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NDT Advance Access published online on July 31, 2007
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfm509
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Blockage of JAK/STAT signalling attenuates renal ischaemia-reperfusion injury in rat

Niansheng Yang1, Mingqian Luo1, Rong Li1, Yuefang Huang2, Rui Zhang1, Qingqing Wu1, Fang Wang1, Youji Li1 and Xueqing Yu1

1Department of Nephrology and 2Department of Pediatrics, First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China

Correspondence and offprint requests to: Prof. Niansheng Yang, Department of Nephrology, First Affiliated Hospital, Sun Yat-Sen University, 58 Zhongshan Road II, Guangzhou 510080, P. R. China. Email: zsuyns{at}163.com or yangnsh{at}mail.sysu.edu.cn



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Background. JAK/STAT signalling is one of the major pathways for cytokine signal transduction. However, the role of JAK/STAT in renal ischaemia/reperfusion (I/R) injury is not clear. The present study investigated the protection against renal I/R injury by in vivo inhibition of JAK2 activation.

Methods. Rats subjected to renal I/R were either treated with daily intraperitoneal injection of selective JAK2 inhibitor tyrphostin AG490 (10 mg/kg) or vehicle alone starting 4 h before, immediately after or until 3 h after I/R. Renal function, histology, infiltration of macrophages, apoptosis, expression of chemokines and adhesion molecules were assessed.

Results. AG490 treatment significantly inhibited the phosphorylation of JAK2 and its downstream molecule STAT1 and STAT3. Rats pretreated with AG490 exhibited improved renal function, attenuated histological lesions and reduced apoptosis of tubular epithelial cells. AG490 significantly inhibited renal expression of MCP-1 and ICAM-1 mRNA, as well as the expression of ICAM-1 protein, accompanied by decreased macrophage accumulation in the kidney. Immediate post-ischaemic treatment of AG490 also significantly ameliorated renal injury. However, delayed post-ischaemic treatment until 3 h after I/R failed to attenuate renal damage.

Conclusions. This study demonstrated the involvement of JAK/STAT signalling in the pathogenesis of renal I/R injury, suggesting that JAK/STAT pathway may serve as a potential target for early intervention in ischaemic acute renal failure.

Keywords: inflammation; ischaemia-reperfusion; kidney; macrophage



   Introduction
Top
 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 
Ischaemic acute renal failure (ARF) is a common clinical event caused by reduced blood supply to the kidney. Despite advances in preventive strategies and supportive measures, this disease continues to be associated with significant morbidity and mortality [1]. Vasoconstriction, endothelial cell injury, oxygen free radicals, activation of NF{kappa}B and apoptosis have been shown to be implicated in the pathogenesis of renal ischaemia/reperfusion (I/R) injury [2]. There is accumulating evidence that ARF is an inflammatory disease, as manifested by the infiltration of leucocytes, up-regulation of chemotactic factors by endothelial cells and generation of proinflammatory mediators by renal tubular epithelial cells [3].

Leucocytes potentiate renal I/R injury after activation by pro inflammatory mediators, including cytokines, reactive oxygen species (ROS) and eicosanoids [3,4]. Infusion of a TNF-{alpha} binding protein decreases TNF-{alpha} bioactivity and neutrophil infiltration and preserves renal function in renal I/R injury [4]. In addition, leucocytes are recruited to the sites of injury by proinflammatory cytokines interleukin-1 (IL-1), {gamma}-interferon (IFN-{gamma}) and TNF-{alpha} [4–6]. Renal tubular epithelial cells also generate proinflammatory cytokines including monocyte chemoattractant protein-1 (MCP-1), TNF-{alpha}, IL-1ß, IL-6, IL-18 and regulated upon activation, normal T-cell expressed and secreted (RENTES) [7–9]. Inhibition of the expression of ICAM-1 and KC/IL-8 (KC, the mouse isoform of IL-8) by {alpha}-melanocyte-stimulating hormone (MSH), an endogenous anti-inflammatory cytokine, suppressed infiltration of neutrophils and improves renal dysfunction and histological lesions following I/R [10].

