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preservation of residual renal function ( rrf ) is important not only for predialysis esrd patients but also for the patients undergoing the dialysis .
rrf is a well - established predictor of the outcome and survival rate in dialysis patients .
prospective randomized trials of dialysis adequacy and many observational studies have confirmed that the loss of rrf is highly associated with the mortality and morbidity in peritoneal dialysis ( pd ) patients [ 2 , 3 ] .
rrf in dialysis patients is pretty helpful in small - solute clearance , fluid balance , phosphorus control , and removal of middle - molecular uremic toxins , especially for the toxins relying on renal metabolism or tubular secretion , such as indoxyl sulfate ( is ) [ 2 , 4 ] .
is is a protein - bound uremic toxin that derives from the metabolism of dietary tryptophan .
however , is can not be efficiently removed by conventional hemodialysis because of its high binding affinity to albumin in advanced chronic kidney disease ( ckd ) .
thus , the urinary excretion of is was considered to occur mainly by tubular secretion and glomerular filtration .
the is accumulated in serum accelerates tubular cell injury and induces subsequent interstitial fibrosis , thus acting as a nephrotoxin [ 5 , 810 ] .
studies also indicated that is could lead to complex redox alterations in mesangial cells ( mcs )   and cell proliferation in vascular smooth muscle cells .
is has been shown to have many pathological roles in uremia - related organ injuries .
for example , it can increase the production of reactive oxygen species ( ros ) and cause vascular wall remodeling and extracellular matrix deposition .
the mc proliferation and subsequent matrix synthesis could result in the glomerular impairment and rrf loss .
cox-2 , an inducible isoform of coxs , is expressed in the macula densa of the juxtaglomerular apparatus , cortical thick ascending limb of henle ( ctal ) in the renal cortex , and interstitial cells in the renal medulla . pge2 as one of five major prostaglandins is synthesized by cox-2-related enzyme cascade and regulates glomerular filtration , renin release in the renal cortex , and tubular absorption of sodium and/or water in the medulla .
accumulating evidence indicated that cox-2 contributes to a number of inflammatory diseases [ 16 , 17 ] possibly via pge2-mediated mechanisms .
recently , a report demonstrated that cox-2 was inducible in the mcs in response to sphingosine 1-phosphate stimulation .
thus , in the present study , we fully studied the roles of is in mcs proliferation and cox-2 regulation , as well as the role of cox-2 in the proliferative process of mcs challenged by is .
dulbecco 's modified eagle 's medium ( dmem ) , fetal bovine serum ( fbs ) , penicillin - streptomycin , and trypsin - edta solution were purchased from gibco ( invitrogen , grand island , ny ) .
cox-2 mouse monoclonal antibody was purchased from cayman chemicals ( ann arbor , mi ) .
anti - gapdh ( ab9485 ) was provided by cell signaling technology ( danvers , ma ) .
the pge2 enzyme immunoassay kit was from cayman chemicals ( ann arbor , mi ) .
the mouse mc line hbzy-1 was obtained from the china center for type culture collection ( cctcc wuhan , china ) .
cells were maintained at 37c in a humidified 5% co2 atmosphere in dmem which contained 5.6  mm glucose , 10% fetal bovine serum ( fbs ; gibco ) , 100  u / ml penicillin , 100  mg / ml streptomycin , 44  mm nahco3 , and 14  mm 4-(2-hydroxy - ethyl)-1-piperazineethanesulfonic acid .
after mcs were cultivated to 60%70% confluence , they were treated with is for 24  h at different doses ( 0 , 250 , and 500  m ) with or without cox-2 inhibitor ns-398 treatment at a dose of 10  m .
mcs were treated with vehicle and the indicated doses of is with or without cox-2 inhibitor in serum - free dmem for 24  h. cells were washed twice with pbs before digestion with 0.25% trypsin and fixed in 70% ethanol for at least 2  h at 4c
. then cells were collected by centrifugation , treated with rnase , and stained with propidium iodide by using cell cycle detection kit ( keygen , shanghai , china ) .
the number of cells in g1 , s , and g2/m cell cycle phases was analyzed by flow cytometry ( bd facs calibur flow cytometer , bedford , ma ) , and data analysis was performed with modifit 3.0 software .
total rna was from cultured mcs by using a trizol reagent ( takara ) according to the manufacturer 's protocol .
