research article constitutional nephrin deficiency...

Hindawi Publishing CorporationInternational Journal of NephrologyVolume 2013 Article ID 457490 15 pageshttpdxdoiorg1011552013457490

Research ArticleConstitutional Nephrin Deficiency in ConditionallyImmortalized Human Podocytes InducedEpithelial-Mesenchymal Transition Supported by120573-CateninNF-kappa B Activation A Consequence ofCell Junction Impairment

Gian Marco Ghiggeri1 Maddalena Gigante2 and Armando Di Donato13

1 Department of Nephrology Gaslini Children Hospital Genoa Italy2 Department of Biomedical Sciences Interdepartmental Research Center BIOAGROMED University of Foggia Italy3 Laboratorio di Nefrologia Istituto G Gaslini Largo G Gaslini 5 16147 Genova Italy

Correspondence should be addressed to Armando Di Donato a-didousanet

Received 11 July 2013 Accepted 11 September 2013

Academic Editor Vladimır Tesar

Copyright copy 2013 Gian Marco Ghiggeri et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

The kidney glomerular podocytes are the cellular target of many chronic nephropathies both determined and acquired geneticallyMutations that affected the expression andor the function of nephrin a key component of the slit-diaphragm are often causes ofthese pathologies Recent findings showed that murine podocytes could undergo epithelial-mesenchymal transformation (EMT)suggesting new hypotheses about the pathogenesis of glomerular fibrosis Here we show that also human podocytes can undergoEMT but more importantly nephrin ablation itself can trigger this phenotypic transformation In fact a model of human podocytewith engineered nephrin deficiency constitutionally expressed high levels of 120572-SMA vimentin fibronectin and other hallmarksof EMT Since it is known that cell contact abrogation is one of the triggers of EMT we reasoned that nephrin loss could accountfor such cell junction disruption and cause the EMTTherefore we demonstrated that also normal podocytes could spontaneouslyundergo EMT if grown in Ca2+-free medium which is known to impair cell contacts The analysis of the main intracellular signaltransduction pathways evidenced some major anomalies consequent with the nephrin abrogation The most intriguing was theactivation of 120573-catenin pathway which plays a critical role in podocyte ontogenesis as well as in the nephrin expression and EMTregulation Also other important signaling proteins like NF-120581B p53 and retinoblastoma protein (RB) showed important activitymodifications Interestingly most of the above indicated signaling pathway alterations were again reproducible by cell junctionrupture induced by Ca2+ deprivation Finally immunofluorescence analysis on kidney sections of patients with NS of Finnish typeconfirmed the constitutive expression of 120572-SMA

1 Introduction

Epithelial-mesenchymal transition (EMT) and its reverseprocess (MET) are physiological processes operative sincethe first days of the embryological development [1 2] Inadults this phenotypic transition is often just a physiologicalresponse to a call for tissue regeneration as in the case ofnormal wounds [3 4] In fact the cells deriving from EMTthe myofibroblasts smooth muscle cells alike are specialized

fibroblasts highly active in the production of extracellularmatrix components (collagens elastin fibronectin metallo-proteinase etc) [5] Unfortunately EMT is often exacerbatedduring pathological events such as organ fibrosis where theprocess occurs out of control and is irreversible Recently ithas been pointed out that this differentiating process couldbe at the basis of the spreading of many epithelial cancerssince the cells resulting from EMT acquire a much highermobility potential [6 7] Among the many organ pathologies

2 International Journal of Nephrology

characterized by a fibrotic outcome the renal nephroticsyndrome (NS) is one of the most studied and with thehighest social impact In the last few years many new aspectsof the pathogenesis of the disease have been unveiled Nowit is clear that at least in the genetic and familiar formsbut also in many acquired cases the filtration defect ofNS depends on the quantitative andor qualitative alterationof the podocyte slit-diaphragm protein components Theslit-diaphragm is a very specialized cell junction with aselective filtration capacity which preserves the organismfrom losing important proteins into the preurine filtrate Themajor culprits have been identified in nephrin (NPHS1) andpodocin (NPHS2) [8ndash13] among the many others Nephrinis a large membrane protein with an IgG like ectodomainbelonging to the IgG superfamily proteins while podocinis an integral membrane protein with one transmembranedomain and a C-terminal cytoplasmic tail The nephrintransmembrane domain and most of the cytoplasmic tail arehomologous to the corresponding regions of the stomatinfamily proteins [14] Nephrin and podocin interact physicallyand functionally linking the cell membrane to the cytoskele-ton and directing its reorganization [15 16] Most of thegenetic forms of NS seem to carry mutations impairing theexpression or the function of these two proteins althoughimpairment of all the other slit-diaphragm components caninduce several degrees of proteinuria up to severe NS or focalsegmental glomerulosclerosis (FSGS) [17ndash19] As a result theglomeruli become unable to perform their selective filtrationfunction leading to massive proteinuria On the other handalso external causes can induce damage to the podocyteslike infective metabolic and immunological agents In thesecases as well the slit-diaphragm seems to be the pathogenetictarget Besides the loss of the filtration function NS developsa progressive and persistent fibrosis leading to the loss offunctional glomeruli So far fibrosis has been mainly seenas consequence of the inflammatory and immunologicalresponse due to the involvement of nonresident cells oftenpresent in the disease Still many investigators agree thatthese factors can account for no more than 50ndash60 of theobserved fibrosis In the case of tubular interstitial sclerosisthe other major outcome of many renal pathologies thefibrosis has been partially attributed to the intrinsic abilityof the renal tubular epithelial cells to undergo EMT [20ndash22] Usually this is seen as consequence of increased activityof TGF-1205731 [23 24] and other cytokines produced duringthese diseases Many theories and in vivo models have beenproposed in this regard Among all a very reasonable andsuggestive hypothesis is that the podocyte itself might beactive producer of the sclerotic tissue in the NS by undergo-ing EMT (personal observation) [25] Other earlier andmorerecent investigations respectively showed the ability of thepodocyte to express low levels of 120572-SMA and undergo EMT[26ndash28] although in the some cases the authors referred toit as glomerular epithelial cells not univocally identifyingthem as podocytes [29] Thus we reasoned that if the slit-diaphragm were pathologically altered like for the nephringenetic deficiency this would lead to an impairment of thecell contact machineryThe loss of the cell contacts is indeedone of the triggeringmechanisms of EMT [30ndash32]Therefore

the nephrin deprived podocyte itself might undergo EMTcontributing to the consequent sclerotic outcome In thepresent paper we tested our hypothesis and showed that aconditionally immortalized human podocyte cell line is ableto undergo EMT upon TGF-1205731 treatment like previouslyshown in a murine model [28]

2 Materials and Methods

21 Cell Lines Conditionally immortalized wild type andnephrin-mutated human podocytes cell lines (kindly sup-plied by Saleem MA Academic and Childrenrsquos RenalUnit University of Bristol Bristol UK) were developed bytransfection with the temperature-sensitive SV40-T gene aspreviously described [33 34]The nephrin-mutated podocytecell line was deprived of nephrin by genetic manipulationthat inserted a constitutive 121delCT frame shift mutationin nephrin transcript [35] Both cell lines proliferate atthe ldquopermissiverdquo temperature (33∘C) After transfer to theldquononpermissiverdquo temperature (37∘C) they enter growth arrestand express markers of differentiated in vivo podocytes Thecells were grown at the appropriate temperature with 5CO

2

in a humidified incubator in a growth medium composedof RPMI 1640 10 fetal bovine serum (FBS) 1 glutamine1 antibiotics 10mgmL insulin 55mgmL transferrin and5 ngmL sodium selenite (purchased as a mix from Sigma-Aldrich St Louis MO USA product code I-3146) Whereindicated the cells were starved for 48 hr in 1 FBS andtreated with 5 ngmL of TGF-1205731 (cod 100-21) which waspurchased from Pepro Tech Rocky Hill NJ USA In theexperiment in absence of calcium the cells were grown asindicated above but in calcium-free RPMI 1640 (Gibco OHUSA) as medium and 1 FBS

22 Protein Extraction and Western Blots Total cell lysateswere prepared in RIPA buffer (50mM Tris-HCL 150mMNaCl 1 Triton X-100 and 1mM ethylen-diamine-tetra-acetic acid EDTA) added with a protease cocktail andthe phosphatase inhibitor cocktails I and II (Sigma MOUSA) The lysates were cleared by 30min centrifugationat 20000timesg Typically 30 120583g of the total cell lysates wasseparated on SDS-PAGE Where indicated the cell extractwas fractionated in membranes cytoplasm and nuclei asfollowing After extensive washing in PBS the cells werescraped in isotonic buffer (40mM HEPES pH 75 5mMMgCl

2 and protease and phosphatase inhibitors) Then they

were passed several times through a 21-gauge needle andfrozen and thawed for three times The nuclear fraction waspelleted at 720timesg for 10min at 4∘C and resuspended inRIPA buffer The supernatant was centrifuged at 30000timesgtimes 30min at 4∘C and the pellet containing the membranefraction was resuspended in RIPA buffer The supernatantcontained the cytoplasm fraction The protein concentrationwas determined using a Blue-Coomassie based assay [36]Then the proteins were blotted to a PVDF Immobilon-P membrane which was previously prepared according tothe manufacturerrsquos protocol (Millipore Bedford MA USA)After blocking in TBS1 BSA (01MNaCl 005M Tris-HCl