Characterization of the ability of IFN-{alpha} in inducing gene expression led to the discovery of Janus kinase-signal transducer and activator of the transcription factor (JAK-STAT) signalling pathway [11]. Subsequent studies found that this pathway plays an important role in transducing signals for a wide array of cytokines and growth factors including IL-2, IL-6, IFN-{gamma} and PDGF [12,13]. JAK activation stimulates cell proliferation, differentiation, cell migration and apoptosis [12,14,15]. These cellular events are critical to haematopoiesis, immune response, inflammation, development, growth and other processes. In general, binding of these cytokines to their receptors induces receptor dimerization, tyrosine phosphorylation of the receptor-associated JAKs, followed by activation of the STATs, including STAT1, 2, 3, 4, 5 and 6. Phosphorylated STATs form dimers and translocate to the nucleus to activate transcription of many target genes [12,14,15].

Given that many proinflammatory cytokines are up-regulated during I/R injury, it is likely that activation of JAK/STAT signalling is involved in development of this pathological condition. Indeed, recent study has shown that JAK/STAT signalling was associated with cardiac dysfunction during ischemia and reperfusion [16]. However, the role of JAK/STAT signalling in the pathogenesis of renal I/R injury remains unknown. In this study, we reported that in vivo treatment of tyrphostin AG490, a selective inhibitor of JAK2 phosphorylation, reduced renal I/R injury in rats.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Acknowledgements
 References
 
Animal model
Male Sprague-Dawley rats weighing 200–250 g were obtained from Animal Center, Sun Yat-Sen University, Guangzhou, China. Rats were fed ad libitum with standard rodent chow and housed in an animal facility at 21°C. Rats were anesthetized using chloral hydrate (400 mg/kg, intraperitoneally) and the abdominal cavity was exposed via a midline incision, and the left renal artery was identified and freed by blunt dissection without hampering blood supply to the adrenal gland. A microvascular clamp was placed on the left renal pedicle to effect complete cessation of blood flow. The core temperature of these animals was maintained at 37°C by placing them on a homeothermic table. After 50 min, the clamp was removed with return of blood flow to the kidney. Visual inspection corroborated successful reperfusion with a change in the colour of kidneys from dark blue to bright red after releasing the clamp before right nephrectomy was performed. The surgical incision was sutured and animals were allowed to recover with free access to chow and water. Rats in the sham group had laparotomy without performing ischaemia or nephrectomy. Animal study was approved by the Animal Care Committee, Sun Yat-Sen University and was performed in accordance to our institutional guideline.

Treatment protocol
Littermates were randomly assigned into either vehicle-treated or AG490-treated groups. In the first set of experiments, 24 rats subjected to ischaemia/reperfusion were either treated with 10 mg/kg of daily intraperitoneal injection of selective JAK2 inhibitor tyrphostin AG490 [(2-cyano-3-(3,4-dihydroxyphenyl)-N-(benzyl)-2-propenamide, Sigma-Aldrich, St Louis, MO)] or vehicle alone starting 4 h before I/R until being sacrificed at 24 or 48 h. AG490 was dissolved in a vehicle containing 45% DMSO and 55% normal saline. The dosage of AG490 was chosen based on significant inhibition of JAK2 phosphorylation in our preliminary experiment (data not shown) and on previous report showing significant efficacy at the dose of 5mg kg–1 d–1 for rat in vivo study [17]. In the second set of experiments another 32 rats subjected to I/R were either treated with 10 mg/kg of daily intraperitoneal injection of AG490 or vehicle alone starting either immediately after or delayed until 3 h after I/R until being sacrificed at 48 h. Groups of rats were sacrificed in either the I/R group or in the sham group at the time point of 24 or 48 h after I/R. Rats were euthanized after ether anaesthesia and a blood sample was collected by cardiac puncture. Part of the left kidney was snap frozen and the remaining part was fixed with neutral-buffered formalin.

Biochemistry
Serum was separated by centrifugation and stored at –20°C until use. Serum creatinine and blood urea nitrogen (BUN) concentrations were measured by standard picric acid-based colorimetric kinetic assay using a commercial Beckman SynchronX3 Clinical System autoanalyzer (Beckman Coulter, Inc., Fullerton, CA 92835, USA).

