Signaling Mechanism of Renal Fibrosis in Unilateral Ureteral Obstructive Kidney Disease in ROCK1 Knockout Mice
Ping Fu*,,
Fang Liu*,,
Spencer Su,
Wansheng Wang,
Xiao R. Huang,,
Mark L. Entman,
Robert J. Schwartz,
Lei Wei|| and
Hui Y. Lan,
* Department of Medicine-Nephrology, West China Hospital of Sichuan University, Chengdu, China; Department of Medicine-Nephrology and Cardiovascular Sciences, Baylor College of Medicine; Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, Texas; Department of Medicine, The University of Hong Kong, Hong Kong, China; and || Herman B. Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana
Address correspondence to: Prof. Hui Y. Lan, Department of Medicine, The University of Hong Kong, Hong Kong, China. Phone: +852-28199745; Fax: +852- 28162905; E-mail: hylan{at}hku.hk
Received for publication December 23, 2005.
Accepted for publication August 10, 2006.
It has been shown that blockade of Rho kinase with pharmacologicinhibitors inhibits renal fibrosis. This study examined therole of Rho kinase in renal fibrosis in the unilateral ureteralobstruction (UUO) model in mice that do not express the ROCK1gene, a critical downstream mediator of Rho GTPase. Unexpected,real-time PCR, Western blot, and immunohistochemistry demonstratedthat, compared with the wild-type mice, mice with ROCK1 knockout(KO) were not protected against renal fibrosis at both the early(day 5) and late (day 10) UUO, as determined by histology andexpression of both mRNA and protein levels of -smooth muscleactin, collagen types I and III, and fibronectin within thediseased kidney. Then the mechanisms of loss of protective effecton renal fibrosis in ROCK1 KO mice were investigated. It isinteresting that mice that lacked ROCK1 did not have alteredexpression of ROCK2 but significantly increased TGF- expressionand Smad2/3 activation (phosphorylation and nuclear translocation)in the diseased kidney at day 5, which remained high at day10 of UUO. Similarly, primary cultures of kidney fibroblaststhat were obtained from both ROCK1 wild-type and KO mice showedthat deletion of ROCK1 did not prevent TGF-–induced activationof Smad2/3 and collagen I expression. This also was observedin the presence of Rho kinase inhibitor Y-27632. Taken together,results from this study suggest that Rho/Rho kinase may notbe a necessary or a central pathway for renal fibrosis in theUUO model. The interplay between the Rho/Rho kinase pathwayand the Smad signaling pathway may be a key mechanism by whichloss of ROCK1 does not prevent renal fibrosis in the UUO model.
Emerging evidence shows that TGF- and its signaling molecules,Smads, play a critical role in renal fibrosis in both experimentaland human chronic kidney diseases (1–3). It now is establishedthat TGF- regulates fibrosis positively by receptor-associatedSmads, including Smad2 and Smad3, but negatively by an inhibitorySmad7. In the context of renal fibrosis, both Smad2 and Smad3are strongly activated in experimental and human diabetic kidneydiseases (4,5) and in the rat models of obstructive kidney disease(6,7) and five-sixths nephrectomy (8). Inhibition of renal fibrosisby blocking activation of Smad2 and Smad3 with overexpressionof Smad7 demonstrates a critical role for TGF- signaling inrenal fibrosis (5–10).
Recently, a number of studies have shown that Rho and its downstreameffector, Rho kinase, are potential mediators of TGF-–associatedrenal fibrosis (11–17). Rho is a small GTPase that isbound to GDP when latent and is bound to GTP when active (18,19).Together with its downstream effector, Rho-associated coiled-coilforming protein serine/threonine kinase (Rho kinase or Rock),Rho has been implicated in a wide variety of cellular functions(18,19). In addition to TGF-, the Rho/Rho kinase signaling cascadeis activated by fibrogenic growth factors angiotensin II (20),PDGF (21), and endothelin-I (22). Evidence for the potentialrole of the Rho kinase pathway in renal fibrosis comes fromstudies that used pharmacologic inhibition of the Rho/Rho kinasesignaling pathway with selective inhibitors Y-27632 and fasudil(11–17). With the use of these agents, the Rho/Rho kinasepathway also has been shown to play an important role in epithelial-mesenchymaltransition (15,23,24), cardiovascular diseases (25–27),and liver fibrosis (28,29).
Although pharmacologic inhibition of Rho kinase has been shownto be protective against renal fibrosis, the biologic functionsof Rho kinase have not been well characterized. Therefore, weexamined the functional role of ROCK1, an isoform of Rho kinase,in the pathogenesis of renal fibrosis in a model of unilateralureteral obstruction (UUO) in mice that have genetic deletionof ROCK1 gene.
