tubulointerstitial   fibrosis ( tif ) is the final manifestation of end stage renal disease ( esrd ) 
 and renal injury is correlated to the degree of renal interstitial fibrosis . 

one of the major pathological characteristics of tif is the activated tubulointerstitial 
 fibroblasts transdifferentiating into myofibroblasts .
furthermore , 
 extracellular matrix ( ecm ) secreted by fibroblasts deposits in the tubular 
 interstitium and results in interstitial fibrosis . transforming growth factor- ( tgf- )
. connective tissue 
 growth factor ( ctgf ) has been receiving more and more attention for being one 
 of the major downstream mediators of tgf-. peroxisome proliferator - activated receptor  ( ppar- ) is a 
 member of the ligand - activated nuclear transcriptional superfamily and is 
 expressed in several tissues which includes kidney [ 25 ] .
ppar- is activated by its ligand , 
 15-deoxy--prostaglandin 
 j2 ( 15d - pgj2 ) , or 
 activators ( synthetic thiazolidinedione ppar- agonists ) and then participates in 
 the regulation of cellular function . apart from maintaining glucose homeostasis
studies have shown that ppar- could control glomerular inflammation , modulate 
 vasodilator substances like prostaglandins and no [ 6 , 7 ] , antagonize 
 glomerulosclerosis of diabetic nephropathy , and improve renal function . in a rat 
 remnant kidney model of renal fibrosis , administration of the ppar- agonist 
 troglitazone
ppar- agonists could exert antifibrotic effects on human 
 ptc in high glucose levels by attenuating the production of ap-1 , tgf-1 , and the downstream production of the 
 extracellular matrix protein fibronectin [ 8 , 9 ] .
ppar- 
 activation also decreases glomerular cell proliferation and suppresses 
 plasminogen activator inhibitor-1 ( pai-1 ) and tgf- expression .
furthermore , it down - regulated 
 tgf-1-induced 
 fibronectin expression in mouse glomerular mesangial cells by inhibiting 
 activator protein-1 ( ap-1 ) [ 1113 ] ; but the relationship between ppar- and 
 tubulointerstitial fibrosis is not clear yet . by studying the effects of ppar- 
 activation on tgf--induced fibrosis and its mechanism , our study 
 demonstrates the potential perspective for the antifibrotic property of ppar- 
 agonists .
nrk-49f , the immortalized rat kidney interstitial fibroblast cells were obtained from the chinese academy   of sciences . 


cells were maintained in dmem / f-12 medium supplemented with 10% 
 heat - inactivated fetal calf serum ( gibco / brl ) in a humidified atmosphere of 5% 
 co2/95% air at 37c . 

the cells were passaged every 4 days and then harvested onto six - well culture 
 plates to 6070% confluence in the complete medium for 16 hours followed by changing to serum - free medium .
tgf-1 , 
 15d - pgj2 , troglitazone , and ciglitazone ( biomol research laboratories , inc . , 

plymouth meeting , pa ) were freshly dissolved in culture media and added to the 
 cultures at the indicated concentrations and for the indicated time periods .
total 
 rna from nrk/49f cells was 
 isolated by using a trizol extraction kit ( gibco / brl ) according to the 
 manufacturer s directions .
first - strand cdna was synthesized using 2  g 
 of rna in 20  l 
 of reaction buffer by reverse transcription using moloney murine leukemia 
 virus - derived reverse transcriptase ( promega ) .
complementary dna was amplified 
 in 100  l 
 total volume which contained 50  mmol / l kcl , 20  mmol / l tris - hcl ( ph 8.0 ) , 
 10  mmol / l deoxynucleoside triphosphate ( dntp ) , 1.5  mmol / l mgcl2 , 1u taq polymerase , and 10  pmol of specific 
 pcr primers .
beta - actin was amplified and yielded a 202  bp pcr product as the internal 
 standard . the number of cycles used allowed quantification without saturation . 

amplification products were separated by electrophoresis on 1.2% agarose gel , 
 followed by ethidium bromide staining , and then photographed .
semiquantitation 
 was done by serial dilution of the input cdna to measure the mrna .
the 
 proportion of specific gene product to -actin 
 product was used for semiquantitive analysis .
extracted 
 protein was loaded on a 10% sds - page gel and transferred onto nitrocellulose 
 membranes .
then proteins were then blocked and incubated with specific 
 antibodies : -actin ( sigma - aldrich ) , fibronectin ( gibco ) , type iii collagen 
 ( sigma ) , smad2/3 , or p - smad2/3 ( santa cruz biotechnology , calif , usa ) .
membranes were 
 subsequently washed , incubated with specific secondary horseradish peroxidase  conjugated 
 antibodies , and revealed with the enhanced chemiluminescence ( ecl ) kit ( life 
 science products , boston , mass , usa ) . 

all 
 experiments were repeated at least three times , and the results are presented as 
 mean  standard deviation ( sd ) .
tgf-1-induced 
 ctgf , fn , and col iii mrna expression in a dose - dependent and time - dependent manner 
 in nrk/49f cells .
the 
 induction of ctgf and ecm expression is a hallmark of renal fibrosis in many 
 types of primary glomerular disease .
therefore , we first examined the effect of 
 tgf-1 on ctgf 
 and ecm expression in cultured nrk/49f cells .
as shown in figures 1and 2 , nrk/49f cells had basal expression of ctgf , fn , and col 
 iii mrna . after stimulating with different concentration of tgf-1 ,
the 
 expression of ctgf , fn , and col iii mrna increased significantly in a dose - dependent 
 manner ( see figure 1 ) . after stimulating at 1  ng / ml tgf-1 for 1 
 hour ,
the expression of ctgf , fn , and col iii mrna began to increase 
 ( p  <  .05 ) , peaked at 5  ng / ml ( p  <  .01 ) , and decreased at 
 10  ng / ml but was 
 still greater than that of the control group ( p  <  .01 ) . as shown in figure 2 , 
 tgf-1 also 
 induced ctgf , fn , col iii mrna expression in a time - dependent manner in nrk/49f cells . after stimulating for 6 hours with 
 tgf-1 ( 5  ng / ml ) , expression of ctgf , fn , and col iii mrna increased , and peaked at 24 hours 
 ( p  <  .01 ) .
therefore , we examined the effect of ppar- 
 activation on -sma , ctgf , col iii , and fn mrna expression in cultured 
 nrk/49f .
as shown 
 in figures 3 and 4 , 10  m 
 15d - pgj2 dramatically suppressed tgf-1-mediated -sma , ctgf , col iii , and fn mrna expression 
 ( p  < 
the synthetic ppar- agonists troglitazone , and ciglitazone also 
 effectively inhibited tgf-1-mediated -sma , ctgf , col iii , and fn mrna expression in nrk/49f .
such expression in the troglitazone- and 
 ciglitazone - treated groups was less than that in the 15d - pgj2 treated group .
figure 5 demonstrates that 15d - pgj2 , troglitazone , and ciglitazone suppressed tgf-1-mediated fibronectin expression in nrk/49f cells .
nrk/49f cells expressed a considerable amount of 
 fibronectin at basal conditions , and tgf-1 significantly induced fn expression .
however , nrk/49f treated with 15d - pgj2 , troglitazone , and ciglitazone 
 counteracted the tgf-1-stimulated overproduction of fibronectin .
however , 
 the level of fibronectin in five ppar- agonists treated groups was not significantly 
 different ( p  > 
after stimulating by tgf-1 for 15 minutes , p - smad2/3 protein expression began 
 to increase , peaked at 1 hour , then began to decrease at 2 hours .
there was no 
 difference of total smad2/3 protein expression . to 
 explore the molecular mechanism by which ppar- agonists inhibit tgf-1-mediated renal fibroblast activation , we studied the 
 effect of 15d - pgj2 , troglitazone , and ciglitazone on smad signalling pathway .
as shown in figure 7 , 
 pretreatment of nrk/49f fibroblasts with 15d - pgj2 , troglitazone , and ciglitazone was able to block smad phosphorylation .
there was no 
 difference in each interfering group ( p  >  .05 ) .
tubulointerstitial 
 fibrosis ( tif ) is the final manifestation of end stage renal disease ( esrd ) . 

the major pathological changes of tif are the proliferation of interstitial 
 fibroblasts , transdifferentiation of fibroblast into myofibroblasts ( the major 
 characteristic of which is the increase 
 of -smooth 
 muscular actin expression ) , and overdepositing of extracellular matrix ( ecm ) 
 such as fibronectin and collagen type i , 
 type ii , and type iv .
of all the cytokines and growth factors involved , tgf-1 plays 
 the most important role .
anti - tgf-1 
 antibody and anti - tgf- receptor antibody could reduce the production of ecm 
 .
as tgf-1 has many biological effects , suppression of tgf-1 
 expression / activation or blocking tgf-1 at its receptors could result in many biological side 
 effects .
therefore , targeting the downstream mediators of the over - activated 
 signaling pathway is a good way to antagonize the progression of esrd and thus become 
 the prime focus of current studies .
connective 
 tissue growth factor ( ctgf ) is one of the downstream mediators of tgf-1-induced 
 fibrosis and it participates in fibroblast proliferation and adhesion and in inducing 
 ecm production .
our study shows that 
 nrk/49f had basal 
 expression of -sma mrna until it is stimulated by tgf-1 , whereupon 
 its expression increased significantly , demonstrating that tgf-1 could 
 induce tubulointerstitial fibroblasts to transdifferentiate into 
 myofibroblasts .
tgf-1 could increase the mrna expression of the downstream 
 mediator ctgf and major constituents of ecm ( collagen type iii and fibronectin ) 
 in a time - dependent and dose - dependent manner .
our study demonstrates that tgf- is one 
 of the major fibrosis - inducing mediators which could induce transdifferentiation 
 of renal fibroblasts and production of ecm .
recently , study by lin et al . showed that activation of smad3/4 was essential for tgf-1induced ctgf 
 transcription and that pentoxifylline ( ptx ) , a potent inhibitor of ctgf , could inhibit 
 ctgf expression by interfering with smad3/4-dependent ctgf transcription 
 through protein kinase a and block the profibrogenic effects of ctgf on renal 
 cells .
ppar- has now 
 become the therapeutic target of research on kidney diseases like diabetic 
 nephropathy , glomerulosclerosis , glomerulonephritis , and hypertensive 
 nephropathy .
15d - pgj2 is the natural ligand of ppar- , and thiazolidinediones ( tzds ) such as troglitazone , and rosiglitazone are its 
 agonists .
it has been proven in animal models ( streptozotocin - induced diabetic 
 nephropathy models and 5/6 nephrectomized models ) that tzds could reduce the 
 expression of extraglomerular matrix and ameliorate renal injury .
tzds and 
 15d - pgj2 could reduce production of collagen type i and fibronectin 
 in rats [ 15 , 16 ] , and inhibit production of proinflammatory cytokines and 
 chemotactic factors ( e.g. , no , cox-2 , mcp-1 , etc . ) [ 17 , 18 ] .  
tubulointerstitial fibroblasts could express basal amounts of ppar-. our unpublished study showed that ppar- could 
 inhibit proliferation of human tubulointerstitial fibroblasts and trigger their 
 apoptosis .
our study found that 15d - pgj2 , troglitazone , and 
 rosiglitazone could reduce tgf--induced production of -sma and ecm ( collagen type iii and fibronectin ) and 
 inhibit expression of ctgf mrna .
these results show that ppar- agonists 
 have an antifibrotic effect through inhibition of tgf--induced 
 renal fibroblast transdifferentiation and ecm production .
such results are 
 similar to those results of studies on glomerulosclerosis and arteriosclerosis 
 [ 1921 ] .
exposure of human cortical fibroblasts to pioglitazone causes an 
 antiproliferative effect and reduces ecm production through mechanisms that 
 include reducing timp activity independent of tgf-1 . different 
 ppar- ligands 
 or agonists may have different mechanisms in different cells and there are also 
 ppar- 
 independent pathways involved . baoling found that pioglitazone , and 15d - pgj2 
 could inhibit expression of fibronectin induced by tgf- in rats 
 and the effects of pioglitazone are due to activation of ppar-. 
 however , there were ppar--independent pathways involved in the mechanism of 
 15d - pgj2 action . whether ppar- inhibits tgf-1-induced fibrosis by activating intracellular ppar--receptors 
 requires further study .
sawano s 
 study shows that 15d - pgj2 could downregulate il-1-induced cox-2 expression and 
 troglitazone / rosiglitazone could not reduce the expression of cox-2 . 