Histology
Formalin-fixed paraffin-embedded renal sections were stained with haematoxylin and eosin (H&E) or periodic acid-Schiff (PAS) reagent. Histological lesions due to tubular necrosis were quantitated by calculation of the percent of renal tubules that displayed loss of brush border and cell necrosis as follows and described by De Greef et al. [18]: score 0, normal tubule; score 1, (limited to) loss of brush border; score 2, <50% tubular damage meaning less than 50% of naked basement membrane; score 3, >50% tubular damage; and score 4, total destruction of all epithelial cells, naked basement membrane. Tubular necrosis lesions in the cortex and outer stripe of medulla (most sensitive zone for ischemic injury) were scored separately. At least 20 microscopic fields (x200) were examined for the cortex or for outer stripe of medulla on each slide. The mean score of tubular necrosis for each animal was generated by dividing sum of score by the number of fields examined. Histological assessment was performed in a blinded fashion.

Detection of apoptotic cells
Apoptotic tubular epithelial cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-labelled UTP nick end labelling (TUNEL) method, using an ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Chemicon International Inc., USA) according to the manufacturer's instructions. Briefly, after deparffinization, digestion with proteinase K and quenching of endogenous peroxidase in 3% H2O2, formalin-fixed renal sections were incubated with the TdT enzyme and digoxigenin-labelled nucleotides at 37°C for 60 min, further incubated with a horseradish peroxidase-conjugated anti-digoxigenin antibody at room temperature for 30 min and finally incubated with a substrate diaminobenzidine (DAB) for 3–6 min. Sections were counter-stained with methyl-green. Negative controls were produced by omitting the TdT enzyme.

Antibodies
The following antibodies were used for immunohistochemistry: mouse anti-rat CD54 (intercellular adhesion molecule-1, ICAM-1) mAb (Serotec, Oxford, UK); ED1: mouse anti-rat CD68 mAb, which recognizes rat macrophages and monocytes (Serotec, Oxford, UK); Envision: a peroxidase-labelled secondary antibody with visualization system for immunohistochemical staining (Dako, Glostrup, Denmark). The following antibodies were used for western blotting: goat anti-JAK2 polyclonal antibody (Santa Cruz, CA, USA); goat anti-p-JAK2 polyclonal antibody (Santa Cruz); rabbit anti-STAT1 polyclonal antibody (Neomarker); mouse anti-p-STAT1 mAb (Santa Cruz); mouse anti-STAT3 mAb (Santa Cruz); mouse anti-p-STAT3 mAb (Santa Cruz) and goat anti-GAPDH polyclonal antibody (Santa Cruz).

Immunohistochemistry
Immunohistochemical staining of macrophages was performed on formalin-fixed paraffin section using a microwave-based technique [19]. Briefly, sections were dewaxed to water. Following with microwave treatment, slides were incubated in 0.3% H2O2 in methanol to inactivate endogenous peroxidase. Each section was incubated with ED1 mAb for 60 min at room temperature, followed by secondary antibody and visualization with 3,3-diaminobenzidine to produce a brown colour according to the manufacturer's instruction. Sections were counterstained with PAS minus hematoxylin and mounted in an aqueous medium. Immunohistochemical staining of ICAM-1 was performed on frozen section without microwave treatment. Briefly, slides were fixed in acetone and were incubated in 0.3% H2O2 in methanol to inactivate endogenous peroxidase. Sections were incubated with mouse anti-CD54 mAb for 60 min at room temperature, followed by secondary antibody and visualization with 3,3-diaminobenzidine. Cryostat sections were not counterstained.

Quantitation of Immunohistochemistry and apotosis
Labelled cells in tissue sections were scored as previously described [19]. Briefly, the number of macrophages (ED1+ cells) and TUNEL+ apoptotic tubular epithelial cells was counted in at least 20 consecutive high power fields (hpf) (x400) in renal cortex and outer stripe of medulla separately, avoiding large vessels. Data ware presented as cells per hpf. The expression of ICAM-1 in cortical renal tubules was semi-quantitatively analysed using a scaling system ranging from 0 to 4: 0 (none), 1 (very mild), 2 (mild), 3 (moderate) and 4 (dense). At least 20 consecutive high power fields were scored and sum of score was divided by the number of fields to yield average score for each animal. The quantitation was performed in a blinded fashion.

