Inhibition of Renal Rho Kinase Attenuates Ischemia/ Reperfusion-Induced Injury

BASIC RESEARCH www.jasn.org

Jai Prakash,* Martin H. de Borst,† Marie Lacombe,‡ Frank Opdam,‡ Pieter A. Klok,† Harry van Goor,† Dirk K.F. Meijer,* Frits Moolenaar,* Klaas Poelstra,* and Robbert J. Kok*§

*Department of Pharmacokinetics and Drug Delivery, Groningen Research Institute for Pharmacy, University of Groningen, and †Department of Pathology and Experimental Medicine, University Medical Centre Groningen, Groningen, ‡Kreatech Biotechnology, Amsterdam, and §Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, Netherlands

ABSTRACT The Rho kinase pathway plays an important role in dedifferentiation of epithelial cells and infiltration of inflammatory cells. For testing of the hypothesis that blockade of this cascade within the kidneys might be beneficial in the treatment of renal injury the Rho kinase inhibitor, Y27632 was coupled to lysozyme, a low molecular weight protein that is filtered through the glomerulus and is reabsorbed in proximal tubular cells. Pharmacokinetic studies with Y27632-lysozyme confirmed that the conjugate rapidly and extensively accumulated in the kidney. Treatment with Y27632-lysozyme substantially inhibited ischemia/reperfusion-induced tubular damage, indicated by reduced staining of the dedifferentiation markers kidney injury molecule 1 and vimentin, and increased E-cadherin relative to controls. Rho kinase activation was inhibited by Y27632-lysozyme within tubular cells and the interstitium. Y27632-lysozyme also inhibited inflammation and fibrogenesis, indicated by a reduction in gene expression of monocyte chemoattractant protein 1, procollagen I␣1, TGF-␤1, tissue inhibitor of metalloproteinase 1, and ␣-smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of ␣-smooth muscle actin, collagen I, collagen III, and fibronectin. In contrast, unconjugated Y27632 did not have these beneficial effects but instead caused systemic adverse effects, such as leukopenia. Neither treatment improved renal function in the bilateral ischemia/reperfusion model. In conclusion, the renally targeted Y27632-lysozyme conjugate strongly inhibits tubular damage, inflammation, and fibrogenesis induced by ischemia/reperfusion injury.

J Am Soc Nephrol 19: 2086–2097, 2008. doi: 10.1681/ASN.2007070794

Renal ischemia-reperfusion (I/R) injury is the activation of RhoGTPases leads to the transdif- main cause of acute renal failure after major renal ferentiation of renal tubular cells to fibroblasts, surgery, trauma, or transplantation.1–3 After I/R which turn into myofibroblasts.9 injury, proximal tubular cells lose their polarity We hypothesized that blockade of the ROCK and detach from the tubules as a result of cytoskeletal reorganization, which is mainly regu- Received July 20, 2007. Accepted May 26, 2008. lated by RhoGTPases.4,5 RhoGTPases belong to the Ras superfamily of GTP-binding proteins and Published online ahead of print. Publication date available at act by stimulating downstream Rho effectors www.jasn.org. such as Rho-associated coiled-coil–forming pro- Correspondence: Dr. Jai Prakash, Department of Pharmacokinet- ics and Drug Delivery, Groningen Research Institute for Phar- tein kinase (ROCK). In addition to cytoskeletal macy, University of Groningen, Antonius Deusinglaan 1, 9713 AV, regulation, RhoGTPases are involved in the infil- Groningen, Netherlands. Phone: ϩ31-50-3636414; Fax: ϩ31-50- tration of inflammatory cells,6,7 which is an im- 3633247; E-mail: [email protected] perative process after I/R injury.8 Moreover, Copyright ᮊ 2008 by the American Society of Nephrology

