peritoneal dialysis ( pd ) is a commonly used renal replacement modality to treat end - stage renal disease patients .
however , long - term pd leads to peritoneal fibrosis in most patients receiving pd .
the progression of peritoneal fibrosis invariably causes membrane failure , leading to volume control failure and loss of dialysis adequacy resulting in the discontinuation of pd.2 , 3 , 4 however , detailed molecular mechanisms underlying peritoneal fibrosis development and progression have not been fully elucidated .
therefore , the strategies to prevent peritoneal fibrosis and to treat established peritoneal fibrosis are limited .
the peritoneal membrane is covered by a single layer of peritoneal mesothelial cells ( pmcs ) , which are supported by a submesothelial connective tissue area composed of fibroblasts , mast cells , and macrophages .
recently , pmcs were suggested to play a key role in the development and progression of peritoneal fibrosis by producing type i and type ii collagen and fibronectin .
the synthesis of these extracellular matrix ( ecm ) proteins by pmcs is accompanied by a complex mechanism called the epithelial - mesenchymal transition ( emt ) .
emt of pmcs and their excessive production of ecm proteins are largely attributed to the activation of the transforming growth factor 1 ( tgf-1 ) pathway .
pd fluid is known to increase the production of tgf-1 by pmcs , and the high glucose level in the peritoneal dialysate has been shown to be a primary cause of this upregulation of tgf-1 in pmcs .
periostin is an ecm protein and is upregulated by the activation of the tgf-1 pathway in several cell types .
it functions as a ligand of v/3 and v/5 integrin to activate integrin - related intracellular signaling pathways , resulting in the increased transcription of ecm genes , such as collagen and fibronectin.12 , 13 , 14 , 15 recently , periostin levels in the pd fluid were demonstrated to be upregulated in patients with encapsulated peritoneal sclerosis , indicating the possibility that periostin contributes to peritoneal fibrosis in patients undergoing pd .
aptamers are single - stranded oligonucleotides that bind to specific target molecules . once bound to a specific target ,
monoclonal antibodies have been applied in the clinical field to suppress the activity of specific proteins . however , using antibodies to treat diseases is accompanied by some critical limitations , such as low bio - stability , the induction of undesirable immune responses , and high production costs .
in contrast , aptamers are produced chemically and are relatively bio - stable , resulting in lower production costs and more convenient storage and distribution compared with antibodies.17 , 18 they are also less immunogenic and therefore are associated with a lower risk of developing hypersensitivity reactions . due to these advantages
, aptamers have been proposed as promising drug candidates to inhibit specific target molecules in various diseases.19 , 20 , 21 previously , a benzyl - d(u)tp - modified dna aptamer , that binds selectively to periostin , was shown to disrupt the interaction with its receptor integrins in breast cancer cells . in this study ,
the effects of this periostin - binding dna aptamer ( pa ) on tgf-1-induced emt and ecm accumulation were evaluated using human pmcs ( hpmcs ) and a murine model of the pd .
first , to verify that periostin is expressed during the emt process of hpmcs , the expression of periostin was evaluated in hpmcs treated with tgf-1 .
hpmcs treated with tgf-1 ( 2  ng / ml ) showed significantly increased expression of periostin mrna and protein . this increase in periostin
mrna expression was accompanied by the upregulation of fibronectin , -smooth muscle actin ( -sma ) , and snail and downregulation of e - cadherin mrna expression .
the protein levels of fibronectin , -sma , snail , and e - cadherin showed a similar expression pattern as the corresponding mrna levels in hpmcs ( figure  1 ) .
these results indicate that tgf-1 administration induces emt in hpmcs along with a significant upregulation of periostin.figure  1expression of periostin and emt - related molecules in hpmcs exposed to tgf-1(a ) periostin , fibronectin , e - cadherin , -sma , and snail mrna levels in hpmcs treated with or without tgf-1 ( 2  ng / ml ) ( n  = 12 ) .
the tgf-1 significantly induced fibronectin , -sma , and snail mrna expression and significantly reduced e - cadherin mrna expression in hpmcs .
( b ) a representative western blot analysis of periostin , fibronectin , e - cadherin , -sma , snail , and -actin protein expression in hpmcs treated with or without tgf-1 ( 2  ng / ml ) ( a representative of four blots ) . the protein expression of periostin , fibronectin , -sma , and snail significantly increased and that of e - cadherin significantly decreased in the tgf-1-stimulated cells ( n  = 12 ) .
expression of periostin and emt - related molecules in hpmcs exposed to tgf-1 ( a ) periostin , fibronectin , e - cadherin , -sma , and snail mrna levels in hpmcs treated with or without tgf-1 ( 2  ng / ml ) ( n  = 12 ) .
the tgf-1 significantly induced fibronectin , -sma , and snail mrna expression and significantly reduced e - cadherin mrna expression in hpmcs .