International Journal of Nephrology 3

pH 74 and 1 BSA pH 75) for 2 hr the membrane wasincubated overnight or for at least 3 hr with the indicatedantibodies The antibody providers are indicated in the rela-tive figures Then the blots were incubated with the properalkaline phosphatase conjugated secondary antibodies anddeveloped with nitro blue tetrazolium chloride5-bromo-4-chloro-3-indolyl phosphate (NBTBCIP) reagents (RocheDiagnostics GmbH Mannheim Germany)

23 Cell Immunofluorescence (Figure 2) Thecells were grownto subconfluence on cover glass (BDH Milan Italy) fixedwith 4 paraformaldheyde The cells were permeabilizedwith 01 saponin The blocking was obtained by incubatingthe slides in 1 BSA The primary antibodies were used ata 1 10 to 1 20 dilution in the blocking solution as indicatedin the relative figures The goat Snai-1 (homologous ofDrosophila Snail) (sc-10433) and anti-Fsp1 (also known asMts-1) (sc-19949) antibodieswere purchased fromSantaCruzBiotechnologies (Santa Cruz CA USA)The secondary anti-bodies were conjugated with Alexa Fluor 488 dye (MolecularProbes Eugene OR USA) and used at 1 100 dilution Theconfocal laser-scanning microscopy was performed using aFocal Scanning System Laser Co MRC 1024 ER (BioRadUSA) The nuclei were previously stained with propidiumiodide

24 Tissue Immunofluorescence (Figure 9) Expression levelsof 120572-SMA WT-1 nephrin and proteins were evaluated byindirect immunofluorescence and confocal microscopy anal-ysis on renal biopsy from patient with nephrotic syndrome ofFinnish type homozygous for pS569R (c1707CgtA) NPHS1mutation and from wild type (WT) subject Briefly the slideswere incubated overnight with the primary antibodies at 4∘C(mouse anti-120572-SMA Sigma-Aldrich 1 100 rabbit anti-WT-1 sc-192 Santa Cruz Biotechnologies 1 100 guinea pig anti-nephrin Progen Biotechnik 1 100) washed in PBS and thenincubated with goat anti-mouse IgG-Alexa Fluor 488 goatanti-rabbit IgG-Alexa Fluor 555 and chicken anti-guinea pigIgG-Alexa Fluor 488 respectively The slides mounted withan antifading aqueous medium (GelMount Biomeda) wereexamined under a fluorescence microscope equipped withappropriate filters (Leica TCS SP5 Leica Wetzlar Germany)Confocal images were taken at 500 nm intervals throughthe 119911-axis of the section encompassing a total of 4 120583m indepth Images from individual optical planes and multipleserial optical sections were analyzed and the images weresequentially scanned Negative controls were performed byreplacing the primary antibody with nonimmune serum atequivalent concentration Each experiment was carried outthree times

25 Nuclear Extracts The cells were collected by scrapingwith a rubber policeman after previous wash with PBSThe cells were pelleted at 600 g for 10min resuspended in200120583L100mm plate of 20mMHEPES pH 79 1mM EDTA1mM dithiothreitol and protease and phosphatase mix (seeSection 22) and kept at 4∘C for 15min The cell suspensionwas then added with 14 vol of 1 NP40 to obtain a final

concentration of 02 NP40 and incubated at 4∘C for 15minThe cell lysate was then centrifuged at 600 g for 15minThe pellet (P1) mostly containing the unbroken nuclei wasresuspended in 1 vol of BLS (20mMHEPES pH 79 15 mMMgCl

2 02mMEDTA 05mMdithiothreitol 05mMPMSF

80mM NaCl and 25 glycerol) After extensive mixing 1vol of BHS (as in BLS but containing instead 09M NaCl)was slowly added and incubated at 4∘C for 30min undermoderate shaking P1 was centrifuged at 16000 g for 30minThe protein concentration was determined using a Blue-Coomassie based assay [36]

26 Electrophoresis Mobility Shift Assay (EMSA) The assaywas performed as previously described [37] using 2ndash5 120583g ofnuclear extract proteins in a total volume of 20 120583L containing20mM Tris-HCl pH 75 01M NaCl 035mM dithiothreitol05mM EDTA 05mM PMSF 10 glycerol and 05 120583L of2mgmL of double stranded poly(dI-dC) (1 120583greaction)The sequences of the oligonucleotides used as double-strandprobe was the following NF-120581B plus 51015840-AGT TGA GGGGAC TTT CC CAG GC-31015840 NF-120581B plus 51015840- gCC TGG GAAAGTCCCCTCAACT-31015840The oligonucleotides utilizedwerelabeled at their 51015840 ends with 120574minus(32P)ATP (NEN Life Science)using T4 polynucleotide kinase The rabbit anti-p65 (C-20)antibody was purchased from Santa Cruz BiotechnologiesThe binding to the double-stranded oligonucleotides wasperformed by incubating at 4∘C for 30min 10 ftmoles of 32P-labeled probes with the previous nuclear extractWhere indi-cated 1 120583L of antibody anti-p65 was preincubated at 4∘C for30min with the nuclear extractTheDNA-protein complexeswere separated by electrophoresis on a 5 polyacrylamide geland detected by autoradiography of the dried gel

3 Results

In the present investigation we used two conditionallyimmortalized human podocyte cell lines One reproducedthe normal podocyte features (wild type podocytes WT)[33] while the other was genetically deprived of nephrinwith a constitutive 121delCT frame shift mutation in nephrintranscript (nephrin-mutated podocytes Nephminus) [35] TheNephminus clone showed in this paper is representative of othertested nephrin deprived clones since they all gave similarresults and the experiments were always performed withat least 3 other similarly obtained clones to rule out aldquocasual clone effectrdquo Both lines were immortalized with aretroviral construct carrying the SV40 large 119879 antigen genecontaining both the tsA58 and the U19mutationsThe imma-ture podocytes were able to differentiate into nonreplicatingmature podocytes only at nonpermissive temperature InFigure 1 (left panel) the effects of TGF-1205731 treatment onwild type human podocytes are shown Differentiation intoa fibroblast-like phenotype is evident Assuming an EMTprocess behind such a phenotype we tested the podocytesfor the expression of 120572-SMA The western blot in the rightpanel of Figure 1 shows that TGF-1205731 indeed induced astrong 10-time increase of 120572-SMA already after 48 hr oftreatment andwas persistent even after five days Noteworthy


Untreated +TGF-1205731TGF-1205731

120572-SMA

120572-SMAGAPDH 003 032 005 057

GAPDH

2 5Days

minus minus+ +

Wild type podocytes

Figure 1 Phenotypic features of wild type (WT) human podocyte cell lines and 120572-SMA expression effects of TGF-1205731 treatment (a) Thecells were grown as indicated in Section 2 Prior to the TGF-1205731 incubation the cells were starved for 48 hr The pictures show the cellsafter 48 hr of TGF-1205731 treatment used at 5 ngmL The optical images were obtained with a Will (Wetzelar) inverted microscopy at a 10xenlargement (b) Western blot detection of 120572-SMA Typically 30 120583g of total cell lysates was loaded on a 10 polyacrylamide gel Westernblot is described in Section 2 GAPDH expression was used to normalize protein loading The anti-120572-SMAmouse monoclonal antibody waspurchased from Sigma-Aldrich (St Louis MO USA) The anti-GAPDH (sc-25778) rabbit polyclonal antibody was purchased from SantaCruz Biotechnologies (Santa Cruz CA USA) To quantify the protein bands we scanned the blots and performed a densitometry analysisusing the software NIH ImageJ v 64 (freeware NIH MD USA)The figure shows a typical experiment of at least three performed under thesame conditions

it is the presence of discrete amounts of 120572-SMA in theuntreated podocytes as previously described in these cells[38] Another report showed that 120572-SMA is expressed inprimary murine podocytes and seems to play a role intheir maturation [39] Yet although constitutively expressedin our experiments TGF-1205731 significantly triggered 120572-SMAexpression in an EMT process fashion To better definewhether we were in the presence of a typical EMT wesearched for a set of other specific EMT markers Thereforewe looked at the downregulation of P-cadherin typicallyexpressed in podocytes instead of the classical E-cadherinIn Figure 2(a) we showed that P-cadherin was almostcompletely absent after TGF-1205731 treatment being stronglydownregulated already after 48 hr In regard to cadherinbehavior an interesting finding was the switch to N-cadherin(Figure 2(b)) which is often observed during EMT occurringin metastasizing epithelial tumors [40] Screening for otherEMTmarkers we looked at LOXL2 amember of LOX family[41 42] recently identified as an upstream inhibitor of theproteasome-dependent degradation of Snail [43 44] whichin turn down regulates E-cadherin In this case too we foundan important upregulation (Figure 2(c)) The same increasedexpression was detected for Slug transcriptional partner ofSnail-1 [45 46] (Figure 2(d) right panel) Figure 2(e) alsoshowed confocal images of Snail-1 and Fsp-1 (mts-1) specificfibroblast marker The figure showed their expressions inbasal conditions or after 5 days of TGF-1205731 treatment andconfirmed that they were upregulated as well So all themain actors of the EMT initiation were affected Takentogether these results suggest that we were in presence of atypical epithelial-mesenchymal transition Since the typicalnephrotic syndrome is characterized by a qualitative andorquantitative impairment of proteins of the slit-diaphragmmainly nephrin andor podocin we thought that the syn-drome might configure a condition of cell contact losswhich by itself has been described as a possible EMT trigger