Western blotting
At time of sacrifice, part of the left kidney from each animal was snap frozen in liquid nitrogen and stored at –80°C. The sample was crushed and lysed in buffer [20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin] (Akt kinase Assay kit, Cell Signaling Techonology, USA) and homogenized, then centrifuged at 14 000 rpm for 20 min. After determination of protein concentration using Bio-Rad protein assay (Bio-Rad, Richmond, CA), the extracted protein was mixed with 2x SDS sample buffer (125 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 10% ß-mercaptoethanol) at a 1:1 ratio, boiled 10 min, resolved on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, and transferred to nitrocellulose membrane (Schleicher & Schuell BioScience Inc., Germany). After transfer, nitrocellulose membrane was washed with 30 ml TBS [10 mmol/l Tris (pH 7.4), 138 mmol/l NaCl] for 5 min three times at room temperature. Membranes were blocked with 5% (wt/vol) skimmed milk powder in TBST (TBS with 0.1% Tween-20, pH 7.4) for 1 h at room temperature. Blots were then washed three times in TBST and incubated with goat anti-JAK2, goat anti-p-JAK2, rabbit anti-STAT1, mouse anti-p-STAT1, mouse anti-STAT3 or anti-p-STAT3 antibody in 5% BSA in TBST overnight at 4°C. Blots were washed three times, then incubated with corresponding horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature, washed three times, and the membrane-bound antibody detected by Phototope®-HRP Western Blot Detection System (Cell Sinaling Techonology, USA) and captured on Kodak XAR film (EastmanKodak Co., Rochester, NY, USA). Protein levels were quantitated densitometrically with a documentation system (Fluor ChemTM8900, Alfa Innotech, USA). GAPDH was used as internal control to calculated relative ratio of optical density and values were compared to those of normal controls.

RT-PCR
Total RNA was isolated from kidneys with Trizol reagent (Invitrogen), according to manufacturer's recommendations. Samples were checked for degradation of total RNA on 2% agarose gel. RNA concentrations were determined by spectrophotometric measurement at wavelengths of 260/280 nm. Reverse transcription was performed by two-step methods (MBI). The following sequence-specific primers were used: rat MCP-1 forward primer: 5'-GTT GTT CAC AGT TGC TGC CT-3', reverse primer: 5'-CTC TGT CAT ACT GGT CAC TTC TAC-3'; rat ICAM-1 forward primer: 5'-GTG AGC GTC CAT ATT TAG GCA TGG-3', reverse primer: 5'-ACA GAC ACT AGA GGA GTG AGC AGG-3'; rat ß-actin forward primer: P1:5'-GAC CGA GCG TGG CTA CAG C-3', reverse primer: 5'-TGT CAG CGA TGC CTG GGT AC-3'. The PCR was performed for 35 cycles at 94°C for 60 s, 55°C for 45 s and 72°C for 45 s for MCP-1, or performed for 30 cycles at 94°C for 45 s, 60°C for 60 s and 72°C for 90 s for ICAM-1 or performed for 35 cycles at 94°C for 50 s, 53°C for 45 s and 72°C for 60 s for ß-actin in Mastercycler PCR Amplifier (Eppendorf, USA). Amplification products were separated by agarose gel electrophoresis and photographed using FluorChemTM 8900 (Alpha Inotech, San Leandro, CA). DNA bands corresponding to MCP-1, ICAM-1 and ß-actin cDNA were quantified and results were normalized for ß-actin levels.

Statistical Analysis
Values were expressed as mean ± SEM for histological scoring data and as mean ± SD for serum creatinine and BUN. Comparison of mean between groups was analysed by analysis of variance (ANOVA). A P-value less than 0.05 was considered statistically significant.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Acknowledgements
 References
 
Effects of AG490 treatment on renal function
In contrast to the sham-operated rats, rats subjected to I/R had dramatic increase in serum creatinine and BUN (P < 0.01), indicating renal dysfunction (Figure 1A and B). Pretreatment of rats with AG490 starting 4 h before I/R significantly lowered serum creatinine and BUN level both at 24 and 48 h after I/R injury compared to the vehicle-treated control (P < 0.05 and P < 0.01, respectively). In the second set of experiments, rats treated with AG490 initiated immediately after I/R also had significant lower serum creatinine and BUN at 48 h after I/R compared to the vehicle-treated control (P < 0.01) (Figure 1C and D). However, there were no significant differences in serum creatinine and BUN between AG490-treated or vehicle-treated group when AG490 treatment was delayed until 3 h after I/R (P > 0.05). In the first set of experiments, no animals died during the study. However, one rat in the AG490-treated group and another in the vehicle-treated group died in the second set of experiments. Survival was not compared between the two groups because of low mortality.