( b ) a representative western blot analysis of periostin , fibronectin , e - cadherin , -sma , snail , and -actin protein expression in hpmcs treated with or without tgf-1 ( 2  ng / ml ) ( a representative of four blots ) . the protein expression of periostin , fibronectin , -sma , and snail significantly increased and that of e - cadherin significantly decreased in the tgf-1-stimulated cells ( n  = 12 ) .
* p  < 0.01 versus untreated . to evaluate the role of periostin in the emt process of hpmcs
, periostin expression was knocked down by small interfering rna ( sirna ) transfection in tgf-1-stimulated hpmcs .
as shown in figure  2a , tgf-1 ( 2  ng / ml ) induced fibroblast - like cell morphological changes characterized by thin spindle - like cell shapes , in contrast to the cuboidal shape of unstimulated hpmcs . interestingly , periostin sirna ( 100  nm ) administration markedly ameliorated the tgf-1-induced cell morphological changes .
this intriguing result prompted the examination of whether periostin knockdown normalized the expression of mesenchymal markers triggered by tgf-1 in hpmcs .
as expected , periostin knockdown by sirna administration effectively inhibited periostin expression induced by tgf-1 treatment . the expression of fibronectin , -sma , and snail mrna also significantly decreased in hpmcs treated with tgf-1 and periostin sirna compared to hpmcs treated with tgf-1 alone , and the decreased e - cadherin mrna level in tgf-1-stimulated hpmcs was significantly restored by periostin sirna transfection . regarding the protein levels
, periostin sirna significantly attenuated the increases in periostin , fibronectin , -sma , and snail expression and the decrease in e - cadherin expression induced by tgf-1 ( figure  2 ) .
together , these results suggest that periostin upregulation is an essential factor in the emt process of tgf-1-stimulated hpmcs and that periostin inhibition can mitigate the pathologic emt of hpmcs.figure  2changes in cell morphology and mrna and protein expression of emt - related molecules following periostin sirna transfection(a ) cell morphology of untreated and tgf-1- ( 2  ng / ml ) stimulated hpmcs with or without periostin sirna ( 100  nm ) transfection .
the tgf-1 treatment provoked fibroblast - like cell morphological changes in hpmcs , and periostin sirna transfection reversed these tgf-1-induced cell morphological changes ( 100 ) .
( b ) mrna levels in tgf-1-stimulated hpmcs treated with or without periostin sirna ( n  = 12 ) .
the periostin sirna significantly attenuated tgf-1-induced increases in fibronectin , -sma , and snail mrna expression and restored the downregulated e - cadherin mrna level in hpmcs treated with tgf-1 .
( c ) a representative western blot analysis of protein expression in tgf-1-stimulated hpmcs with or without periostin sirna transfection ( a representative of four blots ) .
the periostin sirna transfection significantly mitigated the increases in periostin , fibronectin , -sma , and snail protein expression and the decrease in e - cadherin protein expression in tgf-1-stimulated hpmcs .
the negative control scramble sirna transfection did not affect the expression of protein levels ( n  = 12 ) .
( d ) mrna levels in tgf-1-stimulated hpmcs transfected with different doses of periostin sirna ( n  = 12 ) .
the periostin sirna significantly attenuated tgf-1-induced increases in fibronectin , -sma , and snail mrna expression and restored the downregulated e - cadherin mrna level in a dose - dependent manner .
( e ) a representative western blot analysis of protein expression in tgf-1-stimulated hpmcs with different doses of periostin sirna transfection ( a representative of four blots ) .
the periostin sirna transfection significantly mitigated the tgf-1-induced increases in periostin , fibronectin , -sma , and snail protein expression and the decrease in e - cadherin protein expression in a dose - dependent manner ( n  = 12 ) .
un , untreated ; lipo , lipofectamine ; sigapdh , gapdh sirna ; scr , negative control scramble sirna ; siperiostin , periostin sirna . * p  < 0.01 versus untreated ; # p  < 0.01 versus tgf-1 .
changes in cell morphology and mrna and protein expression of emt - related molecules following periostin sirna transfection ( a ) cell morphology of untreated and tgf-1- ( 2  ng / ml ) stimulated hpmcs with or without periostin sirna ( 100  nm ) transfection .
the tgf-1 treatment provoked fibroblast - like cell morphological changes in hpmcs , and periostin sirna transfection reversed these tgf-1-induced cell morphological changes ( 100 ) .