2086 ISSN : 1046-6673/1911-2086 J Am Soc Nephrol 19: 2086–2097, 2008 www.jasn.org BASIC RESEARCH pathway locally within kidneys may be an interesting approach A to treat renal injury. The efficacy of the widely known ROCK NH 2 inhibitor Y27632 has been demonstrated in various I/R injury H models10,11 and in the unilateral ureteral obstruction model12; N Y27632-Lysozyme conjugate however, the ROCK pathway is also involved in the contrac- O N tion of vascular smooth muscle cells and plays an important Y-27632 role in regulation of vascular tone.13–15 Last, Rho kinase signal- B ing is important in migration and activation of immune i.v. injection of Renal filtration of Y27632-Lysozyme the conjugate cells.16–18 Systemic administration of ROCK inhibitors can conjugate therefore lead to adverse effects. Considering the systemic role of Rho signaling, we now Proximal tubular cell propose renal-specific delivery as a targeted approach to inves- Y-27632-Lysozyme conjugate tigate renal effects of ROCK inhibitors. Renal-specific delivery Megalin can avoid interactions with nontarget cells and thereby de- receptor crease adverse effects. Furthermore, renal drug delivery will Drug action enhance the drug’s accumulation in the targeted organ, which Lumen potentially can improve therapeutic efficacy at lower dosages Blood vessel to be used. In previous studies, we showed that renal-specific Released drug drug delivery can be achieved using the low molecular weight Urine protein lysozyme (LZM) as drug carrier.19–21 LZM filters through the glomerulus and is reabsorbed by tubular cells via Figure 1. (A) Y27632 was chemically modified and conjugated to the megalin receptor,22 and we have made use of this process. LZM. (B) Scheme of the renal uptake mechanism of Y27632-LZM in We recently developed a versatile linking technology, the the proximal tubular cells. After an intravenous injection, the conju- so-called Universal Linkage System (ULS), for the coupling of gate filters through the glomerular membrane and is internalized via drugs to carrier systems,20,21,23,24 which we now have applied megalin receptors by proximal tubular cells. Drug is released slowly for the conjugation of the Rho kinase inhibitor Y27632 to from the conjugate within these cells, and the released drug exhibits LZM. We investigated the potential activity of Y27632-LZM in its activity by blocking kinase signaling cascade. the ischemia-reperfusion (I/R) injury model and demonstrated both enhanced activity and increased selectivity of the conjugates, Y27632-LZM accumulated efficiently in the kid- tubular-targeted Rho kinase inhibitor. neys within 1 h after the intravenous injection (Figure 2B). Accumulation of Y27632-LZM in tubular cells was confirmed by anti-LZM immunohistochemical staining on kidney sections (Figure 2C). As underlined already, the uptake of lysozyme is mediated by the megalin receptor present on the RESULTS surface of proximal tubular cells (Figure 1B). Anti-LZM stain- Synthesis and Characterization of Y27632-ULS-LZM ing corresponded closely to the anti-megalin receptor staining Y27632 was conjugated to ULS and then coupled to the carrier in kidneys (Figure 2D). We also examined the accumulation of LZM at a 1:1 drug-to-protein ratio (Figure 1A). Stability stud- Y27632-LZM in other organs. At 2 h after its administration, ies demonstrated that this type of conjugate is highly stable in staining for LZM was strongly positive in kidneys (Figure 2E), buffers and serum for 24 h at 37°C. In contrast, addition of whereas only minor levels of the conjugate were detectable in glutathione to the medium afforded release of drug that was the sinusoidal area of liver (Figure 2F), in lungs, and in the concentration dependent. Approximately 5 and 9% of the spleen (Figure 2, G and H). Spleen accumulation of the conju- bound drug was released in 5 and 50 mM glutathione, respec- gate was mainly localized in the interstitium not correspond- tively, in 24 h. No release was found with serum and PBS in ing with T and B lymphocytes in the white pulp or with the 24 h. From these data, we concluded that the coupled Y27632 localization of macrophages and granulocytes, as visualized by drug can be displaced competitively by endogenous ligands staining for ED-1 and His48 (Supplemental Figure 1, A and B). that are normally present within the cytosol. Anti-LZM and anti–ED-1 staining was present in the same area, but the staining patterns were quite different. The tissue Pharmacokinetics of the Y27632-LZM Conjugate distribution of the conjugate at later time points (t ϭ 6h) We investigated the renal accumulation of Y27632-LZM after showed similarly high levels within the kidney, whereas the intravenous administration to rats. The serum-disappearance staining in other organs was faint (Supplemental Figure 1, C curve of Y27632-LZM, which followed a two-compartment through F). These results indicate that Y27632-LZM was spe- pharmacokinetic model, is shown in Figure 2A. Only carrier- cifically accumulated in kidneys but not deposited in other bound Y27632 was detected in the serum, whereas free drug organs, which is in agreement with the clearance of the conju- was absent. Furthermore, in accordance with other drug-LZM gate via renal elimination.

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A B

100 100

10 10

serum levels (%dose) serum levels 1 Renal levels (% dose) (% Renal levels 1

0.1 0.1 0 60 120 180 240 300 360 0 60 120 180 240 300 360 Time (min) Time (min)

C D

G T G T

Figure 2. (A and B) Serum (A) and renal levels (B) of Y27632-LZM after administration of Y27632-LZM intravenously at a dos- E F age of 20 mg/kg (equivalent to 555 ␮g/kg Y27632) to rats. Symbols represent the percentage dose of Y27632 at each time point. The continuous line represents the pharmacokinetic data-fit curve (two-compartment model). (C) Localization of the conjugate in proximal tubular cells after1hofintrave- nous injection of Y27632-LZM conjugate by anti-LZM staining (red color, arrowheads). G H (D) Anti-megalin receptor staining (arrows) at the brush border of proximal tubular cells; this corresponds to the anti-LZM staining for the internalized conjugate. G, glomerulus; T, renal tubule. (E through H) Anti-LZM staining in kidneys, liver, lungs, and spleen, respectively, at 2 h after administration of the conjugate. Magnifications: ϫ400 in C; ϫ200 in E through H.

Effect of Renal-Delivered Y27632 in the I/R Model in Rats Kidney Weights, Serum Creatinine Levels, and Blood The efficacy of Y27632-LZM was evaluated in the unilateral I/R Cell Counts model because this animal model is characterized by an early After4dofunilateral I/R, the weights of ischemic kidneys were inflammatory phase that finally leads to fibrosis. In addition, significantly increased as compared with contralateral kidneys we evaluated the effects of ROCK inhibition in the bilateral I/R or kidneys of normal rats (Table 1). Interestingly, treatment model. Drug treatment was started simultaneously with the with Y27632-LZM reduced this increase in kidney weight, induction of I/R to intervene in the early inflammatory and whereas free drug did not affect it. In the unilateral I/R model, fibrotic events. Because drug release from the conjugate would renal function was hardly affected as a result of presence of an provide continuous drug levels for a prolonged period of intact contralateral kidney, and we found only a subtle increase time,20 we treated the animals with once-daily doses. in serum creatinine levels of control rats at 4 d after I/R. Treat-

