BASIC RESEARCH

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

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
JASN November 2008, 19 (11) 2086-2097; DOI: https://doi.org/10.1681/ASN.2007070794

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 Iα1, TGF-β1, tissue inhibitor of metalloproteinase 1, and α-smooth muscle actin. Immunohistochemistry revealed reduced macrophage infiltration and decreased expression of α-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.

Renal ischemia-reperfusion (I/R) injury is the main cause of acute renal failure after major renal surgery, trauma, or transplantation.1–3 After I/R injury, proximal tubular cells lose their polarity and detach from the tubules as a result of cytoskeletal reorganization, which is mainly regulated by RhoGTPases.4,5 RhoGTPases belong to the Ras superfamily of GTP-binding proteins and act by stimulating downstream Rho effectors such as Rho-associated coiled-coil–forming protein kinase (ROCK). In addition to cytoskeletal regulation, RhoGTPases are involved in the infiltration of inflammatory cells,6,7 which is an imperative process after I/R injury.8 Moreover, activation of RhoGTPases leads to the transdifferentiation of renal tubular cells to fibroblasts, which turn into myofibroblasts.9

We hypothesized that blockade of the ROCK pathway locally within kidneys may be an interesting approach to treat renal injury. The efficacy of the widely known ROCK inhibitor Y27632 has been demonstrated in various I/R injury models10,11 and in the unilateral ureteral obstruction model12; however, the ROCK pathway is also involved in the contraction of vascular smooth muscle cells and plays an important role in regulation of vascular tone.13–15 Last, Rho kinase signaling is important in migration and activation of immune cells.16–18 Systemic administration of ROCK inhibitors can therefore lead to adverse effects.

Considering the systemic role of Rho signaling, we now propose renal-specific delivery as a targeted approach to investigate renal effects of ROCK inhibitors. Renal-specific delivery can avoid interactions with nontarget cells and thereby decrease adverse effects. Furthermore, renal drug delivery will enhance the drug's accumulation in the targeted organ, which potentially can improve therapeutic efficacy at lower dosages to be used. In previous studies, we showed that renal-specific drug delivery can be achieved using the low molecular weight protein lysozyme (LZM) as drug carrier.19–21 LZM filters through the glomerulus and is reabsorbed by tubular cells via the megalin receptor,22 and we have made use of this process.

We recently developed a versatile linking technology, the so-called Universal Linkage System (ULS), for the coupling of drugs to carrier systems,20,21,23,24 which we now have applied for the conjugation of the Rho kinase inhibitor Y27632 to LZM. We investigated the potential activity of Y27632-LZM in the ischemia-reperfusion (I/R) injury model and demonstrated both enhanced activity and increased selectivity of the tubular-targeted Rho kinase inhibitor.

RESULTS

Synthesis and Characterization of Y27632-ULS-LZM

Y27632 was conjugated to ULS and then coupled to the carrier LZM at a 1:1 drug-to-protein ratio (Figure 1A). Stability studies demonstrated that this type of conjugate is highly stable in buffers and serum for 24 h at 37°C. In contrast, addition of glutathione to the medium afforded release of drug that was concentration dependent. Approximately 5 and 9% of the bound drug was released in 5 and 50 mM glutathione, respectively, in 24 h. No release was found with serum and PBS in 24 h. From these data, we concluded that the coupled Y27632 drug can be displaced competitively by endogenous ligands that are normally present within the cytosol.

Figure 1.
Figure 1.

(A) Y27632 was chemically modified and conjugated to LZM. (B) Scheme of the renal uptake mechanism of Y27632-LZM in the proximal tubular cells. After an intravenous injection, the conjugate filters through the glomerular membrane and is internalized via megalin receptors by proximal tubular cells. Drug is released slowly from the conjugate within these cells, and the released drug exhibits its activity by blocking kinase signaling cascade.