[30 31] To test our hypothesis we analyzed the nephrin-mutated podocytes for the same myofibroblast markers withand without TGF-1205731 treatment First we noticed that theysignificantly differed at a phenotypic level from the wild typepodocytes (compare Figure 1 right panelWT and Figure 3(a)Neph) They showed a more elongated fibroblast-like shapeWestern blots indicated a much higher expression of 120572-SMA as compared to the wild type podocytes (Figure 3(b))In addition fibronectin and vimentin were constitutivelyhighly expressed (Figures 3(c)-3(d)) while they were almostabsent or weakly expressed in the control cells Interestinglyin the same figure (panel (e)) we show that these typicalmesenchymal markers like vimentin and 120572-SMA failed tobe upregulated by TGF-1205731 probably because they are alreadyexpressed at high levels compared to the ones achieved by thewild type podocytes when triggered by this cytokine Takentogether this evidence suggests that the nephrin deficiencyitself induced an EMT process To test whether those featuresdepended on the rupture of the homophilic contacts betweenpodocytes due to the absence of nephrin we reproduceda similar condition in the wild type podocytes by growingthem in Ca2+-free medium According to our hypothesis thewild type podocytes in absence of Ca2+ showed a significantupregulation of120572-SMA (Figure 4) It was already evident after2 days of Ca++ deprivation reaching the same extent of TGF-1205731 or even higher after 5 days (from about 2 times to 5 timesafter 2 and 5 days) The TGF-1205731 did not affect the alreadyhigh level of 120572-SMA Then we decided to study the conse-quences of nephrin deficiency on the podocyte intracellularsignaling Since the EMT induces loosing of cell contacts ofwhich the downregulation of E-cadherin is the most typicalconsequence (P-cadherin in the podocytes case) we lookedat the signaling downstream of it We focused first on 120573-catenin a direct cadherin target As evinced by its expressionand cellular distribution we found indeed a significantactivation of 120573-catenin Figure 5(a) showed in fact a nuclear


TGF minus minus+ +

P-Cadherin

P-CadhGAPDH 022 014 017 005

GAPDH

2 5Days

(a)

minus minus+ +

N-Cadherin

N-CadhGAPDH 002 038 003 025

GAPDH

2 5Days

(b)

TGF minus minus+ +

LOXL2

LOXL2GAPDH 015 058 008 119

GAPDH

2 5Days

(c)

minus minus+ +

Slug

SlugGAPDH 005 058 005 041

GAPDH

2 5Days

(d)

Untreated +TGF-1205731

Snai

Fsp-1 (mts-1)

100120583m

(e)

Figure 2 Analysis of additional EMT marker expressions in wild type human podocytes upon TGF-1205731 treatment The cells were treated asindicated in Figure 1 (a) The rabbit anti-P-cadherin (sc-7893) (b) N-cadherin (sc-7939) (d) anti-Slug (sc-15321) and polyclonal antibodieswere purchased from Santa Cruz Biotechnologies (Santa Cruz CA USA) (c) The rabbit anti-LOXL2 (A01) was purchased from Abnova(Taiwan) To quantify the protein bands we scanned the blots and performed a densitometry analysis using the software NIH ImageJ v64 (freeware NIH MD USA) (e) Confocal laser scanned immunofluorescence of Snail-1 and Fsp-1 expressions The cells were treated asindicated and prepared for immunofluorescence analysis as described in Section 2The goat anti-Snail-1 (sc-10433) and anti-Fsp1 (also knownas Mts-1) (sc-19949) antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz CA USA) The secondary antibodies wereconjugated with Alexa Fluor 488 dye (Molecular Probes Eugene OR USA) Nuclei were previously stained with propidium iodideThe figureshows a typical experiment of at least three performed under the same conditions



100120583m

Nephminus

(a)

WT Nephminus

120572-SMA

120572-SMAGAPDH 025 088

GAPDH

(b)

Membrane Cytosol

NaK ATPase

Fibronectin

GAPDH

WT Nephminus WT Nephminus

(c)

Total cell lysate

Vimentin

VimGAPDH 044 266

GAPDH

WT Nephminus

(d)

TGF minus minus+ +

Vimentin

VimGAPDH 006 0141 0171 0134

GAPDH

120572-SMA

120572-SMAGAPDH 022 074 183 162

GAPDH

WT Nephminus

(e)

Figure 3 Phenotypic features of nephrin-mutated podocytes and EMT marker expression in human wild type and nephrin-mutatedpodocytes effects of TGF-1205731 treatment (a) The cells were grown as indicated in Section 2 Prior to the TGF-1205731 incubation (a) and (e) thecells were starved for 48 hr The pictures show the cells after 48 hr of TGF-1205731 treatment used at 5 ngmL The optical images were obtainedwith a Will (Wetzelar) inverted microscopy at a 10x enlargement (b) 120572-SMA detection in wild type (WT) and nephrin deprived humanpodocytes (Nephminus) The anti-120572-SMA mouse monoclonal antibody was purchased from Sigma-Aldrich (St Louis MO USA) (c) Detectionof fibronectin bound to the membrane fraction and in the cytoplasm Na-K-ATPase-1205721 was used as cell membrane marker to normalize theprotein loading Mouse monoclonal anti-fibronectin (sc-18825) goat anti-Na-K-ATPase-1205721 (sc-16041) polyclonal antibodies were purchasedfrom Santa Cruz Biotechnologies (Santa Cruz CA USA) (d) Detection of vimentin in the total cell lysate The goat anti-vimentin (sc-7557)was purchased from Santa Cruz Biotechnologies (Santa Cruz CA USA) (e) Effects of TGF-1205731 on vimentin and 120572-SMA expression in theindicated podocyte cells To quantify the protein bands we scanned the blots and performed a densitometry analysis using the software NIHImageJ v 64 (freeware NIH MD USA) The figure shows a typical experiment of at least three performed under the same conditions


TGF-1205731 minus minus

minus minus+ +

+ + minus minus

minus minus+ +

+ +

Ca2+

120572-SMA

120572-SMAGAPDH 036 191 060 056

GAPDH

2 5DaysDays

017 071 091 090

Wild type podocytes

Figure 4 Effects of Ca2+ deprivation and TGF-1205731 on 120572-SMA expression in wild type podocytes The cells were exposed to the indicatedtreatment and processed for western blot as specified in Section 2 The antibodies used are described in the previous figures To quantify theprotein bands we scanned the blots and performed a densitometry analysis using the software NIH ImageJ v 64 (freeware NIHMD USA)The figure shows a typical experiment of at least three performed under the same conditions

increase of 120573-catenin translocation and its overall higherexpression (Figure 5(a) Membrane +Nucleus) However theactivation of 120573-catenin was not induced by TGF-1205731 in wildtype podocytes (data not shown) in contrast with previousreports [31] Based on these findings we analyzed the nuclearlevels of TCF-4 and LEF-1 two of the main transcriptionfactors cooperating with 120573-catenin at transcription level InFigure 5(b) it is shown that nuclear TCF-4 and LEF-1 wereincreased respectively three and nine times Wonderingabout the consequences at cell cycle level of E-cadherin-120573-catenin signaling perturbation we investigated the func-tional status of two important tumor suppressor and cellproliferation regulators often acting in coordination p53and pRB Figure 5(c) shows that p53 phosphorylated formis increased both as fraction of total p53 (phospho-p53p53total) and as absolute amount both in the cytoplasm andnucleus (phospho-p53GAPDH or Histone H1) Howeverthe expression of total p53 (phosphorylated and unphos-phorylated) between the wild type and nephrin-mutatedpodocytes was not significantly changed (p53 totalGAPDHor histone H1) Panel (d) of the same figure shows alsoa sensible increased phosphorylation of pRB in nephrin-deficient podocytes meaning an overall less active statusInvestigating other main signaling pathways in agreementwith a recent report [47] we found also a significant upreg-ulated expression and activation of NF-120581B p65 (Figure 6(a)left panel) In fact most of the increase of p65 is localizedin the nuclear compartment (Figure 6(a) right panel) In thesame figure the EMSA for the NF-120581B consensus sequenceis also shown confirming an increase of p65 binding to itsDNA target in nephrin-mutated versus wild type podocytes