Figure 1
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  		Fig. 1. Alterations in renal function following renal I/R. Serum creatinine (A) and BUN (B) concentration were measured 24 h or 48 h following renal I/R in rats pre-treated with either vehicle or AG490 starting 4 h before I/R. In the second experiment, serum creatinine (C) and BUN (D) concentration were measured 48 h following renal I/R in rats treated with either vehicle or AG490 initiated immediately after (0 h) or delayed until 3 h after I/R. Open bar, Sham-operated group (n = 6–8); Black bar, vehicle-treated control (n = 6–8); Gray bar, AG490-treated group (n = 6–8). *P < 0.05, **P < 0.01 vs vehicle-treated control; ## P < 0.01 vs sham-operated rats; NS, not significant vs vehicle-treated control.

 
AG490 treatment attenuated histological lesion
Improvement in renal function was accompanied by attenuation of histological lesions in AG490-pretreated rats. There were significant tubular changes including loss of brush border, dilation of renal tubules, as well as degeneration and necrosis of renal tubular epithelial cells following I/R (Figure 2B) compared to normal rats (Figure 2A). Tubular lesions were more prominent in outer stripe of medulla than those in cortex. In contrast, pretreatment with AG490 significantly ameliorated tubular lesions (Figure 2C). Tubular necrosis score in cortex and outer stripe of medulla were significantly lowered both at 24 and 48 h after I/R (P < 0.05 and P < 0.01) compared to the vehicle-treated rats (Figure 3A and B). More significant reduction in tubular necrosis score was noted in outer stripe of medulla. In rats treated with AG490 immediately after I/R, there was also significant reduction in tubular necrosis score compared to the vehicle-treated group (P < 0.01) (Figure 3C and D). However, no significant improvement in tubular lesion was found when AG490 treatment was delayed until 3 h after I/R (P > 0.05) (Figure 3C and D).


Figure 2
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  		Fig. 2. Histology, apoptosis, infiltration of macrophages and expression of ICAM-1 in the kidney of rats following renal I/R. PAS staining, TUNEL and immunohistochemical staining were performed on formalin-fixed paraffin-embedded or frozen sections of kidney 24 h following renal I/R in sham-operated (A, D, G and J), vehicle-treated (B, E, H and K) or AG490-pretreated rats (C, F, I and L). A–C: PAS staining (x200); D–F: Apoptosis in the tubular epithelial cells identified by TUNEL (brown) (x200); G–I: ED1+ macrophages identified by immunohistochemical staining of CD68 (brown) (x400); J–L: Immunohistochemical staining of ICAM-1 (brown) on frozen sections (x400).

 

Figure 3
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  		Fig. 3. Tubular lesions in the kidney following renal I/R. Tubular lesions in the renal cortex (A) and outer stripe of medulla (OSM) (B) were assessed 24 h or 48 h following renal I/R in rats pretreated with either vehicle or AG490 starting 4 h before I/R. In the second experiment, the tubular lesions in the renal cortex (C) and OSM (D) were assessed 48 h following renal I/R in rats treated with either vehicle or AG490 starting immediately after (0 h) or delayed until 3 h after I/R. Open bar, sham-operated group (n = 6–8); Black bar, vehicle-treated control (n = 6–8); gray bar, AG490-treated group (n = 6–8). *P < 0.05, **P < 0.01 vs vehicle-treated control; ##P < 0.01 vs sham-operated rats; NS, not significant vs vehicle-treated control.

 
Effect of AG490 on apoptosis of tubular epithelial cells
In normal rat kidney, only occasional apoptotic tubular epithelial cells (Figure 2D) were detected. However, the number of apoptotic tubular epithelial cells significantly increased following I/R (Figure 2E). Apoptosis was more prominent in the outer stripe of medulla and was significantly inhibited by pre-ischaemic treatment of AG490 (Figure 2F). The density of apoptotic tubular epithelial cells in AG490-treated rats was significantly lower than that in vehicle-treated controls both at 24 and 48 h after I/R (Table 1).


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  		Table 1. Apoptosis, macrophage infiltration and expression of ICAM-1 in the kidney of rats following I/R

 
AG490 treatment reduced renal macrophage accumulation
As shown in Table 1 and Figure 2G, there were rare ED1+ macrophages accumulated in normal rat kidney. The infiltration of ED1+ macrophages in the renal interstitium significantly increased both at 24 and 48 h following I/R (Figure 2H). The infiltration of ED1+ macrophages was more prominent in the outer stripe of medulla at 48 h after I/R, which was significantly reduced by pre-ischaemic treatment of AG490 (Figure 2I).

AG490 treatment inhibited renal MCP-1 mRNA expression
There was weak expression of chemokine MCP-1 mRNA in normal rat kidney (Figure 4). In agreement with macrophage infiltration, the expression of MCP-1 mRNA was significantly up-regulated both 24 and 48 h following I/R. Pre-treatment of AG490 significantly down-regulated the expression of MCP-1 mRNA (P < 0.01) (Figure 4).


Figure 4
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  		Fig. 4. Expression of MCP-1 mRNA and ICAM-1 mRNA in kidneys of sham-operated rats (A) or rats pre-treated with either vehicle (B and C) or AG490 (D and E) at 24 (B and D) or 48 h (C and E) following I/R. Open bar, Sham-operated group (n = 6); Black bar, vehicle-treated control (n = 6); gray bar, AG490-treated group (n = 6). **P < 0.01 vs vehicle-treated control; ##P < 0.01 vs sham-operated rats.

 
AG490 treatment inhibited renal ICAM-1 mRNA expression
In normal rat kidney, the expression of adhesion molecule ICAM-1 mRNA was weak (Figure 4). Accompanied with renal macrophage infiltration following I/R, there was also marked up-regulation of ICAM-1 mRNA in the kidney 24 and 48 h following I/R. Pre-ischaemic treatment of AG490 significantly inhibited the expression of ICAM-1 mRNA (P < 0.01) (Figure 4).

AG490 treatment inhibited renal ICAM-1 protein expression
Normal rat kidney had weak expression of ICAM-1 protein (Figure 2J). In concert with the up-regulation of ICAM-1 mRNA, immunohistochemical staining showed augmented expression of ICAM-1 protein in the kidney after I/R (Figure 2K). ICAM-1 protein was mainly expressed in the peritubular capillary endothelium, especially in the outer stripe of the medulla. On the other hand, the up-regulation of ICAM-1 expression in the glomeruli was unremarkable. The expression of ICAM-1 protein was significantly reduced by pre-ischaemic treatment of AG490 (Figure 2L). Semiquantitative analysis showed that the score of ICAM-1 expression in AG490-treated rats was significantly lower than that in vehicle-treated rats (P < 0.01) (Table 1).

Blockage of JAK/STAT signalling by AG490
To verify that AG490 exerted its effects via inhibiting JAK/STAT signalling, we performed western blot to analyse phosphorylation of JAK2, STAT1 and STAT3. In the kidney of sham-operated rats, there is a low-grade of phosphorylation for JAK2 (Figure 5). The level of p-JAK2 significantly increased in contrast to total JAK2 in the kidney 24 and 48 h following I/R. Treatment with AG490 in vivo resulted in reduced phosphorylation of JAK2 (p-JAK2), but did not change the expression of JAK2. In the mean time, phosphorylation of STAT1 (p-STAT1) and STAT3 (p-STAT3), downstream molecules of JAK2 cascade, was also significantly inhibited by AG490 treatment (both P < 0.01) (Figure 5).


Figure 5
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  		Fig. 5. Western blot showing renal expression of p-JAK/JAK, p-STAT1/STAT1 and p-STAT3/STAT3 following I/R in sham-operated rats (A) or rats treated with either vehicle (B and D) or AG490 (C and E) at 24h (B and C) or 48 h (D and E) following I/R. Open bar, sham-operated group (n = 6); black bar, vehicle-treated control (n = 6); shaded bar, AG490-treated group (n = 6). **P < 0.01 vs vehicle-treated control; ##P < 0.01 vs sham-operated rats.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Acknowledgements
 References
 
In the present study, we have shown that JAK/STAT signalling pathway was activated in renal I/R injury. The phosphorylation of JAK2 was induced in the kidney after I/R, accompanied by the phosphorylation of downstream protein STAT1 and STAT3. Tyrphostin AG490 is a derivative of benzylidene malononitrile which selectively inhibits the activation of JAK2 [20,21]. Our results demonstrated that treatment with tyrphostin AG490 significantly inhibited the phosphorylation of JAK2 and its downstream molecule STAT1 and STAT3 in rat renal I/R injury. Renal function and histology were significantly improved by AG490 treatment, thereby suggesting that the activation of JAK/STAT signalling pathway is involved in the pathogenesis of ischaemic ARF. This is consistent with a report in cardiac I/R showing that a heart treated with AG490 had reduced myocardial infarct size following global ischaemia and reperfusion and had a recovery in functional haemodynamics of the myocardium [16]. There are few data on the roles of JAK/STAT signalling in renal I/R injury. Leonard et al. [22] reported that 15-epi-16-(para-fluorophenoxy)-lipoxin A4-methyl ester inhibited ICAM-1 and growth regulated oncogene-1 (GRO1, a chemokine) in ischaemic ARF in association with increased renal mRNA levels for suppressors of cytokine signalling-1 (SOCS-1) and SOCS-2, endogenous inhibitors of cytokine-elicited JAK/STAT-signalling pathways. However, our studies provide direct evidence that inhibition of the JAK/STAT signalling pathway ameliorates renal injury in this in vivo model of renal I/R.

Because of the difficulty in predicting ARF in many clinical cases, it is more important to know whether post-ischaemic treatment is beneficial. Therefore, we also investigated the effects of post-ischaemic treatment for renal I/R injury. Treatment of AG490 immediately after I/R also significantly attenuated renal injury. However, delayed treatment until 3 h after I/R failed to show any improvement in renal function. These results suggest that activation of JAK/STAT signalling occurs early in the course of renal I/R injury. Although many agents were reported to be protective in animal models of renal I/R injury on pre-ischaemic treatment [23], only a few were effective if treatment was initiated after ischaemia [24,25]. On the other hand, post-ischaemic treatment with FK409, a nitric oxide donor, at 6 h after reperfusion even aggravated renal injury [26]. Therefore, our results indicate that the timing of treatment is critical for renal I/R and emphasize the need for early intervention in established ARF [27].

Mounting evidence now indicates that apoptosis is the major mechanism of early tubule cell death in both clinical acute renal failure and experimental ischaemia/reperfusion injury [28]. A multitude of pathways, including the intrinsic (Bcl-2 family, cytochrome c and caspase 9), extrinsic (Fas, FADD and caspase 8), and regulatory (p53 and NF-{kappa}B) factors, seem to be activated by ischaemic AKI [28]. In this study, prominent apoptosis occurred in the renal tubular epithelial cells following ischaemia/reperfusion. Pre-treatment of AG490 significantly attenuated the apoptosis of the tubular cells, suggesting that the JAK2/STATs pathway is involved in the pro-apoptotic process. Interferon-{gamma} was reported to induce Fas trafficking to the cell surface of vascular smooth muscle cells and increase Fas-induced apoptosis in vitro, which was abrogated by AG490 [29]. Recently, Arany et al. reported that inhibition of JAK2 or STAT3 by AG490 or dominant-negative STAT3 adenovirus respectively lead to increased ERK activation and survival of mouse proximal tubular epithelial (TKPTS) cells during severe oxidative stress, suggesting a role of tyrosine-phosphorylated STAT3 in the apoptosis of tubular epithelial cells following ischaemia/reperfusion [30].

