Table Of ContentYanetal.DiagnosticPathology2012,7:5
http://www.diagnosticpathology.org/content/7/1/5
RESEARCH Open Access
b
Expression of GSK-3 in renal allograft tissue and
its significance in pathogenesis of chronic
allograft dysfunction
Qiang Yan1, Baoyao Wang2, Weiguo Sui1, Guimian Zou1, Huaizhou Chen1, Shenping Xie1 and Hequn Zou3*
Abstract
Objective: To explore the expression of Glycogen synthase kinase 3 beta (GSK-3b) in renal allograft tissue and its
significance in the pathogenesis of chronic allograft dysfunction.
Methods: Renal allograft biopsy was performed in all of the renal allograft recipients with proteinuria or increased
serum creatinine level who came into our hospital from January 2007 to December 2009. Among them 28 cases
was diagnosed as chronic allograft dysfunction based on pahtological observation, including 21 males with a mean
age of 45 ± 10 years old and 7 females with a mean age of 42 ± 9 years old. The time from kidney transplantation
to biopsy were 1-9 (3.5) years. Their serum creatinine level were 206 ± 122 umol/L. Immunohistochemical assay
and computer-assisted genuine color image analysis system (imagepro-plus 6.0) were used to detect the
expression of GSK-3b in the renal allografts of 28 cases of recipients with chronic allograft dysfunction. Mean area
and mean integrated optical density of GSK-3b expression were calculated. The relationship between expression
level of GSK-3b and either the grade of inflammatory cell infiltration or interstitial fibrosis/tubular atrophy in renal
allograft was analyzed. Five specimens of healthy renal tissue were used as controls.
Results: The expression level of the GSK-3b was significantly increased in the renal allograft tissue of recipients
with chronic allograft dysfunction, compared to normal renal tissues, and GSK-3b expression became stronger
along with the increasing of the grade of either inflammatory cell infiltration or interstitial fibrosis/tubular atrophy
in renal allograft tissue.
Conclusion: There might be a positive correlation between either inflammatory cell infiltration or interstitial
fibrosis/tubular atrophy and high GSK-3b expression in renal allograft tissue.
Virtual slides: The virtual slide(s) for this article can be found here:
http://www.diagnosticpathology.diagnomx.eu/vs/9924478946162998.
Keywords: kidney transplantation, GSK-3β, inflammatory cell infiltration
Introduction clinical medicine. Recent researches have found that
Kidney transplantation isthe optimal renal replacement chronic renal allograft dysfunction is the main factor
therapy for the patients with end stage renal disease influenceonthelongtermsurvivalofrenalallograft.The
(ESRD).Astheapplicationofmoreneweffectiveimmu- main clinical manifestations of chronic renal allograft
nodepressantsandthedevelopmentoftransplanttechni- dysfunctionareserumlevelofcreatinineisslowlycreep-
que, the incidence of acute rejection is obviously ingupwardcompany withproteinuriaandhypertension,
decreasingintheearlystageposttransplantation,butthe and progression into ESRD need kidney transplantation
longtermsurvivalofrenalallograftsisstillachallengeof again or on maintenance dialysis. The main pathogenic
courseofchronicrenalallograftdysfunctionisglomeru-
losclerosis, vasculopathy, atrophy of tubule and chronic
*Correspondence:[email protected]
3InstituteofUrologyandNephrology,TheThirdAffiliatedHospitalof renal interstitial fibrosis. All the pathogenic changesare
SouthernMedicalUniversity,Guangzhou510630,China associated with lymphocyte, plasmocyte and mastocyte
Fulllistofauthorinformationisavailableattheendofthearticle
©2012Yanetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.