panzer found that in experimental glomerulonephritis induced by ats 
 ( antithymus antibody ) , troglitazone , and ciglitazone could upregulate mcp-1 
 expression and increase monocyte / macrophage infiltration and adhesion , however , 
 15d - pgj2 has no effect on mcp-1 expression .
our study shows that 
 troglitazone , and ciglitazone have a more inhibitory effect on -sma 
 expression than 15d - pgj2 . comparing the intervention of different reagents , the 
 expression of ctgf , collagen type iii , and fibronectin shows no difference .
phosphorylated 
 smad2/smad3 ( p - smad2/3 ) is the main signaling pathway of tgf-1 and it 
 participates in the biological effects of tgf- which include cell proliferation , inflammation 
 reaction , and fibrosis [ 24 , 25 ] .
they are essential mediators of tgf--induced 
 endothelial cell transdifferentiation and ecm and ctgf expression .
studies 
 have shown that the tgf-/smads signaling pathway participates in many 
 pathophysiological processes related to kidney diseases like diabetic 
 nephropathy , glomerulonephritis and glomerulosclerosis .
our study shows 
 that tgf-1 induces smad2/3 phosphorylation in a time - dependent 
 manner , which suggests that tgf- could induce smad2/3 phosphorylation in renal 
 fibroblasts .
15d - pgj2 , troglitazone , and ciglitazone could reduce tgf--induced 
 p - smad2/3 protein expression while the total amount of smad2/3 protein did not 
 change .
such results suggest ppar- agonists could inhibit the fibrotic effect of tgf- by 
 interfering in the phosphorylation of smad2/3 .
moreover , all three reagents 
 show no significant difference in inhibiting phosphorylation of smad2/3 .
our 
 study suggests that smad 2/3 signaling pathway is essential in antifibrotic effects 
 of ppar- agonists . 
 while in the study by yang et al .
, smad 2/3 phosphorylation was not 
 inhibited by hepatocyte growth factor ( hgf ) , which acts as a potent inhibitor of 
 the tgf-1-mediated 
 myofibroblastic activation   therefore , further study is required to 
 investigate role of smad signaling pathway in different inhibitors of tgf-1-mediated 
 myofibroblastic activation . in conclusion
such results suggest a 
 perspective for the antifibrotic effects of ppar- and such effects may become the therapeutic target of 
 esrd .
 background .   studies have shown that 
     peroxisome proliferator - activated receptor- ( ppar- ) agonists could ameliorate renal fibrotic lesions in both diabetic nephropathy and nondiabetic chronic kidney diseases . in order to elucidate the antifibrotic mechanism of ppar- agonists , we investigated the effects of ppar- activation on tgf-1-induced renal interstitial fibroblasts . 
  methods . in rat 
 renal interstitial fibroblasts ( nrk/49f ) , the mrna expression of tgf-1-induced 
 -smooth muscle actin ( -sma ) , connective tissue growth factor ( ctgf ) , fibronectin ( fn ) and collagen type iii   ( col iii )   were observed by reverse transcriptase - polymerase chain reaction ( rt - pcr ) . 
 the protein expressions of fn and smads were observed by western blot . 
 
 results . in nrk/49f , 
 tgf-1 enhanced ctgf , fn and col iii mrna expression in a dose- and time - dependent manner . 
 -sma , ctgf , fn and col iii mrna and fn protein expression in 15-deoxy-12,14-prostaglandin j2 ( 15d - pgj2)-troglitazone- and ciglitazone - pretreated groups , respectively , were significantly decreased compared with the tgf-1-stimulated group . 
 tgf-1 ( 5  ng / ml ) enhanced p - smad2/3 protein expression in a time - dependent manner . compared with the tgf-1-stimulated group , p - smad2/3 protein induced by tgf-1 in ppar- agonists - pretreated groups significantly decreased with no statistical difference amongst the three pretreated groups . 
 
 conclusion .   
 ppar- agonists could inhibit tgf-1-induced renal fibroblast activation , ctgf expression and ecm synthesis through abrogating the tgf-1/smads signaling pathway . 
 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISSCUSSION