A Pathogenic Role for c-Jun Amino-Terminal Kinase Signaling in Renal Fibrosis and Tubular Cell Apoptosis
Frank Y. Ma*,
Robert S. Flanc*,,
Greg H. Tesch*,,
Yingjie Han*,,
Robert C. Atkins*,,
Brydon L. Bennett,
Glenn C. Friedman,
Jui-Hsiang Fan and
David J. Nikolic-Paterson*,
* Department of Nephrology and Monash University Department of Medicine, Monash Medical Centre, Clayton, Victoria, Australia; and Celgene, San Diego, California
Address correspondence to: Dr. David J. Nikolic-Paterson, Department of Nephrology, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. Phone: +61-3-9594;3535; Fax: +61-3-9594-6530; E-mail: david.nikolic-paterson{at}med.monash.edu.au
Received for publication June 12, 2006.
Accepted for publication November 24, 2006.
Renal fibrosis and tubular apoptosis are common mechanisms ofprogressive kidney disease. In vitro studies have implicatedthe c-Jun amino-terminal kinase (JNK) pathway in these processes.Both of the major JNK isoforms, JNK1 and JNK2, are expressedin the kidney, but their relative contribution to JNK signalingis unknown. This study investigated the role of JNK signalingin renal fibrosis and tubular apoptosis in the unilateral ureteralobstruction model using two different approaches: (1) Mice thatwere deficient in either JNK1 or JNK2 and (2) a specific inhibitorof all JNK isoforms, CC-401. Western blotting and immunostainingidentified a marked increase in JNK signaling in the obstructedkidney, with substantial redundancy between JNK1 and JNK2 isoforms.Administration of CC-401 blocked JNK signaling in the rat obstructedkidney and significantly inhibited renal fibrosis in terms ofinterstitial myofibroblast accumulation and collagen IV deposition.This effect was attributed to suppression of gene transcriptionfor the profibrotic molecules TGF-1 and connective tissue growthfactor. CC-401 treatment also significantly reduced tubularapoptosis in the obstructed kidney. Genetic deletion of JNK1or JNK2 did not protect mice from renal fibrosis in the unilateralureteral obstruction model, but JNK1 deletion did result ina significant reduction in tubular cell apoptosis. In conclusion,this is the first study to demonstrate that JNK signaling playsa pathogenic role in renal fibrosis and tubular apoptosis. Furthermore,JNK1 plays a nonredundant role in tubular cell apoptosis. Thesestudies identify the JNK pathway as a potential therapeutictarget in progressive kidney disease.
Progressive forms of kidney disease feature common pathologicprocesses such as fibrosis and the loss of intrinsic renal cellsvia apoptosis. These mechanisms operate irrespective of thenature of the initial renal insult and therefore are potentialtargets for therapeutic intervention. The c-Jun amino-terminalkinase (JNK) pathway has been implicated in tubular cell apoptosison the basis of in vitro studies, but the functional role ofJNK signaling in renal fibrosis has not been examined.
A variety of cellular stresses, such as stretch, reactive oxygenspecies, inflammatory cytokines, and osmotic stress, can inducea cascade of signaling events, leading to dual phosphorylationof the Thr-X-Tyr activation motif of JNK (1,2). The active (phosphorylated)form of JNK then can translocate to the nucleus and phosphorylatespecific target proteins to induce a variety of cellular responses,including inflammation and apoptosis. A unique JNK target isthe phosphorylation of Ser63 and Ser73 in the NH2-terminal domainof c-Jun (3–6), which can be used as a surrogate markerof JNK activity.
Three members of the JNK family have been described in mammaliancells (1,2). JNK1 and JNK2 isoforms are widely expressed inmost tissues, including the kidney, but JNK3 is restricted tothe nervous system. Mice that are deficient in Jnk1 or Jnk2are viable, but the double-gene knockout is fetal lethal. Transcriptionof each JNK gene can give rise to multiple 54- and 46-kD proteinsthrough alternative mRNA splicing (1,2).
A proapoptotic role for JNK signaling has been demonstratedin a range of different cell types in vitro, although some studieshave shown that JNK also can exert an antiapoptotic effect undercertain circumstances (7–9). In vivo studies have shownthat excitotoxicity-induced neuron apoptosis largely dependson JNK3 (10), whereas the use of a specific JNK inhibitor reducedhepatocyte apoptosis in liver ischemia/reperfusion injury (11,12).A role for the JNK pathway in the inflammatory response is evidentfrom in vitro studies in which blockade of JNK signaling inhibitsLPS-induced cytokine and nitric oxide production by macrophages(13). These data are supported by in vivo studies in which jointinflammation and erosion in rat adjuvant-induced arthritis wassuppressed by administration of a JNK inhibitor drug (14). Furthermore,gene knockout studies have shown that JNK1 is important in theprimary immune response in which naíive T cells differentiateinto Th1 cells (15,16), whereas JNK2 has been implicated inthe differentiation of CD4+ T cells into Th1 responders (17).
In contrast to inflammation and apoptosis, little is known ofJNK in tissue fibrosis. In vitro studies suggest a role forJNK signaling in TGF-1 induced fibronectin production in fibroblastsand in TGF-1 induced connective tissue growth factor (CTGF)production (18,19). However, a role for JNK signaling in tissuefibrosis has yet to be demonstrated clearly.