reverse transcription was performed using a primescript rt reagent kit ( takara ) according to the manufacturer 's protocol .
oligonucleotides ( cyclin d1 : forward , 5-cgc cct ccg ttt ctt act tc-3 , and reverse , 5-gca gtc agg gga atg gtc t-3 ; cyclin a2 : forward , 5-aag atg ccc tgg ctt tta gtg-3 , and reverse , 5-taacattcactggcttttcgtct-3 ; cyclooxygenase-2 : forward , 5-aggactctgctcacgaagga-3 , and reverse , 5-tgacatggattggaacagca-3 ; and gapdh : forward , 5-gtcttcactaccatggagaagg-3 , and reverse , 5-tcatggatgaccttggccag-3 ) were designed using primer 5 software ( available at http://frodo.wi.mit.edu/ ) and synthesized by invitrogen .
real - time pcr amplification was performed using the abi 7500 real - time pcr detection system ( foster city , ca ) by using sybr premix ex taq ( takara ) .
the cycling program consisted of a preliminary denaturation ( 95c for 10  min ) , followed by 40 cycles ( 95c for 15  s and 60c for 1  min ) .
relative gene expression of mrna was normalized to gapdh and calculated using the ct method . at the indicated time points , mcs were rapidly washed with ice - cold pbs and lysed on ice in lysis buffer containing protease inhibitors .
after centrifugation , the protein level was determined using a micro bca protein assay kit with bovine serum albumin as a standard ( pierce , thermo ) .
sixty micrograms of cellular proteins were separated by sds - page and transferred onto pvdf membranes ( bio - rad ) .
the membranes were blocked by tbs - t ( 0.1% tween 20 in tbs ) containing 5% nonfat milk for 1  h at room temperature , and then incubated with primary antibodies directed against cyclin d1 ( 1  :  1000 ) , cyclin a2 ( 1  :  500 ) , and cox-2 ( 1  :  500 ) by overnight incubation at 4c , followed by the addition of hrp - labeled secondary antibodies at room temperature for 1  h. gapdh was used as an internal standard control . band intensity was measured using image j software ( nih , bethesda , md , usa ) .
the concentration of pge2 in the medium was determined by enzyme immunoassay ( cayman chemical ) , according to the manufacturers ' instructions .
to investigate whether is could induce mc proliferation , we treated the mcs with is at the doses of 250  m and 500  m , respectively .
cell proliferation was firstly examined by direct cell counting under the microscope and dna synthesis rate ( [ h ] thymidine uptake ) . as shown by the data ,
is treatment for 24  h at the doses of 250 and 500  m moderately but significantly increased the total cell number by 21% and 35% , respectively ( figure 1(a ) ) . to further confirm this result , we examined dna synthesis rate .
similarly , the amount of [ h ] thymidine uptake in is - treated cells was also increased in a dose - dependent manner ( figure 1(b ) ) .
the data suggested that is played a role in promoting mesangial cell proliferation . in order to further validate the conclusion shown above , we measured the cell cycle by flow cytometry in mcs exposed to different doses of is .
as shown by data , is caused a moderate but significant decrease of mc numbers in the g1/g0 phase and increase of cell numbers in the s phase ( figures 2(a)2(f ) ) .
cell cycle analysis revealed that is can stimulate cell cycle progression in mcs . to further investigate the is effect on mc proliferation , we measured the expressions of some key cell cycle - related proteins .
here we found that is strikingly increased the mrna levels of cyclin d1 and cyclin a2 in dose- and time - dependent manners determined by qrt - pcr ( figures 3(a)3(d ) ) . by western blotting
, we observed a similar pattern of the protein expressions of cyclin d1 and cyclin a2 as their mrna regulation ( figures 3(e)3(h ) ) . to study the possible involvement of cox-2 in is - induced ms proliferation , we measured cox-2 expression by western blotting and qrt - pcr .
as shown by data , cox-2 protein was dose - dependently elevated ( figures 4(a ) and 4(b ) ) .
for the mrna expression , 500  m but not 250  m increased cox-2 mrna level ( figure 4(c ) ) . using 500 
m is , we found a time - dependent induction of cox-2 mrna expression ( figure 4(d ) ) .