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Table 1. Effect of various treatments on kidney weight/body weight ratio, serum creatinine levels, and blood cell counts after 4 da Parameter Normal I/R ؉ Vehicle I/R ؉ Y27632-LZM I/R ؉ Y27632 Ischemic kidney wt/body wt (%) 0.43 Ϯ 0.01 0.68 Ϯ 0.03b 0.59 Ϯ 0.04c 0.78 Ϯ 0.04 Contralateral kidney wt/body wt (%) 0.43 Ϯ 0.02 0.49 Ϯ 0.06 0.46 Ϯ 0.02 0.54 Ϯ 0.02 Serum creatinine levels (␮mol/L) 17.50 Ϯ 0.29 24.40 Ϯ 0.80b 22.30 Ϯ 0.61d 27.40 Ϯ 0.87e RBC counts (ϫ1012/L) N.D. 6.35 Ϯ 0.19 6.33 Ϯ 0.19 6.33 Ϯ 0.06 WBC counts (ϫ109/L) N.D. 12.09 Ϯ 1.04 12.08 Ϯ 1.99 9.13 Ϯ 0.68e aData are means Ϯ SEM. For red blood cell (RBC) and white blood cell (WBC) count groups, I/R ϩ vehicle (n ϭ 7) and I/R ϩ Y27632-LZM (n ϭ 4). ND, not determined. bP Ͻ 0.001 versus normal. cP Ͻ 0.01, dP Ͻ 0.001 versus I/R ϩ Y27632. eP Ͻ 0.05 versus I/R ϩ vehicle. ment with Y27632-LZM slightly improved serum creatinine normal I/R + vehicle levels, whereas free drug even worsened it. I/R + Y27632-LZM I/R + Y27632 In the bilateral I/R model, both serum creatinine and blood urea nitrogen levels were several-fold increased at days 1 and 3 MCP-1 TGF-β1 after the ischemic insult (Supplemental Figure 2). Rats treated 12 4 with Y27632-LZM showed higher creatinine and blood urea 10 † nitrogen levels at both day 1 and day 3 as compared with the † 3 vehicle-treated group, which was most prominent in one of the 8 animals. Free Y27632 deteriorated renal function and did so in 6 2 a more pronounced manner than Y27632-LZM. These data ## * # ** fold induction fold suggest that ROCK inhibition affected renal function nega- 4 induction fold 1 tively, especially during the first day after bilateral I/R injury. 2 The stronger effects by free Y27632 as compared with Y27632- 0 0 LZM may relate to an effect outside the tubular cells. To investigate whether systemic administration of Y27632 α has hematologic effects, we counted red blood cells and white Procollagen-Iα1 -SMA 4 4 † blood cells at the end of the treatment period in the unilateral †

I/R model (Table 1). We found that free drug significantly 3 3 decreased the number of white blood cells, whereas Y27632- * ## LZM did not display any inhibitory effect. Both free Y27632 2 2 and conjugated drug did not influence red blood cells. These ** ## fold induction fold fold induction fold data already demonstrate the altered pharmacologic profile of 1 1 Y27632-LZM as compared with the free drug. To provide insight into the mechanism of action of Y27632-LZM, we deter- 0 0 mined the expression of inflammatory and fibrotic mediators in the ischemic kidney and stained for markers of tubular dam- TIMP-1 KIM-1 age and inflammation. 14 † 350 Gene Expression in Renal Cortex 12 †† 300 In comparison with normal rats, vehicle-treated unilateral I/R 10 250 ## rats had a significant increase in the gene expression of the 8 * ## 200 inflammation marker monocyte chemoattractant protein 1 6

fold induction fold 150 (MCP-1); tubular injury marker kidney injury molecule 1 4 induction fold (Kim-1); and fibrosis markers ␣ smooth muscle actin 100 2 (␣-SMA), TGF-␤1, procollagen I␣1, and tissue inhibitor of 1 metalloproteinase 1 (TIMP-1; Figure 3). Intriguingly, Y27632- 0 0 LZM inhibited the increase of these genes substantially, Figure 3. Renal gene expression of MCP-1, TGF-␤1, ␣-SMA, whereas free drug at the same low equimolar dosage did not procollagen I␣1, TIMP-1, and Kim-1 in normal rats and vehicle- show any beneficial effects. treated, Y27632-LZM–treated, and Y27632-treated rats after unilateral I/R injury. Data are means Ϯ SEM. †P Ͻ 0.05 and ††P Ͻ Immunohistochemistry 0.01, normal rats versus vehicle-treated I/R rats; *P Ͻ 0.05 and As shown in Figure 4, Kim-1 was highly expressed in tubular **P Ͻ 0.01, versus vehicle-treated I/R rats; #P Ͻ 0.05 and ##P Ͻ cells of vehicle-treated I/R rats, and tubules were largely di- 0.01, versus Y27632-treated I/R rats.

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A B A B

C D C D

E 40 E 4 †† †††

30 3 ** ## * # 20 2

10 1 % KIM-1 expression per expression field KIM-1 %

0 field cells per positive vimentin of score 0 l l le M 32 ma icle ZM 632 rma hic -LZ 76 or eh 2-L 27 No ve 32 Y2 N + v 63 + Y /R + 276 R + I/R Y27 I/R I + Y I/ + I/R I/R