Generation of ROCK1 Knockout Mice
ROCK1 knockout (KO) mice were generated as described previously(30). ROCK1+/– heterozygous mice then were intercrossedto produce homozygous ROCK1–/– mice. The genotypesof the offspring were identified by Southern blot analysis (Figure 1A)and PCR on DNA that was obtained from tails of adult mice asdescribed previously (31). Disruption of the ROCK1 gene alsowas confirmed by Western blot analysis with multiple antibodiesas described previously (30). In addition, deletion of ROCK1gene within the diseased kidney of UUO was confirmed by Westernblot with the anti-ROCK1 antibody (Figure 1B). Mice used inour study had been backcrossed to FVB for at least six generations.The ROCK1 wild-type (WT) and KO mice with FVB background wereused in this study. Homozygous ROCK1 KO mice were viable withneither detectable anatomic abnormalities nor increase in morbidityor mortality, although they were underrepresented (30).
Figure 1. Evidence for deletion of ROCK1 gene in mice. (A) Southern blot shows a deleted band of ROCK1 gene in homozygous mice (–/–), compared with the +/– and +/+ mice. (B) Western blot shows undetectable ROCK1 protein obtained from kidneys with unilateral ureteral obstruction (UUO). Each land represents one mouse. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Obstructive Kidney Disease Model
A UUO kidney disease model was induced in both WT and ROCK1KO mice (20 g body wt, 2 mo of age) by left ureteral ligationas described previously (6,32). For investigation of the roleof ROCK1 in the early and late time points of obstructive kidneydisease, groups of eight ROCK1 KO mice and seven WT mice werekilled on day 5 and groups of 11 KO mice and seven WT mice werekilled on day 10 after the left ureteral ligation. In addition,groups of six normal KO and six WT mice at the same age wereused as normal controls. The experimental procedures were approvedby Animal Experimental Committee at Baylor College of Medicine.Kidney tissue samples were collected at day 5 and day 10 afterUUO for histology, immunohistochemistry, Western blot, and real-timePCR analyses.
Histology and Immunohistochemistry
Changes in renal morphology were examined in methyl Carnoy’s–fixed,paraffin-embedded tissue sections (4 µm) stained withhematoxylin and eosin or periodic acid-Schiff. To retrieve theantigens, 4-µm paraffin sections were microwaved in 0.01M citrate buffer for 10 min as described previously (6,31,33).Sections then were incubated with a mouse mAb against -smoothmuscle actin (-SMA; Sigma, St. Louis, MO); rabbit polyclonalantibodies to phosphorylated Smad2/3, TGF-1, fibronectin (SantaCruz Biotechnology, Santa Cruz, CA); and goat polyclonal antibodiesto collagen I and III (Southern Tech, Birmingham, AL) usinga three-layer peroxidase anti-peroxidase method (6,8). Sectionsthen were developed with diaminobenzidine to produce a browncolor, counterstained with hematoxylin, and coverslipped inan aqueous mounting medium. An isotype-matched mouse mAb (73.5)that recognized human CD45R antigen and goat or rabbit IgG (Sigma)were used as negative controls throughout the study.
Western Blot and Immunoprecipitation Analyses
Protein from kidney tissues was extracted with RIPA lysis buffer(1% Nonidet P-40, 0.1% SDS, 1 mM PMSF, 0.5% sodium deoxycholate,1 mM sodium orthovanadate, and 1 mM sodium fluoride in PBS).After determination of protein concentrations, 20 µg ofthe protein was mixed with an equal amount of 2x SDS loadingbuffer (100 mM Tris-HCl, 4% SDS, 20% glycerol, and 0.2% bromophenolblue) for Western blot analysis as described previously (6,8).Briefly, samples were heated at 99°C for 5 min and thentransferred to a polyvinylidene difluoride membrane. Nonspecificbinding to the membrane was blocked for 1 h at room temperaturewith 5% BSA in Tris-buffered saline buffer (20 mM Tris-HCl,150 mM NaCl, and 0.1% Tween 20). The membranes were then incubatedovernight at 4°C with primary antibodies, including mousemAb against -SMA (Santa Cruz); goat antibodies against collagenI and III; and rabbit antibodies to fibronectin, TGF-1, ROCK1,ROCK2 (Santa Cruz), and glyceraldehyde-3-phosphate dehydrogenase(GAPDH; Chemicon, Temecula, CA). After washing, the membranewas incubated with a peroxidase-conjugated goat anti-mouse IgGor swine anti-goat (or rabbit) IgG in 1% BSA/Tris-buffered salinebuffer. Phosphorylated Smad2/3 was determined by immunoprecipitationwith anti-Smad2/3 antibody (Santa Cruz) followed by immunoblottingand detection with rabbit anti-phosphoserine antibody (6,8).The signals were visualized by an enhanced chemiluminescencesystem (Amersham, Piscataway, NJ).