these results indicated that cox-2 could be induced by is in mcs . to examine the role of cox-2 in is - induced proliferation in mcs
, specific cox-2 inhibitor was applied to mcs . as shown in figures 5(a ) and 5(b ) ,
further , we observed that cox-2 inhibition decreased the cell number in s phase and increased cell number in g1/g0 phase ( figures 5(c)5(j ) ) .
moreover , we also found that cox-2 inhibitor markedly attenuated is - induced cyclin d1 and cyclin a2 expression at mrna levels ( figures 6(a ) and 6(b ) ) .
these data highly suggested cox-2 played an important role in mc proliferation . to further examine the efficiency of cox-2 inhibitor in the study , we measured pge2 production in the medium .
as shown by figure 6(c ) , is treatment increased pge2 level by 3.8-fold , which was entirely abolished by cox-2 inhibition .
studies have demonstrated that the loss of rrf is a powerful predictor of mortality and morbidity in peritoneal dialysis ( pd ) patients [ 13 ] .
rrf is pretty helpful for the removal of middle and larger molecular weight toxins [ 2 , 4 ] .
is , as one of the most known uremic toxins , is markedly accumulated in the serum of dialysis patients and accelerates the progression of disease [ 6 , 19 ] . in this study , we found that is could moderately but significantly stimulate mc proliferation via a cox-2-mediated mechanism , which might contribute to the progressive loss of rrf .
cell proliferation is ultimately regulated at cell cycle process including four stages of g1 , s , g2 , and m with important checkpoints in g1 and g2 .
cyclin d1 controls cell cycle progression through the g1 phase and g1-to - s transition , and cyclin a - associated kinase activity is required for the entry into s , completion of s , and entry into m phase .
thus the expressions of cyclin d1 and cyclin a2 were chosen as markers for cell cycle progression .
dose - response experiments showed that is significantly induced cyclin a2 and cyclin d1 expression in mcs consistent with the results which were demonstrated in rat mcs .
similarly , the increase of cell numbers in the s phase was also observed following is treatment .
these findings highly suggested that is served as a contributor of cell cycle progression and cell proliferation in mcs .
next , we examined the possible mechanism mediating is effect on mc proliferation . by reviewing the literature
, recent evidence indicated that an inducible inflammatory enzyme cox-2 can be induced in mcs in response to sphingosine 1-phosphate stimulation . following this notion , we examined the regulation of cox-2 in mcs following is treatment .
interestingly , is remarkably elevated cox-2 expression at both mrna and protein levels in dose- and time- dependent manners .
these data indicated that is could directly stimulate cox-2 upregulation and pge2 induction in mcs . in order to further define the role of cox-2 in this cell cycle progression ,
meanwhile , reduction of cell cycle - related proteins of cyclin d1 and a2 was also significantly attenuated by cox-2 inhibition in line with a blockade of pge2 induction .
all these data indicated a significant role of is in promoting mc proliferation via activating cox-2 .
the proliferation of mcs has to be attributable to the progressive impairment of residual nephrons , as well as the loss of rrf in esrd patients to some extent .
considering the important role of rrf in maintaining a better homeostasis of internal environment and better life quality of patients , targeting is and cox-2 might be useful for the preservation of rrf .
indoxyl sulfate ( is ) is one of important uremic toxins and is markedly accumulated in the circulation of end stage renal disease ( esrd ) patients , which might contribute to the damage of residual nephrons and progressive loss of residual renal function ( rrf ) . 
 thus this study was undertaken to investigate the role of is in modulating mesangial cell ( mc ) proliferation and the underlying mechanism . 
 the proliferation of mcs induced by is was determined by cell number counting , dna synthase rate , and cell cycle phase analysis . 
 cox-2 expression was examined by western blotting and qrt - pcr , and a specific cox-2 inhibitor ns398 was applied to define its role in is - induced mc proliferation . 
 following is treatment , mcs exhibited increased total cell number , dna synthesis rate , and number of cells in s and g2 phases paralleled with the upregulation of cyclin a2 and cyclin d1 . 
 next , we found an inducible inflammation - related enzyme cox-2 was remarkably enhanced by is , and the inhibition of cox-2 by ns398 significantly blocked is - induced mc proliferation in line with a blockade of pge2 production . 
 these findings indicated that is could induce mc proliferation via a cox-2-mediated mechanism , providing new insights into the understanding and therapies of progressive loss of rrf in esrd .