Pharmacokinetics of the Y27632-LZM Conjugate

We investigated the renal accumulation of Y27632-LZM after intravenous administration to rats. The serum-disappearance curve of Y27632-LZM, which followed a two-compartment pharmacokinetic model, is shown in Figure 2A. Only carrier-bound Y27632 was detected in the serum, whereas free drug was absent. Furthermore, in accordance with other drug-LZM conjugates, Y27632-LZM accumulated efficiently in the kidneys 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 surface of proximal tubular cells (Figure 1B). Anti-LZM staining corresponded closely to the anti-megalin receptor staining in kidneys (Figure 2D). We also examined the accumulation of Y27632-LZM in other organs. At 2 h after its administration, staining for LZM was strongly positive in kidneys (Figure 2E), whereas only minor levels of the conjugate were detectable in the sinusoidal area of liver (Figure 2F), in lungs, and in the spleen (Figure 2, G and H). Spleen accumulation of the conjugate was mainly localized in the interstitium not corresponding with T and B lymphocytes in the white pulp or with the localization of macrophages and granulocytes, as visualized by staining for ED-1 and His48 (Supplemental Figure 1, A and B). Anti-LZM and anti–ED-1 staining was present in the same area, but the staining patterns were quite different. The tissue distribution of the conjugate at later time points (t = 6 h) showed similarly high levels within the kidney, whereas the staining in other organs was faint (Supplemental Figure 1, C through F). These results indicate that Y27632-LZM was specifically accumulated in kidneys but not deposited in other organs, which is in agreement with the clearance of the conjugate via renal elimination.

Figure 2.
Figure 2.

(A and B) Serum (A) and renal levels (B) of Y27632-LZM after administration of Y27632-LZM intravenously at a dosage of 20 mg/kg (equivalent to 555 μg/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 after 1 h of intravenous injection of Y27632-LZM conjugate by anti-LZM staining (red color, arrowheads). (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: ×400 in C; ×200 in E through H.

Effect of Renal-Delivered Y27632 in the I/R Model in Rats

The efficacy of Y27632-LZM was evaluated in the unilateral I/R model because this animal model is characterized by an early inflammatory phase that finally leads to fibrosis. In addition, we evaluated the effects of ROCK inhibition in the bilateral I/R model. Drug treatment was started simultaneously with the induction of I/R to intervene in the early inflammatory and fibrotic events. Because drug release from the conjugate would provide continuous drug levels for a prolonged period of time,20 we treated the animals with once-daily doses.

Kidney Weights, Serum Creatinine Levels, and Blood Cell Counts

After 4 d of unilateral I/R, the weights of ischemic kidneys were significantly increased as compared with contralateral kidneys or kidneys of normal rats (Table 1). Interestingly, treatment with Y27632-LZM reduced this increase in kidney weight, whereas free drug did not affect it. In the unilateral I/R model, renal function was hardly affected as a result of presence of an intact contralateral kidney, and we found only a subtle increase in serum creatinine levels of control rats at 4 d after I/R. Treatment with Y27632-LZM slightly improved serum creatinine levels, whereas free drug even worsened it.

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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

In the bilateral I/R model, both serum creatinine and blood urea nitrogen levels were several-fold increased at days 1 and 3 after the ischemic insult (Supplemental Figure 2). Rats treated with Y27632-LZM showed higher creatinine and blood urea nitrogen levels at both day 1 and day 3 as compared with the vehicle-treated group, which was most prominent in one of the animals. Free Y27632 deteriorated renal function and did so in a more pronounced manner than Y27632-LZM. These data suggest that ROCK inhibition affected renal function negatively, especially during the first day after bilateral I/R injury. The stronger effects by free Y27632 as compared with Y27632-LZM may relate to an effect outside the tubular cells.

To investigate whether systemic administration of Y27632 has hematologic effects, we counted red blood cells and white blood cells at the end of the treatment period in the unilateral I/R model (Table 1). We found that free drug significantly decreased the number of white blood cells, whereas Y27632-LZM did not display any inhibitory effect. Both free Y27632 and conjugated drug did not influence red blood cells. These data already demonstrate the altered pharmacologic profile of Y27632-LZM as compared with the free drug. To provide insight into the mechanism of action of Y27632-LZM, we determined the expression of inflammatory and fibrotic mediators in the ischemic kidney and stained for markers of tubular damage and inflammation.

Gene Expression in Renal Cortex

In comparison with normal rats, vehicle-treated unilateral I/R rats had a significant increase in the gene expression of the inflammation marker monocyte chemoattractant protein 1 (MCP-1); tubular injury marker kidney injury molecule 1 (Kim-1); and fibrosis markers α smooth muscle actin (α-SMA), TGF-β1, procollagen Iα1, and tissue inhibitor of metalloproteinase 1 (TIMP-1; Figure 3). Intriguingly, Y27632-LZM inhibited the increase of these genes substantially, whereas free drug at the same low equimolar dosage did not show any beneficial effects.