Renal I/R leads to increased endothelial expression of adhesion molecules that promote endothelial-leucocyte interaction, including ICAM-1 [31]. By using immunohistochmistry, we found that ICAM-1 was mainly expressed by the peritubular capillary endothelium. There were reports that treatment of renal I/R in the rat with anti-ICAM-1 monoclonal antibody protected the kidney against ischaemic ARF [25]. Moreover, ablation of the ICAM-1 gene rendered mice resistant to ischaemic acute kidney injury [32]. Similarly, inhibition of the surface expression of ICAM-1 by using ICAM-1 antisense oligodeoxyribonucleotides resulted in reduced granulocyte and macrophage infiltration associated with less pathological and functional damage in a rat model of renal I/R injury [33]. In this study, the expression of renal expression of ICAM-1 was significantly down-regulated in AG490-treated rats compared to the vehicle-treated controls, suggesting the protection of I/R injury is also mediated by the inhibition of the expression of adhesion molecules. Several in vitro studies revealed that the expression of ICAM-1 was associated with the activation of JAK/STAT signalling pathway. IFN-{gamma} induced ICAM-1 expression through activation of STAT1 by JAK1/2 in NCI-H292 epithelial cells [34]. Gangliosides, sialic acid-containing glycosphingolipids, were shown to rapidly activate JAK2 and induced phosphorylation of STAT1 and STAT3, resulted in increased transcription of ICAM-1 in rat microglial cells [35]. AG490 treatment significantly inhibited the expression of ICAM-1 in these cells [34,35]. In another report, IFN-{gamma}-dependent ICAM-1 expression in human tracheobronchial epithelial cells was inhibited by using dominant-negative Stat1 [36]. However, in vivo data concerning the relationship between JAK/STAT signal transduction and ICAM-1 expression are scarce. Our results indicate that ICAM-1 expression in the kidney following I/R is associated with in vivo activation of Jak/Stat signalling pathway.

A growing body of evidence indicates that the inflammatory response plays a major role in ischaemic ARF [3,31]. Inflammatory cascades that are initiated by endothelial dysfunction can be augmented dramatically by the generation of a number of potent mediators by the ischaemic renal tubules, especially the thick ascending limbs of the renal tubules, including proinflammatory cytokine TNF-{alpha}, IL-1ß and chemotactic cytokine MCP-1 and RANTES [3,6,37]. Our study demonstrated that renal MCP-1 expression was significantly augmented following I/R accompanied by prominent macrophage accumulation in the renal interstitium. Macrophage accumulation was evident both in the cortex and outer medullary stripe 24 h after I/R injury. This is in consistency with reports that macrophages accumulate in the post-ischaemic kidney in response to up-regulation of MCP-1 in tubule cells [38]. Selective macrophage depletion ameliorates ischaemic acute renal injury [39]. In this study, in vivo treatment of AG490 significantly blunted the expression of MCP-1 following ischaemia and reduced the infiltration of macrophages, indicating the involvement of JAK/STAT signalling in the mediation of inflammatory infiltration. JAK/STAT signalling was shown to mediate MCP-1 expression in cultured brain microglial cells, in human bronchial epithelial cells, gingival fibroblasts and monocytes [35,40–42]. Furthermore, AG490 treatment inhibited MCP-1 expression in brain microglial cells, primary monocytes and human retinal pigment epithelium cells [35,42–43]. Therefore, it is likely that the up-regulation of MCP-1 expression following renal I/R is mediated by the activation of JAK/STAT pathway.

The exact mechanisms of the activation of JAK/STAT signalling in renal I/R injury is not clear. Multiple aspects are involved in the pathogenesis of ischaemic acute kidney injury, including alteration in haemodynamics, tubule cell metabolism, apoptosis, inflammatory response, calcium overload and oxidative stress [28]. One of the possible mechanisms is the activation of signal transduction by reactive oxygen species (ROS) [38,44]. ROS mediates the activation JAK/STAT signalling in renal proximal tubular cells stimulated by albumin [45]. The phosphoralation of STAT3 was enhanced in mice kidney 30 min and 24 h following ischaemia/reperfuision and in mouse proximal tubular epithelial (TKPTS) cells treated with H2O2 in vitro [30]. Considering the fact that almost all cytokines can activate JAK/STAT [46], another possible mechanism of JAK/STAT activation could be the production of cytokines during I/R [3]. Moreover, interaction between multiple signal transduction pathways should also be considered and the protection by AG490 may not be complete. These proposed mechanisms need to be verified in the future studies.

In summary, this study demonstrated that in vivo activation of Jak/Stat signalling is involved in the pathogenesis of renal I/R injury. Our results reported here suggest that intervention targeted at signal transduction pathways with small molecular chemicals such as tyrphostin AG490 may have therapeutic potential for treatment of ischaemic ARF in the human being.



   Acknowledgements
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
References
 
The project was supported by a grant from the National Natural Science Foundation of China (No 30470799), a grant from the Foundation for the Author of National Excellent Doctoral Dissertation of China (No199945) and a grant from the Natural Science Foundation of Guangdong Sciences Committee (No 990075).

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Received for publication: 8.10.06
Accepted in revised form: 3. 7.07


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