JNK activation has been described in a variety of experimentalkidney diseases (20–22), but the functional role of thispathway has not been determined in any model of kidney diseaseto date. We examined the pathologic role of JNK in fibrosisand apoptosis in the obstructed kidney. Unilateral ureteralobstruction (UUO) was selected because there is rapid developmentof renal fibrosis plus tubular apoptosis in this model. Thedevelopment of fibrosis in this model depends on the profibroticgrowth factors TGF-1 and CTGF (23–25). Another importantaspect of this disease model is that renal fibrosis and apoptosisare driven by irreversible mechanical stretch and are independentof the adaptive immune response (26). Two approaches were usedto block JNK activity. First, Jnk1 and Jnk2 gene–deficientmice were examined in the mouse UUO model. Second, a specificJNK inhibitor, CC-401, was administered in the rat UUO model.
Antibodies
The following rabbit antibodies were obtained from Cell Signaling(San Diego, CA): JNK2 (preferentially detects JNK2 over JNK1),phospho-JNK (Tyr183/Tyr185), c-Jun, phospho-c-Jun (Ser63), phospho-c-Jun(Ser73), phospho-p44/42 MAPK (Thr202/Tyr204), and cleaved caspase-3.Other antibodies used were rabbit anti-JNK1/2 and goat anti–glyceraldehyde-3-phosphatedehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA); mouseanti– smooth muscle actin (-SMA; 1A4; (Sigma-Aldrich,Castle Hill, NSW, Australia); goat anti-collagen IV (SouthernBiotechnology Associates, Birmingham, AL); rabbit anti–aquaporin2 (anti-AQP2; Calbiochem, San Diego, CA) as a marker of collectingducts; mouse anti-CD68, which recognizes rat macrophages (ED1),and rat anti-mouse macrophages (F4/80; Serotec, Oxford, UK);and mouse anti-bromodeoxyuridine (Dako, Glostrup, Denmark).Other antibodies included biotinylated goat anti-rabbit IgG,biotinylated rabbit anti-goat IgG, and streptavidin-conjugatedhorseradish peroxidase (all from Zymed-Invitrogen, Carlsbad,CA) and horseradish peroxidase–conjugated goat anti-mouseIgG and mouse peroxidase–conjugated antiperoxidase complexes(PAP; Dako).
Animals
Breeding pairs of MAPK8 (Jnk1+/–) and MAPK9 (Jnk2–/–)gene knockout mice on the C57BL/6J background were importedfrom Jackson Laboratories (Bar Harbor, ME) and bred in-house.Wild-type C57BL/6J mice and Sprague-Dawley rats were obtainedfrom Monash Animal Services (Clayton, VIC, Australia). All animalexperimentation was approved by the Monash Medical Centre AnimalEthics Committee.
Mouse Model of Renal Fibrosis
UUO surgery was performed on groups of eight female mice (22to 25 g) of each genotype. Briefly, mice were anesthetized usingketamine/xylazine; a midline incision made; and the left ureterwas exposed, tied at two points, and then cut between theseties. Animals were killed 7 d after surgery, and tissue wasremoved for analysis. All animals were given 50 mg/kg bromodeoxyuridine(BrdU) by intraperitoneal injection 3 h before being killedto label dividing cells.
Western Blot Analysis
A quarter kidney was homogenized in 0.5 ml of RIPA lysis bufferthen centrifuged to remove debris, and the supernatant storedat –80°C. Samples were separated by SDS-PAGE; electroblottedonto nitrocellulose membranes that were incubated with BlockingBuffer (LiCor Biosciences, Lincoln, NE) before incubation withprimary antibody overnight at 4°C, washing, and then incubationwith Alexa Fluor 680 goat anti-rabbit IgG (Molecular Probes,Eugene, OR) for 30 min; and analyzed by the Odyssey infraredimaging system (LiCor Biosciences). Blots were reprobed forglyceraldehyde-3-phosphate dehydrogenase as a loading control.Results were quantified using the Gel-Pro Analyzer program (MediaCybernetics, Silver Spring, MD).
CC-401
The specific JNK inhibitor CC-401 was synthesized by Celgene(11,12). CC-401 is a potent inhibitor of all three forms ofJNK (Ki of 25 to 50 nM) and has at least 40-fold selectivityfor JNK compared with other related kinases, including p38,extracellular signal–regulated kinase (ERK), inhibitorof B kinase (IKK2), protein kinase C, Lck, zeta-associated proteinof 70 kDa (ZAP70). In cell-based assays, 1 to 5 µmol/LCC-401 provides specific JNK inhibition. CC-401 is preparedin a sodium citrate vehicle and administered to rats by twice-dailygavage at 100 mg/kg. Peak serum levels (approximately 1 µmol/L)occur 3 to 5 h after gavage. Serum levels of CC-401 were analyzedby HPLC.
CC-401 Treatment of Rat Renal Fibrosis
Groups of 10 to 14 Sprague-Dawley rats (150 to 180 g) underwentUUO surgery as described for mice above. Rats received CC-401,vehicle alone, or no treatment beginning 3 h before UUO surgeryand continuing twice daily until being killed on day 7. A groupof normal rats also were examined. Animals were given 50 mg/kgBrdU by intraperitoneal injection 3 h before being killed tolabel dividing cells.
Renal Histology
Paraffin sections (2 µm) of methylcarn-fixed tissue werestained using periodic acid-Schiff and hematoxylin. Renal interstitialarea in the UUO model was assessed by point counting of theentire cortex using x250 fields. All scoring was performed onblinded slides.
