerosis nd interstitil firosis scores, lower glomerulr filtrtion rtes, nd incresed expression of severl moleculr mrkers of dietic nephropthy. Eighteen weeks of fsudil tretment reduced renl ROCK ctivity, nd meliorted dietes-induced structurl chnges in the kidney nd expression of the moleculr mrkers in ssocition with modest nti-proteinuric effect ut no chnge in lood pressure. Lte intervention with fsudil reduced glomerulosclerosis, ut did not influence proteinuri. Most effects of fsudil were comprle to those of losrtn, lthough losrtn lowered lood pressure nd further lowered proteinuri. The comintion of oth tretments ws no different thn losrtn lone. Thus, ROCK inhiition protected the kidney from dietic nephropthy even though it did not reduce the lood pressure. Kidney Interntionl (211) 79, ; doi:1.138/ki ; pulished online 2 Octoer 21 KEYWORDS: connective tissue growth fctor; dietic nephropthy; fsudil; glomerulosclerosis; Rho-ssocited kinses (ROCK); trnsforming growth fctor- Correspondence: Rdko Komers, Division of Nephrology nd Hypertension, Oregon Helth & Science University, PP262, 3314 SW US Veterns Hospitl Rod, Portlnd, Oregon, , USA. E-mil: komersr@ohsu.edu Received 14 Jnury 21; revised 6 August 21; ccepted 7 Septemer 21; pulished online 2 Octoer 21 Despite progress in the prevention nd tretment of dietic nephropthy (DN), reversl nd even stiliztion of the progressive course of DN re still difficult to chieve, nd mny ptients still progress to end-stge renl disese. New pproches tht would roden the spectrum of ville tretments for DN re needed to improve prognosis in these ptients. RhoA, memer of the Rs superfmily of smll GTP-inding proteins, nd its downstrem effectors Rho-ssocited kinses (ROCKs), re signling molecules implicted in vriety of iologicl functions, including cell contrction, cell migrtion, cell dhesion, cell cycle progression, nd gene expression. 1 The RhoA/ROCK pthwy is stimulted y gonists cting vi G-protein-coupled, tyrosine kinse, nd cytokine receptors, cell dhesion nd integrin clustering, s well s y mechnicl stress, which regulte the ctivity of RhoA gunine nucleotide exchnge fctors nd GTP loding of RhoA. 1 RhoA/ROCK hve emerged s importnt plyers in crdiovsculr nd renl pthophysiology. This pthwy is ctivted in the vsculture nd kidney in different models of hypertension, hypertensive end-orgn dmge, nd kidney disese, 2 7 nd studies with ROCK inhiitors (ROCKi), such s fsudil or Y27632, indicte protective renl ctions of these compounds. 4 1 Importntly, severl clinicl studies hve lso documented eneficil effects of ROCKi in ptients with crdiovsculr disorders. 11,12 RhoA/ROCK re ctivted in the kidney nd crdiovsculr system in models of dietes oth in vitro nd in vivo. 6,7,13 16 The pthwy converges numerous pthophysiologicl signls triggered y the dietic milieu, nd medites processes implicted in the pthophysiology of nephropthy, such s upregultion of prosclerotic cytokines, PAI-1 (plsminogen ctivtor inhiitor 1), osteopontin, nd production of extrcellulr/mesngil mtrix (ECM). 6,7,13 17 Bsed on this evidence, the RhoA/ROCK pthwy ppers to e promising trget for phrmcologicl intervention to prevent the development nd progression of nephropthy. To ddress this issue, we explored the nephroprotective potentil nd mechnisms of ction of the ROCKi fsudil in uninephrectomized dietic rts with n ccelerted course 432 Kidney Interntionl (211) 79,

2 R Komers et l.: ROCK inhiition in dietic nephropthy originl rticle of nephropthy, 18 including the effects lter in the course of this disorder. The effects of fsudil were compred with those of the ngiotensin receptor locker (ARB) losrtn, n estlished tretment for oth clinicl nd experimentl DN, nd to comintion of oth gents. RESULTS Physicl nd metolic prmeters As summrized in Tle 1, ll dietic groups demonstrted lower ody weight gins. The right kidney weight ws incresed in dietic nimls, except the group of dietic rts treted with fsudil (; fsudil from Cliochem, Sn Diego, CA), nd the kidney/ody weight rtios were significntly higher in ll groups of dietic rts when compred with non-dietic controls. Dietic rts hd hyperglycemi nd corresponding increses in HBA1c (glycosylted hemogloin) when compred with nondietic rts (Po.1). The metolic prmeters were not influenced y tretment. Blood