Real-Time PCR
Total kidney RNA was isolated using the RNeasy kit, accordingto the manufacturer’s instructions (Qiagen, Valencia,CA). The cDNA was synthesized as described previously (32).Real-time PCR was run with the Opticon real-time PCR machine(MJ Research, Waltham, MA). The specificity of real-time PCRwas confirmed via routine agarose gel electrophoresis and Melting-curveanalysis. Housekeeping gene GAPDH was used as an internal standard.The primers used in this study are as follows: -SMA, forward5'-ACTGGGACGACATGGAAAAG-3', reverse 5'-CATCTCCAGAGTCCAGCACA-3';collagen I, forward 5'-GAGCGGAGAGTACTGGATCG-3', reverse 5'-TACTCGAACGGGAATCCATC-3';collagen III, forward 5'-TGGTCCTCAGGGTGTAAAGG-3', reverse 5'-GTCCAGCATCACCTTTTGGT-3';fibronectin, forward 5'-ACACGGTTTCCCATTACGCCAT-3', reverse 5'-AATGACCACTGCCAAAGCCCAA-3';and GAPDH, forward 5'-TGCTGAGTATGTCGTGGAGTCTA-3' and reverse5'-AGTGGGAGTTGCTGTTGAAATC-3'. Ratios for -SMA/GAPDH, collagenI/GAPDH, collagen III/GAPDH, and fibronectin/GAPDH were calculatedfor each sample and expressed as the means ± SEM.
Quantitative Analyses
Smad2/3 activation was determined by the extent of its nuclearlocalization in stained tissue using antiphosphorylated Smad2/3antibody. The number of positive cells for activated Smad2/3was counted in 20 consecutive tubulointerstitial areas underhigh-power fields (x40) by means of a 0.25-mm2 graticule fittedin the eyepiece of the microscope, excluding the glomerulusand big vessels, and expressed as cells/cm2. The degree of TGF-1expression and accumulation of -SMA, collagen types I and III,and fibronectin in the entire cortical tubulointerstitium (across-section of the kidney) were determined using quantitativeImage Analysis System (Optima 6.5; Media Cybernetics, SilverSpring, MD) as described previously (6,8). Briefly, the examinedarea of the tubulointerstitium was outlined, positive stainingpatterns were identified, and the percentage of positive areain the examined tubulointerstitium was measured. Data are expressedas percentage as positive area examined. All scoring was performedin a blinded manner on coded slides.
In Vitro Studies
To study the mechanisms by which ROCK1 mediates renal fibrosis,we isolated kidney fibroblasts, a key cell type in the pathogenesisof renal fibrosis, from normal renal cortex of ROCK1 WT andKO mice. Briefly, under sterile condition, the cortex of mousekidney was collected, minced, and digested with Liberase Blenzyme3 (0.3 mg/ml; Roche, Palo Alto, CA) for 2 h. The pellet wasincubated in DMEM that contained 10% FBS for 4 h, and then thenonadherent cells were removed. More than 85% of adherent cellswere kidney fibroblasts as identified by elongated morphologyand positive for the anti–fibroblast-specific protein1 antibody (Abcam, Cambridge, MA). Cells under passage 3 wereused for the experiments.
Primary renal fibroblasts were incubated in DMEM that contained10% FBS until confluent. After serum starvation for 24 h, thecells were stimulated with TGF-1 (2.5 ng/ml; R&D Systems,Minneapolis, MN) in the presence or absence of Y-27632 (140nM; Sigma) for 30 min for examination of Smad2/3 phosphorylationand for 24 h for detection of collagen type 1 by Western blotanalysis as described in Western Blot and ImmunoprecipitationAnalyses. Effect of Y-27632 on inhibition of TGF--induced (2.5ng/ml) Rho kinase activities was confirmed by the MBL Rho kinaseAssay Kit (CY-1160) following the manufacturer’s instruction(CycLex Co., Ltd, Negano, Japan).
Statistical Analyses
Data that were obtained from this study are expressed as themean ± SEM. Statistical analyses were performed usingone-way ANOVA from GraphPad Prism 3.0 (GraphPad Software, SanDiego, CA).
Histologic Injury
On periodic acid-Schiff–stained sections, kidney histologywas normal in both normal WT and KO mice (Figure 2, A and B).Five days after the left ureteral ligation, WT mice developedsevere tubulointerstitial damage, including tubular atrophyand interstitial fibrosis (Figure 2C). Unexpected, ROCK1 KOmice also exhibited a similar degree of severe tubulointerstitialdamage (Figure 2D), demonstrating no protection from renal fibrosisin the ROCK1 KO mice. Similar results also were found in bothWT and KO mice at day 10 after UUO (Figure 2, E and F).