( b ) mrna levels in tgf-1-stimulated hpmcs treated with or without periostin sirna ( n  = 12 ) .
the periostin sirna significantly attenuated tgf-1-induced increases in fibronectin , -sma , and snail mrna expression and restored the downregulated e - cadherin mrna level in hpmcs treated with tgf-1 .
( c ) a representative western blot analysis of protein expression in tgf-1-stimulated hpmcs with or without periostin sirna transfection ( a representative of four blots ) .
the periostin sirna transfection significantly mitigated the increases in periostin , fibronectin , -sma , and snail protein expression and the decrease in e - cadherin protein expression in tgf-1-stimulated hpmcs .
the negative control scramble sirna transfection did not affect the expression of protein levels ( n  = 12 ) .
( d ) mrna levels in tgf-1-stimulated hpmcs transfected with different doses of periostin sirna ( n  = 12 ) .
the periostin sirna significantly attenuated tgf-1-induced increases in fibronectin , -sma , and snail mrna expression and restored the downregulated e - cadherin mrna level in a dose - dependent manner .
( e ) a representative western blot analysis of protein expression in tgf-1-stimulated hpmcs with different doses of periostin sirna transfection ( a representative of four blots ) .
the periostin sirna transfection significantly mitigated the tgf-1-induced increases in periostin , fibronectin , -sma , and snail protein expression and the decrease in e - cadherin protein expression in a dose - dependent manner ( n  = 12 ) .
un , untreated ; lipo , lipofectamine ; sigapdh , gapdh sirna ; scr , negative control scramble sirna ; siperiostin , periostin sirna . *
p  < 0.01 versus untreated ; # p  < 0.01 versus tgf-1 . after confirming that periostin played a role in emt induction in hpmcs , it was determined whether a pa could affect the emt process in hpmcs .
first , fluorescence - activated cell sorting ( facs ) analysis was conducted to show that the pa attached specifically to periostin in hpmcs . a fluorescein isothiocyanate ( fitc)-tagged
pa was used to evaluate the amount of the pa that attached to periostin expressed in hpmcs .
compared to the negative control , the fitc - tagged pa - treated cells showed an increase in the fitc - positive fraction of the total hpmc count ( 10.6% ) .
the fitc - positive fraction further increased to 88.2% in tgf-1- ( 10  ng / ml ) stimulated hpmcs treated with the fitc - tagged pa ( 200  nm ) , which was significantly abrogated by periostin sirna ( 100  nm ) transfection ( 24.0% ) .
these findings suggest that the pa specifically binds to periostin expressed by hpmcs ( figure  3).figure  3specific attachment of pa to periostin expressed in hpmcsfacs analysis was performed in hpmcs treated with or without tgf-1 ( 10  ng / ml ) , periostin sirna ( 100  nm ) , or fitc - labeled pa ( 200  nm ) . the fitc - positive fraction was significantly increased in tgf-1-stimulated hpmcs treated with the fitc - labeled pa , which was significantly abrogated by periostin sirna transfection .
un , untreated ; siperiostin , periostin sirna ; sa , negative control scramble aptamer ; pa , periostin - binding dna aptamer . * p  < 0.01 versus untreated ; # p  < 0.01 versus tgf-1
. specific attachment of pa to periostin expressed in hpmcs facs analysis was performed in hpmcs treated with or without tgf-1 ( 10  ng / ml ) , periostin sirna ( 100  nm ) , or fitc - labeled pa ( 200  nm ) .
the fitc - positive fraction was significantly increased in tgf-1-stimulated hpmcs treated with the fitc - labeled pa , which was significantly abrogated by periostin sirna transfection .
un , untreated ; siperiostin , periostin sirna ; sa , negative control scramble aptamer ; pa , periostin - binding dna aptamer . * p  < 0.01 versus untreated ; # p  < 0.01 versus tgf-1 . to determine whether blocking periostin signaling with the pa could affect the emt process ,
the changes in cell morphology and the expression of emt - related molecules were examined after treatment with the pa in tgf-1-stimulated hpmcs .
the spindle - shaped feature of hpmcs exposed to tgf-1 ( 2  ng / ml ) was significantly alleviated by the pa treatment ( figure  4 ) , which was similar to the findings observed in tgf-1-stimulated hpmcs transfected with periostin sirna ( figure  2 ) . when the cells were examined for periostin expression ,
the increases in periostin mrna and protein expression following tgf-1 stimulation were not significantly changed by the pa treatment .
in contrast , the upregulated fibronectin , -sma , and snail expression and the downregulated e - cadherin expression in tgf-1-stimulated hpmcs were significantly attenuated by the pa treatment .
these results indicate that the pa effectively mitigates the tgf-1-induced emt process in hpmcs by inhibiting periostin - related pathway activation rather than periostin expression.figure  4changes in cell morphology and mrna and protein expression following pa treatment(a ) morphologic features of control and tgf-1- ( 2  ng / ml ) stimulated hpmcs with or without pa treatment ( 200  nm ) .
the pa inhibited the fibroblast - like cell morphological changes in hpmcs exposed to tgf-1 ( 100 ) .