pressure nd renl functionl prmeters At seline, there were no differences in systolic lood pressure mong the groups of rts (Tle 2). Systolic lood pressure did not significntly chnge throughout the study in control rts receiving vehicle () nd dietic rts receiving vehicle (), nd in rts. In contrst, rts treted with losrtn oth lone or in comintion with fsudil demonstrted lower systolic lood pressure t ll time points during follow-up, with significnt difference t weeks 12 nd 18 when compred with vehicle-treted rts, nd dietic rts receiving fsudil s lte tretment (lte) t weeks 6 nd 12. As shown in Figure 1, 24-h urinry lumin excretion (U l V) ws incresed in rts when compred with rts t weeks 6 nd 12. Tretment with fsudil ws ssocited with significnt ntiluminuric effect t weeks 6 nd 12, similr to the effects of losrtn nd the comintion of oth gents. However, t the end of the study, significnt reduction in U l V ws oserved only in dietic rts treted with losrtn (; losrtn from Merck, Whitehouse Sttion, NJ). Moreover, U l V ws lower in when compred with rts. The course of U l V in lte ws similr to tht in ; U l V ws higher thn in throughout the study, nd lso higher thn losrtn-treted nimls t weeks 12 nd 18 (Po.5). Compred with non-dietic controls, rts demonstrted lower glomerulr filtrtion rte (GFR), determined s cretinine clernce t week 18 (Figure 2). This decrese in cretinine clernce ws not oserved in dietic groups treted with fsudil, losrtn, or lte fsudil; ll demonstrted significntly higher cretinine clernce vlues thn those oserved in. Histologicl nlysis The glomerulr sclerosis score nd proportion of severely ffected glomeruli were incresed in compred with non-dietic controls (Figure 3). In ll fsudil- nd losrtn-treted groups, the glomerulr sclerosis score nd proportion of severely ffected glomeruli were not different from controls, nd significntly lower when compred with rts. The tuulointerstitil firosis score ws significntly higher in when compred with controls, nd reduced to vlues oserved in y tretment with fsudil, losrtn, or the comintion (Figure 3). Unlike the other groups of rts, in which the tretment ws initited t the onset of dietes, the reduction in tuulointerstitil firosis score in lte rts did not rech sttisticl significnce. Determintion of renl ROCK ctivity Compred with, vehicle-treted dietic rts displyed incresed phosphoryltion of MYPT, downstrem sustrte Tle 2 Effect of fsudil nd losrtn on systolic lood pressure (mm Hg) Group Bseline Week 6 Week 12 Week ±7 133±3 141±7 147±7 148±3 141±6 142±4 147±3 143±6 136±6 142±6 145±8 146±6 127±6 c 125±6, 127±4, +LOS 145±6 127±5 c 122±5,,c 131±4 lte 147±6 147±5 15±4 141±6 Arevitions:, control-vehicle;, dietic-fsudil; +LOS, dieticfsudil+losrtn; lte, dietic-lte fsudil tretment;, dietic-losrtn;, dietic-vehicle. Po.5 vs. Po.5 vs. Po.1 vs. c Po.5 vs lte. Tle 1 Physicl nd metolic prmeters Group BWT (g) RKW (g) RKW/BWT (g per 1 g wt) BG (mg/dl) HBA1c (%) 46±12 2.7±.1.58±.2 72±6 3.2±.1 4±6 w 3.3±.1.82±.3 w 295±27 w 4.9±.2 w 43±9 w 3.1±.1.77±.3 321±2 w 4.9±.2 w 414±8 3.4±.2.83±.6 w 327±18 w 5.±.2 w +LOS 417±12 3.6±.2 w.86±.4 w 319±25 w 5.2±.2 w lte 41±4 w 3.5±.1 w.85±.4 w 291±29 w 5.2±.2 w Arevitions: BG, lood glucose; BWT, ody weight;, control-vehicle;, dietic-fsudil; +LOS, dietic-fsudil+losrtn; lte, dietic-lte fsudil tretment;, dietic-losrtn;, dietic-vehicle; HBA1c, glycosylted hemogloin; RKW, right kidney weight. Po.5. w Po.1 vs. Kidney Interntionl (211) 79,

3 originl rticle R Komers et l.: ROCK inhiition in dietic nephropthy 6 weeks 12 weeks 18 weeks (mg per 24 h) LOS lte +LOS lte d c +LOS lte Figure 1 The 24 h urinry lumin excretion. Non-dietic nd dietic rts receiving vehicle (, ), nd dietic rts treted with fsudil (), losrtn (), comintion of fsudil nd losrtn ( þ LOS), or fsudil s lte tretment initited 12 weeks fter induction of dietes (lte) underwent mesurements of 24-h urinry lumin excretion in metolic cges t weeks 6, 12, nd 18. The dt re expressed s geometric mens /C tolernce fctor. Po.5 vs ; Po.5 vs ; Po.5 vs nd þ LOS; c Po.5 vs ; d Po.5 vs. (ml/min) LOS lte (connective tissue growth fctor (CTGF), collgens I nd IV, osteopontin, nd p65 nucler fctor (NF)-kB) did not rech sttisticl significnce. Tretment with fsudil reduced fironectin mrna expression when compred with, ut no