Figure 2. Effect of deletion of ROCK1 on histologic injury in the UUO model. (A) A normal kidney from a wild-type (WT) mouse. (B) A normal kidney from a knockout (KO) mouse. (C) A UUO kidney from a WT mouse at day 5. (D) A UUO kidney from a KO mouse at day 5. (E) A UUO kidney from a WT mouse at day 10. (F) A UUO kidney from a KO mouse at day 10. Representative kidney tissue sections stained with periodic acid-Schiff show that compared with normal mice, severe histologic damage to the kidney, including tubular atrophy and tubulointerstitial fibrosis, develops in both WT and KO mice at both day 5 and day 10 after UUO. However, mice that lack ROCK1 (D and F) are not protected against renal histologic injury compared with the WT mice (C and E). Magnification, x200.
Effect of Deletion of ROCK1 on Extracellular Matrix mRNA Expression
Consistent with the histologic findings, compared with the normalkidney, real-time PCR demonstrated that a 10- to 20-fold increasein mRNA expression of -SMA, collagen I, collagen III, and fibronectinwas found in the diseased kidney in both WT and KO mice at day5 and day 10 after UUO (all P < 0.05; Figure 3). It is interestingthat deletion of ROCK1 reduced (but not significantly) -SMAmRNA expression and significantly increased collagen III mRNAat day 5 after UUO (Figure 3, A and C). However, there was nodifference in the levels of increased extracellular matrix (ECM)mRNA expression of -SMA, collagen types I or III, or fibronectinwithin the diseased kidney between the WT and KO mice at day10 after UUO (all P > 0.05; Figure 3).
Figure 3. Effect of deletion of ROCK1 on extracellular matrix (ECM) mRNA expression in the UUO model. Real-time PCR shows that compared with normal mice, a marked increase in ECM mRNA expression including -smooth muscle actin (-SMA; A), collagen types I (B) and III (C), and fibronectin (D), is evident in the obstructive kidney in both WT and KO mice at both day 5 and day 10 after UUO (all P < 0.05). However, mice that lack ROCK1 show no protection against ECM mRNA expression. Note that deletion of ROCK1 significantly enhances collagen III expression at UUO kidney at day 5 compared with the WT UUO mice (C). Each bar represents the mean ± SEM for at least six mice.
Effect of Deletion of ROCK1 on ECM Protein Expression
Similar to the results found in mRNA expression, immunohistochemistryshowed that compared with the normal kidney, a marked increasein accumulation of -SMA, collagen I, collagen III, and fibronectinoccurred in the UUO kidney in both WT and KO mice at day 5 andday 10 after UUO (all P < 0.001; Figures 4 through 7). However,compared with the WT mice, ROCK1 KO mice were not protectedagainst renal fibrosis as demonstrated by similar accumulationof tubulointerstitial -SMA, collagen types I and III, and fibronectinwithin the diseased kidney at day 10 (P > 0.05; Figures 4 through 7,E through G). Unexpected, deletion of ROCK1 significantly enhancedcollagen I, collagen III, and fibronectin accumulation at day5 after UUO (Figures 5 through 7, C, D, and G).
Figure 4. Effect of deletion of ROCK1 on -SMA accumulation in UUO model. (A) A normal kidney from a WT mouse. (B) A normal kidney from a KO mouse. (C) A UUO kidney from a WT mouse at day 5. (D) A UUO kidney from a KO mouse at day 5. (E) A UUO kidney from a WT mouse at day 10. (F) A UUO kidney from a KO mouse at day 10. (G) Semiquantitative analysis. Immunohistochemistry shows that -SMA–positive myofibroblasts are apparent in tubulointerstitium, resulting in severe tubulointerstitial fibrosis in both WT and KO mice in the diseased kidney at day 5 and day 10 after UUO compared with normal WT or KO mice (P < 0.001; A and B). However, compared with the diseased WT mice (C and E), deletion of ROCK1 shows no protection against -SMA–positive cell accumulation within the diseased kidney at both day 5 and day 10. Each bar represents the mean ± SEM for at least 6 mice. Magnification, x200.