Figure 4. (A through D) Representative photomicrographs of Figure 5. (A through D) Representative photomicrographs of Kim-1 immunostaining (periodic acid-Schiff [PAS] counterstained) vimentin staining in normal (A), vehicle-treated (B), Y27632-LZM– in normal (A), vehicle-treated (B), Y27632-LZM–treated (C), and treated (C), and Y27632-treated (D) rats. Pictures show that ex- Y27632-treated (D) rats. Kim-1 staining indicates injured tubular pression of vimentin, a dedifferentiation marker in tubular cells, cells. Pictures indicate that Kim-1 expression was increased after was significantly increased after I/R, particularly in corticomedul- I/R, which was subsequently reduced after the treatment with lary areas. Moreover, arrowheads show that tubular cells were Y27632-LZM. No effect of free Y27632 was observed. Counter- shed from tubules and accumulated in the tubular lumen. Treat- staining with PAS indicates the morphology of tubular cells. Treat- ment with Y27632-LZM markedly reduced the expression of vi- ment with Y27632-LZM clearly prevented the I/R-induced dilation mentin and prevented the shedding of cells, whereas free Y27632 of tubules. (E) Semiquantification of Kim-1 expression per inter- showed no effects. PAS counterstaining was used for vimentin. (E) stitial field. †††P Ͻ 0.001, normal rats versus vehicle-treated I/R Scoring for vimentin staining. ††P Ͻ 0.01, normal rats versus rats; *P Ͻ 0.05, versus vehicle-treated I/R rats; #P Ͻ 0.05, versus vehicle-treated I/R rats; **P Ͻ 0.01, versus vehicle-treated I/R rats; Y27632-treated I/R rats. Magnification, ϫ200. ##P Ͻ 0.01, versus Y27632-treated I/R rats. Magnification, ϫ200. lated. The degree of tubular dilation was well correlated with tin expression and showed loss of E-cadherin, similar to the Kim-1 expression in tubular cells. Treatment with Y27632- vehicle-treated I/R rats. LZM reduced this Kim-1 expression by Ͼ30% (Figure 4, C and Renal inflammation was clearly detectable in the damaged kid- E). In contrast, free drug did not inhibit Kim-1 expression. We neys. There was an enormous influx of monocytes/macrophages investigated the effect of Y27632-LZM on the epithelial-mes- in the renal cortex and the corticomedullary area at 4 d after I/R enchymal transition of tubular cells by staining for the dedif- (Figure 8A). Treatment with Y27632-LZM reduced this inflam- ferentiation marker vimentin (Figure 5) and the epithelial cell matory cell influx by 50 and 65% compared with vehicle-treated marker E-cadherin (Figure 6). In addition, we double-stained and Y27632-treated groups, respectively (Figure 8, A and B). for these markers and the tubular marker megalin (Figure 7). These data are in good agreement with the observed inhibition of Vimentin expression was strongly upregulated and E-cadherin MCP-1 gene expression by Y27632-LZM. expression was diminished in tubular cells at day 4 after I/R We investigated the onset of fibrotic process in ischemic kid- injury. This phenomenon was more pronounced in tubular ney injury by immunostaining for ␣-SMA, collagen I and III, and cells of the corticomedullary region as compared with renal fibronectin. Expression of ␣-SMA, indicating fibroblast activa- cortex (data not shown). We also observed that vimentin pos- tion, was appreciably increased in the tubulointerstitium after I/R, itive tubular cells detached from tubules and accumulated in which was subsequently attenuated by Y27632-LZM (Figure 8, A the tubular lumen as indicated in Figure 5 (arrows). Y27632- and C). In contrast, free drug did not show any effect. Further- LZM substantially inhibited expression of vimentin (Figures 5 more, we detected an intense deposition of extracellular matrix and 7) and prevented loss of E-cadherin in tubular cells (Fig- proteins collagen I, collagen III, and fibronectin in the ischemic ures 6 and 7). On the contrary, free drug did not reduce vimen- kidneys of control rats, underscoring that fibrosis was established

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Vimentin (blue) / E-cadherin (blue) / A B megalin (red) megalin (red) Normal

C D I/R + vehicle I/R

E 5

*** ### 3 I/R + Y27632-LZM I/R ††† 2

0 + Y27632 I/R score for e-cadherin expression per field al le 2 rm hic LZM 763 No ve 32- Y2 /R + 276 R + I + Y I/ I/R

Figure 6. (A through D) Representative photomicrographs of Figure 7. Representative photomicrographs for the double im- E-cadherin staining in normal (A), vehicle-treated (B), Y27632- munostainings of vimentin/megalin (left column) and E-cadherin/ LZM–treated (C), and Y27632-treated (D) rats. Pictures show megalin (right column) in normal, vehicle-treated, Y27632-LZM– that E-cadherin expression, an epithelial cell marker, was de- treated, and Y27632-treated I/R rats. In the left column, blue creased after I/R. Treatment with Y27632-LZM reverted the loss staining indicates the co-localization of vimentin expression in of expression. Light brown color of E-cadherin staining indi- proximal tubular cells visualized with red color staining for mega- cates proximal tubular cells, and dark brown color indicates lin at its brush border. In the right column, E-cadherin (blue distal tubular cells. Hematoxylin counterstaining was used to staining) is co-localized with proximal tubular cells (red color). ϫ stain nuclei. (E) Scoring for E-cadherin staining. †††P Ͻ 0.001, Magnification, 400. normal rats versus vehicle-treated I/R rats; ***P Ͻ 0.001, versus vehicle-treated I/R rats; ###P Ͻ 0.001, versus Y27632-treated DISCUSSION I/R rats. Magnification, ϫ200. The ROCK pathway is an endogenous regulator of proliferation, migration, and apoptosis of renal tubular cells25 and par- at 4 d after the I/R insult (Figure 9). Y27632-LZM significantly ticipates in the infiltration of inflammatory cells and the dedif- reduced the deposition of both types of collagen, whereas free ferentiation of epithelial tubular cells.6,7,9 Because all of these drug reduced collagen expression to a lesser extent. In addition, processes are important aspects of I/R-induced tubular dam- conjugate attenuated the expression of fibronectin, but free drug age, ROCK inhibition within proximal tubular cells may be an did not reduce it (Figure 9). attractive strategy to treat I/R-induced renal injury. Such a To investigate whether beneficial effects of Y27632-LZM strategy will increase the renal accumulation of the drug in the were related to inhibition of Rho kinase pathway in tubular kidneys, thereby increasing its efficacy, and will avoid other cells, we immunostained for the phosphorylation of the down- cells that may respond to ROCK inhibition. This is the first stream Rho-kinase substrate myosin light chain-2 (p-MLC2 study to demonstrate the delivery of the ROCK inhibitor staining; Figure 10). p-MLC2 expression (blue color) was sub- Y27632 to a specific cell type by means of a drug-carrier con- stantially enhanced in proximal tubular cells and co-localized jugate. In addition, our novel drug-linking technology pro- with megalin expression (red color; Figure 10B). Treatment vides a slow and prolonged intracellular release of drug within with Y27632-LZM significantly reduced Rho kinase activity in target cells, which can impede the activated ROCK pathway for tubular cells and interstitial fibroblasts as illustrated by re- a prolonged duration. duced p-MLC2 expression (Figure 10, C and E). No inhibition Low molecular weight proteins such as LZM filter freely with Y27632 alone was observed. Taken together, these data through the glomerular membrane and are taken up by indicate that Y27632-LZM affected I/R-induced tubular dam- megalin/gp330 receptor present at brush border of proxi- age, renal inflammation, and fibrosis by inhibiting the Rho mal tubular cells. The high renal expression levels of mega- kinase pathway. lin and its high internalization rate and easy accessibility