effects were detected on other genes. The losrtn-treted nimls demonstrted lower mrna expression of TGF-, fironectin, collgens I nd III mrna, nd p65 NF-kB. The nlysis of profirotic fctors nd ECM proteins t the protein level showed dietes-induced increses in renl undnce of TGF-, CTGF, nd fironectin (Figure 5). All these prmeters, s well s collgen I protein, were reduced in oth fsudil- nd losrtn-treted rts when compred with vehicle-treted counterprts. Figure 2 The 24 h cretinine clernce (CCr). Non-dietic nd dietic rts receiving vehicle (, ), nd dietic rts treted with fsudil (), losrtn (), comintion of fsudil nd losrtn ( þ LOS), or fsudil s lte tretment initited 12 weeks fter induction of dietes (lte) underwent mesurements of CCr in metolic cges t week 18. Po.5; Po.5 vs. of ROCKs tht ws nlyzed s mrker of RhoA/ROCK ctivity, in renl corticl homogentes (Figure 4). The phosphorylted myosin phosphtse trget suunit 1 (P-MYT) ws rely detectle in fsudil-treted nimls, indicting effective lockde of RhoA/ROCK. In ddition, significnt reduction in phosphorylted myosin phosphtse trget suunit 1 expression ws lso oserved in rts. Renl expression of meditors nd mrkers of DN To determine the mechnisms nd possile differences in protective effects of ROCKi nd ARB in the dietic kidney, we next determined the effects of fsudil nd losrtn on mrna nd protein expression of estlished meditors nd mrkers of DN. mrna nd protein expression studies were performed in control nd dietic vehicle-treted rts, nd in rts with fsudil or losrtn monotherpies. As shown in Tle 3, displyed increses in trnsforming growth fctor- (TGF-) nd collgen III mrna expression, wheres the increses in expression of other genes Renl expression of mrkers ssocited with epithelil mesenchyml trnsition (EMT) To further investigte the mechnisms of ctions of fsudil, in prticulr in the tuulointerstitil comprtment, we determined expression of the EMT mrkers, firolst-specific protein 1 (FSP1, lso known s S1A4), -smooth muscle ctin (-SMA), vimentin, nd E-cdherin. Screening renl corticl smples reveled mrked upregultion of mrna for the erly mrker FSP1 19 in dietic nimls (Tle 3), which ws reduced in nd rts. In, immunohistochemicl nlysis loclized FSP1 in occsionl interstitil nd tuulr cells. In contrst, rts demonstrted mssive ccumultions of FSP1-positive cells in the corticl interstitium (Figure 6), frequently ssocited with res of tuulr trophy nd surrounding firosis. The FSP1 interstitil immunorectivity ws lso detected in nd rts, ut only in smll clusters or single cells. -SMA ws detected in vsculr structures in ll groups, wheres tuulointerstitil immunorectivity ws detectle in rts (Figure 6), to lesser extent in rts, nd prcticlly undetectle in nd nimls. In ddition to glomerulr locliztion of vimentin, oserved in ll groups of rts, displyed undnt tuulointerstitil vimentin immunorectivity, which ws reduced in oth fsudil- nd losrtn-treted dietic kidneys (Figure 6). E-cdherin ws loclized predominntly in distl tuules 434 Kidney Interntionl (211) 79,

4 R Komers et l.: ROCK inhiition in dietic nephropthy originl rticle +LOS lte Glomerulosclerosis score Advnced glomerulr lesions Tuulointerstitil firosis +LOS lte (% of ll glomeruli) LOS lte (% of corticl re) 1 5 +LOS lte Figure 3 Anlysis of glomerulr sclerosis score (GSS), proportion of severely ffected glomeruli, nd tuulointerstitil firosis score (TIFS). () Representtive microphotogrphs of glomeruli stined with periodic cid Schiff (PAS) with different degrees of glomerulosclerosis nd () interstitil regions stined with trichrome showing res of vrious degrees of interstitil firosis nd tuulr trophy. (c) Br grph presenttion of quntittive evlution of GSS, (d) proportion of severely ffected glomeruli, nd (e) TIFS., control-vehicle;, dietic-fsudil; þ LOS, dietic-fsudil þ losrtn; lte, dietic-lte fsudil tretment;, dietic-losrtn;, dietic-vehicle. w Po.1 vs ; Po.5; Po.1 vs. in ll groups of rts without pprent effects of dietes or tretments. The immunohistochemicl findings were mirrored y mesurements of protein undnce of EMT mrkers y immunolotting (Figure 7) indicting increses in FSP1, - SMA, nd vimentin in when compred with nondietic nimls, nd reductions in rts receiving fsudil or losrtn tretments. In contrst, there were no significnt differences in E-cdherin