(Figure 6(b) left panel) In Figure 6(b) right panel the p65identity in the major DNA-protein complex detected is alsoconfirmed since a specific anti-p65 antibody induced asupershift of the radiolabelled band Since NF-120581B is centralto many transduction pathways we wondered whether theresults regarding p53 and pRB and maybe 120573-catenin were insome way dependent on NF-120581B activation To this purposewe blocked the I120581B-120572 proteasome-dependent degradationwith Bay-117082 an inhibitor of I120581B-120572 phosphorylation [48]inducing therefore degradation of NF-120581B In these condi-tions we screened both podocyte cell lines for 120573-catenin Ph-p53 Ph-RB and some of themesenchymalmarkers which areoverexpressed in nephrin-mutated podocytes In Figure 7 itis shown that Bay-117082 lowered p65 expression as expectedbut also p53 phosphorylation was diminished and 120572-SMAwas down-regulated in both podocyte cell lines In the figurewe showed that even vimentin level was almost shut down bythe treatment in the nephrin-mutated podocytes indicatinga sort of reversion of EMT Conversely pRB phosphorylationand 120573-catenin nuclear translocation remained unaffectedas well as the nuclear level of TCF-4 a 120573-catenin targetStrangely Bay-117082 upregulated TCF4 in the wild typepodocytes We do not have an explanation for that at thismoment Thus not all the altered intracellular signaling ofthe nephrin-mutated podocyte can be ascribed to NF-kBactivation since 120573-catenin and pRB are not affected It doesthough have an effect on the mesenchymal transformationsince 120572-SMA and vimentin are down-regulated upon itsinhibition This has been described in other models [49ndash52] To reinforce our original hypothesis we tested whetheralso these altered pathways could be reproduced in the wild


Membrane Cytoplasm NucleusWT Nephminus WT Nephminus WT Nephminus

120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)

Figure 5 120573-Catenin TCF-4 LEF-1 p53 and pRB distribution and expression in wild type and nephrin-mutated podocytes The cells werelysated fractionated and processed for western blot as described in Section 2 (a) Distribution and expression of 120573-catenin in cell membranesand nuclear compartment The goat anti-120573-catenin (sc-1496) antibody was purchased from Santa Cruz Biotechnologies (Santa Cruz CAUSA) (b) Expression of LEF-1 and TCF-4 in nuclear compartment The goat anti-LEF-1 (sc-8591) and anti-TCF-4 (sc-8631) antibodies werepurchased from Santa Cruz Biotechnologies (Santa Cruz CA USA) (c) Phosphorylated and total p53 distribution and expression in nucleusand cytoplasm The mouse monoclonal anti-p53 IC12 (no 2524) and the rabbit polyclonal anti-Phospho-p53 (ser15) (no 9284) antibodieswere purchased from Cell Signaling Technologies (Boston MA USA) (d) Phosphorylated-RB expression in total cell lysate The rabbit anti-phospho-RB (ser780) (no 9307) antibody was purchased from Cell Signaling Technologies (Boston MA USA) To quantify the proteinbands we scanned the blots and performed a densitometry analysis using the software NIH ImageJ v 64 (freeware NIH MD USA)Where the relative amount of protein is indicated as generic ldquoratiordquo instead of ratio to a specific housekeeping protein it is because theprotein quantization referred to the different cell compartment housekeepers indicated at the bottom of each blot The figure shows a typicalexperiment of at least three performed under the same conditions

type human podocytes by Ca2+ deprivation Figure 8 indeedshows that 120573-catenin nuclear translocation rate and p53and pRB phosphorylation levels mirrored their behavior innephrin-mutated podocytes At this point it is evident thatnephrin deprivation and therefore cell contact rupture mustinduce a wide and complex series of cross-interacting signalsprobably EMT-dependent A natural question arising from

this study is whether these in vitro findings reflected a phys-iopathological occurrence in human chronic nephropathiesdependent on nephrin impairment like for NPHS1 Toanswer the question we analyzed by immunofluorescenceand confocal microscopy renal biopsies from patients withnephrotic syndrome of Finnish type The one shown hereis homozygous for pS569R (c1707CgtA) NPHS1 mutation


Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)

NF-120581B cold probe minus minus ++

Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)

Figure 6 NF-120581B p65 expression distribution and EMSA for NF-120581B consensus in wild type and nephrin-mutated podocytes (a) WT andNephminus podocytes were grown fractionated and processed for western blot as described in Section 2 The rabbit anti-p65 (sc-372) polyclonalantibody was purchased from Santa Cruz Biotechnologies (Santa Cruz CA USA)The sheep anti-histoneH1 (H5110) polyclonal antibody waspurchased from US Biological (Swampscott MA USA) To quantify the protein bands we scanned the blots and performed a densitometryanalysis using the software NIH ImageJ v 64 (freeware NIHMD USA) (b) Nuclear extracts fromWT andNephminus podocytes were preparedas indicated in Section 2 EMSA was performed using NF-120581B consensus [32P]-labeled double-strand oligonucleotide The sequence of theprobe and the EMSA technique are described in Section 2 The anti-p65 antibody is the same as in part A of the present figure but 10 timesmore concentrated (sc-372X) (Santa Cruz Biotechnology Santa Cruz CA USA) The figure shows a typical experiment of at least threeperformed under the same conditions

The results in Figure 9 demonstrated that 120572-SMA was highlyexpressed in the glomeruli of the tested NPHS1 biopsyas compared to a kidney biopsy from a normal tissue InFigure 9 panel (A) (compare (a) versus (b)) we showedthe absence of nephrin expression in the NPHS1 glomerulias expected The panel (C) of the same figure showed thecolocalization of 120572-SMAwithWT1 typical podocyte markerto indicate its specific podocyte expression (compare (a)(b) and (c) versus (d) (e) and (f)) Such a colocalizationis better shown in panel (D) which is an enlargement of aparticular of panel (C) In panel (B) of Figure 9 the negativecontrol reaction for the immunofluorescence of 120572-SMA andWT1 is shown as indicated In panel (E) of the same figurewe showed the expression of 120572-SMA in a different renalpathology that is not based on nephrin impairment theIgA nephropathy In this case the expression was much lesspronounced as compared to the biopsy of a healthy subject

and more importantly did not seem to affect the podocytearea of the glomerulus Therefore the bioptic analysis gavefurther support to our cellular studies

4 Discussion

Overall our investigation demonstrated that human podo-cytes lines are prone to undergo EMT They responded tothe most classical EMT inducer the TGF-1205731 The P-cadherinwas down-regulated and the expression of 120572-SMA typicalmyofibroblast marker was upregulated and so all the majorplayers of EMT Our study agrees with other investigatorfindings in similar mouse cell systems [28 29 39] Webelieve that our findings corroborate the hypothesis ofpodocyte EMT as a putative mechanism underlying thesclerotic reactions that characterize human nephroticsyndromes and other glomerular-based pathologies More


WT Nephminus

Bay-11 minus minus+ +

120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin

VimentinGAPDH 015 004

GAPDH

Nephminus

Total cell lysate

Bay-11

Figure 7 Effects of Bay-117082 on the distribution and expression of p53 and its phosphorylated form phosphorylated pRB 120573-catenin a-SMA vimentin and p65 in wild type and nephrin-mutated podocytes Where indicated the cells were treated with Bay-117082 (CalbiochemUSA) at 05mM for 4 days (in the figure indicated as Bay-11) Afterwards they were lysated fractionated and processed for western blotas described in Section 2 The antibodies used have been described in the previous figures To quantify the protein bands we scanned theblots and performed a densitometry analysis using the software NIH ImageJ v 64 (freeware NIH MD USA) The figure shows a typicalexperiment of at least three performed under the same conditions

interestingly we showed that genetic ablation of nephrininduced a constitutional EMT as well with most of theabove indicated molecular hallmarks Interestingly TGF-1205731barely affected if at all the expression of mesenchymalmarkers in nephrin-mutated podocytes just as expected ina myofibroblast phenotype These results prompted us to testthe hypothesis that the impairment of the slit-diaphragmand in general of the cell contacts might have a role innephrin deficiency-dependent EMT In fact the rupture ofthe intercellular junctions by Ca2+ deprivation induced in

the human wild type podocytes phenotypic and molecularexpression changes very similar to the EMT observed in thenephrin-mutated podocytes This finding might explain aswell glomerulosclerosis occurring in nephrotic syndromesin which components of the slit-diaphragm other thannephrin are absent or functionally impaired Amore detailedinvestigation on the molecular consequences of nephrinablation showed complex alterations of critical pathwaysinvolving 120573-catenin NF120581B p53 and pRB Consideringthe indubitable upsetting of the slit-diaphragm induced by


Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus

Figure 8 Growth of wild type podocytes in Ca2+-free medium induced the same effects of nephrin ablation on 120573-catenin p53 and phospho-pRB expressions and distributionThe cells were lysated fractionated and processed for western blot as described in Section 2The antibodiesused have been described in the previous figures To quantify the protein bands we scanned the blots and performed a densitometry analysisusing the software NIH ImageJ v 64 (freeware NIH MD USA)The figure shows a typical experiment of at least three performed under thesame conditions

nephrin ablation and EMT-dependent downregulation ofP-cadherin we postulated that 120573-catenin among otherswas likely the most affected protein in the network betweenthe cell membrane and the cytoskeleton In fact manyinvestigations reported concomitant 120573-catenin activationupon E-cadherin knocking down or inhibition [53ndash55]Although primary 120573-catenin activation seems to be ableto indirectly downregulate E-cadherin by activating Snailwhile it is known that Snail in turn can also downregulatenephrin [56] in our model it is more likely that 120573-cateninactivation is secondary to the cell contact impairmentand consequent loss of cadherin However Wnt-1120573-catenin pathway has been recently highly considered inthe podocytopathies as a new important pathogenetic orfavoring factor to be taken into account [57 58] Definitelyit is more difficult to understand the p53 activation andpRB downmodulation by hyperphosphorylation We donot know yet what their role is in the economy of this kindof podocyte damage mainly because we did not find anyapoptosis increase However the absence of apoptosis couldstill be explained by the survival signaling coming from120573-catenin activation pathway As matter of fact a case of p53activation and increase of pRB phosphorylation has beendescribed as dependent on nutlin-3 a p53 indirect activator[59] Moreover previous observations indicated 120572-SMA tobe positively regulated by p53 [60] Also p53 activation inaggressive fibromatosis together with 120572-SMA expression [61]and in another case RB depletion both induced E-cadherindownregulation and EMT [62] Adding further complexityto the observed pathway picture we confirmed the activationof NF-120581B in agreement with a recent investigation on

the same nephrin-deficient cell line [47] Based on thepresented findings we would like to express a differentpoint of view at this regard since we believe that NF-120581Bactivation is more likely dependent on the EMTP-cadherindownregulation process as previously noted in a modelof malignant melanoma [51] rather than being induceddirectly by nephrin ablation However Hussain et Al [47]while finding a NF-120581B activation did not describe a nephrindeficiency-dependent EMT like in our case As matterof fact NF-120581B must play a role in what we observed Infact Bay-117082-dependent inhibition of NF-120581B affectednegatively p53 phosphorylation as well as vimentin and 120572-SMA expressions We are in presence of a complex signalingnetwork that must be triggered at the slit-diaphragm levelbut it is hard to say what is due just to nephrin absence andwhat is dependent on the cell contact impairment as wholeIt could well be that nephrin ablation on one side and cellcontacts loss on the other trigger a cascade of overlappingand interfering signals probably orchestrated by the EMTprocess Our in vitro study found also confirmation in kidneybiopsy from a patient carrying a NPHS1 mutation In fact wefound an overexpression of 120572-SMA at podocytes level Thisis very strong evidence that the in vivo pathology follows apathway that is not different from what we observed in vitroNevertheless we are fully aware that more cases of NPHS1should have been analyzed but due to the difficulties to findthose cases we were not able to perform more immunoflu-orescence experiments Though it is quite suggestive thatonly the nephrotic syndrome caused by nephrin deficiencyshowed high levels of 120572-SMA while in a IgA nephropathybiopsy [63] the slight increase of 120572-SMAwasmainly detected


Nephrin

WT

pS5

69R

Neg

ativ

e CTR

120572-SMA WT1 Merge

WT

pS5

69R

WT

pS5

69R

pS5

69R

Staining with nonimmune serum (negative control)


(A)

(B)

(C)

(D)

(E)


Staining with specific antibodies

Enlargements from the indicated square areas in B

Healthy IgAN Anti-SMA negctr

120572-SMA staining

Figure 9 Nephrin and 120572-SMA expression in kidney biopsies from a normal individual and a patient carrying a Finnish mutation pS569R(c1707CgtA) (NPHS1) and in IgAN Immunofluorescence and confocal analysis were carried out as described in Section 2 (A) Nephrinexpression in normal (WT) (a) andNPHS1 kidney (b) (c) shows the antibody negative control (B) Negative control for 120572-SMA (a) expressionand WT1 (b) antibodies and their merged signals (d) (C) 120572-SMA and WT1 expression in normal (WT) and NPHS1 kidney (WT a-b andpS569R d-e) and their merged signal (c-f) 120572-SMA colocalizes with WT1 indicating its podocyte origin (D) Enlarged particular of panel(c) showing the colocalization of 120572-SMA and WT1 in NPHS1 kidney tissue and their merged signals (a b and c) (E) 120572-SMA expression inhealthy or IgA nephropaty (IgAN) renal biopsy The technical details are described in Section 2 The figure shows a typical experiment of atleast three performed under the same conditions


outside the glomerular area Finally we showed that Ca2+deprivation reproduced the described pathway alterationseven in the wild type podocytes Although we are aware thatCa2+ deprivation might induce more complex metaboliceffects the impairment of the cell contacts is one of the mostevident consequences and this method has been previouslyused to the same purpose [30 31] Therefore we feel that thislatter result is reinforcing our hypothesis that the cell-cellinteractions might play an important and probably primaryrole in the phenomena here described

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported byThe Italian Ministry of ScientificResearch The author would like to thank Dr MA Saleem(Academic and Childrenrsquos Renal Unit University of BristolBristol UK) for providing us with the cell lines and his pre-cious suggestions and Dr Tullio Florio and StefanoThellung(Department of Oncology and Genetic Biology University ofGenoa Genoa Italy) for the confocal imaging analysis

References

[1] C L Chaffer E WThompson and E DWilliams ldquoMesenchy-mal to epithelial transition in development and diseaserdquo CellsTissues Organs vol 185 no 1ndash3 pp 7ndash19 2007

[2] L Saxen and I Thesleff ldquoEpithelial-mesenchymal interactionsinmurine organogenesisrdquoCiba Foundation symposium vol 165pp 183ndash198 1992

[3] G J Allan J Beattie and D J Flint ldquoEpithelial injuryinduces an innate repair mechanism linked to cellular senes-cence and fibrosis involving IGF-binding protein-5rdquo Journal ofEndocrinology vol 199 no 2 pp 155ndash164 2008

[4] A Desmouliere C Chaponnier and G Gabbiani ldquoTissuerepair contraction and the myofibroblastrdquo Wound Repair andRegeneration vol 13 no 1 pp 7ndash12 2005

[5] G Gabbiani ldquoThe biology of the myofibroblastrdquo Kidney Inter-national vol 41 no 3 pp 530ndash532 1992

[6] L Larue and A Bellacosa ldquoEpithelial-mesenchymal transitionin development and cancer role of phosphatidylinositol 31015840kinaseAKT pathwaysrdquo Oncogene vol 24 no 50 pp 7443ndash7454 2005

[7] J M Lee S Dedhar R Kalluri and E W Thompson ldquoTheepithelial-mesenchymal transition new insights in signalingdevelopment and diseaserdquo Journal of Cell Biology vol 172 no7 pp 973ndash981 2006

[8] H Holthofer H Ahola M-L Solin et al ldquoNephrin localizes atthe podocyte filtration slit area and is characteristically splicedin the human kidneyrdquo The American Journal of Pathology vol155 no 5 pp 1681ndash1687 1999

[9] A-M Kuusniemi J Merenmies A-T Lahdenkari et alldquoGlomerular sclerosis in kidneys with congenital nephroticsyndrome (NPHS1)rdquo Kidney International vol 70 no 8 pp1423ndash1431 2006

[10] L B Holzman P L S T John I A Kovari R Verma HHolthofer and D R Abrahamson ldquoNephrin localizes to the slitpore of the glomerular epithelial cell rapid communicationrdquoKidney International vol 56 no 4 pp 1481ndash1491 1999

[11] N Boute O Gribouval S Roselli et al ldquoNPHS2 encoding theglomerular protein podocin is mutated in autosomal recessivesteroid-resistant nephrotic syndromerdquo Nature Genetics vol 24no 4 pp 349ndash354 2000

[12] Y Frishberg C Rinat O Megged E Shapira S Feinstein andA Raas-Rothschild ldquoMutations in NPHS2 encoding podocinare a prevalent cause of steroid-resistant nephrotic syndromeamong Israeli-Arab childrenrdquo Journal of the American Society ofNephrology vol 13 no 2 pp 400ndash405 2002

[13] S Roselli O Gribouval N Boute et al ldquoPodocin localizes inthe kidney to the slit diaphragm areardquoThe American Journal ofPathology vol 160 no 1 pp 131ndash139 2002

[14] I Kadurin S Hubert and S Grunder ldquoA single conserved pro-line residue determines the membrane topology of stomatinrdquoBiochemical Journal vol 418 no 3 pp 587ndash594 2009

[15] T B Huber M Simons B Hartleben et al ldquoMolecular basisof the functional podocin-nephrin complex mutations in theNPHS2 gene disrupt nephrin targeting to lipid raft micro-domainsrdquo Human Molecular Genetics vol 12 no 24 pp 3397ndash3405 2003

[16] T B Huber M Kottgen B Schilling G Walz and T BenzingldquoInteractionwith podocin facilitates nephrin signalingrdquo Journalof Biological Chemistry vol 276 no 45 pp 41543ndash41546 2001

