Prakoura, Niki

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The role of Calreticulinin renal fibrosisNiki PrakouraSection of Histology, Center of Basic Research, Biomedical Research Foundation of the Academy of Athens, Greece22-25 March 2012, 24th ERCSG meeting, Arnhem, The NetherlandsThe Unilateral Ureteral Obstruction (UUO) model of kidney fibrosis•in vivo model•encompasses many aspects of other models of kidney fibrosis•there are features that occur within 1 week•there is evidence that animal models with UUO are reflective of the molecular changes in human situations•leaves one kidney intact•mimics in a short time a situation that can take years in humansProteomic analysisSham operated 2d UUO 2d Sham operated 8d UUO 8d UUO  2d UUO 8d Kypreou K. et al., Proteomics, 2008Venn diagram of identified proteinsSham / Ligated 8Sham / Ligated 2Ligated 2 / Ligated 8 285124918CALRETICULINLigated 2 daysSham operated 2 daysSham operated 8 days Ligated 8 daysCalreticulin is a multifunctional protein• First isolated in 1974 as ahigh-affinity Ca2+-bindingprotein of the ERThere is no correlation with fibrotic processes yet !TRENDS in Cell Biology, Vol.11, 2001Confirmation of Calreticulin upregulationin fibrotic samplesShamoperatedLigated 2 daysRatio of calreticulin expression/tubulin expressionLigated 8 days00.10.20.30.40.50.60.70.80.9Quantification of calreticulinprotein expressionFold of calreticulin mRNA expression normalized to 18S rRNA 050150200250300ShamoperatedLigated 2daysLigated 8 days100Quantification of calreticulinmRNA expressionKypreou K. et al., Proteomics, 2008Sham Ligated 2 daysCalreticulinbeta-tubulinSham Ligated 8 daysCalreticulin is upregulated in the tubular cells of the kidneySham operated Ligated 2 days Ligated 8 daysWHAT IS THE ROLE OF CALRETICULIN IN FIBROSIS?o Phenotypic observations in a tubular cell line after Calreticulin overexpressionCalreticulin overexpression in HK-2 cells HK-2 cell lines, which stably express the mouse Calreticulin cDNA.Calreticulinβ-actin0,000,501,001,502,002,503,003,504,004,50control overexpression 1 overexpression 2Calreticulinto β-actinratioCalreticulin/β-αctin expression***Calreticulin-overexpressing cells acquire an altered morphologyControl Overexpression 2Differentially expressed proteins in Calreticulin-overexpressing HK-2 cellsE-cadherinβ-actinVinculinVimentinβ-actin0,000,200,400,600,801,001,20Control Overexpression 1 Overexpression 2E-cadherin to β-actin ratioE-cadherin/β-actin expression*****0,000,501,001,502,002,503,00control overexpression1overexpression2Vinculin to β-actin ratioVinculin/β-actin expression****0,000,501,001,502,002,50control overexpression1overexpression2Vimentin to β-actin ratioVimentin/β-actin expression*****Differentially expressed genes in Calreticulin-overexpressing cells0,005,0010,0015,0020,0025,00E-cadherin Vimentin Acta2 Snai1 Snai2Relative mRNA expression normalized to 18s rRNAExpression of genes related to epithelial and mesenchymal phenotypeControlOverexpression 1Overexpression 2*** ************************β-catenin expression/localization in the Calreticulin-overexpressing cellsb-catenindapimergepcDNA (control)mCRT-16 (overexpression 1)mCRT-17 (overexpression 2)Calreticulin overexpression induces the activity of the TCF-luc-reporter pTOPFlash0,005000,0010000,0015000,0020000,0025000,0030000,0035000,0040000,00pcDNA mCRT-16 mCRT-17RLU/b-galpTOPFlash activityCalreticulin overexpressing cells have an increased secretory profileFibronectinβ-actin0,000,501,001,50Control Overexpression1Overexpression2Fibronectin to β-actin ratio Fibronectin/β-actin****COLIVmerge with DAPIControl Overexpression 1 Overexpression 20,000,501,001,502,002,503,003,50Control Overexpression 1 Overexpression 2Relative mRNA expression normalized to 18S rRNACOL4A1 mRNA expression***0,001,002,003,004,005,006,00Control Overexpression 1 Overexpression 2Relative mRNA expression normalized to 18S rRNAFibronectin mRNA expression****Collagen I expression levels are not increased by Calreticulin overexpressionCOLIA1Collagen I secretion in conditioned mediumPonceau S0,000,200,400,600,801,001,201,40Control Overexpression 1 Overexpression 2Relative mRNA expression normalized to 18SrRNACOL1A1 mRNA expression**Calreticulin overexpression enhances the migratory capacity of HK-2 cells0,0020,0040,0060,0080,00100,00120,000h 24h 48h 72h% wounded areatime after wound induction (h)Wound-healing assaycontroloverexpression 1overexpression 2*** ********Calreticulin-overexpressing cells have lower proliferation capacity than the control cells0,0010,0020,0030,0040,0050,0060,00control overexpression 1 overexpression 2% BrdU/DAPI counts Proliferation assay****Calreticulin-overexpressing cells exhibit increased cellular stressBiP (GRP78)β-αctin0,000,501,001,502,002,503,003,504,004,505,00Control Overexpression 1 Overexpression 2GRP78 to β-actinratioGRP78/β-actin***Calreticulin overexpression enhances dramatically cell sensitivity to apoptosis0,00200,00400,00600,00800,001000,001200,001400,00Control Overexpression 1 Overexpression 2% TUNEL counts/DAPI vs. controlTUNEL assay after treatment with H2O2******050100150200250300Control Overexpression 2% TUNEL counts/DAPI vs. controlTUNEL assay***0,02,04,06,08,010,012,014,016,018,020,0normal medium H2O2 50μM% activated caspase 3/DAPI Activated caspase 3 expressionControlOverexpression 1Overexpression 2***** **Conclusions  Calreticulin overexpression in HK-2 cells:-increases the fibronectin and collagen IV production-increases significantly the ER stress and the migration capacity-reduces the proliferation rate while increases the cell sensitivity toapoptosis,all of which represent characteristics of fibrosis.All the above points introduce Calreticulin as a novel potential central molecule in the fibrotic processes. Calreticulin overexpression in HK-2 cells:-reduces E-cadherin expression-increases vimentin and vinculin expression-induces Snail1/2 expression and β-catenin activation-reduces aSMA expression while not affecting ColI productionthus resulting in a decline of the epithelial cell phenotype, without inducingcomplete EMT.AcknowledgementsDr. Aris CharonisDr. Panos PolitisValeria KalteziotiFani KaragianniPanos KavvadasThanassis StergiopoulosZozefina FoskolouLila KaltsaDafni AntoniouElena FrangouPrincipal investigatorsLab membersCenter for Experimental SurgeryMichalis KatsiboulasHistochemistry Core FacilityAnna AgapakiFundingBRFAAEuropean Social Fund: NSRF-Heracleitus IICollaboratorsDr. Yoshito Ihara, WakayamaMedical University, JapanDr. Marek Michalak, Universityof Alberta, CanadaPetros MoustardasMaria Liousia

The role of Calreticulin   in renal fibrosis

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The role of Calreticulin in renal fibrosis

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