expression etween the groups of rts. Renl expression of nephrin nd vsculr endothelil growth fctor (VEGF) Further nlyses determined renl corticl expression of nephrin nd VEGF, the importnt determinnts of glomerulr filtrtion rrier The mrna expression of the podocyte protein nephrin ws decresed in compred with non-dietic nimls, nd restored y fsudil nd prtilly y losrtn (Figure 8). Although this dietesinduced nephrin downregultion ws not oserved t the protein level, fsudil-treted rts demonstrted incresed nephrin protein undnce when compred with oth dietic nd non-dietic controls (Figure 8). Unlike nephrin, VEGF protein expression ws similr mong the groups (Figure 8c). DISCUSSION In the present studies, uninephrectomized dietic rts displyed more rpid course in the development of luminuri, reduction in GFR, nd progressive glomerulosclerosis nd interstitil firosis when compred with nondietic uninephrectomized nimls. Tretment with the ROCKi fsudil, when initited t the onset of dietes, ws nephroprotective s ssessed y its effects on spectrum of structurl, functionl, nd moleculr chrcteristics of DN. These eneficil effects were oserved despite the sence of n effect on lood pressure, in ccord with previous evidence in non-dietic models of hypertensive end-orgn injury 2,4,5,8,9,23 nd in models of DN. 7 The dietes-induced increse in MYPT phosphoryltion, mesured s mrker of RhoA/ROCK ctivtion, ws suppressed y fsudil, indicting effective inhiition of the pthwy. Kidney Interntionl (211) 79,

5 originl rticle R Komers et l.: ROCK inhiition in dietic nephropthy P-MYPT Totl MYPT P-MYPT/ctin (fold ) kd 15 kd Figure 4 Renl protein expression of phosphorylted nd totl MYPT. MYPT phosphoryltion (Thr853) ws determined in conjunction with totl MYPT protein y western lotting s n indictor of renl Rho-ssocited kinse (ROCK) ctivity. Dt re presented s protein/ctin rtios plotted on y xis. The upper pnel shows representtive lots., control-vehicle;, dietic-fsudil;, dietic-losrtn;, dietic-vehicle. Po.5 vs ; Po.1 vs. Tle 3 Gene expression of meditors nd mrkers of dietic nephropthy Genes n TGF- 1.±.2 1.8±.4 1.8±.6.7±.2,c CTGF 1.±.2 2.4± ±.3 2.±.4 Fironectin 1.±.1.8±.3.3±.1.3±.1 Collgen I 1.±.3 2.1± ±.5 w.8±.2 c Collgen III 1.±.3 2.6±.3 3.±.4 1.±.2,c Collgen IV3 1.±.2 2.±.6 1.±.2 1.±.3 Osteopontin 1.±.2 3.5± ±.4 1.4±.4 P65 NFkppB 1.±.3 1.9±.7 2.2±.5.7±.1 FSP1 1.±.2 27.± ±.6 1.6±.5 Arevitions: CTGF, connective tissue growth fctor;, control-vehicle;, dietic-fsudil;, dietic-losrtn;, dietic-vehicle; FSP1, firolstspecific protein 1; TGF-, trnsforming growth fctor-. The dt re expressed s fold induction compred with. Po.5. w Po.1 vs. Po.5 vs. Po.1 vs. c Po.5 vs. Studies exploring the long-term effects of ROCKi in models of DN hve only recently strted to emerge. Nephroprotective effects of fsudil hve een explored in erlier stges of nephropthy in streptozotocin-dietic rts 7 nd in d/d mice, 6 model of type 2 dietes mellitus. Both studies documented eneficil effects of fsudil on the development of mesngil expnsion, proteinuri, nd expression of ECM proteins, ssocited with reductions of dietes-induced increses in RhoA/ROCK ctivity. Our oservtions re in ccord with the ove-mentioned studies, nd lso extend the evidence in severl directions tht hve not een previously explored in models of type 1 dietes: evlution of ROCKi in more dvnced stges of nephropthy; evlution of ROCKi in comintion with RAS inhiition; nd evlution of the effects of ROCKi initited lter in the course of the disese. In ddition to the effects on glomerulr rchitecture, this design llowed us to document eneficil effects of fsudil on tuulointerstitil firosis, nd preservtion of renl function to levels comprle to those in non-dietic nimls. It should e noted tht similr design hs een previously pplied in study exploring the effects of fsudil in OLETF (Otsuk Long-Evns Tokushim ftty) rts, 24 model of type 2 dietes. However, in tht study the renoprotective effects of fsudil (1 mg/kg) were ssocited with meliortion of dietes nd could e therefore ttriutle to improved metolic sttus rther thn more direct effect of the drug. In prllel with the eneficil effects on renl rchitecture, we oserved eneficil effects of fsudil on luminuri t weeks 6 nd 12, lthough the effect ws not pprent t the end of the study. Notly, the U l V plteued in rts t the end of the follow-up, most likely ecuse of the decresing GFR, s some of these nimls displyed lower U l V t week 18 compred with week 12, nd to incresed luminuri over time in nimls. This decrese in GFR in rts is ttriutle to oth more dvnced glomerulosclerosis nd tuulointerstitil sclerosis, which is n importnt determinnt of GFR t lter stges of chronic kidney disese. 