[17] T Palmen S Lehtonen A Ora et al ldquoInteraction of endoge-nous nephrin and CD2-associated protein in mouse epithelialM-1 cell linerdquo Journal of the American Society of Nephrology vol13 no 7 pp 1766ndash1772 2002

[18] Q Fan Y Xing J Ding N Guan and J Zhang ldquoThe relation-ship among nephrin podocin CD2AP and 120572-actininmight notbe a true ldquointeractionrdquo in podocyterdquo Kidney International vol69 no 7 pp 1207ndash1215 2006

[19] H Kawachi K Suzuki N Miyauchi T Hashimoto Y Otakiand F Shimizu ldquoSlit diaphragm dysfunction in proteinuricstates identification of novel therapeutic targets for nephroticsyndromerdquo Clinical and Experimental Nephrology vol 13 no 4pp 275ndash280 2009

[20] J Yang and Y Liu ldquoDissection of key events in tubularepithelial to myofibroblast transition and its implications inrenal interstitial fibrosisrdquo The American Journal of Pathologyvol 159 no 4 pp 1465ndash1475 2001

[21] I Mucsi and L Rosivall ldquoEpithelial-mesenchymal transition inrenal tubular cells in the pathogenesis of progressive tubulo-interstitial fibrosisrdquoActa PhysiologicaHungarica vol 94 no 1-2pp 117ndash131 2007

[22] W C Burns P Kantharidis and M C Thomas ldquoThe role oftubular epithelial-mesenchymal transition in progressive kid-ney diseaserdquo Cells Tissues Organs vol 185 no 1ndash3 pp 222ndash2312007

[23] Y Chihara H Ono T Ishimitsu et al ldquoRoles of TGF-1205731 andapoptosis in the progression of glomerulosclerosis in humanIgA nephropathyrdquo Clinical Nephrology vol 65 no 6 pp 385ndash392 2006

[24] M Hwang H-J Kim H-J Noh et al ldquoTGF-1205731 siRNA sup-presses the tubulointerstitial fibrosis in the kidney of ureteralobstructionrdquo Experimental andMolecular Pathology vol 81 no1 pp 48ndash54 2006


[25] ROleggini N Gastaldo andA di Donato ldquoPodocytes undergoepithelial-mesenchymal transition (EMT) in vitrordquo in Proceed-ings of the 6th International Podocyte Conference HelsinkiFinland June 2006

[26] J Bariety G S Hill C Mandet et al ldquoGlomerural epithelial-mesenchymal transdifferentiation in pauci-immune crescenticglomerulonephritisrdquo Nephrology Dialysis Transplantation vol18 no 9 pp 1777ndash1784 2003

[27] C Wang X Liu Z Ke et al ldquoMesangial medium from IgAnephropathy patients induces podocyte epithelial-to-mesen-chymal transition through activation of the phosphatidylinositol-3-kinaseakt signaling pathwayrdquo Cell Physiol Biochemvol 29 no 5-6 pp 743ndash752 2012

[28] Y Li Y S Kang C Dai L P Kiss X Wen and Y LiuldquoEpithelial-to-mesenchymal transition is a potential pathwayleading to podocyte dysfunction and proteinuriardquo The Ameri-can Journal of Pathology vol 172 no 2 pp 299ndash308 2008

[29] R Sam LWanna K P Gudehithlu et al ldquoGlomerular epithelialcells transform to myofibroblasts early but not late removal ofTGF-1205731 reverses transformationrdquo Translational Research vol148 no 3 pp 142ndash148 2006

[30] L Fan A Sebe Z Peterfi et al ldquoCell contact-dependent reg-ulation of epithelial-myofibroblast transition via the Rho-Rhokinase-phospho-myosin pathwayrdquoMolecular Biology of the Cellvol 18 no 3 pp 1083ndash1097 2007

[31] A Masszi L Fan L Rosivall et al ldquoIntegrity of cell-cellcontacts is a critical regulator of TGF-1205731-induced epithelial-to-myofibroblast transition role for 120573-cateninrdquo The AmericanJournal of Pathology vol 165 no 6 pp 1955ndash1967 2004

[32] D B N Lee E Huang and H J Ward ldquoTight junction biologyand kidney dysfunctionrdquo American Journal of PhysiologymdashRenal Physiology vol 290 no 1 pp F20ndashF34 2006

[33] M A Saleem M J OrsquoHare J Reiser et al ldquoA conditionallyimmortalized human podocyte cell line demonstrating nephrinand podocin expressionrdquo Journal of the American Society ofNephrology vol 13 no 3 pp 630ndash638 2002

[34] P Mundel J Reiser and W Kriz ldquoInduction of differentiationin cultured rat and human podocytesrdquo Journal of the AmericanSociety of Nephrology vol 8 no 5 pp 697ndash705 1997

[35] R JM Coward G IWelsh A Koziell et al ldquoNephrin is criticalfor the action of insulin on human glomerular podocytesrdquoDiabetes vol 56 no 4 pp 1127ndash1135 2007

[36] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976

[37] R Barresi M Sirito G Karsenty and R Ravazzolo ldquoA negativecis-actingG-fer element participates in the regulation of expres-sion of the human H-ferritin-encoding gene (FERH)rdquo Genevol 140 no 2 pp 195ndash201 1994

[38] M A Saleem J Zavadil M Bailly et al ldquoThe molecularand functional phenotype of glomerular podocytes reveals keyfeatures of contractile smooth muscle cellsrdquo American Journalof PhysiologymdashRenal Physiology vol 295 no 4 pp F959ndashF9702008

[39] C-A Chen J-C Hwang J-Y Guh J-C Tsai and H-C ChenldquoTGF-1205731 and integrin synergistically facilitate the differenti-ation of rat podocytes by increasing 120572-smooth muscle actinexpressionrdquo Translational Research vol 148 no 3 pp 134ndash1412006

[40] M Maeda K R Johnson and M J Wheelock ldquoCadherinswitching essential for behavioral but not morphological

changes during an epithelium-to-mesenchyme transitionrdquo Jour-nal of Cell Science vol 118 no 5 pp 873ndash887 2005

[41] C Jourdan-Le Saux O le Saux C Gleyzal P Sommer and KCsiszar ldquoThe mouse lysyl oxidase-like 2 gene (mLOXL2) mapsto chromosome 14 and is highly expressed in skin lung andthymusrdquoMatrix Biology vol 19 no 2 pp 179ndash183 2000

[42] H M Kagan and W Li ldquoLysyl oxidase properties specificityand biological roles inside and outside of the cellrdquo Journal ofCellular Biochemistry vol 88 no 4 pp 660ndash672 2003

[43] H Peinado M D C Iglesias-de la Cruz D Olmeda et alldquoA molecular role for lysyl oxidase-like 2 enzyme in Snailregulation and tumor progressionrdquoThe EMBO Journal vol 24no 19 pp 3446ndash3458 2005

[44] H Peinado F Portillo and A Cano ldquoSwitching on-off snailLOXL2 versus GSK3120573rdquo Cell Cycle vol 4 no 12 pp 1749ndash17522005

[45] C Castro Alves E Rosivatz C Schott et al ldquoSlug is overex-pressed in gastric carcinomas and may act synergistically withSIPI and Snail in the down-regulation of E-cadherinrdquo Journal ofPathology vol 211 no 5 pp 507ndash515 2007

[46] J Choi Y P Sun and C-K Joo ldquoTransforming growth factor-1205731 represses E-cadherin production via Slug expression in lensepithelial cellsrdquo Investigative Ophthalmology and Visual Sciencevol 48 no 6 pp 2708ndash2718 2007

[47] S Hussain L Romio M Saleem et al ldquoNephrin deficiencyactivates NF-120581B and promotes glomerular injuryrdquo Journal of theAmerican Society of Nephrology vol 20 no 8 pp 1733ndash17432009

[48] J W Pierce R Schoenleber G Jesmok et al ldquoNovel inhibitorsof cytokine-induced I120581B120572 phosphorylation and endothelial celladhesion molecule expression show anti-inflammatory effectsin vivordquo Journal of Biological Chemistry vol 272 no 34 pp21096ndash21103 1997

[49] M A Huber N Azoitei B Baumann et al ldquoNF-120581B isessential for epithelial-mesenchymal transition and metastasisin a model of breast cancer progressionrdquo Journal of ClinicalInvestigation vol 114 no 4 pp 569ndash581 2004

[50] M A Huber H Beug and T Wirth ldquoEpithelial-mesenchymaltransition NF-120581B takes center stagerdquo Cell Cycle vol 3 no 12pp 1477ndash1480 2004

[51] S Kuphal I Poser C Jobin C Hellerbrand and A KBosserhoff ldquoLoss of E-cadherin leads to upregulation of NF120581Bactivity in malignant melanomardquo Oncogene vol 23 no 52 pp8509ndash8519 2004

[52] Q Li B-C Liu L-L Lv K-L Ma X-L Zhang and AO Phillips ldquoMonocytes induce proximal tubular epithelial-mesenchymal transition through NF-kappa B dependentupregulation of ICAM-1rdquo Journal of Cellular Biochemistry vol112 no 6 pp 1585ndash1592 2011

[53] S Z Yang N Kohno A Yokoyama K Kondo H Hamadaand K Hiwada ldquoDecreased E-cadherin augments beta-cateninnuclear localization studies in breast cancer cell linesrdquo Interna-tional Journal of Oncology vol 18 no 3 pp 541ndash548 2001

[54] C J Gottardi E Wong and B M Gumbiner ldquoE-cadherinsuppresses cellular transformation by inhibiting 120573-catenin sig-naling in an adhesion-independent mannerrdquo Journal of CellBiology vol 153 no 5 pp 1049ndash1060 2001

[55] L Ma J Young H Prabhala et al ldquoMiR-9 a MYCMYCN-activated microRNA regulates E-cadherin and cancer metas-tasisrdquo Nature Cell Biology vol 12 no 3 pp 247ndash256 2010


[56] I Matsui T Ito H Kurihara E Imai T Ogihara and M HorildquoSnail a transcriptional regulator represses nephrin expressionin glomerular epithelial cells of nephrotic ratsrdquo LaboratoryInvestigation vol 87 no 3 pp 273ndash283 2007

[57] C Dai D B Stolz L P Kiss S P Monga L B Holzman and YLiu ldquoWnt120573-catenin signaling promotes podocyte dysfunctionand albuminuriardquo Journal of theAmerican Society ofNephrologyvol 20 no 9 pp 1997ndash2008 2009

[58] Y Liu ldquoNew insights into epithelial-mesenchymal transition inkidney fibrosisrdquo Journal of the American Society of Nephrologyvol 21 no 2 pp 212ndash222 2010

[59] W Du J Wu E M Walsh Y Zhang C Y Chen and Z-X J Xiao ldquoNutlin-3 affects expression and function of retino-blastoma protein Role of Retinoblastoma protein in cellularresponse to nutlin-3rdquo Journal of Biological Chemistry vol 284no 39 pp 26315ndash26321 2009

[60] K A Comer P A Dennis L Armstrong J J Catino M BKastan and C C Kumar ldquoHuman smooth muscle 120572-actin geneis a transcriptional target of the p53 tumor suppressor proteinrdquoOncogene vol 16 no 10 pp 1299ndash1308 1998

[61] C Gebert J Hardes C Kersting et al ldquoExpression of 120573-cateninand p53 are prognostic factors in deep aggressive fibromatosisrdquoHistopathology vol 50 no 4 pp 491ndash497 2007

[62] Y Arima Y Inoue T Shibata et al ldquoRb depletion results inderegulation of E-cadherin and induction of cellular phenotypicchanges that are characteristic of the epithelial-to-mesenchymaltransitionrdquo Cancer Research vol 68 no 13 pp 5104ndash5112 2008

[63] M V Agrez ldquoCell adhesion molecules and colon cancerrdquoAustralian and New Zealand Journal of Surgery vol 66 no 12pp 791ndash798 1996

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


characterized by a fibrotic outcome the renal nephroticsyndrome (NS) is one of the most studied and with thehighest social impact In the last few years many new aspectsof the pathogenesis of the disease have been unveiled Nowit is clear that at least in the genetic and familiar formsbut also in many acquired cases the filtration defect ofNS depends on the quantitative andor qualitative alterationof the podocyte slit-diaphragm protein components Theslit-diaphragm is a very specialized cell junction with aselective filtration capacity which preserves the organismfrom losing important proteins into the preurine filtrate Themajor culprits have been identified in nephrin (NPHS1) andpodocin (NPHS2) [8ndash13] among the many others Nephrinis a large membrane protein with an IgG like ectodomainbelonging to the IgG superfamily proteins while podocinis an integral membrane protein with one transmembranedomain and a C-terminal cytoplasmic tail The nephrintransmembrane domain and most of the cytoplasmic tail arehomologous to the corresponding regions of the stomatinfamily proteins [14] Nephrin and podocin interact physicallyand functionally linking the cell membrane to the cytoskele-ton and directing its reorganization [15 16] Most of thegenetic forms of NS seem to carry mutations impairing theexpression or the function of these two proteins althoughimpairment of all the other slit-diaphragm components caninduce several degrees of proteinuria up to severe NS or focalsegmental glomerulosclerosis (FSGS) [17ndash19] As a result theglomeruli become unable to perform their selective filtrationfunction leading to massive proteinuria On the other handalso external causes can induce damage to the podocyteslike infective metabolic and immunological agents In thesecases as well the slit-diaphragm seems to be the pathogenetictarget Besides the loss of the filtration function NS developsa progressive and persistent fibrosis leading to the loss offunctional glomeruli So far fibrosis has been mainly seenas consequence of the inflammatory and immunologicalresponse due to the involvement of nonresident cells oftenpresent in the disease Still many investigators agree thatthese factors can account for no more than 50ndash60 of theobserved fibrosis In the case of tubular interstitial sclerosisthe other major outcome of many renal pathologies thefibrosis has been partially attributed to the intrinsic abilityof the renal tubular epithelial cells to undergo EMT [20ndash22] Usually this is seen as consequence of increased activityof TGF-1205731 [23 24] and other cytokines produced duringthese diseases Many theories and in vivo models have beenproposed in this regard Among all a very reasonable andsuggestive hypothesis is that the podocyte itself might beactive producer of the sclerotic tissue in the NS by undergo-ing EMT (personal observation) [25] Other earlier andmorerecent investigations respectively showed the ability of thepodocyte to express low levels of 120572-SMA and undergo EMT[26ndash28] although in the some cases the authors referred toit as glomerular epithelial cells not univocally identifyingthem as podocytes [29] Thus we reasoned that if the slit-diaphragm were pathologically altered like for the nephringenetic deficiency this would lead to an impairment of thecell contact machineryThe loss of the cell contacts is indeedone of the triggeringmechanisms of EMT [30ndash32]Therefore

the nephrin deprived podocyte itself might undergo EMTcontributing to the consequent sclerotic outcome In thepresent paper we tested our hypothesis and showed that aconditionally immortalized human podocyte cell line is ableto undergo EMT upon TGF-1205731 treatment like previouslyshown in a murine model [28]

2 Materials and Methods

21 Cell Lines Conditionally immortalized wild type andnephrin-mutated human podocytes cell lines (kindly sup-plied by Saleem MA Academic and Childrenrsquos RenalUnit University of Bristol Bristol UK) were developed bytransfection with the temperature-sensitive SV40-T gene aspreviously described [33 34]The nephrin-mutated podocytecell line was deprived of nephrin by genetic manipulationthat inserted a constitutive 121delCT frame shift mutationin nephrin transcript [35] Both cell lines proliferate atthe ldquopermissiverdquo temperature (33∘C) After transfer to theldquononpermissiverdquo temperature (37∘C) they enter growth arrestand express markers of differentiated in vivo podocytes Thecells were grown at the appropriate temperature with 5CO

2

in a humidified incubator in a growth medium composedof RPMI 1640 10 fetal bovine serum (FBS) 1 glutamine1 antibiotics 10mgmL insulin 55mgmL transferrin and5 ngmL sodium selenite (purchased as a mix from Sigma-Aldrich St Louis MO USA product code I-3146) Whereindicated the cells were starved for 48 hr in 1 FBS andtreated with 5 ngmL of TGF-1205731 (cod 100-21) which waspurchased from Pepro Tech Rocky Hill NJ USA In theexperiment in absence of calcium the cells were grown asindicated above but in calcium-free RPMI 1640 (Gibco OHUSA) as medium and 1 FBS

22 Protein Extraction and Western Blots Total cell lysateswere prepared in RIPA buffer (50mM Tris-HCL 150mMNaCl 1 Triton X-100 and 1mM ethylen-diamine-tetra-acetic acid EDTA) added with a protease cocktail andthe phosphatase inhibitor cocktails I and II (Sigma MOUSA) The lysates were cleared by 30min centrifugationat 20000timesg Typically 30 120583g of the total cell lysates wasseparated on SDS-PAGE Where indicated the cell extractwas fractionated in membranes cytoplasm and nuclei asfollowing After extensive washing in PBS the cells werescraped in isotonic buffer (40mM HEPES pH 75 5mMMgCl

2 and protease and phosphatase inhibitors) Then they

were passed several times through a 21-gauge needle andfrozen and thawed for three times The nuclear fraction waspelleted at 720timesg for 10min at 4∘C and resuspended inRIPA buffer The supernatant was centrifuged at 30000timesgtimes 30min at 4∘C and the pellet containing the membranefraction was resuspended in RIPA buffer The supernatantcontained the cytoplasm fraction The protein concentrationwas determined using a Blue-Coomassie based assay [36]Then the proteins were blotted to a PVDF Immobilon-P membrane which was previously prepared according tothe manufacturerrsquos protocol (Millipore Bedford MA USA)After blocking in TBS1 BSA (01MNaCl 005M Tris-HCl