Figure 3.
Figure 3.

Renal gene expression of MCP-1, TGF-β1, α-SMA, procollagen Iα1, TIMP-1, and Kim-1 in normal rats and vehicle-treated, Y27632-LZM–treated, and Y27632-treated rats after unilateral I/R injury. Data are means ± SEM. †P < 0.05 and ††P < 0.01, normal rats versus vehicle-treated I/R rats; *P < 0.05 and **P < 0.01, versus vehicle-treated I/R rats; #P < 0.05 and ##P < 0.01, versus Y27632-treated I/R rats.

Immunohistochemistry

As shown in Figure 4, Kim-1 was highly expressed in tubular cells of vehicle-treated I/R rats, and tubules were largely dilated. The degree of tubular dilation was well correlated with Kim-1 expression in tubular cells. Treatment with Y27632-LZM reduced this Kim-1 expression by >30% (Figure 4, C and E). In contrast, free drug did not inhibit Kim-1 expression. We investigated the effect of Y27632-LZM on the epithelial-mesenchymal transition of tubular cells by staining for the dedifferentiation marker vimentin (Figure 5) and the epithelial cell marker E-cadherin (Figure 6). In addition, we double-stained for these markers and the tubular marker megalin (Figure 7). Vimentin expression was strongly upregulated and E-cadherin expression was diminished in tubular cells at day 4 after I/R injury. This phenomenon was more pronounced in tubular cells of the corticomedullary region as compared with renal cortex (data not shown). We also observed that vimentin positive tubular cells detached from tubules and accumulated in the tubular lumen as indicated in Figure 5 (arrows). Y27632-LZM substantially inhibited expression of vimentin (Figures 5 and 7) and prevented loss of E-cadherin in tubular cells (Figures 6 and 7). On the contrary, free drug did not reduce vimentin expression and showed loss of E-cadherin, similar to the vehicle-treated I/R rats.

Figure 4.
Figure 4.

(A through D) Representative photomicrographs of Kim-1 immunostaining (periodic acid-Schiff [PAS] counterstained) in normal (A), vehicle-treated (B), Y27632-LZM–treated (C), and Y27632-treated (D) rats. Kim-1 staining indicates injured tubular cells. Pictures indicate that Kim-1 expression was increased after I/R, which was subsequently reduced after the treatment with Y27632-LZM. No effect of free Y27632 was observed. Counterstaining with PAS indicates the morphology of tubular cells. Treatment with Y27632-LZM clearly prevented the I/R-induced dilation of tubules. (E) Semiquantification of Kim-1 expression per interstitial field. †††P < 0.001, normal rats versus vehicle-treated I/R rats; *P < 0.05, versus vehicle-treated I/R rats; #P < 0.05, versus Y27632-treated I/R rats. Magnification, ×200.

Figure 5.
Figure 5.

(A through D) Representative photomicrographs of vimentin staining in normal (A), vehicle-treated (B), Y27632-LZM–treated (C), and Y27632-treated (D) rats. Pictures show that expression of vimentin, a dedifferentiation marker in tubular cells, was significantly increased after I/R, particularly in corticomedullary areas. Moreover, arrowheads show that tubular cells were shed from tubules and accumulated in the tubular lumen. Treatment with Y27632-LZM markedly reduced the expression of vimentin and prevented the shedding of cells, whereas free Y27632 showed no effects. PAS counterstaining was used for vimentin. (E) Scoring for vimentin staining. ††P < 0.01, normal rats versus vehicle-treated I/R rats; **P < 0.01, versus vehicle-treated I/R rats; ##P < 0.01, versus Y27632-treated I/R rats. Magnification, ×200.

Figure 6.
Figure 6.

(A through D) Representative photomicrographs of E-cadherin staining in normal (A), vehicle-treated (B), Y27632-LZM–treated (C), and Y27632-treated (D) rats. Pictures show that E-cadherin expression, an epithelial cell marker, was decreased after I/R. Treatment with Y27632-LZM reverted the loss of expression. Light brown color of E-cadherin staining indicates proximal tubular cells, and dark brown color indicates distal tubular cells. Hematoxylin counterstaining was used to stain nuclei. (E) Scoring for E-cadherin staining. †††P < 0.001, normal rats versus vehicle-treated I/R rats; ***P < 0.001, versus vehicle-treated I/R rats; ###P < 0.001, versus Y27632-treated I/R rats. Magnification, ×200.

Figure 7.
Figure 7.

Representative photomicrographs for the double immunostainings of vimentin/megalin (left column) and E-cadherin/megalin (right column) in normal, vehicle-treated, Y27632-LZM–treated, and Y27632-treated I/R rats. In the left column, blue staining indicates the co-localization of vimentin expression in proximal tubular cells visualized with red color staining for megalin at its brush border. In the right column, E-cadherin (blue staining) is co-localized with proximal tubular cells (red color). Magnification, ×400.

Renal inflammation was clearly detectable in the damaged kidneys. There was an enormous influx of monocytes/macrophages in the renal cortex and the corticomedullary area at 4 d after I/R (Figure 8A). Treatment with Y27632-LZM reduced this inflammatory cell influx by 50 and 65% compared with vehicle-treated and Y27632-treated groups, respectively (Figure 8, A and B). These data are in good agreement with the observed inhibition of MCP-1 gene expression by Y27632-LZM.

Figure 8.
Figure 8.

(A) Representative photomicrographs of ED-1 and α-SMA immunostainings in normal, vehicle-treated, Y27632-LZM–treated, and Y27632-treated I/R rats. In the first column, brown dots indicate ED-1–positive cells (infiltrated macrophages) in tubulointerstitial area of cortex and corticomedullary regions. In the second column, brown color staining for α-SMA in I/R rats demonstrates the accumulation of activated fibroblasts in the tubulointerstitial space of the renal cortex. PAS counterstaining depicts the renal morphology. (B and C) Semiquantification of the number of infiltrated macrophages and α-SMA–positive area per tubulointerstitial field. †††P < 0.001, normal rats versus vehicle-treated I/R rats; *P < 0.05, versus vehicle-treated I/R rats; #P < 0.05, versus Y27632-treated I/R rats. Magnification, ×100.

We investigated the onset of fibrotic process in ischemic kidney injury by immunostaining for α-SMA, collagen I and III, and fibronectin. Expression of α-SMA, indicating fibroblast activation, was appreciably increased in the tubulointerstitium after I/R, which was subsequently attenuated by Y27632-LZM (Figure 8, A and C). In contrast, free drug did not show any effect. Furthermore, we detected an intense deposition of extracellular matrix proteins collagen I, collagen III, and fibronectin in the ischemic kidneys of control rats, underscoring that fibrosis was established at 4 d after the I/R insult (Figure 9). Y27632-LZM significantly reduced the deposition of both types of collagen, whereas free drug reduced collagen expression to a lesser extent. In addition, conjugate attenuated the expression of fibronectin, but free drug did not reduce it (Figure 9).

Figure 9.
Figure 9.

(A) Representative photomicrographs of collagen I, collagen III, and fibronectin staining in normal (n = 4), vehicle-treated (n = 8), Y27632-LZM–treated (n = 7), and Y27632-treated (n = 6) rats. Pictures show that fibrotic markers collagen I and III and fibronectin were highly deposited in the tubulointerstitium at 4 d after unilateral I/R. Treatment with Y27632-LZM strongly reduced the deposition of both collagen types and fibronectin, whereas free Y27632 showed less reduction. Hematoxylin counterstaining was used to stain nuclei. (B) Semiquantification data for interstitial deposition of collagens and fibronectin. ††P < 0.01, †††P < 0.001, normal rats versus vehicle-treated I/R rats; *P < 0.05, versus vehicle-treated I/R rats. Magnification, ×100.

To investigate whether beneficial effects of Y27632-LZM were related to inhibition of Rho kinase pathway in tubular cells, we immunostained for the phosphorylation of the downstream Rho-kinase substrate myosin light chain-2 (p-MLC2 staining; Figure 10). p-MLC2 expression (blue color) was substantially enhanced in proximal tubular cells and co-localized with megalin expression (red color; Figure 10B). Treatment with Y27632-LZM significantly reduced Rho kinase activity in tubular cells and interstitial fibroblasts as illustrated by reduced p-MLC2 expression (Figure 10, C and E). No inhibition with Y27632 alone was observed. Taken together, these data indicate that Y27632-LZM affected I/R-induced tubular damage, renal inflammation, and fibrosis by inhibiting the Rho kinase pathway.

Figure 10.
Figure 10.

(A through D) Representative photomicrographs of double staining for p-MLC2 and megalin in normal (A), vehicle-treated (B), Y27632-LZM–treated (C), and Y27632-treated (D) rats. Pictures show that p-MLC2 expression (blue color), an indicator of the activation of the Rho kinase pathway, was increased in tubular cells (visualized by red color staining for megalin) and in tubulointerstitium after I/R injury. Treatment with Y27632-LZM inhibited the activation of the Rho kinase pathway in tubular cells and in adjacent interstitial cells. No effect of only Y27632 was found. (E) Semiquantification data for p-MLC2 staining. †††P < 0.001, normal rats versus vehicle-treated I/R rats; *P < 0.05, versus vehicle-treated I/R rats; ##P < 0.01, versus Y27632-treated I/R rats. Magnification, ×400.

DISCUSSION

The ROCK pathway is an endogenous regulator of proliferation, migration, and apoptosis of renal tubular cells25 and participates in the infiltration of inflammatory cells and the dedifferentiation of epithelial tubular cells.6,7,9 Because all of these processes are important aspects of I/R-induced tubular damage, ROCK inhibition within proximal tubular cells may be an attractive strategy to treat I/R-induced renal injury. Such a strategy will increase the renal accumulation of the drug in the kidneys, thereby increasing its efficacy, and will avoid other cells that may respond to ROCK inhibition. This is the first study to demonstrate the delivery of the ROCK inhibitor Y27632 to a specific cell type by means of a drug-carrier conjugate. In addition, our novel drug-linking technology provides a slow and prolonged intracellular release of drug within target cells, which can impede the activated ROCK pathway for a prolonged duration.

Low molecular weight proteins such as LZM filter freely through the glomerular membrane and are taken up by megalin/gp330 receptor present at brush border of proximal tubular cells. The high renal expression levels of megalin and its high internalization rate and easy accessibility from the lumen of tubules make it an ideal receptor for tubular cell–specific drug delivery. The renal-specific conjugate Y27632-LZM accumulated rapidly and extensively in the kidneys and, more important, within the proximal tubular cells, in accordance with our previous studies with drug-LZM conjugates.20,21,26 It is important to note that Y27632 was not released from the conjugate in serum as determined by in vitro drug release studies as well as by pharmacokinetics data in vivo. Thus, free drug is not formed during the residence of the conjugate in the blood stream, and effects of the conjugate will be confined to the kidney in which the conjugate is accumulated. A pronounced extrarenal activity of the conjugate is therefore not expected, as also can be deduced from the absence of effects of Y27632-LZM on the number of white blood cells.

Renal I/R is a common cause of acute renal failure and is associated with tubular damage accompanied by tubulointerstitial inflammation and fibrosis.27,28 Vimentin, a marker for epithelial-mesenchymal transdifferentiation, is expressed in a cellular and segmental pattern after I/R,29–31 which was also observed in this study. E-cadherin is an adhesive junction protein that maintains the structural integrity and polarity of renal epithelial cells32 and is lost after I/R in these cells.33 Kim-1 is a membrane protein maximally upregulated in proliferating and dedifferentiated tubular epithelial cells after renal ischemia and is associated with their loss of polarity and development of interstitial fibrosis.34,35 The observed effects of Y27632-LZM on the tubular expression of vimentin, E-cadherin, and Kim-1 strongly demonstrate that the drug-LZM conjugate becomes pharmacologically active within the kidneys. Furthermore, we now demonstrate that such a local inhibition is related to an inhibition of ROCK, as reflected by the reduced p-MLC2 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 conjugate 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 parameters.12,37,38 Studies with isolated perfused kidneys have demonstrated that Y27632 strongly affects the vascular tone of the afferent arteriole.39,40 The observed negative effects of Y27632 might relate to an effect on renal blood flow, resulting in a loss of autoregulation after the ischemic insult. Effects of the renally delivered drug should relate to drug originating from Y27632-LZM conjugate and accumulating within the proximal tubular cells. How this may have affected renal blood flow or renal function needs to be investigated further.