25 The effects of fsudil were compred with the ARB losrtn, compound with estlished nephroprotective effects in oth clinicl 26 nd experimentl 27 settings. The structurl effects of fsudil were comprle to those of losrtn. However, losrtn ppered to e more effective in reducing luminuri, especilly t the end of the study. The more prominent effects of losrtn on luminuri re most likely ttriutle to its effects on lood pressure, n importnt driving force for proteinuri. Strtegies tht might enhnce the therpeutic efficcy of RAS inhiitors re likely to hve n importnt impct on nephroprotection in dietes. Studies hve suggested possile synergism in high glucose- nd ngiotensin II (AngII)- induced RhoA/ROCK signling in mesngil cells, 28 ttenution of glomerulr microvsculr ctions of AngII y ROCKi, 29,3 nd eneficil effects of ROCKi on AngII- nd ldosterone-induced renl injury. 4,1 Therefore, we hypothesized tht the comintion of ROCKi nd ARB could enhnce the eneficil effects of the monotherpies. However, we detected no further protective effect of fsudil when comined with losrtn. This phenomenon corresponds to nother oservtion in the present study, the significnt reduction in MYPT phosphoryltion in losrtn-treted rts, suggesting tht AngII inhiition might interfere with RhoA/ 436 Kidney Interntionl (211) 79,

6 R Komers et l.: ROCK inhiition in dietic nephropthy originl rticle TGF-β protein/ctin rtio (fold ) CTGF protein/ctin rtio (fold ) FN protein/ctin rtio (fold ) TGF-β CTGF FN Col-I 5 kd 5 kd 22 kd 15 kd ROCK signling. Interestingly, similr, lthough not significnt, phenomenon ws reported in rts treted with enlpril. 7 Another mechnism explining this phenomenon hs een suggested y Svoi et l., 31 who demonstrted tht stimultion of AngII AT2 receptors could led to ROCK inhiition. Indeed, enhnced ctivtion of the AT2 receptor could e expected during losrtn tretment ecuse of incresed vilility of AngII to stimulte this receptor sutype. On the other hnd, it should e stressed tht the RAS inhiitors re usully so effective in rodent models of DN tht it is difficult to detect dditive effects of comintion tretments. We cnnot exclude the possiility tht in different model or, cliniclly, the comintion of ROCKi with ARB might e proven more eneficil thn ARB monotherpy. In clinicl dietes, tretment is most often initited in ptients who hve lredy developed evidence of nephropthy. Therefore, we lso evluted the effects of strting fsudil fter 12 weeks without tretment. In these nimls, we still detected eneficil effects on glomerulr lesions nd GFR, ut tretment did not significntly influence the tuulointerstitil firosis score, nd completely lcked ntiproteinuric efficcy. During the course of nephropthy, the lesions in individul glomeruli develop t vrile rte. After 12 weeks of dietes, uninephrectomized rts disply lesions spnning from norml to completely sclerotic glomeruli. Bsed on recent oservtions y Teles et l., 27 who hve descried long-term dynmics in the development of glomerulosclerosis in dietic rts, the prtil protection oserved fter delyed fsudil my e ttriutle to its effects on norml or mildly ffected glomeruli. However, the tretment did not reverse estlished glomerulr lesions nd interstitil firosis. To identify the moleculr sis of fsudil s oserved effects nd possile differences etween the ctions of fsudil nd those of losrtn, we further investigted mrna nd protein expression of spectrum of mrkers nd meditors known to e upregulted t vrious stges of DN We detected dietes-induced expression of TGF- nd collgen III mrna. Tretment with fsudil reduced renl fironectin Col-l protein/ctin rtio (fold ) Figure 5 Renl corticl protein expression of trnsforming growth fctor- (TGF-), connective tissue growth fctor (CTGF), fironectin (FN), nd collgen I. Protein expression ws nlyzed y western lotting in renl corticl homogentes. The grphs show densitometric nlysis of western lots. The insets show representtive lots of, non-dietic nimls (lnes 1 nd 2);, the vehicle-treted dietic rts (lnes 3 nd 4);, fsudil-treted dietic nimls (lnes 5 nd 6); nd, losrtn-treted dietic nimls (lnes 7 nd 8). Fsudil nd losrtn were oth effective in reducing dietes-induced increses in renl TGF- (), CTGF (), nd FN expression (c), nd reduced undnce of collgen I (d). Dt re presented s protein/ctin rtios., control-vehicle;, dietic-fsudil;, dietic-losrtn;, dietic-vehicle. Po.5, w Po.1 vs ; Po.5, Po.1 vs. Kidney Interntionl (211) 79,