3 Results




120572-SMA

120572-SMAGAPDH 003 032 005 057

GAPDH

2 5Days

minus minus+ +

Wild type podocytes





TGF minus minus+ +

P-Cadherin

P-CadhGAPDH 022 014 017 005

GAPDH

2 5Days

(a)

minus minus+ +

N-Cadherin

N-CadhGAPDH 002 038 003 025

GAPDH

2 5Days

(b)

TGF minus minus+ +

LOXL2

LOXL2GAPDH 015 058 008 119

GAPDH

2 5Days

(c)

minus minus+ +

Slug

SlugGAPDH 005 058 005 041

GAPDH

2 5Days

(d)


Snai

Fsp-1 (mts-1)

100120583m

(e)




100120583m

Nephminus

(a)

WT Nephminus

120572-SMA

120572-SMAGAPDH 025 088

GAPDH

(b)

Membrane Cytosol

NaK ATPase

Fibronectin

GAPDH


(c)

Total cell lysate

Vimentin

VimGAPDH 044 266

GAPDH

WT Nephminus

(d)

TGF minus minus+ +

Vimentin

VimGAPDH 006 0141 0171 0134

GAPDH

120572-SMA

120572-SMAGAPDH 022 074 183 162

GAPDH

WT Nephminus

(e)




minus minus+ +

+ + minus minus

minus minus+ +

+ +

Ca2+

120572-SMA

120572-SMAGAPDH 036 191 060 056

GAPDH

2 5DaysDays

017 071 091 090

Wild type podocytes






120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)





Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























3 Results




120572-SMA

120572-SMAGAPDH 003 032 005 057

GAPDH

2 5Days

minus minus+ +

Wild type podocytes





TGF minus minus+ +

P-Cadherin

P-CadhGAPDH 022 014 017 005

GAPDH

2 5Days

(a)

minus minus+ +

N-Cadherin

N-CadhGAPDH 002 038 003 025

GAPDH

2 5Days

(b)

TGF minus minus+ +

LOXL2

LOXL2GAPDH 015 058 008 119

GAPDH

2 5Days

(c)

minus minus+ +

Slug

SlugGAPDH 005 058 005 041

GAPDH

2 5Days

(d)


Snai

Fsp-1 (mts-1)

100120583m

(e)




100120583m

Nephminus

(a)

WT Nephminus

120572-SMA

120572-SMAGAPDH 025 088

GAPDH

(b)

Membrane Cytosol

NaK ATPase

Fibronectin

GAPDH


(c)

Total cell lysate

Vimentin

VimGAPDH 044 266

GAPDH

WT Nephminus

(d)

TGF minus minus+ +

Vimentin

VimGAPDH 006 0141 0171 0134

GAPDH

120572-SMA

120572-SMAGAPDH 022 074 183 162

GAPDH

WT Nephminus

(e)




minus minus+ +

+ + minus minus

minus minus+ +

+ +

Ca2+

120572-SMA

120572-SMAGAPDH 036 191 060 056

GAPDH

2 5DaysDays

017 071 091 090

Wild type podocytes






120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)





Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120572-SMA

120572-SMAGAPDH 003 032 005 057

GAPDH

2 5Days

minus minus+ +

Wild type podocytes





TGF minus minus+ +

P-Cadherin

P-CadhGAPDH 022 014 017 005

GAPDH

2 5Days

(a)

minus minus+ +

N-Cadherin

N-CadhGAPDH 002 038 003 025

GAPDH

2 5Days

(b)

TGF minus minus+ +

LOXL2

LOXL2GAPDH 015 058 008 119

GAPDH

2 5Days

(c)

minus minus+ +

Slug

SlugGAPDH 005 058 005 041

GAPDH

2 5Days

(d)


Snai

Fsp-1 (mts-1)

100120583m

(e)




100120583m

Nephminus

(a)

WT Nephminus

120572-SMA

120572-SMAGAPDH 025 088

GAPDH

(b)

Membrane Cytosol

NaK ATPase

Fibronectin

GAPDH


(c)

Total cell lysate

Vimentin

VimGAPDH 044 266

GAPDH

WT Nephminus

(d)

TGF minus minus+ +

Vimentin

VimGAPDH 006 0141 0171 0134

GAPDH

120572-SMA

120572-SMAGAPDH 022 074 183 162

GAPDH

WT Nephminus

(e)




minus minus+ +

+ + minus minus

minus minus+ +

+ +

Ca2+

120572-SMA

120572-SMAGAPDH 036 191 060 056

GAPDH

2 5DaysDays

017 071 091 090

Wild type podocytes






120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)





Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















TGF minus minus+ +

P-Cadherin

P-CadhGAPDH 022 014 017 005

GAPDH

2 5Days

(a)

minus minus+ +

N-Cadherin

N-CadhGAPDH 002 038 003 025

GAPDH

2 5Days

(b)

TGF minus minus+ +

LOXL2

LOXL2GAPDH 015 058 008 119

GAPDH

2 5Days

(c)

minus minus+ +

Slug

SlugGAPDH 005 058 005 041

GAPDH

2 5Days

(d)


Snai

Fsp-1 (mts-1)

100120583m

(e)




100120583m

Nephminus

(a)

WT Nephminus

120572-SMA

120572-SMAGAPDH 025 088

GAPDH

(b)

Membrane Cytosol

NaK ATPase

Fibronectin

GAPDH


(c)

Total cell lysate

Vimentin

VimGAPDH 044 266

GAPDH

WT Nephminus

(d)

TGF minus minus+ +

Vimentin

VimGAPDH 006 0141 0171 0134

GAPDH

120572-SMA

120572-SMAGAPDH 022 074 183 162

GAPDH

WT Nephminus

(e)




minus minus+ +

+ + minus minus

minus minus+ +

+ +

Ca2+

120572-SMA

120572-SMAGAPDH 036 191 060 056

GAPDH

2 5DaysDays

017 071 091 090

Wild type podocytes






120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)





Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















100120583m

Nephminus

(a)

WT Nephminus

120572-SMA

120572-SMAGAPDH 025 088

GAPDH

(b)

Membrane Cytosol

NaK ATPase

Fibronectin

GAPDH


(c)

Total cell lysate

Vimentin

VimGAPDH 044 266

GAPDH

WT Nephminus

(d)

TGF minus minus+ +

Vimentin

VimGAPDH 006 0141 0171 0134

GAPDH

120572-SMA

120572-SMAGAPDH 022 074 183 162

GAPDH

WT Nephminus

(e)




minus minus+ +

+ + minus minus

minus minus+ +

+ +

Ca2+

120572-SMA

120572-SMAGAPDH 036 191 060 056

GAPDH

2 5DaysDays

017 071 091 090

Wild type podocytes






120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)





Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















minus minus+ +

+ + minus minus

minus minus+ +

+ +

Ca2+

120572-SMA

120572-SMAGAPDH 036 191 060 056

GAPDH

2 5DaysDays

017 071 091 090

Wild type podocytes






120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)





Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















120573-Catenin

120573-Cat ratio 152 194 nd nd 046 156

NaK ATPase GAPDH H1

GAPDH

(a)

WT Nephminus

LEF-1

LEFH1 002 018

TCF-4

TCFH1 016 043

H1

Nucleus

(b)


Ph-p53

Ph-p53p53 tot 049 104 061 140

p53 total

Ph-p53 ratio 012 023 105 180

H1

p53 tot ratio 023 022 171 130

Nucleus Cytoplasm

(c)

WT Nephminus

Ph-RB

Ph-RBGAPDH 023 062

GAPDH

Total cell lysate

(d)





Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Total cell lysate

p65 ratio 014 060

WT Nephminus

Cytoplasm Nucleus

p65

052 115 005 034

H1GAPDH


(a)


Nuclear extracts

WT

WT

Nep

hminus

Nep

hminus Nephminus

minus +Ab p65

Super shift

(b)




4 Discussion



WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















WT Nephminus


120573-Catenin

120573-CatH1 058 042 170 140

Ph-p53

Ph-p53H1 090 032 260 092

p65

p65H1 078 030 213 070

TCF-4

TCF-4H1 040 095 400 400

H1

Nucleus

WT Nephminus

minus minus+ +

Ph-pRB

030 022 115 080 Ph-pRBGAPDH

Ph-p53

290 111 310 070 Ph-p53GAPDH

p65

095 037 170 042 p65GAPDH

120572-SMA

129 086 200 059 120572-SMAGAPDH

GAPDH

Cytoplasm

None

Vimentin


GAPDH

Nephminus

Total cell lysate

Bay-11





Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Ca++ + minus

Ph-RB

Ph-RBGAPDH 004 010

Ph-p53

Ph-p53GAPDH 064 082

GAPDH

Cytoplasm

+ minus

120573-Catenin

120573-CatH1

Ph-p53

Ph-p53H1 012 046

008 019

H1

Nucleus





Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nephrin

WT

pS5

69R

Neg

ativ

e CTR


WT

pS5

69R

WT

pS5

69R

pS5

69R



(A)

(B)

(C)

(D)

(E)





120572-SMA staining






Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References








































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article constitutional nephrin deficiency...

Documents