( b ) mrna levels in tgf-1-stimulated hpmcs treated with or without pa ( n  = 12 ) .
the pa significantly attenuated tgf-1-induced fibronectin , -sma , and snail mrna expression and significantly restored the decrease in e - cadherin mrna expression in hpmcs stimulated with tgf-1 .
( c ) a representative western blot analysis of protein expression in hpmcs treated with or without pa ( a representative of four blots ) .
the pa significantly mitigated the increases in fibronectin , -sma , and snail protein expression and the decrease in e - cadherin protein expression in tgf-1-stimulated hpmcs .
the negative control scramble aptamer treatment did not affect the expression of protein levels ( n  = 12 ) .
un , untreated ; sa , negative control scramble aptamer ; pa , periostin - binding dna aptamer . * p  < 0.01 versus untreated ; # p  < 0.01 versus tgf-1 .
changes in cell morphology and mrna and protein expression following pa treatment ( a ) morphologic features of control and tgf-1- ( 2  ng / ml ) stimulated hpmcs with or without pa treatment ( 200  nm ) .
the pa inhibited the fibroblast - like cell morphological changes in hpmcs exposed to tgf-1 ( 100 ) .
( b ) mrna levels in tgf-1-stimulated hpmcs treated with or without pa ( n  = 12 ) .
the pa significantly attenuated tgf-1-induced fibronectin , -sma , and snail mrna expression and significantly restored the decrease in e - cadherin mrna expression in hpmcs stimulated with tgf-1 .
( c ) a representative western blot analysis of protein expression in hpmcs treated with or without pa ( a representative of four blots ) .
the pa significantly mitigated the increases in fibronectin , -sma , and snail protein expression and the decrease in e - cadherin protein expression in tgf-1-stimulated hpmcs .
the negative control scramble aptamer treatment did not affect the expression of protein levels ( n  = 12 ) .
un , untreated ; sa , negative control scramble aptamer ; pa , periostin - binding dna aptamer . *
p  < 0.01 versus untreated ; # p  < 0.01 versus tgf-1 . to investigate the in  vivo effect of the pa on the development of peritoneal fibrosis , the pa was administered to a mouse model of the pd . the peritoneal morphology and emt - related molecules were examined in mice treated with the pd solutions for 4  weeks with or without the intraperitoneal pa administration .
masson s trichrome staining and immunohistochemical staining of the peritoneum revealed significantly higher ecm accumulation and periostin expression in mice treated with the pd solution relative to untreated mice .
the changes in the expression of emt - related molecules in the peritoneum of the pd mice were also significantly ameliorated by the administration of the pa ( figure  5 ) .
furthermore , fibronectin , -sma , and snail mrna and protein expression levels were significantly higher and e - cadherin expression was significantly lower in mice treated with the pd solution compared to untreated mice , and these changes were significantly attenuated by the pa treatment ( figure  6 ) .
these findings suggest that the pa can effectively prevent the pd - induced peritoneal fibrosis.figure  5masson s trichrome staining and immunohistochemical staining of periostin , fibronectin , e - cadherin , -sma , and snail in the peritoneum of untreated , untreated  + pa , pd , and pd  + pa micemasson s trichrome staining and immunohistochemical staining of the peritoneum revealed significantly higher ecm accumulation and periostin expression in mice treated  with pd solution relative to control mice , and pa treatment significantly abrogated ecm accumulation and periostin expression in pd mice .
the changes in the expression of emt - related molecules in the peritoneum of the pd mice were also significantly ameliorated by the administration of the pa ( 200 ) . for each group ,
un , untreated ; pd , peritoneal dialysis ; pa , periostin - binding dna aptamer . *
p  < 0.01 versus untreated ; # p  < 0.01 versus pd.figure  6expression of periostin and emt - related molecules in the peritoneum of untreated , untreated  + pa , pd , and pd  + pa mice(a ) fibronectin , -sma , and snail mrna expression was significantly higher and e - cadherin expression was significantly lower in mice treated with pd solution compared to control mice , and these changes were significantly attenuated by pa treatment ( n  = 12 ) .