Figure 5. Effect of deletion of ROCK1 on collagen I accumulation in UUO model. (A) A normal kidney from a WT mouse. (B) A normal kidney from a KO mouse. (C) A UUO kidney from a WT mouse at day 5. (D) A UUO kidney from a KO mouse at day 5. (E) A UUO kidney from a WT mouse at day 10. (F) A UUO kidney from a KO mouse at day 10. (G) Semiquantitative analysis. Immunohistochemistry shows that a marked increase in tubulointerstitial collagen I accumulation is associated with severe tubulointerstitial fibrosis in both WT and KO mice at day 5 and day 10 after UUO compared with normal mice (P < 0.001; A and B). However, compared with the diseased WT mice (C and E), deletion of ROCK1 shows no protection against tubulointerstitial collagen II expression within the diseased kidney at both day 5 and day 10. Note that deletion of ROCK1 results in a significant increase in collagen I expression at day 5 after UUO (D and G). *P < 0.05 versus the time-matched mice. Each bar represents the mean ± SEM for at least six mice. Magnification, x200.
Figure 6. Effect of deletion of ROCK1 on collagen III accumulation in UUO model. (A) A normal kidney from a WT mouse. (B) A normal kidney from a KO mouse. (C) A UUO kidney from a WT mouse at day 5. (D) A UUO kidney from a KO mouse at day 5. (E) A UUO kidney from a WT mouse at day 10. (F) A UUO kidney from a KO mouse at day 10. (G) Semiquantitative analysis. Immunohistochemistry shows that a marked increase in tubulointerstitial collagen III accumulation is associated with severe tubulointerstitial fibrosis in both WT and KO mice at day 5 and day 10 after UUO, when compared with normal mice (P < 0.001; A and B). However, compared with the diseased WT mice (C and E), deletion of ROCK1 shows no protection against tubulointerstitial collagen III expression within the diseased kidney at both day 5 and day 10. Note that deletion of ROCK1 results in a significant increase in collagen III expression at day 5 after UUO (D and G). *P < 0.05 versus the time-matched animals. Each bar represents the mean ± SEM for at least six mice. Magnification, x200.
Figure 7. Effect of deletion of ROCK1 on fibronectin accumulation in UUO model. (A) A normal kidney from a WT mouse. (B) A normal kidney from a KO mouse. (C) A UUO kidney from a WT mouse at day 5. (D) A UUO kidney from a KO mouse at day 5. (E) A UUO kidney from a WT mouse at day 10. (F) A UUO kidney from a KO mouse at day 10. (G) Semiquantitative analysis. Immunohistochemistry shows that a marked increase in tubulointerstitial fibronectin expression is associated with severe tubulointerstitial fibrosis in both WT and KO mice at day 5 and day 10 after UUO, when compared with normal mice (P < 0.001; A and B). However, deletion of ROCK1 shows no protection against tubulointerstitial fibronectin expression within the diseased kidney at both day 5 and day 10, when compared with the diseased WT mice (C and E). Moreover, deletion of ROCK1 results in a significant increase in fibronectin expression at day 5 after UUO (D and G). ***P < 0.001 versus the time-matched mice. Each bar represents the mean ± SEM for at least six mice. Magnification, x200.
Effect of deletion of ROCK1 on ECM protein expression in theentire obstructive kidney also was determined by Western blotanalysis of total kidney proteins. As shown in Figures 8 and9, accumulation of -SMA, collagen types I and III, and fibronectinlargely was increased in the UUO kidney at day 5 and day 10in both WT and KO mice (all P < 0.01), whereas there wasno difference in the extent of increased ECM expression in thediseased kidney between WT and KO animals (all P > 0.05).
Figure 8. Effect of deletion of ROCK1 on ECM expression in UUO model at day 5 demonstrated by Western blot analysis. Western blot analysis shows that there is a significant increase in the levels of -SMA, collagen III, and fibronectin (FN) expression in both ROCK1 KO and WT UUO kidney at day 5. However, deletion of ROCK1 does not alter the levels of -SMA, collagen III, and FN expression within the obstructive kidney compared with the diseased WT mice (all P > 0.05). Each band represents one mouse kidney from either normal or diseased mice. Each bar represents the mean ± SEM for at least five mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal mice.
Figure 9. Effect of deletion of ROCK1 on ECM expression in UUO model at day 10 demonstrated by Western blot analysis. Western blot analysis shows that there is a significant increase in the levels of -SMA, collagen I, collagen III, and FN expression in both ROCK1 KO and WT UUO kidney at day 10. However, deletion of ROCK1 does not alter the levels of -SMA, collagen I and III, and FN expression within the obstructive kidney, when compared with the diseased WT mice (all P > 0.05). Each band represents one mouse kidney from either normal or diseased mice. Each bar represents the mean ± SEM for at least five mice. ** P < 0.01, ***P < 0.001 versus normal animals.