During I/R injury, infiltration of monocytes/macrophages plays an important role in initiation of acute renal failure.41 MCP-1 is a potent chemoattractant for monocytes/macrophages and has been well correlated with macrophage recruitment in postischemic kidneys.42 Various studies have shown that ROCK inhibition blunted monocytes/macrophage infiltration and interstitial fibrosis in renal disease models and related this to direct effects in macrophages or local renal effects.12,43,44 Because Y27632-LZM strongly accumulates in the kidney but not in immune cells, we now provide further insight into the local renal effects, suggesting that inhibition of macrophage infiltration was due to local inhibition of MCP-1 in kidneys. Furthermore, Y27632-LZM significantly reduced the gene expression of other mediators, such as the profibrotic factors TGF-β1, TIMP-1, procollagen Iα1, and α-SMA, and substantially reduced the interstitial deposition of extracellular proteins collagen I and III and fibronectin. In addition, we found that Y27632 attenuated TGF-β1–induced procollagen Iα1 gene expression in HK-2 renal tubular cells in vitro (unpublished data). These data indicate that activated ROCK signaling within tubular epithelial cells may be crucial for the regulation of inflammation and fibrosis during I/R injury; and cell-selective inhibition of this pathway is now possible with our targeting strategy. The absence of renal effects of the free drug might be due to the relatively low dosage (0.5 mg/kg per d intravenously), which was equivalent to the amount of drug in the Y27632-LZM conjugate. Although Y27632 is a hydrophilic compound that may be eliminated via the kidney, it is unlikely that the free drug will accumulate within tubular cells because it is not a known substrate for active tubular reabsorption.

In conclusion, this study demonstrated that renal-specific delivery of ROCK inhibitor Y27632 can be successfully achieved using Y27632-LZM conjugate, and blockade of the ROCK pathway locally within renal tubular cells can be a promising approach to inhibit renal injury because the conjugate exhibited significant pharmacologic effects by inhibiting both inflammatory and fibrotic factors.

CONCISE METHODS

Synthesis and Characterization of Y27632-LZM Conjugate

Y27632 [(+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride monohydrate] was purchased from Tocris (Bristol, UK). LZM was obtained from Sigma-Aldrich (St. Louis, MO). Cis-[Pt(ethylenediamine)nitrate-chloride] (ULS) was freshly prepared from cis-[Pt(ethylenediamine)dichloride as described previously.45 Y27632 (2.95 μmol, 10 mg/ml in water) was basified with 1 M NaOH to pH 8 and reacted with cisULS (4.43 μmol) at 50°C overnight. Consumption of the starting drug and formation of the products were followed by HPLC and liquid chromatography–mass spectrometry. The reaction mixture was evaporated to dryness under reduced pressure, affording a pale yellow solid (yield 50%) that consisted of the desired 1:1 reaction product. Y27632-ULS was subsequently reacted with LZM according to a similar protocol as described for the synthesis of SB202190-ULS-LZM previously.20 In brief, LZM (0.7 μmol) equipped with surface-exposed methionine groups by the Boc-l-methionine hydroxysuccinimide ester reagent was dissolved in 0.02 M tricine/sodium nitrate buffer (pH 8.5). After addition of Y27632-ULS (3.5 μmol), the mixture was reacted at 37°C for 24 h. The product was purified by dialysis against water for 48 h, filtered, lyophilized, and stored at −20°C. Mass spectrometry analysis confirmed the formation of Y27632-LZM conjugate. The amount of conjugated drug was quantified after competitive displacement of the drug from the conjugate by overnight incubation with 0.5 M potassium thiocyanate in PBS at 80°C, by HPLC analysis as described in the next section. The absence of free drug in the preparation was investigated by HPLC analysis of freshly prepared dilutions of the conjugate in PBS.

Y27632-LZM was characterized for its stability and drug-release properties. The conjugate (100 μg/ml) was incubated in different media: 0.1 M PBS, 5 and 50 mM glutathione (reduced form) in PBS, and serum. After 24 h of incubation at 37°C, 100-μl aliquots were taken and processed immediately for HPLC analysis of Y27632.

HPLC Analysis of Y27632

Y27632 was analyzed on a Waters liquid chromatograph (Waters, Milford, MA). Separations were performed on a C18 reversed-phase SunFire column (Waters) using a mobile phase of water-methanol-trifluoroacetic acid (86:14:0.1, vol/vol/vol; pH 2.0) at a flow rate of 1 ml/min. The effluents were monitored at 270 nm. Y27632 eluted at a retention time of 5.7 min. Serum and kidney homogenates (200 μl) were treated with potassium thiocyanate as explained already to release bound drug and extracted twice with 2 ml of diethyl ether after addition of 100 μl of 2 N NaOH. After freezing of the aqueous layer in liquid nitrogen, the organic layer was decanted and evaporated at 50°C. The residue was reconstituted in 200 μl of eluents, and 50 μl was injected into the HPLC. The peak heights were recorded and related to calibration curves in the corresponding biologic matrices to quantify drug levels.

Animal Experiments

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, Netherlands).

Pharmacokinetics of Y27632-LZM

Rats (N = 5) were administered an intravenous injection of a single dose of the Y27632-LZM conjugate (20 mg/kg equivalent to 555 μg/kg of Y27632, dissolved in 5% glucose). Injections were performed in the penile vein under anesthesia (2% isoflurane in 2:1 O2/N2O, 1 L/min). Rats were killed (n = 1) at 5, 30, 60, 120, and 360 min under anesthesia. Blood samples were collected by heart puncture, and kidneys were isolated after gentle flushing of the organs with saline through the abdominal aorta. Kidneys were weighed, homogenized (1:3 wt/vol, PBS) and then stored at −80°C. To determine Y27632 in serum or tissues, samples were incubated with 0.5 M potassium thiocyanate at 80°C for 24 h to release Y27632 from the linker followed by HPLC analysis. Anti-LZM staining was performed on the frozen kidney sections to detect the cellular localization of the conjugate in the kidneys.

Effect of Y27632-LZM in Unilateral Renal I/R Model in Rats

The pharmacologic efficacy of Y27632-LZM was evaluated in the unilateral I/R rat model. Rats were divided into four groups: Untreated normal rats (n = 4), vehicle-treated I/R rats (5% glucose; n = 12), Y27632-LZM + I/R (20 mg/kg equivalent to 555 μg/kg Y27632; n = 7), Y27632 + I/R (555 μg/kg; n = 6). At 2 h before the ischemia procedure, rats were administered a preinjection of these compounds. Compounds were administered intravenously via the penis vein as described already. Rats were allowed to recover and placed back into the cages until the induction of renal ischemia. Rats were incised from the abdomen under anesthesia, and the left renal artery and vein were clamped for 45 min to stop renal blood flow. Clamps were removed, and reperfusion of the kidney was observed before closing of the wound. Rats were administered another injection of the compounds at 24, 48, and 72 h after I/R. Twenty-four hours after the last dose, the rats were killed and blood samples were collected from abdominal aorta. Kidneys were isolated after gentle perfusion with saline and weighed, and small pieces were snap-frozen in liquid nitrogen for mRNA isolation and put in ice-cold isopentane for preparation of cryosections or in 4% formalin solution to make paraffin-embedded sections.

Determination of mRNA Expression

Total RNA was isolated from renal cortex using Bio-Rad's Aurum Total RNA Mini kit (Bio-Rad, Hercules, CA). RNA content was measured by a NanoDrop UV-detector (NanoDrop Technologies, Wilmington, DE). cDNA was synthesized from similar amounts of RNA using the Superscript III first-strand synthesis kit (Invitrogen, Carlsbad, CA). Gene expression levels for the various genes were measured by quantitative real-time reverse transcription–PCR (Applied Biosystems, Foster City, CA) as explained previously.20 The primers (Table 2) for rat species were obtained from Sigma-Genosys (Haverhill, UK). SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) was used as a fluorescence probe for real-time reverse transcription–PCR. The threshold cycle number (Ct) was calculated for each gene, and relative gene expressions (fold change) were calculated after normalizing for the expression of the control gene GAPDH using ΔΔCt method.

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Table 2.