Immunohistochemistry
Immunoperoxidase staining for phosphorylated JNK (p-JNK), c-Jun,p-c-Jun Ser63, p-c-Jun Ser73, p-ERK, and AQP2 was performedon 4-µm sections of formalin-fixed tissue using antigenretrieval (microwave oven heating in 0.1 M sodium citrate [pH6.0] for 10 min) followed by a three-layer avidin-biotin peroxidasecomplex (ABC) staining method (27). Immunoperoxidase stainingfor cleaved caspase-3 and ED1+ macrophages was performed onformalin-fixed tissue sections without antigen retrieval andusing a three-layer PAP staining method. Immunoperoxidase stainingfor -SMA used a three-layer PAP method on methylcarn-fixed tissue,and immunostaining for collagen IV used frozen sections withthe avidin-biotin peroxidase complex method. Two-color immunostainingfor AQP2 and p-JNK used formalin-fixed tissue sections as describedpreviously (27).
Quantification of Immunostaining
The interstitial area of -SMA and collagen IV immunostainingwas quantified in x250 power fields that covered at least 90%of the cortex by image analysis using Image-Pro software (MediaCybernetics). Large blood vessels were excluded from the analysis.
The number of interstitial ED1+ macrophages was counted in theentire cortex using high-power fields (x400). In addition, thenumber of tubular epithelial cells and interstitial cells thatstained for cleaved caspase-3 was scored in the entire cortexunder high power. All scoring was performed on blinded slides.
Real-Time Reverse Transcriptase–PCR
Total RNA was extracted from whole kidney samples using theRiboPure reagent (Ambion, Austin, TX) and reverse-transcribedusing the Superscript First-Strand Synthesis kit (Invitrogen,Carlsbad, CA) with random primers. Real-time PCR was performedon Rotor-Gene 3000 system (Corbett Research, Sydney, Australia)with thermal cycling conditions of 37°C for 10 min and 95°Cfor 5 min, followed by 50 cycles of 95°C for 15 s, 60°Cfor 20 s, and 68°C for 20 s. The primer pairs and FAM-labeledminor groove binder probes used were as follows: TGF-1 forwardGGA CAC ACA GTA CAG CAA, reverse GAC CCA CGT AGT AGA CGA T andprobe ACA ACC AAC ACA ACC C); CTGF forward CGT TTG TGC CTA TTGTTC TTG T, reverse GAT CCA TTG CTT TAC CGT CTA C, and probeCAA ACT CCA AAC ACC A); and collagen IV forward GGC GGT GCACAG TCA GAC CAT, reverse GGA ATA GCC AAT CCA CAG TGA, and probeCAG TGC CCC AAC GGT). The relative amount of mRNA was calculatedusing comparative Ct (Ct) method. All specific amplicons werenormalized against 18S RNA, which was amplified in the samereaction as an internal control using commercial assay reagents(Applied Biosystems, Scoresby, Australia).
Tubular Cell Culture
Human HK-2 proximal tubular epithelial cells (American TypeCulture Collection, Manassas, VA) were cultured in DMEM/F12media (Sigma-Aldrich) supplemented with 10% FCS, 10 ng/ml EGF(Sigma-Aldrich), and 10 µg/ml bovine pituitary extract(Life Technologies Invitrogen, Carlsbad, CA). For Western blotstudies, cells were seeded into six-well plates and allowedto adhere overnight, and medium was changed to DMEM/F12 supplementedwith only 0.5% FCS for 24 h, by which time cells were confluent.CC-401 was prepared in citric acid (pH 5.5) and added to theconfluent cells 1 h before the addition of 300 mM sorbitol,and cells were harvested 30 min later using urea-RIPA bufferas described previously. Three experiments were performed, eachwith two replicates per condition. For ELISA experiments, HK-2cells were seeded into 24-well plates, allowed to adhere overnight,cultured in DMEM/F12 with 0.5% FCS for 24 h, and then incubatedwith CC-401 or vehicle for 60 min before stimulation with 1µM angiotensin II (AngII; Sigma-Aldrich). Supernatantswere harvested 48 h later and assayed for TGF-1 content usinga commercial ELISA kit (Promega, Annandale, NSW, Australia).Three experiments were performed, each using six replicatesper condition.
Statistical Analyses
Data are presented as means ± 1 SD. Analysis betweengroups of animals was performed by ANOVA using Tukey post testfor multiple comparisons (GraphPad 4.0 Software, San Diego,CA).
JNK Activation in Normal and Obstructed Mouse Kidney
Western blotting with an antibody that recognizes both JNK1and JNK2 (JNK1/2) shows abundant JNK1/2 protein in the normalkidney from wild-type, Jnk1–/–, and Jnk2–/–mice (Figure 1A). An anti-JNK2 antibody, which preferentiallyrecognizes JNK2 over JNK1, detects strong JNK bands in wild-typeand Jnk1–/– mice, which largely are absent in Jnk2–/–mice (Figure 1B). The high level of JNK1 and JNK2 expressionis not affected after 7 d of UUO. A weak signal for p-JNK isevident in normal kidney from wild-type, Jnk1–/–,and Jnk2–/– mice. UUO results in a three- to five-foldincrease in JNK phosphorylation in all three genotypes, indicatingsubstantial redundancy in signaling through JNK1 and JNK2 inthe obstructed kidney (Figure 1, C and D), although the levelof JNK phosphorylation in the obstructed kidney of Jnk1–/–mice was significantly less than that seen in wild-type andJnk2–/– mice (P < 0.001 for both).