( b ) a representative western blot analysis of periostin , fibronectin , e - cadherin , -sma , snail , and -actin protein expression in the peritoneum ( a representative of four blots ) . the protein expression levels of emt - related molecules showed a similar pattern as the corresponding mrna expression levels ( n  = 12 ) .
un , untreated ; pd , peritoneal dialysis ; pa , periostin - binding dna aptamer . *
masson s trichrome staining and immunohistochemical staining of periostin , fibronectin , e - cadherin , -sma , and snail in the peritoneum of untreated , untreated  + pa , pd , and pd  + pa mice masson s trichrome staining and immunohistochemical staining of the peritoneum revealed significantly higher ecm accumulation and periostin expression in mice treated  with pd solution relative to control mice , and pa treatment significantly abrogated ecm accumulation and periostin expression in pd mice .
the changes in the expression of emt - related molecules in the peritoneum of the pd mice were also significantly ameliorated by the administration of the pa ( 200 ) . for each group ,
un , untreated ; pd , peritoneal dialysis ; pa , periostin - binding dna aptamer . *
expression of periostin and emt - related molecules in the peritoneum of untreated , untreated  + pa , pd , and pd  + pa mice ( a ) fibronectin , -sma , and snail mrna expression was significantly higher and e - cadherin expression was significantly lower in mice treated with pd solution compared to control mice , and these changes were significantly attenuated by pa treatment ( n  = 12 ) .
( b ) a representative western blot analysis of periostin , fibronectin , e - cadherin , -sma , snail , and -actin protein expression in the peritoneum ( a representative of four blots ) . the protein expression levels of emt - related molecules showed a similar pattern as the corresponding mrna expression levels ( n  = 12 ) .
un , untreated ; pd , peritoneal dialysis ; pa , periostin - binding dna aptamer . *
in the present study , periostin was found to play a role in tgf-1-induced emt in cultured hpmcs .
the inhibition of periostin function by the pa resulted in the abrogation of tgf-1-induced emt .
in addition , the intraperitoneal administration of the pa successfully inhibited peritoneal fibrosis in an animal model of the pd . in pd , long - term dialysate exposure results in fibrotic changes in the peritoneum , decreasing the efficiency of solute diffusion and salt removal from the peritoneal capillary vessels to the peritoneal space , a condition termed ultrafiltration failure.22 , 23 this ultrafiltration failure is a major factor that limits the widespread use of the pd as a long - term renal replacement modality .
the results of this study indicate the possible effectiveness of the intraperitoneal administration of a pa in the pd patients to prevent ultrafiltration failure .
moreover , due to the advantages of aptamers compared to protein- or small - molecule - based treatment options , such as lower manufacturing costs and fewer side effects , the current findings demonstrate that treatment with the pa is a practical and effective strategy to inhibit pd - induced peritoneal fibrosis and has potential clinical applications .
continual exposure to dialysate solutions as well as recurrent peritonitis episodes cause peritoneal inflammation and injury , eventually leading to peritoneal fibrosis .
pmcs have been implicated as one of the major cellular elements in the pathogenesis of this peritoneal fibrosis . in the past , resident
peritoneal fibroblasts and infiltrating inflammatory cells have been considered the key players promoting peritoneal fibrosis .
however , recent studies have shown that the emt of mesothelial cells is an important process in the induction of fibrosis and subsequent deterioration of peritoneal function.6 , 24 , 26 it has also been demonstrated to be a key process in embryonic development , malignant cell metastasis , and the promotion of organ fibrosis . in the case of peritoneal fibrosis , pmcs have been found to lose their epithelial cytokeratins and gain the expression of mesenchymal cellular molecules upon exposure to dialysate . in the present study , tgf-1 treatment of cultured hpmcs significantly downregulated e - cadherin expression and clearly upregulated -sma and snail expression , which was accompanied by a significant increase in fibronectin expression . a similar finding
these findings suggest that emt is involved in the course of the pd - induced peritoneal fibrosis and that pmcs play a significant role in this process , which is in accordance with the results of previous studies.29 , 30 tgf-1 is a well - known profibrotic cytokine that plays key pathological roles , including emt promotion , in peritoneal fibrosis in patients undergoing the pd .
tgf-1 activates smad2 and smad3 , which consequently induces a molecular transition that results in the downregulation of the intracellular adhesion molecule e - cadherin and upregulation of mesenchymal - associated molecules , such as snail , -sma , and fibronectin .
clinical studies have shown that the levels of tgf-1 detected in the pd fluid correlated with the risk of technical failure in the pd patients.32 , 33 furthermore , overexpression of tgf-1 by the transfection of adenovirus vectors into the peritoneal cavity led to peritoneal fibrosis , angiogenesis , and ultrafiltration failure in an animal model of the pd .
in addition , stimulation of pmcs in  vitro with dialysate - containing culture media resulted in an increase in tgf-1 mrna and protein expression .
based on these findings , tgf-1 has been considered a key inducer of fibrotic changes in the peritoneum of patients undergoing the pd , and , therefore , to simulate the pd environment associated with emt and fibrosis , cultured hpmcs were treated with tgf-1 for the in  vitro experiments in this study .
previously , strategies to inhibit tgf-1 were evaluated to prevent organ fibrosis in various disease conditions .
the administration of tgf-1 antibodies has been considered as an option for tgf-1 inhibition and has shown promising anti - fibrotic results in some animal models.36 , 37 , 38 moreover , substances having an inhibitory effect on tgf-1 , such as decorin and pirfenidone [ 5-methyl-1-phenyl-2(1h)-pyridone ] , have been tested for their efficacies in various diseases , such as systemic sclerosis and glomerulosclerosis.39 , 40 regarding peritoneal fibrosis , in  vitro studies on pmcs transfected with tgf-1 small hairpin rna ( shrna)-expressing vectors demonstrated the amelioration of ecm protein synthesis in these cells . however , in addition to promoting pathological fibrotic changes in disease states , tgf-1 is also involved in various physiological functions , such as wound healing and immune reactions . furthermore , because tgf-1 is located upstream of the tgf-1-induced fibrosis signaling cascade , inhibition of tgf-1 blocks numerous downstream cell signaling pathways critical for cell survival .
for these reasons , the clinical applications of anti - tgf-1 therapies have been limited .
therefore , recent investigations have focused on molecular targets farther downstream of the cell - signaling cascade to induce anti - fibrotic effects .
it should be noted that the inhibition of periostin , a terminally located protein in the tgf-1-induced fibrosis pathway , successfully attenuated the pd - related fibrosis process in the peritoneum .
previous studies have found that periostin acts as a ligand and exerts intracellular signaling through the activation of integrin v/3 and v/5 .
the activation of integrin is known to involve key cell signaling pathways that contribute to emt and fibrosis , such as akt and p38 .
the periostin - specific binding property of the pa has been previously shown to inhibit the periostin - integrin interaction , thereby suppressing the integrin signaling pathways .
this is supported by the results for the periostin protein levels in this study , which were not changed following the pa treatment despite its anti - fibrotic effects .
further investigations on the cellular pathways that are affected by the pa are needed to confirm this conclusion .
in addition , because periostin expression itself is also known to be regulated by integrin activation , a higher dose of the pa could ultimately downregulate the expression of periostin .
the possibility of periostin level attenuation with a higher pa dose treatment could explain the discrepancy found between in  vivo and in  vitro experiments in this study .
treatment with the pa significantly attenuated the immunohistochemical staining of periostin in the peritoneum of the pd mice , while a clear amelioration of periostin level was not found in cell studies .
, the results of this study suggest that periostin acts as a key mediator of peritoneal fibrosis in the pd patients , and treatment with the pa can be considered an effective treatment modality for peritoneal fibrosis in these patients .
primary hpmcs were isolated in accordance with the process reported by stylianou et  al . using human omenta specimens from patients who had undergone abdominal surgery and agreed to provide consent .
the specimens were washed three times using sterile pbs and incubated in a solution containing 0.05% trypsin and 0.02% edta for 20  min at 37c , with continuous shaking . after an incubation period , free hpmc - containing suspension
subsequently , the pellet was re - suspended into m199 media supplemented with 10% fetal bovine serum ( fbs ) , 100  u / ml penicillin , 100  mg / ml streptomycin , and 26  mm nahco3 and seeded on cell culture dishes .
the cells were cultured in the same media at 37c in humidified 5% co2 in air .
the media were exchanged 24  hr after seeding and then every 3  days .
next , the media were exchanged to fbs - free m199 media for the control group and media with tgf-1 ( 2  ng / ml ) ( r&d systems ) for the intervention group .
hpmcs were harvested for rna and protein analyses 72  hr after media change . for periostin
periostin sirna was transfected with lipofectamine 2000 ( invitrogen ) according to the manufacturer s protocol .
briefly , 6  l of lipofectamine 2000 was diluted in 1  ml of opti - mem i reduced serum medium ( invitrogen ) , incubated for 15  min at room temperature ( rt ) , and mixed with periostin sirna .
after a 15  min incubation at rt , the mixture was added to each well of hpmcs , which were plated at a density of 5   10 cells / well into 6-well plates the day before , and the medium was changed after 24  hr . the doses of tgf-1 and periostin sirna for this study were determined based on preliminary experimental results .
to select periostin - specific aptamers , a modified dna selex procedure was used.21 , 46 the selected pa and negative control scramble aptamer were constructed by aptamer sciences inc . as previously reported .