Primer sequences for real-time PCR analysisa

Immunohistochemistry

For detection of LZM, megalin, ED-1 (Figure 2H), collagen I, collagen III, fibronectin, and p-MLC2 in tissues, cryostat sections (4 μm thick) were stained as described previously20 except that for p-MLC2, sections were incubated with primary antibody overnight at 4°C. Primary antibodies such as rabbit anti-LZM polyclonal antibody (Chemicon, Temecula, CA), goat anti-megalin polyclonal antibody (Santa Cruz Biotechnologies, Santa Cruz, CA), goat anti–collagen I (Southern Biotech, Birmingham, AL), goat anti–collagen III (Southern Biotech), rabbit anti-fibronectin (Telios Pharmaceutical, San Diego, CA), and mouse phospho-myosin light chain (Ser19) mAb (Cell Signaling Technology, Beverly, MA) were used. Double stainings were performed on frozen tissues by incubating sections with primary antibodies and then their respective secondary antibodies, one labeled with horseradish peroxidase to produce red color with 3-amino-9-ethylcarbazole and the other labeled with alkaline phosphatase to produce blue color with naphthol AS phosphate and fast blue RR mixture. Other immunohistochemical stainings were performed on 3-μm-thick paraffin-embedded sections. Sections were deparaffinized in xylene and rehydrated in alcohol and distilled water. Heat-induced antigen retrieval was achieved by incubation in a microwave for 15 min at 300 W with 1 mM EDTA (pH 8.0) for vimentin, 15 min at 300 W with 0.1 M Tris/HCl (pH 9.0) for E-cadherin, or overnight at 80°C in 0.1 M Tris/HCl buffer (pH 9.0) for ED-1 and α-SMA. Endogenous peroxidase activity was blocked with 0.03% H2O2/sodium azide for 5 min. Slides were then incubated with primary antibodies against Kim-1 (antibody against the intracellular domain of Kim-1: Peptide 9; Biogen, Cambridge, MA), vimentin (DAKO, Glostrup, Denmark), E-cadherin (BD Transduction Labs, San Diego, CA), α-SMA (clone 1A4, Sigma, MO), or the rat macrophage marker ED-1 (Serotec, Oxford, UK). Binding was detected by sequential incubation with peroxidase-labeled secondary antibodies (DAKO) in the presence of 1% normal rat serum for 30 min. The peroxidase activity was visualized using 3,3′-diaminobenzidine tetrahydrochloride (DAKO) for 10 min. Periodic acid-Schiff base staining was performed by standard protocol to observe renal morphology. The extent of tubular Kim-1 and interstitial α-SMA expression was determined in approximately 30 interstitial rectangular fields per section using computerized morphometry at the magnification of ×100 and ×200, respectively. For determination of interstitial macrophage influx, ED-1–positive cells were counted using computerized morphometry in approximately 30 fields per section at the magnification of ×200. For all analyses, both cortex and corticomedullary regions were considered, and in the case of α-SMA and ED-1, glomeruli and vascular areas were manually excluded. The amount of brown precipitate was measured and presented either as a percentage of the total selected area (α-SMA and Kim-1) or as estimated number of macrophages in the selected fields. Vimentin, E-cadherin, collagen I and III, and p-MLC2 staining were scored with five grades as follows: 0.5, very weak; 1, weak; 2, moderate; 3, strong; and 4, very strong. Grades were given by analysis of complete kidney section in different fields at the magnification of ×40. All morphometric measurements and gradings were performed in a blinded manner.

Statistical Analysis

Statistical analyses were performed using the unpaired t test. P < 0.05 was considered as the minimal level of significance. Data are presented as means ± SEM. Pharmacokinetic analysis of the serum Y27632 concentrations was performed using the Multifit program (Department of Pharmacokinetics and Drug Delivery, University of Groningen, Groningen, Netherlands).

DISCLOSURES

None.

Acknowledgments

This work was supported by a grant from the European Framework program FP6 (DIALOK, LSHB-CT-2007-036644).

We are grateful to Annemiek van Loenen-Weemaes, Catharina Reker-Smit, and Mieke Zeinstra-Smith (Department of Pharmacokinetics and Drug Delivery) and Marian Bulthuis and N.J. Kloosterhuis (Department of Pathology) for technical assistance. Colleagues at Kreatech are acknowledged for critical reading of the manuscript. We are thankful to Prof. R.H. Henning for scientific discussions.

Footnotes

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Inhibition of Renal Rho Kinase Attenuates Ischemia/Reperfusion-Induced Injury
Jai Prakash, Martin H. de Borst, Marie Lacombe, Frank Opdam, Pieter A. Klok, Harry van Goor, Dirk K.F. Meijer, Frits Moolenaar, Klaas Poelstra, Robbert J. Kok
JASN Nov 2008, 19 (11) 2086-2097; DOI: 10.1681/ASN.2007070794
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