Mechanisms of Deletion of ROCK1 Fail to Prevent Renal Fibrosis in Obstructive Kidney
We next examined the mechanisms by which deletion of ROCK1 failsto prevent renal fibrosis in the obstructive kidney by examiningthe expression of ROCK2. As shown in Figure 10, Western blotanalysis demonstrated an increase, although not statisticallysignificant, in the expression of ROCK2 in the obstructive kidneyat day 10 in both WT and KO mice. However, deletion of ROCK1did not alter the levels of ROCK2 expression compared with thediseased WT mice (P > 0.05).
Figure 10. Effect of deletion of ROCK1 on ROCK2 expression in UUO model. Western blot analysis shows that compared with the normal mice, an increase in expression of ROCK2 is evident in the diseased kidney at day 10 after UUO, but this is not statistically significant. However, deletion of ROCK1 does not alter the level of ROCK2 expression within the diseased kidney at day 10 compared with the WT mice. Each band represents one mouse kidney from either normal or diseased mice. Each bar represents the mean ± SEM for at least five mice.
We then asked whether the loss of protective effect on renalfibrosis in ROCK1 KO mice is associated with TGF- expression.Immunohistochemistry and Western blot analyses showed that expressionof TGF-1 protein was markedly upregulated in the UUO kidneyin both WT and KO mice at day 5 and day 10 (Figures 11 and 12).Upregulation of TGF-1 was confined largely in the tubulointerstitiumof the UUO kidney, where severe tubulointerstitial fibrosiswas evident (Figure 11, C through F). It is interesting thatWestern blot analysis demonstrated that deletion of ROCK1 significantlyenhanced renal TGF-1 protein expression at day 5 after UUO whencompared with the diseased WT animals (Figure 12A). However,when disease became progressive at the later time point on day10, levels of TGF-1 expression within the diseased kidney betweenWT and KO mice were not significantly different (Figure 12B).
Figure 11. Effect of deletion of ROCK1 on TGF-1 protein expression in UUO model demonstrated by immunohistochemistry. (A) A normal kidney from a WT mouse. (B) A normal kidney from a KO mouse. (C) A UUO kidney from a WT mouse at day 5. (D) A UUO kidney from a KO mouse at day 5. (E) A UUO kidney from a WT mouse at day 10. (F) A UUO kidney from a KO mouse at day 10. (G) Semiquantitative analysis. Immunohistochemistry shows that TGF-1 is strongly expressed in normal glomeruli but not in tubulointerstitium in both normal WT and KO mice (A and B). In contrast, marked upregulation of TGF-1 is found in the tubulointerstitial area with severe fibrosis in both WT and KO mice at day 5 and day 10 after UUO (C through G). Compared with the WT mice, deletion of ROCK1 does not alter the levels of TGF-1 expression within the normal and the diseased kidney at both day 5 and day 10 after UUO. Each bar represents the mean ± SEM for at least six mice. Magnification, x200
Figure 12. Effect of deletion of ROCK1 on TGF- expression and Smad2/3 phosphorylation in UUO model at day 5 (A) and day 10 (B) mice. Western blot analysis shows that compared with normal controls, there is a significant increase in TGF-1 expression and Smad2/3 phosphorylation in the obstructive kidney. It is interesting that compared with the WT mice, deletion of ROCK1 shows a further increase in TGF-1 expression within the diseased kidney at day 5 and day 10, which is associated with a significant increase in the level of Smad2/3 phosphorylation (p-S2/3). Each band represents one mouse kidney from either normal or diseased mice. Each bar represents the mean ± SEM for normal (n = 4), diseased WT (n = 6), and diseased KO (n = 6) mice. *P < 0.05, **P < 0.01, ***P < 0.001 versus normal; #P < 0.05, ##P < 0.01 versus as indicated.
We next examined activation of the TGF-/Smad signaling pathwaywithin the diseased kidney by Western blot and immunohistochemistry.As shown in Figures 12 and 13, both Smad2/3 phosphorylation(p-Smad2/3) and nuclear translocation were markedly increasedin the UUO kidney in both WT and KO mice at day 5 and day 10.Importantly, deletion of ROCK1 significantly enhanced furtherphosphorylation levels of p-Smad2/3 (Figure 12) and p-Smad2/3nuclear translocation within the diseased kidney at day 5 andday 10 after UUO (Figure 13), demonstrating that deletion ofROCK1 enhanced Smad signaling in the obstructed kidney.