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A ED-1 -SMA A Collagen-I Collagen-III Fibronectin Normal Normal I/R + vehicle I/R I/R + vehicle I/R I/R + Y27632-LZM I/R I/R + Y27632 I/R I/R + Y27632-LZM I/R

Collagen-I B 4 ††† Collagen-III Fibronectin ††† ††

3 *

I/R + Y27632 I/R 2

B C scoreaverage per field 1 200 25 ††† 180 0 160 20 al le 2 rm hic LZM 763 # No ve 32- Y2 /R + 276 /R + 140 I + Y I ††† I/R 120 15 100 Figure 9. (A) Representative photomicrographs of collagen I, 80 * # 10 collagen III, and fibronectin staining in normal (n ϭ 4), vehicle- 60 treated (n ϭ 8), Y27632-LZM–treated (n ϭ 7), and Y27632-treated

per tubulointerstitial field tubulointerstitial per 40 5 ϭ 20 positive-SMA tubulointerstitial area (n 6) rats. Pictures show that fibrotic markers collagen I and III α number of infiltrated macrophages macrophages infiltrated of number

0 % 0 and fibronectin were highly deposited in the tubulointerstitium at l e 2 l e 2 rma icl ZM 63 ma icl ZM 63 No veh 2-L Y27 Nor veh 2-L Y27 + 763 + + 763 + I/R Y2 I/R I/R Y2 I/R 4 d after unilateral I/R. Treatment with Y27632-LZM strongly R + R + I/ I/ reduced the deposition of both collagen types and fibronectin, Figure 8. (A) Representative photomicrographs of ED-1 and whereas free Y27632 showed less reduction. Hematoxylin coun- ␣-SMA immunostainings in normal, vehicle-treated, Y27632- terstaining was used to stain nuclei. (B) Semiquantification data †† Ͻ LZM–treated, and Y27632-treated I/R rats. In the first column, for interstitial deposition of collagens and fibronectin. P 0.01, ††† Ͻ Ͻ brown dots indicate ED-1–positive cells (infiltrated macrophages) P 0.001, normal rats versus vehicle-treated I/R rats; *P ϫ in tubulointerstitial area of cortex and corticomedullary regions. In 0.05, versus vehicle-treated I/R rats. Magnification, 100. the second column, brown color staining for ␣-SMA in I/R rats demonstrates the accumulation of activated fibroblasts in the the kidneys and, more important, within the proximal tu- tubulointerstitial space of the renal cortex. PAS counterstaining bular cells, in accordance with our previous studies with depicts the renal morphology. (B and C) Semiquantification of the drug-LZM conjugates.20,21,26 It is important to note that number of infiltrated macrophages and ␣-SMA–positive area per Y27632 was not released from the conjugate in serum as tubulointerstitial field. †††P Ͻ 0.001, normal rats versus vehicle- treated I/R rats; *P Ͻ 0.05, versus vehicle-treated I/R rats; #P Ͻ determined by in vitro drug release studies as well as by 0.05, versus Y27632-treated I/R rats. Magnification, ϫ100. pharmacokinetics data in vivo. Thus, free drug is not formed during the residence of the conjugate in the blood stream, from the lumen of tubules make it an ideal receptor for and effects of the conjugate will be confined to the kidney in tubular cell–specific drug delivery. The renal-specific con- which the conjugate is accumulated. A pronounced extra- jugate Y27632-LZM accumulated rapidly and extensively in renal activity of the conjugate is therefore not expected, as

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A B staining in tubular cells. The importance of ROCK signaling in the transdifferentiation of renal tubular cells has already been demonstrated in vitro,9 and we now confirm this by in vivo local inhibition of the pathway. The beneficial effects of Y27632-LZM on tubular cells was also shown by the effect on tubular dilation; however, the con- D C jugate did not improve renal function in the unilateral model and showed even slightly negative effects in the bilateral model, whereas untargeted Y27632 in both studies deteriorated renal function. Most in vivo studies with Y27632, except one study,36 reported antifibrotic effects without reporting renal function E parameters.12,37,38 Studies with isolated perfused kidneys have 5 demonstrated that Y27632 strongly affects the vascular tone of ††† 4 the afferent arteriole.39,40 The observed negative effects of Y27632 might relate to an effect on renal blood flow, resulting 3 * ## in a loss of autoregulation after the ischemic insult. Effects of 2 the renally delivered drug should relate to drug originating from Y27632-LZM conjugate and accumulating within the 1 proximal tubular cells. How this may have affected renal blood score for pMLC2 expression per field per pMLC2 expression for score

0 flow or renal function needs to be investigated further. al le 2 rm hic LZM 763 No ve 32- Y2 /R + 276 /R + I + Y I I/R During I/R injury, infiltration of monocytes/macrophages plays an important role in initiation of acute renal Figure 10. (A through D) Representative photomicrographs of failure.41 MCP-1 is a potent chemoattractant for mono- double staining for p-MLC2 and megalin in normal (A), vehicle- cytes/macrophages and has been well correlated with mac- treated (B), Y27632-LZM–treated (C), and Y27632-treated (D) rats. 42 Pictures show that p-MLC2 expression (blue color), an indicator of rophage recruitment in postischemic kidneys. Various the activation of the Rho kinase pathway, was increased in tubular studies have shown that ROCK inhibition blunted mono- cells (visualized by red color staining for megalin) and in tubu- cytes/macrophage infiltration and interstitial fibrosis in re- lointerstitium after I/R injury. Treatment with Y27632-LZM inhib- nal disease models and related this to direct effects in mac- ited the activation of the Rho kinase pathway in tubular cells and rophages or local renal effects.12,43,44 Because Y27632-LZM in adjacent interstitial cells. No effect of only Y27632 was found. strongly accumulates in the kidney but not in immune cells, (E) Semiquantification data for p-MLC2 staining. †††P Ͻ 0.001, we now provide further insight into the local renal effects, normal rats versus vehicle-treated I/R rats; *P Ͻ 0.05, versus suggesting that inhibition of macrophage infiltration was ## Ͻ vehicle-treated I/R rats; P 0.01, versus Y27632-treated I/R due to local inhibition of MCP-1 in kidneys. Furthermore, ϫ rats. Magnification, 400. Y27632-LZM significantly reduced the gene expression of other mediators, such as the profibrotic factors TGF-␤1, also can be deduced from the absence of effects of Y27632- TIMP-1, procollagen I␣1, and ␣-SMA, and substantially re- LZM on the number of white blood cells. duced the interstitial deposition of extracellular proteins Renal I/R is a common cause of acute renal failure and is collagen I and III and fibronectin. In addition, we found associated with tubular damage accompanied by tubuloin- that Y27632 attenuated TGF-␤1–induced procollagen I␣1 terstitial inflammation and fibrosis.27,28 Vimentin, a marker gene expression in HK-2 renal tubular cells in vitro (unpub- for epithelial-mesenchymal transdifferentiation, is ex- lished data). These data indicate that activated ROCK sig- pressed in a cellular and segmental pattern after I/R,29–31 naling within tubular epithelial cells may be crucial for the which was also observed in this study. E-cadherin is an ad- regulation of inflammation and fibrosis during I/R injury; hesive junction protein that maintains the structural integ- and cell-selective inhibition of this pathway is now possible rity and polarity of renal epithelial cells32 and is lost after I/R with our targeting strategy. The absence of renal effects of in these cells.33 Kim-1 is a membrane protein maximally the free drug might be due to the relatively low dosage (0.5 upregulated in proliferating and dedifferentiated tubular mg/kg per d intravenously), which was equivalent to the epithelial cells after renal ischemia and is associated with amount of drug in the Y27632-LZM conjugate. Although their loss of polarity and development of interstitial fibro- Y27632 is a hydrophilic compound that may be eliminated sis.34,35 The observed effects of Y27632-LZM on the tubular via the kidney, it is unlikely that the free drug will accumu- expression of vimentin, E-cadherin, and Kim-1 strongly late within tubular cells because it is not a known substrate demonstrate that the drug-LZM conjugate becomes phar- for active tubular reabsorption. macologically active within the kidneys. Furthermore, we In conclusion, this study demonstrated that renal-specific now demonstrate that such a local inhibition is related to an delivery of ROCK inhibitor Y27632 can be successfully inhibition of ROCK, as reflected by the reduced p-MLC2 achieved using Y27632-LZM conjugate, and blockade of the

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ROCK pathway locally within renal tubular cells can be a calibration curves in the corresponding biologic matrices to quantify promising approach to inhibit renal injury because the conju- drug levels. gate exhibited significant pharmacologic effects by inhibiting Animal Experiments both inflammatory and fibrotic factors. All experimental protocols for animal studies were approved by the Animal Ethics Committee of the University of Groningen. Normal male Wistar rats (220 to 240 g) were obtained from Harlan (Zeist, CONCISE METHODS Netherlands).