Figure 1. Detection of c-Jun amino-terminal kinase (JNK) phosphorylation in wild-type (WT), Jnk1–/–, and Jnk2–/– mice by Western blotting of whole-kidney lysates. (A) Detection of total JNK1 and JNK2 (JNK1/2) in WT normal mice and in WT, Jnk1–/–, and Jnk2–/– mice with 7 d of unilateral ureteric obstruction (UUO). (B) Detection of JNK2 in the same groups as in A. (C) Detection of phosphorylated-JNK (p-JNK) in normal and UUO kidney from WT, Jnk1–/–, and Jnk2–/– mice. Blots also were probed for glyceraldehyde-3-phosphate dehydrogenase as a loading control. (D) Graph summarizing quantification of Western blotting results for p-JNK. Data are means ± SD for groups of eight mice with statistical analysis by ANOVA.
Immunostaining localized p-JNK to collecting ducts in the cortexand the medulla and to some parietal epithelial cells in normalkidney (Figure 2A), with occasional glomerular cells also stained.Double immunolabeling confirmed co-localization of p-JNK stainingin AQP2-stained collecting ducts (data not shown). This stainingpattern for p-JNK was not altered in the kidney of normal Jnk1–/–or Jnk2–/– mice (data not shown). UUO induced amarked increase in JNK phosphorylation with p-JNK staining prominentin dilated, atrophic tubules as well as in morphologically normaltubules and in interstitial cells (Figure 2D). A similar increasein p-JNK staining is seen in the obstructed kidney in Jnk1–/–and Jnk2–/– mice (Figure 2, G and J), indicatingthat both JNK isoforms are activated in the damaged kidney.
Figure 2. Detection of JNK activation by immunoperoxidase staining in mouse UUO. (A) Immunostaining for phosphorylated JNK (p-JNK) in normal mouse kidney shows a positive signal in the cytoplasm and nucleus of collecting duct cells in the renal cortex. No signal is apparent for c-Jun (B) or p-c-Jun Ser63 (C) in normal mouse kidney. (D) A marked increase in p-JNK immunostaining is apparent in UUO in WT mice, with staining of 30 to 50% of cortical tubules, many of which show marked dilation. (E) There is a dramatic induction of c-Jun in WT UUO kidney with many tubular cells showing strong nuclear staining, including dilated tubules, with a similar distribution to that of p-JNK staining. (F) Serial section to E shows immunostaining for p-c-Jun Ser63 that exhibits a pattern of nuclear staining that correlates very closely to that of total c-Jun, except that fewer cells are stained. UUO in Jnk1–/– mice induced a very similar increase in p-JNK (G), c-Jun (H), and p-c-Jun Ser63 (I) immunostaining as that observed in UUO in WT mice. Similarly, UUO in Jnk2–/– induced a very similar increase in p-JNK (J), c-Jun (K), and p-c-Jun Ser63 (L) immunostaining as that observed in UUO in WT mice. Magnification, x250.
We examined JNK activity by immunostaining for phosphorylationof c-Jun at Ser63, considered a JNK-specific target (3–5).c-Jun protein largely is absent from normal kidney (Figure 2B),but it is prominent in both damaged and intact tubules and ininterstitial cells in the obstructed kidney (Figure 2E). Lackof either Jnk1 or Jnk2 did not affect the induction of c-Junin the obstructed kidney (Figure 2, H and K). Although absentin normal kidney, phosphorylation of c-Jun at Ser63 was evidentin dilated, atrophic tubules as well as in intact tubules andinterstitial cells in the obstructed kidney (Figure 2, C andF) in a pattern that corresponds exactly with total c-Jun expressionand is similar to that of JNK phosphorylation. The pattern ofimmunostaining for p-c-Jun Ser63 in the obstructed kidney ofJnk1–/– and Jnk2–/– mice was not differentfrom that seen in wild-type mice (Figure 2, I and L), againdemonstrating redundancy in signaling. As an additional control,nuclear staining for phosphorylation of c-Jun at Ser73 (anotherspecific JNK target) followed the same pattern as that of phosphorylationof c-Jun at Ser63 (data not shown).
Renal Fibrosis and Apoptosis in the Obstructed Mouse Kidney
UUO in wild-type mice results in marked tubular dilation andan increase in interstitial volume with development of renalfibrosis in terms of interstitial accumulation of -SMA+ myofibroblastsand deposition of collagen IV (Figure 3, A through C). Jnk1–/–mice showed a minor reduction in interstitial volume and inthe accumulation of -SMA+ myofibroblasts, but there was no changein the deposition of collagen IV after UUO (Figure 3, A throughC). Jnk2–/– mice showed no protection from renalfibrosis.
Figure 3. Quantification of renal fibrosis and apoptosis in mouse UUO. UUO was induced in groups of WT, Jnk1–/–, and Jnk2–/– mice and compared with normal mice for interstitial area (A), area of -smooth muscle actin (-SMA) immunostaining (B), area of collagen IV immunostaining (C), and the number of proliferating tubular cells (D) and interstitial cells (E), on the basis of immunostaining for bromodeoxyuridine (BrdU) incorporation. Data are means ± SD for groups of eight mice with statistical analysis by ANOVA.
Apoptosis in the obstructed kidney was identified by immunostainingfor cleaved caspase-3. Although immunostaining of serial sectionswas able to identify JNK activation (p-c-Jun Ser63 staining)in some apoptotic cells (Figure 4, A and B), most apoptoticcells did not show JNK activation. Jnk1–/– miceshowed significant protection from apoptosis of tubular epithelialcells and interstitial cells in the UUO model, whereas Jnk2–/–mice were not protected (Figure 4, C and D). Finally, substantialproliferation of both tubular and interstitial cells was evidentin the wild-type obstructed kidney on the basis of immunostainingfor BrdU-labeled cells, and this was not altered in Jnk1–/–or Jnk2–/– UUO mice (Figure 3, D and E).
Figure 4. Apoptosis in mouse UUO. UUO was induced in groups of WT, Jnk1–/–, and Jnk2–/– mice, and apoptotic cells were identified by immunostaining for cleaved caspase-3. Immunostaining of serial sections of WT UUO kidney shows a cleaved caspase-3+ tubular cell (A, arrow), and the same cell is stained for p-c-Jun Ser63 (B, arrow). Graphs show quantification of the number of tubular cells (C) and interstitial cells (D), stained for cleaved caspase-3. Data are means ± SD for groups of eight mice with statistical analysis by ANOVA (C and D). Magnification, x400.
JNK Blockade Suppresses Renal Fibrosis in the Obstructed Rat Kidney
Given the redundancy in JNK1 and JNK2 signaling that was observedin the obstructed mouse kidney, we sought to block the activityof both JNK isoforms. For this purpose, we used CC-401, a smallmolecule that is a specific inhibitor of all three JNK isoforms(11,12). CC-401 competitively binds the ATP binding site inJNK, resulting in inhibition of the phosphorylation of the N-terminalactivation domain of the transcription factor c-Jun. The specificityof this inhibitor was tested in vitro using osmotic stress ofthe HK-2 human tubular epithelial cell line. CC-401 inhibitedsorbitol-induced phosphorylation of c-Jun in a dosage-dependentmanner (Figure 5). However, CC-401 did not prevent sorbitol-inducedphosphorylation of JNK, p38, or ERK. In vivo studies using CC-401were performed in the rat UUO model because of the poor pharmacokineticsof CC-401 in the mouse. However, the pathologic changes andmechanisms of fibrosis in the obstructed kidney are very similarin the rat and the mouse.
Figure 5. CC-401 inhibits c-Jun phosphorylation in tubular epithelial cells. Human HK-2 tubular epithelial cells were incubated with various concentrations of CC-401 and then given an osmotic shock with 300 mM sorbitol for 30 min. (A) Cell lysates were examined by Western blotting for p-c-Jun Ser63, p-JNK, p-p38, and phosphorylated extracellular signal–regulated kinase (p-ERK), and blots were reprobed for tubulin. (B through E) Graphs of the various phosphorylated proteins relative to tubulin showing pooled data from three independent experiments. Data are means ± SD with statistical analysis by ANOVA.
Ureter ligation of the rat kidney induced a marked increasein p-JNK staining with the induction of c-Jun expression anddetection of phosphorylation of c-Jun on Ser63 in a patternvery similar to that in the obstructed mouse kidney (Figure 6).Administration of CC-401 partially reduced JNK phosphorylation(Figure 6I), and there was an almost complete absence of phosphorylationof c-Jun at Ser63 in the obstructed kidney (Figure 6J). As anadditional control, we performed immunostaining with an antibodyagainst p-c-Jun Ser73, which is another JNK-specific target,although this antibody also recognizes phosphorylation of JunDat Ser100. Normal rat kidney shows positive staining with theantibody against p-c-Jun Ser73 in the luminal surface of collectingduct cells (Figure 6C). Because c-Jun is absent from tubularcells in normal kidney, this staining presumably reflects phosphorylationof JunD at Ser100 given that JunD is expressed constitutivelyin normal kidney (28). In the ligated kidney, nuclear stainingfor p-c-Jun Ser73 followed a virtually identical pattern tothat seen for p-c-Jun Ser63 (Figure 6G). The nuclear p-c-JunSer73 staining was abrogated by CC-401 treatment, although weakluminal staining of dilated tubules was apparent. This luminalstaining presumably reflects p-JunD Ser100 because it is knownthat JunD is upregulated in the injured kidney (28). We alsoexamined activation of ERK because this kinase is related closelyto JNK and ERK can phosphorylate JunD at Ser100 (5). Immunostainingshowed p-ERK in collecting ducts in normal rat kidney, and therewas marked p-ERK staining in damaged and normal tubules andin interstitial cells in the obstructed kidney, which was unalteredby CC-401 treatment (Figure 6, D, H, and L). Western blot analysisof whole-kidney tissue confirmed a substantial increase in thephosphorylation of JNK, ERK, and p38 kinases in the rat UUOkidney (all P < 0.05 versus normal). However, no significantdifferences were seen in phosphorylation of JNK, ERK, or p38kinases between the no-treatment, vehicle-treated, and CC-401–treatedUUO groups (Figure 7). Animals were killed 2 h after the lastdrug administration, at which time serum CC-401 levels were1.65 ± 1.17 µM, indicating that peak blood levelswere in the target range in which the drug is highly specificfor inhibiting JNK activity.
Figure 6. Detection of JNK and ERK activation by immunoperoxidase staining in rat UUO. Immunostaining of normal rat kidney detects staining for p-JNK in the cytoplasm and nucleus of collecting duct cells in the renal cortex (A); no signal for p-c-Jun Ser63 (B); luminal staining for p-c-Jun Ser73 in collecting ducts (C), and nuclear and cytoplasmic staining for p-ERK in collecting ducts (D). Immunostaining of day 7 UUO shows a marked increase in p-JNK with staining of cortical tubules, many of which show marked dilation, and some interstitial cells (E); nuclear staining for p-c-Jun Ser63 in many tubules, with a distribution similar to that for p-JNK (F); nuclear staining for p-c-Jun Ser73 in many tubules in a pattern very similar to that of p-c-Jun Ser63 (G); and nuclear staining for p-ERK in many tubules, including damaged tubules (H). Immunostaining of CC-401–treated day 7 UUO shows a partial reduction in p-JNK immunostaining compared with untreated UUO, but increased p-JNK staining is still evident in dilated tubules (I); an almost complete abrogation of p-c-Jun Ser63 staining (J); an almost complete abrogation of nuclear p-c-Jun Ser73 staining, although luminal staining is evident in damaged tubules (K); and no effect on the increase in p-ERK staining in the UUO kidney (L). Magnification, x250.
Figure 7. CC-401 does not affect phosphorylation of mitogen-activated protein kinases (MAPK) in rat UUO. (A) Western blotting of whole-kidney lysates shows increased phosphorylation of JNK, ERK, and p38 in rat UUO compared with normal kidney. No difference is seen with vehicle (Veh) or CC-401 treatment of UUO. Quantification of the ratio of phosphorylated MAPK to tubulin is shown for p-JNK (B), p-ERK (C), and p-p38 (D). For each individual MAPK, all UUO groups were increased significantly compared with normal kidney (P < 0.05), but no difference was seen between the various UUO groups. Data are means ± SD for groups of six rats with statistical analysis by ANOVA.
UUO in untreated or vehicle-treated rats resulted in markedtubular dilation with an increase in interstitial volume asa result, in part, of interstitial accumulation of -SMA+ myofibroblastsand deposition of collagen IV (Figure 8). A significant increasein kidney mRNA levels for collagen IV and for the profibroticgrowth factors TGF-1 and CTGF was evident in untreated and vehicle-treatedUUO (Figure 9). Despite ongoing ureter ligation that causestubular dilation, CC-401 treatment significantly reduced interstitialvolume, -SMA+ myofibroblast accumulation, and the depositionof collagen IV (Figure 8). These effects were associated witha partial reduction in renal TGF-1 and collagen IV mRNA levelsand normalization of renal CTGF mRNA levels (Figure 9).
Figure 8. CC-401 suppresses renal fibrosis in rat UUO. Groups of rats underwent UUO surgery and received CC-401, vehicle (Veh), or no treatment (No Tx) and were killed 7 d later. Periodic acid-Schiff–stained sections show that compared with normal rat kidney (A), ureter obstruction in the No Tx group resulted in tubular dilation and atrophy with an increase in interstitial area (B). CC-401 treatment of UUO did not prevent tubular dilation, but it did suppress the increase in interstitial area. (D) Graph quantifying the interstitial area in all animal groups. Immunostaining for -SMA identified arteries and arterioles in normal rat kidney (E), whereas in the untreated UUO group is a dramatic accumulation of interstitial -SMA+ myofibroblasts (F), which was reduced by CC-401 treatment (G). (H) Graph quantifying the area of interstitial -SMA immunostaining in all animal groups. Immunostaining shows collagen IV in glomerular and tubular basement membranes in normal rat kidney (I). A marked increase in interstitial collagen IV is evident in untreated UUO (J), which was prevented in part by CC-401 treatment (K). (L) Graph quantifying the area of interstitial collagen IV immunostaining in all animal groups. Data are means ± SD for groups of 10 to 14 rats with statistical analysis by ANOVA. Magnifications: x250 in A through C; x160 in E through G and I through K.
Figure 9. CC-401 suppresses the expression of profibrotic growth factors in rat UUO. Groups of rats underwent UUO surgery and received CC-401, vehicle (Veh), or no treatment (No Tx) and were killed 7 d later. Real-time reverse transcriptase–PCR was used to analyze renal levels of TGF-1 (A), connective tissue growth factor (CTGF) (B), and collagen IV (C) mRNA relative to 18S rRNA. Data are means ± SD for groups of eight rats with statistical analysis by ANOVA.
To investigate whether JNK blockade directly inhibits TGF-1production, we examined the effect of CC-401 on AngII–inducedTGF-1 secretion by the proximal tubular epithelial cell lineHK-2. The addition of 1 µM AngII induced a two-fold increasein TGF-1 secretion, which was suppressed in a dosage-dependentmanner by CC-401, with a significant reduction observed with4 µM CC-401 (Figure 10).
Figure 10. CC-401 inhibits angiotensin II (AngII)-induced TGF-1 secretion in vitro. Stimulation of cultured HK-2 proximal tubules with 1 µM AngII for 48 h caused a two-fold increase TGF-1 secretion (P < 0.001) as quantified by ELISA. CC-401 inhibited TGF-1 secretion in a dosage-dependent manner. One experiment of three is shown, all of which showed similar results. Replicates of six were used in each condition, and results were analyzed by ANOVA.
Tubular epithelial cell apoptosis was evident in the obstructedkidney as assessed by immunostaining for cleaved caspase-3,and this was reduced by approximately 50% with CC-401 treatment(Figure 11A). Apoptosis of interstitial cells also was evidentin the obstructed kidney. This was reduced by CC-401 treatment,but this failed to reach statistical significance (Figure 11B).A marked increase in the proliferation of interstitial and tubularcells was apparent in the obstructed kidney (Figure 11, C andD). Although CC-401 reduced the mean number of BrdU+ proliferatinginterstitial cells, this did not reach statistical significance.Last, interstitial accumulation of ED1+ macrophages in the obstructedkidney was not affected by CC-401 treatment (Figure 11E).
Figure 11. CC-401 suppresses tubular epithelial cell apoptosis in rat UUO. Groups of rats underwent UUO surgery and received CC-401, vehicle (Veh), or no treatment (No Tx) and were killed 7 d later. Tubular apoptosis (A) and interstitial cells apoptosis (B) were assessed by scoring cells that were stained for cleaved caspase-3. Proliferation of tubular epithelial cells (C) and interstitial cells (D) was assessed by immunostaining for BrdU incorporation. (E) Interstitial ED1+ macrophages were assessed by immunostaining. Data are means ± SD for groups of 10 to 14 rats with statistical analysis by ANOVA.
This study examined the relative contribution of JNK1 and JNK2isoforms to JNK signaling in normal or diseased kidney. We foundsubstantial redundancy between JNK1 and JNK2 in regard to JNKsignaling in both normal kidney and the obstructed kidney. Indeed,the distribution pattern of the marked increase in JNK phosphorylationand of p-c-Jun Ser63 and p-c-Jun Ser73 in the obstructed kidneywas unaltered in Jnk1–/– and Jnk2–/–mice, although Western blotting did reveal a significant reductionin total JNK phosphorylation in the obstructed kidney in Jnk1–/–mice.
Phosphorylation of JNK was identified in collecting ducts andparietal epithelial cells in normal mouse and rat kidney. Itis not clear whether JNK signaling in the normal kidney simplyreflects osmotic or mechanical stress or plays an importantphysiologic function. A number of in vitro studies have demonstratedthat mechanical and osmotic stresses can activate the JNK pathway(29–34). However, JNK activation does not seem to be criticalfor normal renal function because the use of CEP-1347, a drugthat blocks several signaling pathways, including JNK, has beenshown to be safe and well tolerated in short-term (35) and long-termclinical trials in Parkinson’s disease (Clinical TrialNCT0004002).
This is the first study to examine JNK signaling in renal fibrosis.For examination of this question, it is important to recognizethat JNK signaling is involved in the innate and adaptive immuneresponse that often is the cause of tissue injury to which fibrosisis the response. Therefore, to delineate the role of JNK signalingin the fibrotic response without the potential complicationof modifying the immune response, we examined the UUO modelin which fibrosis develops in response to a nonimmune, mechanicalinsult in a lymphocyte-independent manner (26).
Given the extensive redundancy between JNK1 and JNK2 isoforms,it was not surprising that neither Jnk1–/– nor Jnk2–/–mice were protected from the development of renal fibrosis inthe UUO model. However, administration of CC-401, which inhibitsall JNK isoforms, provided substantial protection from renalfibrosis in the rat model on the basis of reduced collagen IVdeposition and -SMA+ myofibroblast accumulation. Fibrosis inthe UUO is model depends on the profibrotic growth factors TGF-1and CTGF (23–25), common mechanisms that drive fibrosisin other forms of kidney disease. Upregulation of TGF-1 in theUUO model depends on AngII (36). We found that CC-401 inhibitedAngII-induced TGF-1 secretion by cultured tubular epithelialcells in a dosage-dependent manner, showing a significant effectin the concentration range that was achieved in vivo. Theseresults are consistent with previous in vitro studies that demonstratedthat AngII-induced TGF- production in mesangial cells operatesin JNK-dependent manner (37). Therefore, the reduction in TGF-1mRNA levels probably is a major mechanism by which CC-401 treatmentsuppresses renal fibrosis in the UUO model. The identificationof an activating protein-1 (AP-1) binding site in the TGF- genepromoter provides a mechanism whereby JNK blockade can inhibitTGF- gene transcription directly (38). The reduction in collagenIV mRNA levels is consistent with a reduction in TGF-1 activity.Of note, JNK activation was identified in both tubular epithelialcells and interstitial -SMA+ myofibroblasts in the obstructedkidney—the two cell types that were identified as beingresponsible for the increase in renal TGF-1 mRNA levels in thismodel (36,39,40). CC-401 treatment also prevented an increasein CTGF gene transcription in the obstructed kidney. This probablyreflects an indirect effect of the reduction in TGF-1 mRNA levels.However, CC-401 may have inhibited the CTGF response in a directmanner because TGF-1–induced CTGF production in humanlung fibroblasts is JNK dependent (19).
The reduction in -SMA+ myofibroblast accumulation that was seenwith CC-401 treatment in the UUO model could operate via indirector direct mechanisms. The reduction in TGF-1 production thatwas seen with CC-401 treatment may be an indirect mechanismto reduce -SMA+ myofibroblast accumulation because TGF-1 isassociated with the migration of fibrocytes from the circulationinto sites of tissue injury (41), TGF-1 can induce the differentiationof local fibroblasts into -SMA+ myofibroblasts (42), and TGF-1can promote the transition of tubular epithelial cells into-SMA+ myofibroblasts (43,44). Local proliferation also contributesto interstitial myofibroblast accumulation in the obstructedkidney. In vitro studies that used antisense oligonucleotidesimplicated JNK2 in promoting proliferation of murine fibroblasts(45). CC-401 treatment reduced interstitial cell proliferationin rat UUO, but this failed to reach statistical significance,whereas interstitial cell proliferation was unaltered in Jnk1–/–or Jnk2–/– UUO, indicating that JNK signaling doesnot promote renal fibroblast or myofibroblast proliferationin vivo.
A direct link between JNK activation and tubular epithelialcell apoptosis was identified by immunostaining of serial sectionsfor cleaved caspase-3 and p-c-Jun Ser63. However, most apoptoticcells did not exhibit JNK signaling, indicating either thatthe c-Jun protein had been de-phosphorylated/degraded beforeactivation of caspase-3 or that other pathways contribute toapoptosis in this model. Tubular cell apoptosis was suppressedby JNK blockade with CC-401. Furthermore, studies in mouse UUOshowed a nonredundant role for JNK1 but not JNK2 in the apoptosisof tubular epithelial cells and interstitial cells. This isthe first in vivo study to demonstrate that renal cell apoptosisdepends, at least in part, on the JNK signaling pathway. Therole of individual JNK forms in apoptotic cell death are celltype dependent. Both Jnk1 and Jnk2 gene–deficient micehave reduced apoptosis of myocytes after cardiac ischemia/reperfusioninjury (46); however, JNK1 but not JNK2 is involved in TNF-–inducedapoptosis of fibroblasts (47). These data also are consistentwith studies in liver ischemia/reperfusion injury in which CC-401treatment reduced hepatocyte apoptosis and improved liver function(11,12).
The finding that JNK signaling plays a critical role in tubularepithelial cell apoptosis in the obstructed kidney confirmsprevious in vitro studies that showed that oxidative stress–inducedtubular cell apoptosis is mediated in part through JNK signaling(48,49). Similarly, stretch-induced apoptosis of cultured tubularepithelial cells also depends, in part, on JNK signaling (50).Macrophages also have been postulated as mediators of tubularepithelial cell apoptosis (51). CC-401 treatment had no effecton interstitial macrophage accumulation in the obstructed kidney;however, we cannot rule out that JNK signaling may have a rolein macrophage activation that leads to tubular epithelial cellapoptosis in the UUO model, because previous adoptive transferstudies in rat anti–glomerular basement membrane diseasehave shown that blockade of JNK signaling in macrophages canreduce substantially macrophage-mediated renal injury withoutany effect on macrophage recruitment into the injured kidney(52).
JNK blockade using CC-401 did not completely abrogate renalfibrosis in the obstructed rat kidney, suggesting that otherpathways contribute to the pathogenesis of interstitial fibrosis.The closely related pathway p38 mitogen activated protein kinasehas been shown to promote renal fibrosis in the UUO model (53).It is interesting that p38 blockade caused a partial reductionin interstitial -SMA+ myofibroblast accumulation and collagenIV deposition without affecting the upregulation of TGF-1 mRNAlevels (53). The distinct differences between JNK and p38 signalingpathways in regard to transcription of the TGF-1 gene raisesthe question as to whether combined inhibition of JNK and p38pathways could provide an additive benefit in suppressing renalfibrosis in this model.
The specificity of CC-401 for the JNK pathway was examined inseveral ways in this study. First, phosphorylation of c-Junat Ser63 and Ser73 was blocked efficiently by CC-401. Althoughthe specificity of this reaction has been questioned (54), severalstudies have argued strongly that ERK is unable to phosphorylatethe amino terminus of c-Jun (3–6). Furthermore, blockadeof ERK signaling in the mouse UUO model using the MEK1 inhibitorUO126 failed to prevent the phosphorylation of c-Jun at Ser63or Ser73 (Y.H. and D.J.N.-P., unpublished data). Second, thepeak serum level of CC-401 was in the target range of 0.5 to2.0 µM, at which CC-401 has at least 40-fold selectivityfor JNK compared with a panel of related kinases. Third, CC-401treatment had no effect on phosphorylation of ERK or p38 kinasesin the UUO model or in cell culture studies. However, in studiesof this type, it is impossible to exclude the possibility thatthe drug being examined exerts effects on some unidentifiedpathway.
This is the first study to demonstrate that JNK signaling playsa pathogenic role in renal fibrosis and tubular apoptosis. Whereasthe JNK1 and JNK2 isoforms showed redundancy with respect tothe development of renal fibrosis in the obstructed kidney,JNK1 was identified to play a nonredundant role in tubular cellapoptosis. These studies identify the JNK pathway as a potentialtherapeutic target in progressive kidney disease.
Celgene in part funded these studies, and D.N.-P. acts as aconsultant for Celgene.
Acknowledgments
This study was funded by the National Health and Medical ResearchCouncil (NHMRC) of Australia and by Celgene. We acknowledgea NHMRC Scholarship (R.F.), a NHMRC/Kidney Health Australia/Australianand New Zealand Society of Nephrology Career Development Award(G.T.), and a NHMRC Senior Research Fellowship (D.N.-P.).
Part of these studies were presented at the 38th annual meetingof the American Society of Nephrology; November 8 through 13,2005; Philadelphia, PA.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
1 and connective tissue growth factor. CC-401 treatment also significantly reduced tubular apoptosis in the obstructed kidney. Genetic deletion of JNK1 or JNK2 did not protect mice from renal fibrosis in the unilateral ureteral obstruction model, but JNK1 deletion did result in a significant reduction in tubular cell apoptosis. In conclusion, this is the first study to demonstrate that JNK signaling plays a pathogenic role in renal fibrosis and tubular apoptosis. Furthermore, JNK1 plays a nonredundant role in tubular cell apoptosis. These studies identify the JNK pathway as a potential therapeutic target in progressive kidney disease. 
smooth muscle actin (
B kinase (IKK2), protein kinase C, Lck, zeta-associated protein of 70 kDa (ZAP70). In cell-based assays, 1 to 5 µmol/L CC-401 provides specific JNK inhibition. CC-401 is prepared in a sodium citrate vehicle and administered to rats by twice-daily gavage at 100 mg/kg. Peak serum levels (approximately 1 µmol/L) occur 3 to 5 h after gavage. Serum levels of CC-401 were analyzed by HPLC. 