Figure 13. Effect of deletion of ROCK1 on phosphorylated Smad2/3 nuclear translocation in UUO model at day 5 and day 10. (A) A normal kidney from a WT mouse. (B) A normal kidney from a KO mouse. (C) A UUO kidney from a WT mouse at day 5. (D) A UUO kidney from a KO mouse at day 5. (E) A UUO kidney from a WT mouse at day 10. (F) A UUO kidney from a KO mouse at day 10. (G) Semiquantitative analysis. Immunohistochemistry shows that there is a marked increase in nucleated p-Smad2/3 in both WT and KO mouse kidney after UUO at day 5 and day 10 compared with the normal kidney (all P < 0.001). Note that compared with the WT mice (C and E), deletion of ROCK1 does not alter the levels of nucleated p-Smad2/3 in the normal mouse kidney (A, B, and G), but significantly enhances p-Smad2/3 nuclear location in the diseased kidney at day 5 and day 10 after UUO (D, F, and G). Each bar represents the mean ± SEM for at least 6 mice. **P < 0.01, ***P < 0.001 versus WT mice at time-matched mice. Magnification, x200.
To confirm whether deletion of ROCK1 can influence the TGF-/Smadsignaling pathway, we isolated kidney fibroblasts, a major celltype in production of collagen matrix, from the normal renalcortex of ROCK1 KO and WT mice and stimulated them with TGF-1in the presence or absence of Rho kinase inhibitor Y-27632.As shown in Figure 14, deletion of ROCK1 did not prevent TGF-–inducedp-Smad2/3 and collagen I expression. Unexpected, addition ofRho kinase inhibitor produced no inhibitory effect on eitherp-Smad2/3 or collagen I expression in response to TGF-1, althoughit inhibited TGF-–induced Rho kinase activities in a dose-dependentmanner (30 to 50% reduced) as determined by the Rho Kinase AssayKit.
Figure 14. Deletion of ROCK1 on kidney fibroblasts does not prevent TGF-1–induced Smad2/3 phosphorylation and collagen I expression in vitro. (A) Western blot analysis. (B) Semiquantification of p-Smad2/3. (C) Semiquantification of collagen I. Results show that additional TGF-1 (2.5 ng/ml) causes a marked Smad2/3 phosphorylation and collagen I expression in both ROCK1 KO and WT kidney fibroblasts (all P < 0.05), which is not blocked by addition of Rho kinase inhibitor (Y-27632, 140 nM; P > 0.05). **P < 0.01 versus normal mice. Data represent the mean ± SEM for at least three independent experiments.
This study showed that deletion of ROCK1 was not protectiveagainst renal fibrosis in a mouse model of UUO. Moreover, deletionof ROCK1 enhanced further collagen matrix and fibronectin expressionwithin the diseased kidney at day 5 after UUO, although no significantdifference in the kidney histology and fibrogenic parameters,including -SMA, collagen types I and III, and fibronectin, wasobserved in both ROCK1 KO and WT mice at day 10 after UUO. Thesefindings indicate that the Rho kinase pathway may not be a keypathway in the pathogenesis of renal fibrosis as previouslythought.
ROCK1 and ROCK2 are isoforms of Rho kinase, both of which areactive in cytoskeletal arrangement (18,19), cardiovascular remodeling(20,25–27), liver fibrosis (28,29), and epithelial-mesenchymaltransition (15,23,24). Evidence for the role of Rho kinase inrenal fibrosis comes from recent reports that the use of selectiveinhibitors for both isoforms of Rho kinase, Y-27632 and Fasudil,is able to inhibit tubulointerstitial fibrosis in the mousemodel of UUO nephropathy (11,12), ischemia/reperfusion-inducedacute renal failure (13), and glomerulosclerosis in spontaneoushypertensive rats (14). Unexpected, results that were obtainedfrom our study did not support the findings as previously reportedusing pharmacologic inhibitors (11–15) and found thatdeletion of ROCK1 produced no protective effect on renal fibrosisin UUO. The use of specific targeting of the ROCK1 gene in ourstudy versus the use of pharmacologic inhibitors in the othersmay be the major reason for this discrepancy. It is possiblethat the use of Rho kinase inhibitors is able to inhibit bothROCK1 and ROCK2 activities, thereby inhibiting renal fibrosis,whereas specific deletion of ROCK1 gene alone may not be adequateto prevent fibrosis in the UUO model. Could the failure of ROCK1deletion to protect against renal fibrosis stem from an increasein ROCK2 activities? Using Western blot analysis of diseasedkidneys, we showed that deletion of ROCK1 did not alter expressionof ROCK2. This is consistent with the finding that expressionof ROCK2 does not increase to compensate for the loss of ROCK1deletion in the ROCK1 KO mice, although deletion of ROCK1 exhibitsa protective effect on cardiac fibrosis (30). Nevertheless,the interplay between the ROCK1 and ROCK2 pathways in termsof renal fibrosis in obstructive kidney disease remains largelyunknown. It remains unclear whether deletion of both ROCK1 andROCK2 may be required for protection from tubulointerstitialfibrosis, as is accomplished by the pharmacologic inhibitionof Rho kinase by Y-27632 or fasudil. Alternatively, comparedwith studies that used Rho kinase inhibitors, the inabilityof deletion of ROCK1 to prevent renal fibrosis also may indicatethat the Rho kinase inhibitors may not be entirely specificin inhibition of Rho kinase activities or may have additionalactivities beyond the selective inhibition of Rho kinase. Inaddition, the failure of deletion of ROCK1 to prevent UUO kidneyfrom fibrosis is inconsistent with the finding in a pressureoverload heart model in the same phenotype of mice (30). Thismay be associated with the nature of disease in which the UUOmodel is much more aggressive compared with the pressure overloadheart model.