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infiltration in the renal tissue. Researches have proved including hematoxylin/feosin stain, periodic-acid schiff
thatchronicinflammatorywasthekeypathogeniccourse (PAS) stain, masson trichrome and periodic schiff-
of nephron loss in various of kidney disease, including methenamine (PASM) stain. The slices were observed
chronic renal allograft dysfunction. Recently, researches under microscope in a blind manner, including inflam-
discoveredthat GSK-3bmediatedchronic inflammatory matory cell infiltration in renal tissues, increased mesan-
related to deterioration of renal allograft function, but gial matrix and extracellular matric, proliferation of
the mechanism has not been fully interpreted. In this mesangial cells, epithelium and endothelium, adhesions
study, we mainly detected the expression of GSK-3b in and sclerosis, thickening of glomerular basement mem-
thetissueofrenalallograft,andanalyzedtherelationship branes and double track sign; thickening of peritubular
betweentheexpressionofGSK-3bandinflammatorycell capillaries basement membrane, interstitial fibrosis,
infiltration in renal interstitium, interstitial fibrosis and inflammatory cell infiltration, tubular atrophy and inti-
tubule atrophy. The role of GSK-3b in chronic allograft mal thickening of artery.
dysfunctionwasdiscussed.
Pathologic diagnosis
Methods Diagnosis criteria of chronic allograft dysfunction:
Clinical data thickening and splitting appearance of glomerular base-
The renal biopsy samples were collected from kidney ment membrane, thickening of basement membrane of
transplant patients with proteinuria and elevated serum peritubular capillaries, and/or diffuse monocyte infiltra-
creatine from January, 2007to December2009in Guilin tion of mononuclearcell, interstitial fibrosis/diffuse tub-
No.181hospital.Inthe28casesconsistentedwithclinical ular atrophy, and/or thickening of artery intima,
diagnosis of chronic allograft dysfunction, 21 cases were excluding acute rejection, nephrotoxicity of immuno-
males(age45±10years)and7caseswerefemales(age42 suppressant, obstruction/back- streaming of ureter and
±9years).Thedurationsafterkidneytransplantationwere other diseases.
1~9years(mean3.5years)andthe meanlevelsof serum Thedegreeofinflammatorycellinfiltrationintubuloin-
creatinewere206±122μmol/L.Thetripleimmunosup- terstitial was described as mild, midrange and severe.
pressant protocols were cyclosporine + mycophenolate There werefew andscatter inflammatorycell infiltration
mofetil + prednisone in 18 patients and tacrolimus + in10-25%ofparenchymawasdefinedasmild;inflamma-
mycophenolatemofetil+prednisonein9patientsandsir- torycellinfiltrationin26-50%ofparenchymawasdefined
olimus+.mycophenolatemofetil+prednisonein1patient. asmidrange;inflammatorycellinfiltrationin>50%ofpar-
Beforerenalbiopsy,colorDopplersupersonicdetectionin enchymawasdefinedasserve[1].
renal allograft and serum drug levels were performed to Degree of interstitial fibrosis and tubular atrophy: IF/
exclude acute rejection, nephrotoxicity of immunosup- TA-I, as mild interstitial fibrosis and tubular atrophy
pressant,obstruction/backstreamingofureter,thrombosis (<25%ofcorticalarea);IF/TA-II,asmoderateinterstitial
orembolisminrenalarteriesorveinsandotherdiseases. fibrosis and tubular atrophy (26-50% of cortical area);
The donors andrecipients were matching inABO blood IF/TA-III, as Severe interstitial fibrosisandtubular atro-
type, andtwoor more matchinginHLA,and the results phy/loss(>50%ofcorticalarea)[1].