Synthesis and Characterization of Y27632-LZM Pharmacokinetics of Y27632-LZM Conjugate Rats (N ϭ 5) were administered an intravenous injection of a single dose Y27632 [(ϩ)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexan- of the Y27632-LZM conjugate (20 mg/kg equivalent to 555 ␮g/kg of ecarboxamide dihydrochloride monohydrate] was purchased from Toc- Y27632, dissolved in 5% glucose). Injections were performed in the ris (Bristol, UK). LZM was obtained from Sigma-Aldrich (St. Louis, penile vein under anesthesia (2% isoflurane in 2:1 O2/N2O, 1 L/min). MO). Cis-[Pt(ethylenediamine)nitrate-chloride] (ULS) was freshly Rats were killed (n ϭ 1) at 5, 30, 60, 120, and 360 min under anesthe- prepared from cis-[Pt(ethylenediamine)dichloride as described previ- sia. Blood samples were collected by heart puncture, and kidneys were ously.45 Y27632 (2.95 ␮mol, 10 mg/ml in water) was basified with 1 M isolated after gentle flushing of the organs with saline through the NaOH to pH 8 and reacted with cisULS (4.43 ␮mol) at 50°C overnight. abdominal aorta. Kidneys were weighed, homogenized (1:3 wt/vol, Consumption of the starting drug and formation of the products were PBS) and then stored at Ϫ80°C. To determine Y27632 in serum or followed by HPLC and liquid chromatography–mass spectrometry. The tissues, samples were incubated with 0.5 M potassium thiocyanate at reaction mixture was evaporated to dryness under reduced pressure, 80°C for 24 h to release Y27632 from the linker followed by HPLC affording a pale yellow solid (yield 50%) that consisted of the desired analysis. Anti-LZM staining was performed on the frozen kidney sec- 1:1 reaction product. Y27632-ULS was subsequently reacted with LZM tions to detect the cellular localization of the conjugate in the kidneys. according to a similar protocol as described for the synthesis of 20 ␮ SB202190-ULS-LZM previously. In brief, LZM (0.7 mol) equipped Effect of Y27632-LZM in Unilateral Renal I/R Model in with surface-exposed methionine groups by the Boc-L-methionine hy- Rats droxysuccinimide ester reagent was dissolved in 0.02 M tricine/sodium The pharmacologic efficacy of Y27632-LZM was evaluated in the uni- nitrate buffer (pH 8.5). After addition of Y27632-ULS (3.5 ␮mol), the lateral I/R rat model. Rats were divided into four groups: Untreated mixture was reacted at 37°C for 24 h. The product was purified by dialysis normal rats (n ϭ 4), vehicle-treated I/R rats (5% glucose; n ϭ 12), against water for 48 h, filtered, lyophilized, and stored at Ϫ20°C. Mass Y27632-LZM ϩ I/R (20 mg/kg equivalent to 555 ␮g/kg Y27632; n ϭ spectrometry analysis confirmed the formation of Y27632-LZM conju- 7), Y27632 ϩ I/R (555 ␮g/kg; n ϭ 6). At 2 h before the ischemia gate. The amount of conjugated drug was quantified after competitive procedure, rats were administered a preinjection of these compounds. displacement of the drug from the conjugate by overnight incubation Compounds were administered intravenously via the penis vein as with 0.5 M potassium thiocyanate in PBS at 80°C, by HPLC analysis as described already. Rats were allowed to recover and placed back into described in the next section. The absence of free drug in the preparation the cages until the induction of renal ischemia. Rats were incised from was investigated by HPLC analysis of freshly prepared dilutions of the the abdomen under anesthesia, and the left renal artery and vein were conjugate in PBS. clamped for 45 min to stop renal blood flow. Clamps were removed, Y27632-LZM was characterized for its stability and drug-release and reperfusion of the kidney was observed before closing of the properties. The conjugate (100 ␮g/ml) was incubated in different me- wound. Rats were administered another injection of the compounds dia: 0.1 M PBS, 5 and 50 mM glutathione (reduced form) in PBS, and at 24, 48, and 72 h after I/R. Twenty-four hours after the last dose, the serum. After 24 h of incubation at 37°C, 100-␮l aliquots were taken rats were killed and blood samples were collected from abdominal and processed immediately for HPLC analysis of Y27632. aorta. Kidneys were isolated after gentle perfusion with saline and weighed, and small pieces were snap-frozen in liquid nitrogen for mRNA HPLC Analysis of Y27632 isolation and put in ice-cold isopentane for preparation of cryosections Y27632 was analyzed on a Waters liquid chromatograph (Waters, or in 4% formalin solution to make paraffin-embedded sections.

Milford, MA). Separations were performed on a C18 reversed-phase SunFire column (Waters) using a mobile phase of water-methanol- Determination of mRNA Expression trifluoroacetic acid (86:14:0.1, vol/vol/vol; pH 2.0) at a flow rate of 1 Total RNA was isolated from renal cortex using Bio-Rad’s Aurum Total ml/min. The effluents were monitored at 270 nm. Y27632 eluted at a RNA Mini kit (Bio-Rad, Hercules, CA). RNA content was measured by a retention time of 5.7 min. Serum and kidney homogenates (200 ␮l) NanoDrop UV-detector (NanoDrop Technologies, Wilmington, DE). were treated with potassium thiocyanate as explained already to re- cDNA was synthesized from similar amounts of RNA using the Super- lease bound drug and extracted twice with 2 ml of diethyl ether after script III first-strand synthesis kit (Invitrogen, Carlsbad, CA). Gene ex- addition of 100 ␮l of 2 N NaOH. After freezing of the aqueous layer in pression levels for the various genes were measured by quantitative real- liquid nitrogen, the organic layer was decanted and evaporated at time reverse transcription–PCR (Applied Biosystems, Foster City, CA) as 50°C. The residue was reconstituted in 200 ␮l of eluents, and 50 ␮l was explained previously.20 The primers (Table 2) for rat species were ob- injected into the HPLC. The peak heights were recorded and related to tained from Sigma-Genosys (Haverhill, UK). SYBR Green PCR Master