this aptamer was prepared in three forms : cy3-labeled aptamer , fitc - labeled aptamer , and polyethylene glycol ( peg)-conjugated aptamer . cy3-labeled
pa and fitc - labeled pa were prepared by labeling cy3 and fitc to the 5 end of the aptamer , respectively .
fitc - labeled pa was used to evaluate the periostin - specific aptamer attachment in the facs analysis studies .
peg - conjugated pa was prepared by conjugating 40  kda peg to the 5 end .
in addition , the peg - conjugated pa contained an inverted deoxythymidine ( dt ) to the 3 end to increase in  vivo biological stability .
the peg - conjugated pa was applied for animal studies.table 1periostin - binding dna aptamer sequencesequencecy3-labled pacy3-acgagyygycgcaygygcggyycagycyggyccyycagcaccgyacaacaafitc - labeled pafitc - acgagyygycgcaygygcggyycagycyggyccyycagcaccgyacaacaapeg - conjugated papeg - acgagyygycgcaygygcggyycagycyggyccyycagcaccgyacaacaa - idty , benzyl - d(u)tp ; idt , inverted dt .
periostin - binding dna aptamer sequence y , benzyl - d(u)tp ; idt , inverted dt .
hpmcs were centrifuged at 500 g for 5  min and washed twice in an isotonic , calcium , and magnesium free - pbs buffer to remove residual growth factors in the medium .
a final concentration of 1   10  cells / ml was re - suspended and incubated with 200 
nmol / l fitc - labeled pa containing binding buffer ( pbs including 5  mm mgcl2 ) .
samples of 100  l were used per assay ( 1   10 cells ) into each 1.5  ml tube for staining .
flow cytometric analysis of surface staining intensity was performed using an facsverse system ( bd biosciences ) and signals were obtained from a blue laser .
the protocols for animal experiments were approved by the committee for the care and use of laboratory animals at yonsei university college of medicine , seoul , korea .
all of the animal experiments were conducted in accordance with the principles of laboratory animal care ( nih publication no .
85 - 23 , revised 1985 ) . 48 male c57bl/6 mice weighing 2025  g were used . to establish the pd model , pd catheter and ports ( cat # mmp-4s-061108a ; access technologies and solomon scientific ) were inserted , and the wounds were observed for 1  week . at
1  week after catheter insertion , the untreated group received 2  ml of saline instillation once a day .
the pd group was infused intraperitoneally with 2  ml of peritoneal dialysate daily ( physioneal pd solution with 4.25% dextrose , ph 7.4 ; baxter international ) for 4  weeks . in the untreated  + pa group , the pa was delivered at 500  g / kg / d mixed with saline . in the pd  + pa group , the pa was delivered at 500  g / kg / d mixed with the pd fluid .
the mixtures were given through the pd catheter daily in a final volume of 2  ml .
preliminary investigations evaluating the amount of the pa detected in the peritoneum , evaluated by real - time qpcr were used to select the dosage of the pa delivered ( figure  s1 ) .
after 4  weeks of treatment , all of the mice were sacrificed , and the parietal peritoneum was collected from each animal.table 2primer sequences used for real - time qpcrsequence ( 53)periostin ( mouse and human)senseggcaccaaaaagaaatactantisenseggaaggtaagagtatatfibronectin ( mouse)sensetgacaactgccgtagacctggantisensetactggttgtaggtgtggccgfibronectin ( human)senseaacctacggatgactcgtgcantisensetgaatcacatctgaaatgaccace - cadherin ( mouse)sensetttctacagcatcaccggcantisensecactttcagccagcctgtce - cadherin ( human)sensecacagacgcggacgatgatantisenseaggatcttggctgaggatggt-sma ( mouse)sensectgacagaggcaccactgaaantisensecatctccagagtccagcaca-sma ( human)sensegccttggtgtgtgacaatggantisenseaaaacagccctgggagcatpa detectionsenseacgagttgtcgcatgtgcggtantisensetgttgtacggtgctgaaggacca primer sequences used for real - time qpcr the rna extraction methods from hpmcs and the peritoneum were described in a previous study . for animal samples , a total of 0.1  g peritoneum tissue for each animal was used to extract rna . a boehringer mannheim cdna synthesis kit ( boehringer mannheim )
was used to obtain first - strand cdna . using the abi prism 7700 sequence detection system ( applied biosystems ) ,
a total volume of 20  l mixture in each well was used containing 10  l of sybr green pcr master mix ( applied biosystems ) , 5  l of cdna , and 5 pmol sense and antisense primers .
the primer concentrations were determined by preliminary experiments that analyzed the optimal concentrations of each primer .
the pcr conditions were as follows : 35 cycles of denaturation for 30  min at 94.5c , annealing for 30  s at 60c , and extension for 1  min at 72c .