7 originl rticle R Komers et l.: ROCK inhiition in dietic nephropthy FSP1 α-sma Vimentin E-cdherin ni Figure 6 Immunohistochemicl expression nd locliztion of mrkers ssocited with epithelil mesenchyml trnsition (EMT) in renl cortex. Representtive microphotogrphs ( 2) show corticl sections from non-dietic nimls (), vehicle-treted dietic rts (), fsudil-treted dietic nimls (), nd dietic nimls treted with losrtn () proed with primry ntiodies rised ginst firolst-specific protein 1 (FSP1), -smooth muscle ctin (-SMA), vimentin, nd E-cdherin. Imges of sections stined with nonimmune serum re shown for rts (ni). Arrows show tuulointerstitil nd rrowheds show vsculr immunorectivity of proteins of interest. mrna expression, wheres losrtn seemed to e more effective on the mrna expression of TGF-, fironectin, collgens I nd III mrna, nd p65 NF-kB gene expression. At the protein level, oth tretments were effective in reducing TGF-, CTGF, fironectin, nd collgen I protein undnce. These reductions in ECM protein undnces re in ccord with recently pulished long-term effects of fsudil in DN, 6,7 wheres fsudil-induced suppression of TGF- nd CTGF expression corresponds to findings in models of non-dietic progressive glomerulosclerosis nd interstitil firosis. 4,9,1 Both TGF- nd CTGF stimulte EMT, 36,37 process closely linked with the development of renl firosis. Although the role of EMT in mediting renl firosis in 438 Kidney Interntionl (211) 79,

8 R Komers et l.: ROCK inhiition in dietic nephropthy originl rticle FSP1 1 kd α-sma FSP1/ctin rtio (fold ) α-sma/ctin rtio (fold ) Vimentin 5 kd E-cdherin 1 kd Vimentin/ctin rtio (fold ) E-cdherin/ctin rtio (fold ). Figure 7 Immunolot nlysis of mrkers ssocited with epithelil mesenchyml trnsition (EMT) in renl cortex. Protein expression ws quntified y western lotting in renl corticl homogentes. The grphs show densitometric nlysis of western lots. The upper insets show representtive lots of firolst-specific protein 1 (FSP1), -smooth muscle ctin (-SMA), vimentin, nd E-cdherin in non-dietic nimls (, lnes 1 nd 2); vehicle-treted dietic rts (, lnes 3 nd 4); fsudil-treted dietic nimls (, lnes 5 nd 6); nd dietic nimls treted with losrtn (, lnes 7 nd 8). Dt re presented s protein/ctin rtios. Po.5; w Po.1 vs ; Po.5; Po.1 vs dietes is still deted, prticulrly in vivo, there is incresing evidence supporting role for this process The Rho/ROCK pthwy hs een shown to medite EMT in non-dietic context. 41,42 Consequently, we next studied EMT indictors. The dt show tht uninephrectomized dietic rts disply mrkers of EMT, such s FSP1, -SMA, nd vimentin. The expression of these mrkers ws ttenuted y ROCKi in conjunction with eneficil effects on renl firosis nd reduced expression of TGF- nd CTGF. Fsudil reduced EMT to n extent similr to tht of losrtn. The effect of losrtn is in ccord with descried role of AngII in stimulting EMT in vitro, nd vlidtes those oservtions in vivo. Loss of E-cdherin, phenomenon trditionlly ssocited with EMT, 45 ws not detected in dietic kidneys. This finding corresponds to the recently descried prtil EMT phenotype, 46 nd emerging evidence tht this process might operte in the dietic kidney. 47 It hs een postulted tht the entire clssicl EMT progrm oserved primrily in vitro, with the ssocited decrese in E-cdherin, my not e criticl for the development nd progression of tuulointerstitil firosis. 47 Both TGF- nd CTGF increse ECM expression independently of E-cdherin. TGF- lowers E-cdherin, wheres CTGF ( driver of prtil EMT) increses E-cdherin expression. Moreover, n increse in E-cdherin hs een recently reported in rts with ureterl ostruction, clssicl model of EMT-medited kidney firosis. 48 The lood pressure-independent ntiproteinuric effect of fsudil suggests eneficil ctions on the glomerulr filtrtion rrier. In this context, we investigted the impct of this tretment on nephrin expression. Although loss of nephrin in glomerulr epithelil cells is ssocited with severe proteinuri, studies in experimentl dietes hve produced conflicting dt showing decresed, 2 unchnged, 22 or incresed 49 glomerulr nephrin mrna nd protein. In the present study, kidneys from rts displyed mrked reductions in nephrin mrna, which were restored y fsudil nd prtilly y tretment with losrtn. The dietesinduced downregultion of nephrin ws not oserved t the protein level; however, similr to the effects on mrna, oth fsudil nd losrtn incresed nephrin renl corticl protein undnce. This suggests tht modultion of nephrin expression might e ssocited with the modest ntiproteinuric effects of fsudil, phenomenon corresponding to recently reported eneficil effects of this compound on effcement of foot processes. 7 Furthermore, we oserved no effect of dietes nd tretments on renl VEGF protein expression. Although VEGF my e elevted in the initil phses of DN, 5 our finding most likely reflects more dvnced stge of DN, when this increse my not e mintined s chronic firotic chnges occur in the kidney. 51,52 Some differences etween the groups in moleculr mrkers nd meditors of nephropthy were more prominent Kidney Interntionl (211) 79,

9 originl rticle R Komers et l.: ROCK inhiition in dietic nephropthy Nephrin protein/ctin (fold ) Nephrin mrna (fold ) Nephrin VEGF p42 VEGF p21 t the protein rther thn the mrna level. Although this phenomenon could e ttriutle to dietes-induced impirment in protein degrdtion, 53 mrna ctivtion erlier in the course of the disese, or the influence of uninephrectomy driving the expression of pro-growth genes lso in non-dietic kidneys, 54 it lso suggests posttrnscriptionl mechnisms. Indeed, this notion is supported y nlysis of p7s6k (phosphoryltion of the 7-kD S6 protein kinse) nd 4E-BP1 (eif4e-inding protein-1), the pivotl intermedites in process of initition of mrna 15 kd 2 kd Figure 8 Renl corticl expression of nephrin nd vsculr endothelil growth fctor (VEGF). Nephrin gene expression () ws nlyzed in renl corticl smples y rel-time PCR (rtpcr). The dt re shown s fold induction compred with. Protein expression ws nlyzed y western lotting in renl corticl homogentes. () Densitometric nlysis nd representtive imges of nephrin western lots. Dt re presented s protein/ ctin rtios plotted on y xis. (c) Representtive imges for VEGF western lots (, non-dietic nimls (lnes 1 nd 2);, the vehicle-treted dietic rts (lnes 3 nd 4);, fsudil-treted dietic nimls (lnes 5 nd 6); nd, losrtn-treted dietic nimls (lnes 7 nd 8)). Po.5 vs ; Po.5 vs. trnsltion, 55 which suggests incresed ctivity of this pthwy in the dietic kidney nd its meliortion y fsudil or losrtn (Supplementry Figure 1 online). Of note, ctivtion of mrna trnsltion hs een previously implicted in enhnced ECM synthesis in the dietic kidney, nd represents promising trget for future studies in this field of ROCK pthoiochemistry in dietes. In conclusion, ROCKi with fsudil initited t the onset of dietes hd lood pressure-independent eneficil effects on the development of glomerulosclerosis nd interstitil firosis in uninephrectomized dietic rts, ssocited with modest ntiproteinuric effect. These eneficil structurl effects were comprle to those of the ARB losrtn, nd were ssocited with reduced expression of prosclerotic cytokines nd ECM proteins, prtil suppression of the EMT phenotype, nd upregultion of nephrin. However, the study did not document dditive eneficil effects of comintion ROCKi/ARB when compred with ARB monotherpy. MATERIALS AND METHODS The dietic rt model Mle Sprgue-Dwley rts were sujected to right nephrectomy under Brevitl nesthesi (5 mg/kg intrperitonel) t the ge of 8 weeks. After 2 weeks, some rts were mde dietic y intrperitonel injection of streptozotocin (Sigm Chemicl, St Louis, MO; 55 mg per kg ody weight). The uninephrectomized dietic rt model ws employed to ccelerte the development of renl injury. 