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Table 2. Primer sequences for real-time PCR analysisa Product Primer Forward Reverse (bp) MCP-1 5Ј-TCC TCC ACC ACT ATGCAGGT-3Ј 5Ј-TTC CTT ATTGGG GTCAGCAC-3Ј 255 TGF-␤15Ј-ATACGC CTG AGTGGCTGT CT 5Ј-TGGGAC TGA TCC CATTGA TT-3Ј 153 ␣-SMA 5Ј-GACACCAGGGAG TGATGG TT-3Ј 5Ј-GTTAGCAAG GTCGGATGC TC-3Ј 202 Procollagen I␣15Ј-AGC CTGAGCCAGCAGATTGA-3Ј 5Ј-CCAGGTTGC AGC CTTGGTTA-3Ј 145 TIMP-1 5Ј-GAGAGC CTC TGTGGATAT GT-3Ј 5Ј-CAGCCAGCA CTA TAGGTC TT-3Ј 334 Kim-1 5Ј-GTC TGT ATT GTTGCCGAGTG-3Ј 5Ј-GGT CTT GTTGGAGGACTT GT-3Ј 106 GAPDH 5Ј-CGCTGG TGC TGA GTATGTCG-3Ј 5Ј-CTGTGG TCA TGAGCCCTTCC-3Ј 179 aGAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Mix (Applied Biosystems, Warrington, UK) was used as a fluorescence flux, ED-1–positive cells were counted using computerized morphome- probe for real-time reverse transcription–PCR. The threshold cycle num- try in approximately 30 fields per section at the magnification of ϫ200. ber (Ct) was calculated for each gene, and relative gene expressions (fold For all analyses, both cortex and corticomedullary regions were consid- change) were calculated after normalizing for the expression of the con- ered, and in the case of ␣-SMA and ED-1, glomeruli and vascular areas were trol gene GAPDH using ⌬⌬Ct method. manually excluded. The amount of brown precipitate was measured and presented either as a percentage of the total selected area (␣-SMA and Kim-1) Immunohistochemistry For detection of LZM, megalin, ED-1 (Figure 2H), collagen I, collagen III, or as estimated number of macrophages in the selected fields. Vimentin, E- fibronectin, and p-MLC2 in tissues, cryostat sections (4 ␮m thick) were cadherin, collagen I and III, and p-MLC2 staining were scored with five stained as described previously20 except that for p-MLC2, sections were grades as follows: 0.5, very weak; 1, weak; 2, moderate; 3, strong; and 4, very incubated with primary antibody overnight at 4°C. Primary antibodies strong. Grades were given by analysis of complete kidney section in different ϫ such as rabbit anti-LZM polyclonal antibody (Chemicon, Temecula, fields at the magnification of 40. All morphometric measurements and CA), goat anti-megalin polyclonal antibody (Santa Cruz Biotechnolo- gradings were performed in a blinded manner. gies, Santa Cruz, CA), goat anti–collagen I (Southern Biotech, Birming- ham, AL), goat anti–collagen III (Southern Biotech), rabbit anti-fi- Statistical Analysis bronectin (Telios Pharmaceutical, San Diego, CA), and mouse phospho- Statistical analyses were performed using the unpaired t test. P Ͻ 0.05 myosin light chain (Ser19) mAb (Cell Signaling Technology, Beverly, was considered as the minimal level of significance. Data are pre- MA) were used. Double stainings were performed on frozen tissues by sented as means Ϯ SEM. Pharmacokinetic analysis of the serum incubating sections with primary antibodies and then their respective Y27632 concentrations was performed using the Multifit program secondary antibodies, one labeled with horseradish peroxidase to pro- (Department of Pharmacokinetics and Drug Delivery, University of duce red color with 3-amino-9-ethylcarbazole and the other labeled with Groningen, Groningen, Netherlands). alkaline phosphatase to produce blue color with naphthol AS phosphate and fast blue RR mixture. Other immunohistochemical stainings were performed on 3-␮m-thick paraffin-embedded sections. Sections were ACKNOWLEDGMENTS deparaffinized in xylene and rehydrated in alcohol and distilled water. Heat-induced antigen retrieval was achieved by incubation in a micro- This work was supported by a grant from the European Framework wave for 15 min at 300 W with 1 mM EDTA (pH 8.0) for vimentin, 15 program FP6 (DIALOK, LSHB-CT-2007-036644). min at 300 W with 0.1 M Tris/HCl (pH 9.0) for E-cadherin, or overnight We are grateful to Annemiek van Loenen-Weemaes, Catharina at 80°C in 0.1 M Tris/HCl buffer (pH 9.0) for ED-1 and ␣-SMA. Endog- Reker-Smit, and Mieke Zeinstra-Smith (Department of Pharmacoki- enous peroxidase activity was blocked with 0.03% H O /sodium azide 2 2 netics and Drug Delivery) and Marian Bulthuis and N.J. Kloosterhuis for 5 min. Slides were then incubated with primary antibodies against (Department of Pathology) for technical assistance. Colleagues at Kim-1 (antibody against the intracellular domain of Kim-1: Peptide 9; Kreatech are acknowledged for critical reading of the manuscript. We Biogen, Cambridge, MA), vimentin (DAKO, Glostrup, Denmark), E- are thankful to Prof. R.H. Henning for scientific discussions. cadherin (BD Transduction Labs, San Diego, CA), ␣-SMA (clone 1A4, Sigma, MO), or the rat macrophage marker ED-1 (Serotec, Oxford, UK). Binding was detected by sequential incubation with peroxidase-labeled DISCLOSURES secondary antibodies (DAKO) in the presence of 1% normal rat serum None. for 30 min. The peroxidase activity was visualized using 3,3Ј-diamino- benzidine tetrahydrochloride (DAKO) for 10 min. Periodic acid-Schiff base staining was performed by standard protocol to observe renal mor- REFERENCES phology. The extent of tubular Kim-1 and interstitial ␣-SMA expression was determined in approximately 30 interstitial rectangular fields per 1. Sharples EJ, Thiemermann C, Yaqoob MM: Mechanisms of disease: ϫ section using computerized morphometry at the magnification of 100 Cell death in acute renal failure and emerging evidence for a protec- and ϫ200, respectively. For determination of interstitial macrophage in- tive role of erythropoietin. Nat Clin Pract Nephrol 1: 87–97, 2005

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