initial heating for 9  min at 95c and final extension for 7  min at 72c were performed for all pcr reactions .
each sample was run in triplicate in separate tubes and a control without cdna was also run in parallel with each assay .
after real - time pcr , the temperature was increased from 60c to 95c at a rate of 2c / min to construct a melting curve .
the cdna content of each specimen was determined using a comparative ct method with 2 .
the results were given as the relative expression normalized to the expression of 18s rrna and expressed in arbitrary units .
a total of 0.2  g peritoneum tissue for each animal was used to extract protein .
blots were incubated overnight at 4c with polyclonal antibodies to periostin ( abcam ) , e - cadherin ( bd bioscience ) , -sma ( abcam ) , fibronectin ( dako ) , snail ( abcam ) , gapdh ( abcam ) , or -actin ( sigma chemical ) .
the membranes were washed three times for 10  min in 1   pbs with 0.1% tween-20 and incubated in buffer a containing a 1:1,000 dilution of horseradish peroxidase - linked goat anti - rabbit or anti - mouse igg ( santa cruz biotechnology ) .
tina image software ( raytest ) was used to measure the band densities , and the changes in the optical densities of bands from the treated groups relative to untreated cells or tissues were used for analysis . to fix the peritoneum samples ,
paraffin - embedded tissues were processed in 5  m - thick sections for immunohistochemical staining .
tissue sections were deparaffinized , rehydrated in ethyl alcohol , and washed in tap water .
antigen retrieval was performed in 10  mm sodium citrate buffer for 20  min using a black & decker vegetable steamer .
slides were blocked with 10% donkey serum for 30  min at rt and then washed using pbs .
primary antibodies for fibronectin , e - cadherin , -sma , and snail were diluted to the proper concentrations with 2% casein in bsa , added to the slides , and then incubated overnight at 4c .
after washing , the slides were treated with a secondary antibody for 1  hr at rt .
diaminobenzidine was added for 2  min , and hematoxylin was used to counterstain the slides . for masson
s trichrome staining , paraffin - embedded tissues processed in 5  m - thick sections were deparaffinized , rehydrated in ethyl alcohol , washed in tap water , and re - fixed in bouin s solution for 1  hr at 56c . after rinsing in running tap water for 10  min and staining with weigert s iron hematoxylin working solution for 10  min ,
the slides were stained with biebrich scarlet - acid fuchsin solution for 15  min and washed in tap water .
the sections were differentiated in phosphomolybdic - phosphotungstic acid solution for 15  min , transferred to aniline blue solution , and stained for 10  min .
after rinsing briefly in tap water , the slides were reacted with 1% acetic acid solution for 5  min .
a semiquantitative score for staining intensity was assessed by examining at least five fields in each section under 400 magnification and with digital image analysis ( metamorph version 4.6r5 , universal imaging ) .
one - way anova with post hoc testing using bonferroni s correction was used for multiple comparisons .
b.y.n . , j.t.p . , j.p.l . , and s .- w.k . conceived the project . b.y.n . , y.e.k . ,
s.k . , j.p . , j.e.u . , and m.w . performed the experiments .
 peritoneal fibrosis is a major complication in peritoneal dialysis ( pd ) patients , which leads to dialysis discontinuation . 
 periostin , increased by transforming growth factor 1 ( tgf-1 ) stimulation , induces the expression of extracellular matrix ( ecm ) genes . 
 aberrant periostin expression has been demonstrated to be associated with pd - related peritoneal fibrosis . 
 therefore , the effect of periostin inhibition by an aptamer - based inhibitor on peritoneal fibrosis was evaluated . in  vitro , 
 tgf-1 treatment upregulated periostin , fibronectin , -smooth muscle actin ( -sma ) , and snail expression and reduced e - cadherin expression in human peritoneal mesothelial cells ( hpmcs ) . periostin small interfering rna ( sirna ) treatment ameliorated the tgf-1-induced periostin , fibronectin , -sma , and snail expression and restored e - cadherin expression in hpmcs . 
 similarly , the periostin - binding dna aptamer ( pa ) also attenuated fibronectin , -sma , and snail upregulation and e - cadherin downregulation in tgf-1-stimulated hpmcs . in mice treated with pd solution for 4  weeks 
 , the expression of periostin , fibronectin , -sma , and snail was significantly increased in the peritoneum , whereas e - cadherin expression was significantly decreased . 
 the thickness of the submesothelial layer and the intensity of masson s trichrome staining in the pd group were significantly increased compared to the untreated group . 
 these changes were significantly abrogated by the intraperitoneal administration of pa . 
 these findings suggest that pa can be a potential therapeutic strategy for peritoneal fibrosis in pd patients . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest