epithelial - mesenchymal transition ( emt ) and its reverse process ( met ) are physiological processes operative since the first days of the embryological development [ 1 , 2 ] . in adults , this phenotypic transition is often just a physiological response to a call for tissue regeneration , as in the case of normal wounds [ 3 , 4 ] . in fact , the cells deriving from emt , the myofibroblasts , smooth muscle cells alike , are specialized fibroblasts highly active in the production of extracellular matrix components ( collagens , elastin , fibronectin , metalloproteinase , etc . ) . unfortunately , emt is often exacerbated during pathological events such as organ fibrosis , where the process occurs out of control and is irreversible . recently , it has been pointed out that this differentiating process could be at the basis of the spreading of many epithelial cancers , since the cells resulting from emt acquire a much higher mobility potential [ 6 , 7 ] . among the many organ pathologies characterized by a fibrotic outcome , the renal nephrotic syndrome ( ns ) is one of the most studied and with the highest social impact . in the last few years , many new aspects of the pathogenesis of the disease have been unveiled . now it is clear that , at least in the genetic and familiar forms , but also in many acquired cases , the filtration defect of ns depends on the quantitative and/or qualitative alteration of the podocyte slit - diaphragm protein components . the slit - diaphragm is a very specialized cell junction , with a selective filtration capacity , which preserves the organism from losing important proteins into the preurine filtrate . the major culprits have been identified in nephrin ( nphs1 ) and podocin ( nphs2 ) [ 813 ] , among the many others . nephrin is a large membrane protein with an igg like ectodomain , belonging to the igg superfamily proteins , while podocin is an integral membrane protein with one transmembrane domain and a c - terminal cytoplasmic tail . the nephrin transmembrane domain and most of the cytoplasmic tail are homologous to the corresponding regions of the stomatin family proteins . nephrin and podocin interact physically and functionally , linking the cell membrane to the cytoskeleton and directing its reorganization [ 15 , 16 ] . most of the genetic forms of ns seem to carry mutations impairing the expression or the function of these two proteins , although impairment of all the other slit - diaphragm components can induce several degrees of proteinuria , up to severe ns or focal segmental glomerulosclerosis ( fsgs ) [ 1719 ] . as a result , the glomeruli become unable to perform their selective filtration function , leading to massive proteinuria . on the other hand , also external causes can induce damage to the podocytes , like infective , metabolic , and immunological agents . in these cases , as well , the slit - diaphragm seems to be the pathogenetic target . besides the loss of the filtration function , ns develops a progressive and persistent fibrosis leading to the loss of functional glomeruli . so far , fibrosis has been mainly seen as consequence of the inflammatory and immunological response due to the involvement of nonresident cells , often present in the disease . still , many investigators agree that these factors can account for no more than 5060% of the observed fibrosis . in the case of tubular interstitial sclerosis , the other major outcome of many renal pathologies , the fibrosis has been partially attributed to the intrinsic ability of the renal tubular epithelial cells to undergo emt [ 2022 ] . usually , this is seen as consequence of increased activity of tgf-1 [ 23 , 24 ] and other cytokines produced during these diseases . , a very reasonable and suggestive hypothesis is that the podocyte itself might be active producer of the sclerotic tissue in the ns , by undergoing emt ( personal observation ) . other earlier and more recent investigations , respectively , showed the ability of the podocyte to express low levels of -sma and undergo emt [ 2628 ] although , in the some cases , the authors referred to it as glomerular epithelial cells , not univocally identifying them as podocytes . thus , we reasoned that , if the slit - diaphragm were pathologically altered , like for the nephrin genetic deficiency , this would lead to an impairment of the cell contact machinery . the loss of the cell contacts is , indeed , one of the triggering mechanisms of emt [ 3032 ] . therefore , the nephrin deprived podocyte itself might undergo emt , contributing to the consequent sclerotic outcome . in the present paper , we tested our hypothesis and showed that a conditionally immortalized human podocyte cell line is able to undergo emt upon tgf-1 treatment , like previously shown in a murine model . conditionally immortalized wild type and nephrin - mutated human podocytes cell lines ( kindly supplied by saleem , ma , academic and children 's renal unit , university of bristol , bristol , uk ) were developed by transfection with the temperature - sensitive sv40-t gene , as previously described [ 33 , 34 ] . the nephrin - mutated podocyte cell line was deprived of nephrin by genetic manipulation that inserted a constitutive 121delct frame shift mutation in nephrin transcript . after transfer to the  nonpermissive  temperature ( 37c ) , they enter growth arrest and express markers of differentiated in vivo podocytes . the cells were grown at the appropriate temperature , with 5% co2 in a humidified incubator in a growth medium composed of rpmi 1640 , 10% fetal bovine serum ( fbs ) , 1% glutamine , 1% antibiotics , 10  mg / ml insulin , 5.5  mg / ml transferrin , and 5  ng / ml sodium selenite ( purchased as a mix from sigma - aldrich , st . louis , mo , usa , product code i-3146 ) . where indicated , the cells were starved for 48  hr in 1% fbs and treated with 5  ng / ml of tgf-1 ( cod . 100 - 21 ) , which was purchased from pepro tech , rocky hill , nj , usa . in the experiment in absence of calcium , the cells were grown as indicated above but in calcium - free rpmi 1640 ( gibco , oh , usa ) as medium and 1% fbs . total cell lysates were prepared in ripa buffer ( 50  mm tris - hcl , 150  mm nacl , 1% triton x-100 , and 1  mm ethylen - diamine - tetra - acetic acid , edta ) , added with a protease cocktail and the phosphatase inhibitor cocktails i and ii ( sigma , mo , usa ) . typically , 30  g of the total cell lysates was separated on sds - page . where indicated , the cell extract was fractionated in membranes , cytoplasm , and nuclei as following . after extensive washing in pbs , the cells were scraped in isotonic buffer ( 40  mm hepes , ph 7.5 , 5  mm mgcl2 , and protease and phosphatase inhibitors ) . then they were passed several times through a 21-gauge needle and frozen and thawed for three times . the nuclear fraction was pelleted at 720  g for 10  min at 4c and resuspended in ripa buffer . 30  min at 4c , and the pellet containing the membrane fraction was resuspended in ripa buffer . then , the proteins were blotted to a pvdf immobilon - p membrane , which was previously prepared according to the manufacturer 's protocol ( millipore , bedford , ma , usa ) . after blocking in tbs/1% bsa ( 0.1  m nacl , 0.05  m tris - hcl , ph 7.4 , and 1% bsa , ph 7.5 ) for 2  hr , the membrane was incubated overnight or for at least 3  hr with the indicated antibodies . then , the blots were incubated with the proper alkaline phosphatase conjugated secondary antibodies and developed with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate ( nbt / bcip ) reagents ( roche diagnostics gmbh , mannheim , germany ) . the cells were grown to subconfluence on cover glass ( bdh , milan , italy ) fixed with 4% paraformaldheyde . the primary antibodies were used at a 1  :  10 to 1  :  20 dilution in the blocking solution as indicated in the relative figures . the goat snai-1 ( homologous of drosophila snail ) ( sc-10433 ) and anti - fsp1 ( also known as mts-1 ) ( sc-19949 ) antibodies were purchased from santa cruz biotechnologies , ( santa cruz , ca , usa ) . the secondary antibodies were conjugated with alexa fluor 488 dye ( molecular probes , eugene , or , usa ) and used at 1  :  100 dilution . the confocal laser - scanning microscopy was performed using a focal scanning system laser co mrc 1024 er ( biorad , usa ) . expression levels of -sma , wt-1 , nephrin , and proteins were evaluated by indirect immunofluorescence and confocal microscopy analysis on renal biopsy from patient with nephrotic syndrome of finnish type homozygous for p.s569r ( c.1707c > a ) nphs1 mutation and from wild type ( wt ) subject . briefly , the slides were incubated overnight with the primary antibodies at 4c ( mouse anti--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 then incubated with goat anti - mouse igg - alexa fluor 488 , goat anti - rabbit igg - alexa fluor 555 , and chicken anti - guinea pig igg - alexa fluor 488 , respectively . the slides , mounted with an antifading aqueous medium ( gel / mount , biomeda ) , were examined under a fluorescence microscope equipped with appropriate filters ( leica tcs sp5 ; leica , wetzlar , germany ) . confocal images were taken at 500  nm intervals through the z - axis of the section , encompassing a total of 4  m in depth . images from individual optical planes and multiple serial optical sections were analyzed , and the images were sequentially scanned . negative controls were performed by replacing the primary antibody with nonimmune serum at equivalent concentration . the cells were collected by scraping with a rubber policeman , after previous wash with pbs . the cells were pelleted at 600  g for 10  min , resuspended in 200  l/100  mm plate of 20  mm hepes , ph 7.9 , 1  mm edta , 1  mm dithiothreitol , and protease and phosphatase mix ( see section 2.2 ) , and kept at 4c for 15  min . of 1% np40 to obtain a final concentration of 0.2% np40 and incubated at 4c for 15  min . the pellet ( p1 ) mostly containing the unbroken nuclei was resuspended in 1 vol . of bls ( 20  mm hepes , ph 7.9 , 1.5 mm mgcl2 , 0.2  mm edta , 0.5  mm dithiothreitol , 0.5  mm pmsf , 80  mm nacl , and 25% glycerol ) . of bhs ( as in bls , but containing instead 0.9  m nacl ) was slowly added and incubated at 4c for 30  min under moderate shaking . the assay was performed as previously described   using 25  g of nuclear extract proteins in a total volume of 20  l containing 20  mm tris - hcl , ph 7.5 , 0.1  m nacl , 0.35  mm dithiothreitol , 0.5  mm edta , 0.5  mm pmsf , 10% glycerol , and 0.5  l of 2  mg / ml of double stranded poly(di - dc ) ( 1  g / reaction ) . the sequences of the oligonucleotides used as double - strand probe was the following : nf-b plus : 5-agt tga ggg gac ttt cc cag gc-3 ; nf-b plus : 5- gcc tgg gaa agt ccc ctc aac t-3. the oligonucleotides utilized were labeled at their 5 ends with (p)atp ( nen life science ) using t4 polynucleotide kinase . the rabbit anti - p65 ( c-20 ) antibody was purchased from santa cruz biotechnologies . the binding to the double - stranded oligonucleotides was performed by incubating at 4c for 30  min 10 ftmoles of p - labeled probes with the previous nuclear extract . where indicated , 1  l of antibody anti - p65 the dna - protein complexes were separated by electrophoresis on a 5% polyacrylamide gel and detected by autoradiography of the dried gel . one reproduced the normal podocyte features ( wild type podocytes : wt ) , while the other was genetically deprived of nephrin with a constitutive 121delct frame shift mutation in nephrin transcript ( nephrin - mutated podocytes : neph ) . the neph clone showed in this paper is representative of other tested nephrin deprived clones , since they all gave similar results and the experiments were always performed with at least 3 other similarly obtained clones , to rule out a  casual clone effect  . both lines were immortalized with a retroviral construct carrying the sv40 large t antigen gene containing both the tsa58 and the u19 mutations . the immature podocytes were able to differentiate into nonreplicating mature podocytes only at nonpermissive temperature . in figure 1 ( left panel ) , the effects of tgf-1 treatment on wild type human podocytes are shown . assuming an emt process behind such a phenotype , we tested the podocytes for the expression of -sma . the western blot in the right panel of figure 1 shows that tgf-1 , indeed , induced a strong 10-time increase of -sma already after 48  hr of treatment and was persistent even after five days . noteworthy , it is the presence of discrete amounts of -sma in the untreated podocytes , as previously described in these cells . another report showed that -sma is expressed in primary murine podocytes and seems to play a role in their maturation . yet , although constitutively expressed , in our experiments , tgf-1 significantly triggered -sma expression in an emt process fashion . to better define whether we were in the presence of a typical emt , we searched for a set of other specific emt markers . therefore , we looked at the downregulation of p - cadherin , typically expressed in podocytes , instead of the classical e - cadherin . in figure 2(a ) , we showed that p - cadherin was almost completely absent after tgf-1 treatment , being strongly downregulated already after 48  hr . in regard to cadherin behavior , an interesting finding was the switch to n - cadherin ( figure 2(b ) ) , which is often observed during emt occurring in metastasizing epithelial tumors . screening for other emt markers , we looked at loxl2 , a member of lox family [ 41 , 42 ] , recently identified as an upstream inhibitor of the proteasome - dependent degradation of snail [ 43 , 44 ] , which in turn , down regulates e - cadherin . in this case too , we found an important upregulation ( figure 2(c ) ) . the same increased expression was detected for slug , transcriptional partner of snail-1 [ 45 , 46 ] ( figure 2(d ) , right panel ) . figure 2(e ) also showed confocal images of snail-1 and fsp-1 ( mts-1 ) , specific fibroblast marker . the figure showed their expressions in basal conditions or after 5 days of tgf-1 treatment and confirmed that they were upregulated as well . taken together , these results suggest that we were in presence of a typical epithelial - mesenchymal transition . since the typical nephrotic syndrome is characterized by a qualitative and/or quantitative impairment of proteins of the slit - diaphragm , mainly nephrin and/or podocin , we thought that the syndrome might configure a condition of cell contact loss , which 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 , with and without tgf-1 treatment . first , we noticed that they significantly differed at a phenotypic level from the wild type podocytes ( compare figure 1 , right panel wt and figure 3(a ) neph ) . western blots indicated a much higher expression of -sma as compared to the wild type podocytes ( figure 3(b ) ) . in addition , fibronectin and vimentin were constitutively highly expressed ( figures 3(c)-3(d ) ) , while they were almost absent or weakly expressed in the control cells . interestingly , in the same figure ( panel ( e ) ) we show that these typical mesenchymal markers , like vimentin and -sma , failed to be upregulated by tgf-1 , probably because they are already expressed at high levels , compared to the ones achieved by the wild type podocytes when triggered by this cytokine . taken together , this evidence suggests that the nephrin deficiency itself induced an emt process . to test whether those features depended on the rupture of the homophilic contacts between podocytes , due to the absence of nephrin , we reproduced a similar condition in the wild type podocytes , by growing them in ca - free medium . according to our hypothesis , the wild type podocytes in absence of ca showed a significant upregulation of -sma ( figure 4 ) . it was already evident after 2 days of ca deprivation , reaching the same extent of tgf-1 , or even higher , after 5 days ( from about 2 times to 5 times after 2 and 5 days ) . then , we decided to study the consequences of nephrin deficiency on the podocyte intracellular signaling . since the emt induces loosing of cell contacts , of which the downregulation of e - cadherin is the most typical consequence ( p - cadherin in the podocytes case ) , we looked at the signaling downstream of it . as evinced by its expression and cellular distribution , we found , indeed , a significant activation of -catenin . figure 5(a ) showed , in fact , a nuclear increase of -catenin translocation and its overall higher expression ( figure 5(a ) : membrane + nucleus ) . however , the activation of -catenin was not induced by tgf-1 in wild type podocytes ( data not shown ) , in contrast with previous reports . based on these findings , we analyzed the nuclear levels of tcf-4 and lef-1 , two of the main transcription factors cooperating with -catenin at transcription level . in figure 5(b ) , it is shown that nuclear tcf-4 and lef-1 were increased , respectively , three and nine times . wondering about the consequences at cell cycle level of e - cadherin--catenin signaling perturbation , we investigated the functional status of two important tumor suppressor and cell proliferation regulators , often acting in coordination : p53 and prb . figure 5(c ) shows that p53 phosphorylated form is increased , both as fraction of total p53 ( phospho - p53/p53 total ) and as absolute amount , both in the cytoplasm and nucleus ( phospho - p53/gapdh or /histone however , the expression of total p53 ( phosphorylated and unphosphorylated ) between the wild type and nephrin - mutated podocytes was not significantly changed ( p53 total / gapdh or /histone panel ( d ) of the same figure shows also a sensible increased phosphorylation of prb in nephrin - deficient podocytes , meaning an overall less active status . investigating other main signaling pathways , in agreement with a recent report , we found also a significant upregulated expression and activation of nf-b p65 ( figure 6(a ) , left panel ) . in fact , most of the increase of p65 is localized in the nuclear compartment ( figure 6(a ) , right panel ) . in the same figure , the emsa for the nf-b consensus sequence is also shown , confirming an increase of p65 binding to its dna target in nephrin - mutated versus wild type podocytes ( figure 6(b ) , left panel ) . in figure 6(b ) , right panel , the p65 identity in the major dna - protein complex detected is also confirmed , since a specific anti - p65 antibody induced a supershift of the radiolabelled band . since nf-b is central to many transduction pathways , we wondered whether the results regarding p53 and prb , and maybe -catenin , were in some way dependent on nf-b activation . to this purpose , we blocked the ib- proteasome - dependent degradation with bay-117082 , an inhibitor of ib- phosphorylation , inducing , therefore , degradation of nf-b . in these conditions , we screened both podocyte cell lines for -catenin , ph - p53 , ph - rb , and some of the mesenchymal markers which are overexpressed in nephrin - mutated podocytes . in figure 7 , it is shown that bay-117082 lowered p65 expression as expected , but also p53 phosphorylation was diminished and -sma was down - regulated in both podocyte cell lines . in the figure , we showed that even vimentin level was almost shut down by the treatment in the nephrin - mutated podocytes , indicating a sort of reversion of emt . conversely , prb phosphorylation and -catenin nuclear translocation remained unaffected , as well as the nuclear level of tcf-4 , a -catenin target . strangely , bay-117082 upregulated tcf4 in the wild type podocytes . thus , not all the altered intracellular signaling of the nephrin - mutated podocyte can be ascribed to nf - kb activation , since -catenin and prb are not affected . it does , though , have an effect on the mesenchymal transformation , since -sma and vimentin are down - regulated upon its inhibition . this has been described in other models [ 4952 ] . to reinforce our original hypothesis , we tested whether also these altered pathways could be reproduced in the wild type human podocytes by ca deprivation . figure 8 , indeed , shows that -catenin nuclear translocation rate and p53 and prb phosphorylation levels mirrored their behavior in nephrin - mutated podocytes . at this point , it is evident that nephrin deprivation and , therefore , cell contact rupture must induce a wide and complex series of cross - interacting signals probably emt - dependent . a natural question arising from this study is whether these in vitro findings reflected a physiopathological occurrence in human chronic nephropathies , dependent on nephrin impairment , like for nphs1 . to answer the question , we analyzed by immunofluorescence and confocal microscopy renal biopsies from patients with nephrotic syndrome of finnish type . the one shown here is homozygous for p.s569r ( c.1707c > a ) nphs1 mutation . the results in figure 9 demonstrated that -sma was highly expressed in the glomeruli of the tested nphs1 biopsy , as compared to a kidney biopsy from a normal tissue . in figure 9 , panel ( a ) ( compare ( a ) versus ( b ) ) , we showed the absence of nephrin expression in the nphs1 glomeruli , as expected . the panel ( c ) of the same figure showed the colocalization of -sma with wt1 , typical podocyte marker , to indicate its specific podocyte expression ( compare ( a ) , ( b ) , and ( c ) versus ( d ) , ( e ) , and ( f ) ) . such a colocalization is better shown in panel ( d ) , which is an enlargement of a particular of panel ( c ) . in panel ( b ) of figure 9 the negative control reaction for the immunofluorescence of -sma and wt1 is shown , as indicated . in panel ( e ) of the same figure , we showed the expression of -sma in a different renal pathology that is not based on nephrin impairment , the iga nephropathy . in this case , the expression was much less pronounced as compared to the biopsy of a healthy subject and , more importantly , did not seem to affect the podocyte area of the glomerulus . the p - cadherin was down - regulated , and the expression of -sma , typical myofibroblast marker , was upregulated and so all the major players of emt . our study agrees with other investigator findings in similar mouse cell systems [ 28 , 29 , 39 ] . we believe that our findings corroborate the hypothesis of podocyte emt as a putative mechanism underlying the sclerotic reactions that characterize human nephrotic syndromes and other glomerular - based pathologies . more interestingly , we showed that genetic ablation of nephrin induced a constitutional emt as well , with most of the above indicated molecular hallmarks . interestingly , tgf-1 barely affected , if at all , the expression of mesenchymal markers in nephrin - mutated podocytes , just as expected in a myofibroblast phenotype . these results prompted us to test the hypothesis that the impairment of the slit - diaphragm , and in general , of the cell contacts , might have a role in nephrin deficiency - dependent emt . in fact , the rupture of the intercellular junctions by ca deprivation induced , in the human wild type podocytes , phenotypic and molecular expression changes very similar to the emt observed in the nephrin - mutated podocytes . this finding might explain as well glomerulosclerosis occurring in nephrotic syndromes in which components of the slit - diaphragm other than nephrin are absent or functionally impaired . a more detailed investigation on the molecular consequences of nephrin ablation showed complex alterations of critical pathways , involving -catenin , nfb , p53 , and prb . considering the indubitable upsetting of the slit - diaphragm induced by nephrin ablation and emt - dependent downregulation of p - cadherin , we postulated that -catenin , among others , was likely the most affected protein in the network between the cell membrane and the cytoskeleton . in fact , many investigations reported concomitant -catenin activation upon e - cadherin knocking down or inhibition [ 5355 ] . although primary -catenin activation seems to be able to indirectly downregulate e - cadherin , by activating snail , while it is known that snail , in turn , can also downregulate nephrin , in our model , it is more likely that -catenin activation is secondary to the cell contact impairment and consequent loss of cadherin . however , wnt-1/-catenin pathway has been recently highly considered in the podocytopathies as a new important pathogenetic or favoring factor to be taken into account [ 57 , 58 ] . definitely , it is more difficult to understand the p53 activation and prb downmodulation by hyperphosphorylation . we do not know yet what their role is in the economy of this kind of podocyte damage , mainly because we did not find any apoptosis increase . however , the absence of apoptosis could still be explained by the survival signaling coming from -catenin activation pathway . as matter of fact , a case of p53 activation and increase of prb phosphorylation has been described as dependent on nutlin-3 , a p53 indirect activator . moreover , previous observations indicated -sma to be positively regulated by p53 . also , p53 activation in aggressive fibromatosis together with -sma expression , and in another case , rb depletion , both induced e - cadherin downregulation and emt . adding further complexity to the observed pathway picture , we confirmed the activation of nf-b , in agreement with a recent investigation on the same nephrin - deficient cell line . based on the presented findings , we would like to express a different point of view at this regard , since we believe that nf-b activation is more likely dependent on the emt / p - cadherin downregulation process , as previously noted in a model of malignant melanoma , rather than being induced directly by nephrin ablation . however , hussain et al . , while finding a nf-b activation , did not describe a nephrin deficiency - dependent emt , like in our case . as matter of fact in fact , bay-117082-dependent inhibition of nf-b affected negatively p53 phosphorylation , as well as vimentin and -sma expressions . we are in presence of a complex signaling network that must be triggered at the slit - diaphragm level , but it is hard to say what is due just to nephrin absence and what is dependent on the cell contact impairment as whole . it could well be that nephrin ablation on one side and cell contacts loss on the other trigger a cascade of overlapping and interfering signals , probably orchestrated by the emt process . our in vitro study found also confirmation in kidney biopsy from a patient carrying a nphs1 mutation . this is very strong evidence that the in vivo pathology follows a pathway that is not different from what we observed in vitro . nevertheless , we are fully aware that more cases of nphs1 should have been analyzed , but due to the difficulties to find those cases , we were not able to perform more immunofluorescence experiments . though , it is quite suggestive that only the nephrotic syndrome caused by nephrin deficiency showed high levels of -sma , while in a iga nephropathy biopsy , the slight increase of -sma was mainly detected outside the glomerular area . finally , we showed that ca deprivation reproduced the described pathway alterations even in the wild type podocytes . although we are aware that ca deprivation might induce more complex metabolic effects , the impairment of the cell contacts is one of the most evident consequences , and this method has been previously used to the same purpose [ 30 , 31 ] . therefore , we feel that this latter result is reinforcing our hypothesis that the cell - cell interactions might play an important and probably primary role in the phenomena here described .
the kidney glomerular podocytes are the cellular target of many chronic nephropathies both determined and acquired genetically . 
 mutations that affected the expression and/or the function of nephrin , a key component of the slit - diaphragm , are often causes of these 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 undergo emt , but more importantly nephrin ablation itself can trigger this phenotypic transformation . 
 in fact , a model of human podocyte with engineered nephrin deficiency constitutionally expressed high levels of -sma , vimentin , fibronectin , and other hallmarks of emt . 
 since it is known that cell contact abrogation is one of the triggers of emt , we reasoned that nephrin loss could account for such cell junction disruption and cause the emt . 
 therefore , we demonstrated that also normal podocytes could spontaneously undergo emt if grown in ca2 + -free medium , which is known to impair cell contacts . 
 the analysis of the main intracellular signal transduction pathways evidenced some major anomalies consequent with the nephrin abrogation . 
 the most intriguing was the activation of -catenin pathway , which plays a critical role in podocyte ontogenesis as well as in the nephrin expression and emt regulation . also other important signaling proteins , like nf-b , p53 , and retinoblastoma protein ( rb ) , showed important activity modifications . 
 interestingly , most of the above indicated signaling pathway alterations were again reproducible by cell junction rupture , induced by ca2 + deprivation . 
 finally , immunofluorescence analysis on kidney sections of patients with ns of finnish type confirmed the constitutive expression of -sma .