18 After 2 dys, induction of dietes ws confirmed y mesurement of til lood glucose level (One Touch II; Lifescn, Milpits, CA). Dietic rts received dily evening injections of long-cting glrgine insulin (Lntus; Eli Lilly, Indinpolis, IN) in doses individully djusted to mintin lood glucose t B3 mg/dl (17 mmol/l). Blood glucose levels were monitored t lest weekly in ll dietic rts. Uninephrectomized rts injected with phosphte uffer were studied s non-dietic controls. These studies were pproved y the Portlnd VA institutionl niml cre nd use sucommittee. Study design After confirmtion of hyperglycemi in dietic rts, the nimls were rndomized into the following groups: (1) (tp wter) (n ¼ 9); (2) (n ¼ 1); (3) (Cliochem; fsudil 3 mg/kg/dy; 23 n ¼ 1); (4) (Merck; losrtn 2 mg/kg/ dy; 59 n ¼ 1); (5) dietic rts receiving oth fsudil nd losrtn (n ¼ 1, þ LOS); nd (6) dietic rts receiving fsudil s lte tretment initited 12 weeks fter induction of dietes (n ¼ 11, lte). All drugs were dministered in the drinking wter. The rts were mintined on these tretments for 18 weeks, except the lte rts tht received fsudil for 6 weeks. Mesurements of systolic lood pressure (til plethysmogrphy), lood glucose, nd U l V were performed t weeks 6, 12, nd 18. Plsm levels nd urinry cretinine excretion rtes llowing clcultion of 24-h cretinine clernce were mesured t week 18. Within 2 to 3 dys fter the lst urinry collection, the nimls were nesthetized with Inctin (1 mg/kg intrperitonel) nd lood smples were otined for determintion of HBA1c. The left kidney ws then hrvested for morphologicl studies nd moleculr nlyses. 44 Kidney Interntionl (211) 79,

10 R Komers et l.: ROCK inhiition in dietic nephropthy originl rticle Glomerulosclerosis score The excised kidneys were immersed in 1% formlin, then dehydrted through grded series of ethnols, emedded in prffin, sectioned t 4 mm thickness, nd plced onto glss slides. The glomerulr sclerosis score ws determined on periodic cid Schiff-stined sections using scle rnging from to 4 for norml (), 1 ¼ 25% sclerosis, 2 ¼ 5% sclerosis, 3 ¼ 75% sclerosis, nd 4 ¼ 1% sclerosis s previously descried. 6 On verge, 2 glomeruli were evluted per rt. In ddition to trditionl scoring, the proportion of glomeruli with dvnced glomerulosclerotic lesions chrcterized y confluent res of dense deposition of periodic cid Schiff-positive mteril t the glomerulr tuft, cpillry loop occlusions, nd dhesions of the glomerulr tuft to Bowmn s cpsule ws determined for ech kidney section. Tuulointerstitil firosis score Mesurements were performed on Msson-trichrome-stined sections. The 1 squre grid ws pplied t low mgnifiction ( 5) on consecutive microscopic corticl fields strting t renl hili in clockwise direction. The numer of squres contining stined firous tissue or trophic tuules ws recorded for ech field nd verged for ech kidney. Perivsculr spces nd glomeruli were not counted. On verge, 18 fields were evluted per kidney. All structurl nlyses were conducted in lind mnner. Isoltion of totl RNA, synthesis of cdna, nd quntittive rel-time PCR These methods were performed s previously pulished 61 nd detiled in Supplementry Mteril. The primer sequences re listed in Supplementry Tle 1 online. Immunolotting Western lot nlysis ws performed s previously descried. 62,63 The description nd list of primry ntiodies re provided in Supplementry Mteril. Immunohistochemistry For immunohistochemicl nlysis, the formlin-fixed, prffinemedded kidneys were processed s previously descried. 62 More detiled informtion is provided in Supplementry Mteril. Anlyticl methods Urinry lumin concentrtions were determined using the Nephrt kit (Exocell, Phildelphi, PA). Serum nd urine cretinine concentrtions were mesured y spectrophotometric ssy (Biovision, Mountin View, CA). HBA1c ws determined y ffinity column chromtogrphy (Glyco-Gel B; Pierce Chemicl, Rockford, IL). Sttisticl nlysis Comprisons of vriles etween control nd dietic groups were nlyzed y one-wy nlysis of vrince followed y the Bonferoni test. Sttisticl significnce ws defined s Po.5. Dt were expressed s mens±s.e.m. Dt for luminuri were nlyzed fter logrithmic trnsformtion nd the dt expressed s geometric mens /C tolernce fctor. DISCLOSURE All the uthors declred no competing interests. ACKNOWLEDGMENTS These studies were supported y the Juvenile Dietes Reserch Foundtion (RK ). SUPPLEMENTARY MATERIAL Figure S1. Renl corticl expression nd phosphoryltion of p7s6k nd 4e-BP1. Tle S1. Primer sequences used for rtpcr. 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