A wide array of studies have established that TGF- signals throughits downstream Smad signaling pathway to mediate renal fibrosis(1–4). Blockade of Smad2/3 activation by overexpressingan inhibitory Smad7 inhibits renal fibrosis in rat models ofUUO and remnant kidney disease (6–8). The finding thatmice null for Smad3 are protected against obstructive nephropathyindicates that Smad signaling plays a critical role in renalfibrosis (34). Beyond the Smad signaling pathway, Rho kinasealso has been identified as a non–Smad-dependent pathwayand plays a role in fibrosis in response to TGF- (35). In additionto the Smad signaling pathway, it has been shown that TGF- isable to activate the Rho/Rho kinase pathway to induce epithelial-mesenchymaltransition and cell growth arrest involving an initial inhibitionof Cdc25A enzymatic activity (36). This is followed by upregulationof Cdk inhibitory proteins and downregulation of c-Myc and Cdc25A(36). In addition, the Rho/Rho kinase pathway is important inTGF-–mediated Smad-dependent growth inhibition in humanMCF10CA1h breast carcinoma cells (35). It is interesting thattreatment of MCF10CA1h cells with the Rho kinase inhibitor Y27632alone significantly increases the basal levels of phosphorylatedSmad3 (35). This suggests that the Rho/Rho kinase pathway negativelyregulates the TGF-/Smad signaling. This observation leads usto hypothesize that the failure of a protective effect on renalfibrosis in mice that lack ROCK1 may be associated with theenhancement of TGF-/Smad signaling. Indeed, expression of TGF-1was markedly upregulated in the obstructive kidney in both WTand KO mice. Importantly, at the early time point of UUO atday 5, we found that deletion of ROCK1 caused a further increasein TGF-1 expression, thereby enhancing further TGF-/Smad signalingas demonstrated by an increase in phosphorylation of Smad2/3and p-Smad2/3 nuclear translocation and resulting in more severerenal fibrosis compared with WT mice. Therefore, an increasein TGF-/Smad signaling in ROCK1 KO mice with UUO may be a mechanismwhereby deletion of ROCK1 produced no protective effect on renalfibrosis in obstructive kidney disease. This also is supportedby the finding at the late time point of UUO at day 10 and bythe in vitro finding that kidney fibroblasts that lack ROCK1or addition of Rho kinase inhibitor could not prevent TGF-–inducedSmad2/3 activation and collagen matrix production. Taking together,these findings suggest that there may be a redundant mechanismbetween the Rho/Rho kinase pathway and the TGF-/Smad signalingpathway in TGF-–mediated renal fibrosis in the UUO model.Deletion of ROCK1 may result in an increase in expression ofTGF-–and Smad-dependent renal fibrosis. This may be onemechanism by which ROCK1 KO fails to exhibit its protectiverole on renal fibrosis in the UUO model. However, our studycannot exclude the role of ROCK2 in renal fibrosis. Future studywith ROCK2 KO is needed to confirm the role of the Rho/Rho kinasepathway in renal fibrosis in vivo.
Acknowledgments
This study was supported by grants from Research Grant Councilof Hong Kong (HKU7592/06) and National Institutes of Health/NationalInstitute of Diabetes and Digestive and Kidney Diseases (P50DK64233 and R01DK062828) to H.Y.L. and NIHR01-HL72897 to L.W.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
-smooth muscle actin, collagen types I and III, and fibronectin within the diseased kidney. Then the mechanisms of loss of protective effect on renal fibrosis in ROCK1 KO mice were investigated. It is interesting that mice that lacked ROCK1 did not have altered expression of ROCK2 but significantly increased TGF-
expression and Smad2/3 activation (phosphorylation and nuclear translocation) in the diseased kidney at day 5, which remained high at day 10 of UUO. Similarly, primary cultures of kidney fibroblasts that were obtained from both ROCK1 wild-type and KO mice showed that deletion of ROCK1 did not prevent TGF-