of lymphocytotoxicity test were less than 10%, and the
results of panel reaction antibody (PRA) were negative. Immunohistochemisty examination
The renal samples of 5 cases of control were collected Immunohistochemisty with EnVision was used to detect
from routine donor renal biopsy before transplantation, the expression of GSK-3b protein in the renal allograft
and the pathologic manifestation of which was normal. tissue. 3-μm-thick tissue sections were hydrated through
Informed consents were obtained from all patients that graded ethanol, and endogenous peroxidase was blocked
participatedtothestudy.Thisstudywasperformedunder with 3% peroxide. GSK-3b was repaired with microwave
thesupervisionofInstitutionalReviewBoardofSouthern for 15 minute. We incubated primary antibody (rabbit
Medical University, and abided the Helsinki Declaration anti-human GSK polyclonal antibody, 1:100, Wuhan
onethicalprinciplesformedicalresearchinvolvinghuman Boster Biological Technology, Ltd) to detect GSK-3b at
subjects. overnight at 4°C. After washing with PBS, we incubated
the tissue sections with the second antibody (mice anti-
Histological examination rabbit monoclonal antibody, Maixin Biological Technol-
The paraffin-embedded kidney sections were incised ogy Development Co. Fujian) for 30 min at 37°C, and
into 3-μm-thick tissue sections that were deparafinized then washing with PBS, DAB coloration and re-stained
through xylene and hydrated through graded ethanol with hematoxylin.
(100%, 96%, 90%, and 70%) and distilled water. The sec- Immunohistochemisty test of IgG, IgA, IgM, Clq, C4c
tions were stained by standard histology procedures, and C4d were performed in the same way.
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Image analysis Table 1The area andintegrated opticaldensity of
Imageswere acquiredusinga Leica DMR-Xmicroscope GSK-3b expression inrenal tissue withdifferent degree
coupledtoaLeicaDC500digitalcamera(Leica,Wetzlar, ofinflammatory infiltration
Germany), using the image analysis system Quantimet n AreaofGSKexpression(%) GSK(IOD)
Q550 (Leica Imaging Systems). Ten randomly selected ControlGroup 5 17.45±19.63 21757±23765
discontinuous fields (400 x) per kidney were evaluated, MildGroup 11 33.15±9.16⋇ 48331±20335⋇
including tubulointerstitial in renal cortex, medulla and ModerateGroup 10 45.77±14.56⋇⋆ 70702±24891⋇⋆
the conjunction region, (but not including glomulular SevereGroup 7 60.83±13.53⋇⋆♦ 96300±28169⋇⋆♦
and vessels). More than 60 tubulars in each biopsy sec- ⋇:p<0.05,vsControlGroup;⋆:p<0.05,vsMildGroup;♦:p<0.05,vs
tion. The positive area was yellowly stained and Image- ModerateGroup.
Pro Plus software was used to quantify the integrated GSK:glycogensynthasekinase;IOD:integratedopticaldensity.
optical density. The ratio of positive area to total tubu-
lointerstitial area (not including the area of tubular Discussion
lumina) presented the relative amount of the substance Renal allograft chronic dysfunction is the main factor
expressionintubuloinstitial. influence on the long term survival of renal allograft,
and is due to multiple causes. The clinical manifesta-
Statistic analysis tions are gradually loss of renal allograft function and
Results are expressed as means ± SD. Statistical analysis serum creatinine slowly creeping upward. The risk fac-
was performed using SPSS 13.0 (SPSS, Chicago, IL). tors of renal allograft dysfunction include immune fac-
Measurement data was analyzed with one way ANOVA, tors, such as times of early acute rejection and matching
and the relationship was analyzed with linear correlation of HLA and protocol of immunosuppression, and non-
(Pearson). Statistic significance was set at P < 0.05 level. immune factors such as ischemia/reperfusion injury,
delayed graft function, nephrotoxicity of calcineurin
Results inhibitors, hypertension and dyslipidemia, and progres-
Histological manifestation sively develop into end stage renal disease. Chronic
The main histological manifestation of renal allograft in renal allograft dysfunction morphological appearance as
the 28 cases of chronic renal allograft dysfunction as: thickening of glomerular basement membrane as double
interstitial fibrosis and tubular atrophy accompany with contours sign, thickening of the basement membrane of
infiltration of inflammatory cells including lymphocyte peritubular capillary, and/or diffuse mononuclear cell
and monocyte, enlarge of some tubular lumens, thicken- infiltration and fibrosis in interstitium and difusse tubu-
ing of glomerular basement membrane as double trace lar atrophy, and/or thickening of artery intima.
sign and focal segmental glomerulosclerosis, thickening Our study has found that GSK-3b was weakly
of artery intimae. Discrepancy to the presentation of expressed in normal renal tissue, and was strongly
acute rejection, nephrotoxicity of immunosuppressant expressed in chronic dysfunction renal allograft tissue,
and obstruction uropathy/reflux nephropathy. and was stronger in the more severity of pathological
changes. The IOD of GSK-3b was higher than that in
The expression of GSK-3b in renal tissue normal renal tissue, and increasing with the severity of
There was weak expression of GSK-3b in the cytoplasm inflammatory infiltration, interstitial fibrosis and tubular
of tubular epithelia cell in normal renal tissue. However, atrophy, which suggest that the correlation of GSK-3b
in the dysfunction renal allograft tissue, there were expression and interstitial inflammatory infiltration,
strong cytoplasm GSK-3b expression in tubular epithelia interstitial fibrosis/tubular atrophy. It was necessary to
cell, and stronger expression in the patients with more further study the pathogenic mechanism of GSK-3b in
severity tubulointerstitial damage. (Shown in table 1,
table 2 and Figure 1, 2, 3, 4, Figure 5, 6, 7, 8).
Table 2The area andintegrated opticaldensity of
GSK-3b expression inrenal tissue withdifferent degree
The relationship of GSK-3b expression and ofinterstitial fibrosis andtubular atrophy
inflammatory infiltration, interstitial fibrosis and
n GSK(%) GSK(IOD)
tubularatrophy
Controlgroup 5 17.45±19.63 21757±23765
In the renal tissue of chronic renal allograft dysfunction, IF/TA-Igroup 12 35.48±11.90⋇ 52017±23216⋇
the integrated optical density (IOD) was higher than IF/TA-IIgroup 11 48.50±16.51⋇⋆ 741235±26216⋇
normal renal tissue by analysis with Image-Pro software, IF/TA-IIIgroup 5 57.80±14.40⋇⋆ 94615±33855⋇⋆
and the highest of IOD in more severity of inflamma-
⋇:p<0.05,vsControlGroup;⋆:p<0.05,vsIF/TA-IGroup;♦:p<0.05,vsIF/
tory infiltration, interstitial fibrosis and tubular atrophy
TA-IIGroup.
(Figure 9, 10). GSK:glycogensynthasekinase;IOD:integratedopticaldensity.
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Figure 1 GSK-3b expression in renal tissue with different Figure 3 GSK-3b expression in renal tissue with different
degreeofinflammatoryinfiltration:controlgroup. degreeofinflammatoryinfiltration:moderateinflammatory
infiltration.
the course of interstitial inflammatory infiltration, inter-
stitial fibrosis and tubular atrophy. inflammatory diseases by controling the production of
Recent researches have found that the GSK-3b family inflammatoryfactorsandinvolvementintoNF-(cid:1)Bsignal
of serine/threonine kinases extensively existed in the pathway, and the activation of NF-(cid:1)B is the center step
eucaryon, and mainly express in the lung, kidney and ofinflammatory reaction.GiannopoulouM. etal. found
brain, and influence on construction, growth, activation in cultured renal tubular epithelium that hepatocyte
and apoptosis of the cells by involving the basic cellular growth factor (HGF) has a potent suppressive effect on
processessuchassignaltransduction,regulationofinsu- NFkappaB activation, which was mediated by GSK-3b.
lin, gene transcription andtranslation. The activation of HGF can inhibit renal inflammation and proinflamma-
GSK-3bmainlythroughphosphorylationofserine-9near torychemokineexpressionbydisruptingNF-kBsignaling
theN-terminus,andisassociatedtotheregulationofsig- [2]. Adminis- tration of a GSK-3 inhibitor potently sup-
nal pathway of Wnt, protein kinase B/Akt and NF-(cid:1)B. pressedthe proinflammatory response inmice receiving
TheabnormalactivatinofGSK-3bisassociatedwithsev- lipopolysaccharide and mediated protection from endo-
eral kidney diseases, such as diabetic nephropathy, toxinshock[3].StudyofGongandcolleagueshadproved
mesangioproliferativeglomerulonephritis,lupusnephritis thatGSK-3bwasanewmarkerofchronicrenalallograft
and chronic allograft nephropathy. Recently, several dysfunctionandmediatingproinflammatoryNF-kBacti-
researches revealed that GSK-3b involved in human vation and renal inflammation. GSK-3b over expression
Figure 2 GSK-3b expression in renal tissue with different Figure 4 GSK-3b expression in renal tissue with different
degreeofinflammatoryinfiltration:milddegreeofinflammatory degreeofinflammatoryinfiltration:severeinflammatory
infiltration. infiltration.
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Figure 5 GSK-3b expressions in renal tissue with different Figure 7 GSK -3b expressions in renal tissue with different
degreeofinterstitialfibrosis/tubularatrophy:controlgroup. degreeofinterstitialfibrosis/tubularatrophy:IF/TA-IIGroup.
inthedamagedtubularepithelialcellsandthemagnitude vitro and in vivo experiments shown that GSK-3b
of GSK-3b phosphorylation (p-GSK, inactivation state) involved in inflammatory diseases and inhibition of
was inversely correlated with the degree of injury as GSK-3b retarded the progression of these diseases. In
assessed by Banff criteria. The higher level of p-GSK the diabetic nephropathy mice experiment model, trogli-
expression, the lower level oftubular atrophy and inter- tazone a peroxisome proliferator -activated receptor
stitialfibrosis[4]. gamma (PPAR gamma) agonists, ameliorate renal fibro-
Chronic renal allograft dysfunction is an important tic lesions through reverse high glucose induced expres-
factor influence on the long term survival of transplant sions of GSK-3b and inhibition epithelial-mesenchymal
kidney, but the pathogenic mechanism is still not fully transition [5]. In the rats with experimental mesangial
interpreted. Infiltration of mononuclear cell (monocyte proliferative glomerulonephritis, inhibitors of cyclin-
and lymphocyte) often presents in the renal tissue of dependent kinase (CDK)/GSK-3b were the effective
chronic dysfunction and relate to endarteritis and inter- antagonists of proliferating and immunity renal disease
stitial fibrosis. Impairment of renal tubular epithelium [6]. CDK/GSK-3b inhibitors suppress pathogenic prolif-
and interstitial inflammatory infiltration play an impor- eration, apoptosis, and inflammation, and promote
tant role in various kidney diseases. Inflammatory fac- regeneration of injured tissue in parenchymal renal dis-
tors induce secretion of extracellular matrix by eases [7]. Sinha D and his colleague had studied the
interstitial fibrocyte and result in interstitial fibrosis. In proximal tubular cells cultured in the absence of growth
Figure 6 GSK -3b expressions in renal tissue with different Figure 8 GSK -3b expressions in renal tissue with different
degreeofinterstitialfibrosis/tubularatrophy:IF/TA-IGroup. degreeofinterstitialfibrosis/tubularatrophy:IF/TA-IIIGroup.
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Acknowledgements
Wewouldliketoexpressourgratitudetoallthesubjectswhoparticipated
inthestudy.ThisstudysupportedbyKidneyTranplantationandDialysis
CenterofPLANO.181Hospital,ClinicalCollegeofSouthernMedical
University,Guilin,Guangxi,P.R.China.
Authordetails
1KidneyTranplantationandDialysisCenterofPLANO.181Hospital,Clinical
CollegeofSouthernMedicalUniversity,Guilin524001,Guangxi,P.R.China.
2SouthernMedicalUniversity,Guangzhou510630,Guangdong,P.R.China.
3InstituteofUrologyandNephrology,TheThirdAffiliatedHospitalof
SouthernMedicalUniversity,Guangzhou510630,China.
Authors’contributions
HZandQYdesignthestudy,BWandGZperformedresearchandwrotethe
firstdraftofthemanuscript,WS,HCandSXparticipatedinthestatistical
analyses.Alltheauthorsreadandapprovedthefinalmanuscript.
Figure 9 The correlation between GSK-3b expression and Competinginterests
inflammatoryinfiltration(r=0.688,p<0.001). Theauthorsdeclarethattheyhavenocompetinginterests.
Received:11October2011 Accepted:13January2012
factors, found that GSK-3b inhibitor might improve the Published:13January2012
survival of proximal tubular cells. The main mechanism
References
was by activation of Wnt signaling, which promoted the 1. SolezK,ColvinRB,RacusenLC,etal:Banff07classificationofrenal
repair of damaged renal parenchyma [8]. allograftpathology:updatesandfuturedirections.AmJTransplant2008,
Summary, there is intensive expression of GSK-3b in 8(4):753-760.
2. GiannopoulouM,DaiC,TanX,etal:Hepatocytegrowthfactorexertsits
the renal tissue of the patients with chronic allograft anti-inflammatoryactionbydisruptingnuclearfactor-kappaBsignaling.
dysfunction, and is associated with inflammatory cell AmJPathol2008,173(1):30-41.
3. MartinM,RehaniK,JopeRS,etal:Toll-likereceptor-mediatedcytokine
infiltration of interstitium and interstitial fibrosis/tubular
productionisdifferentiallyregulatedbyglycogensynthasekinase3.Nat
atrophy. Further studies are needed to investigate the Immunol2005,6(8):777-784.
pathogenic mechanism of GSK-3b expression in the 4. GongR,GeY,ChenS,etal:Glycogensynthasekinase3beta:anovel
markerandmodulatorofinflammatoryinjuryinchronicrenalallograft
renal tissue of chronic allograft dysfunction. Inhibition
disease.AmJTransplant2008,8(9):1852-1863.
the activity of GSK-3b may be a new treatment target of 5. LeeYJ,HanHJ:Troglitazoneameliorateshighglucose-inducedEMTand
prevention chronic renal allograft dysfunction. dysfunctionofSGLTsthroughPI3K/Akt,GSK-3{beta},Snail1,and{beta}-
catenininrenalproximaltubulecells.AmJPhysiolRenalPhysiol2009.
6. PippinJW,QuQ,MeijerL,etal:Directinvivoinhibitionofthenuclear
cellcyclecascadeinexperimentalmesangialproliferative
glomerulonephritiswithRoscovitine,anovelcyclin-dependentkinase
antagonist.JClinInvest1997,100(10):2512-2520.
7. ObligadoSH,Ibraghimov-BeskrovnayaO,ZukA,etal:CDK/GSK-3inhibitors
astherapeuticagentsforparenchymalrenaldiseases.KidneyInt2008,
73(6):684-690.
8. SinhaD,WangZ,RuchalskiKL,etal:LithiumactivatestheWntand
phosphatidylinositol3-kinaseAktsignalingpathwaystopromotecell
survivalintheabsenceofsolublesurvivalfactors.AmJPhysiolRenal
Physiol2005,288(4):F703-713.
doi:10.1186/1746-1596-7-5
Citethisarticleas:Yanetal.:ExpressionofGSK-3binrenalallograft
tissueanditssignificanceinpathogenesisofchronicallograft
dysfunction.DiagnosticPathology20127:5.
Figure 10 The correlation between GSK-3b expression and
interstitialfibrosisandtubularatrophy(r=0.584,p<0.001).
