sirtuin 1 ( sirt1 ) , a member of the evolutionarily conserved family of nad - dependent histone deacetylase , is implicated in a wide range of cellular functions involved in the pathogenesis of diseases including cardiovascular diseases , kidney disease . recently , emerging evidences has suggested that sirt1 is a novel target for prevention of kidney diseases [ 24 ] . kidney - specific overexpression of sirt1 rescues cisplatin - induced acute kidney injury ( aki ) in transgenic mice , while sirt1 knockout in diabetes and aki mice shows aggravation of renal changes occurring . it is well known that aki is characterized by structural alterations in renal tubule epithelia , and concomitant with impaired urinary concentration and sodium and water reabsorption . aquaporins ( aqps ; a family of water channel proteins ) , especially aqp2 , plays a major role in water reabsorption in the renal inner medullary collecting duct ( imcd ) . aqp2 has been shown to be decreased markedly in the proximal tubule and imcd in rats with aki induced by ischemia / reperfusion injury or sepsis . however , there is no direct evidence to demonstrate the relationship between sirt1 and aqp2 . therefore , this study was conducted to explore the possible effect of sirt1 on aqp2 expression in renal imcd cells induced by tnf-. furthermore , it s well accepted that the renoprotective properties of sirt1 are closely related to the regulation of nuclear transcription factor ( nf-b ) . it has been shown that nf-b is involved in aqp2 expression in renal collecting duct principal cells , in mouse cortical collecting duct cells , and kidneys of rats with sepsis - induced acute renal failure . so , we also investigated whether nf-b pathway contributed to the regulation of sirt1 on aqp2 expression in renal imcd cells . srt1720 and ex527 were obtained from selleck chemicals ( houston , tx , usa ) . sirtinol and pyrrolidine dithiocarbamate ( pdtc ) were purchased from sigma - aldrich ( st . louis , mo , usa ) . sirt1 sirna , nf-bp65 sirna , and the scrambled negative controls were purchased from genepharma ( shanghai , china ) . , the cells cultured in 6-well plates were transfected with recombinant plasmid pcdna3.1(+)-sirt1 . to knockdown endogenous sirt1 and nf-b transfection was carried by using lipofectamine 2000 ( invitrogen , ny , usa ) according to the manufacturer s protocol . the cells were harvested 48 h after transfection to assess the expression of sirt1 or nf-bp65 . primary cultures enriched in imcd cells were prepared from male sprague - dawley rats ( 120140 g body wt ) , as previously described . all experimental protocols were approved by the review committee for the use of human or animal subjects of yanshan university . after kidney excision , the renal inner medullas including papilla were rapidly removed , cut into small pieces , and digested in phosphate - buffered saline ( pbs ) containing 1 mg / ml hyaluronidase and 2.2 mg / ml collagenase type cls - ii at 37c under continuous agitation for 90 min . after centrifugation , the pellet was washed in prewarmed culture medium ( high glucose dulbecco s modified eagle s medium containing 100 mm nacl , 100 mm urea , 1% non - essential amino acids , 1% ultroser , 500 m dbcamp , 20 u / ml nystatin and 0.25 g / ml gentamicin ) . the imcd cell suspension was then seeded in collagen type iv - coated chamber slides for immunocytochemical analysis or in a 60-mm culture dish for immunoblot analysis . after 24 h , wash cells twice with 600 mosmol hypertonic culture medium ( high glucose dulbecco s modified eagle s medium containing 100 mm nacl and 100 mm urea ) and add fresh medium at 37c in 5% co2 and 95% air for 3 days and then in medium without dbcamp and nystatin for 24 h before starting the experiment on day 6 . the fixed cell membrane was fenestrated with 0.5% triton x-100 in pbs for 5 min , and then blocked with 5% goat serum for 30 min . the cells were incubated with polyclonal anti - rabbit aqp2 antibody ( sc-28629 , 1 : 100 , santa cruz , ca , usa ) overnight at 4c , and then incubated with a tritc - conjugated goat anti - rabbit igg polyclonal antibody ( 1 : 100 dilutions ) for 2 h. after the nuclei were stained with 4-6-diamidino-2-phenylindole ( dapi ) dye ( 1 : 800 dilutions ) for 15 min , the stained cells were investigated using a confocal laser scanning microscopy ( nikon , tokyo , japan ) . cell samples were collected and lysed at 4c in ripa buffer with the protease inhibitor cocktail ( roche , mannheim , germany ) . the protein was separated on 12% sds page gels and then transferred onto polyvinylidene difluoride membranes ( millipore ) . the membranes were blocked with 5% non - fat milk for 1 h and then incubated at 4c overnight with rabbit polyclonal anti - sirt1 ( sc-15404 , 1 : 500 , santa cruz , ca , usa ) , anti - aqp2 ( sc-28629 , 1 : 100 , santa cruz , ca , usa ) , anti - acetyl - nf-bp65 ( lys 310 ) ( ac - p65 ) ( ab19870 , 1 : 400 , abcam , cambridge , ma , uk ) , anti - nf-bp65 ( ab16502 , 1 : 400 , abcam ) , and anti - gapdh ( tdy052 , 1 : 5000 , tdy biotech , beijing , china ) respectively . then , the membranes were incubated with hrp - conjugated secondary antibodies ( s004 , 1 : 10000 , tdy biotech ) for 45 min . protein signals were subsequently detected using the enhanced chemiluminescence ( pierce , rockford , usa ) . total rna was isolated from imcd cells using trizol reagent ( life technologies , foster , ca , usa ) . complementary dna was synthesized using a primescript rt reagent kit ( takara , dalian , china ) . qrt - pcr was performed using the sybr green pcr master mix ( takara ) on a bio - rad cfx connect real - time system ( hercules , ca , usa ) according to the manufacturer s instructions . all primers were as follows : sirt1 forward 5-cagttccagccatctctgtg-3and reverse 5-gcaacctgc tccaaggtatc-3 ; aqp2 forward 5-ttccttcgagctgccttcta-3and reverse 5-ttgtggagagcattgacagc-3 ; gapdh forward 5-acagcaacagggtgg tggac-3and reverse 5-tttgagggtgcagcgaactt-3. gapdh was used as an internal control . quantification of relative gene expression was calculated with 2 method as described by the manufacturer . data were analysed by one - way anova , followed by a post hoc test . srt1720 and ex527 were obtained from selleck chemicals ( houston , tx , usa ) . sirtinol and pyrrolidine dithiocarbamate ( pdtc ) were purchased from sigma - aldrich ( st . louis , mo , usa ) . sirt1 sirna , nf-bp65 sirna , and the scrambled negative controls were purchased from genepharma ( shanghai , china ) . , the cells cultured in 6-well plates were transfected with recombinant plasmid pcdna3.1(+)-sirt1 . to knockdown endogenous sirt1 and nf-b transfection was carried by using lipofectamine 2000 ( invitrogen , ny , usa ) according to the manufacturer s protocol . the cells were harvested 48 h after transfection to assess the expression of sirt1 or nf-bp65 . primary cultures enriched in imcd cells were prepared from male sprague - dawley rats ( 120140 g body wt ) , as previously described . all experimental protocols were approved by the review committee for the use of human or animal subjects of yanshan university . after kidney excision , the renal inner medullas including papilla were rapidly removed , cut into small pieces , and digested in phosphate - buffered saline ( pbs ) containing 1 mg / ml hyaluronidase and 2.2 mg / ml collagenase type cls - ii at 37c under continuous agitation for 90 min . after centrifugation , the pellet was washed in prewarmed culture medium ( high glucose dulbecco s modified eagle s medium containing 100 mm nacl , 100 mm urea , 1% non - essential amino acids , 1% ultroser , 500 m dbcamp , 20 u / ml nystatin and 0.25 g / ml gentamicin ) . the imcd cell suspension was then seeded in collagen type iv - coated chamber slides for immunocytochemical analysis or in a 60-mm culture dish for immunoblot analysis . after 24 h , wash cells twice with 600 mosmol hypertonic culture medium ( high glucose dulbecco s modified eagle s medium containing 100 mm nacl and 100 mm urea ) and add fresh medium at 37c in 5% co2 and 95% air for 3 days and then in medium without dbcamp and nystatin for 24 h before starting the experiment on day 6 . the fixed cell membrane was fenestrated with 0.5% triton x-100 in pbs for 5 min , and then blocked with 5% goat serum for 30 min . the cells were incubated with polyclonal anti - rabbit aqp2 antibody ( sc-28629 , 1 : 100 , santa cruz , ca , usa ) overnight at 4c , and then incubated with a tritc - conjugated goat anti - rabbit igg polyclonal antibody ( 1 : 100 dilutions ) for 2 h. after the nuclei were stained with 4-6-diamidino-2-phenylindole ( dapi ) dye ( 1 : 800 dilutions ) for 15 min , the stained cells were investigated using a confocal laser scanning microscopy ( nikon , tokyo , japan ) . cell samples were collected and lysed at 4c in ripa buffer with the protease inhibitor cocktail ( roche , mannheim , germany ) . the protein was separated on 12% sds page gels and then transferred onto polyvinylidene difluoride membranes ( millipore ) . the membranes were blocked with 5% non - fat milk for 1 h and then incubated at 4c overnight with rabbit polyclonal anti - sirt1 ( sc-15404 , 1 : 500 , santa cruz , ca , usa ) , anti - aqp2 ( sc-28629 , 1 : 100 , santa cruz , ca , usa ) , anti - acetyl - nf-bp65 ( lys 310 ) ( ac - p65 ) ( ab19870 , 1 : 400 , abcam , cambridge , ma , uk ) , anti - nf-bp65 ( ab16502 , 1 : 400 , abcam ) , and anti - gapdh ( tdy052 , 1 : 5000 , tdy biotech , beijing , china ) respectively . then , the membranes were incubated with hrp - conjugated secondary antibodies ( s004 , 1 : 10000 , tdy biotech ) for 45 min . protein signals were subsequently detected using the enhanced chemiluminescence ( pierce , rockford , usa ) . total rna was isolated from imcd cells using trizol reagent ( life technologies , foster , ca , usa ) . complementary dna was synthesized using a primescript rt reagent kit ( takara , dalian , china ) . qrt - pcr was performed using the sybr green pcr master mix ( takara ) on a bio - rad cfx connect real - time system ( hercules , ca , usa ) according to the manufacturer s instructions . all primers were as follows : sirt1 forward 5-cagttccagccatctctgtg-3and reverse 5-gcaacctgc tccaaggtatc-3 ; aqp2 forward 5-ttccttcgagctgccttcta-3and reverse 5-ttgtggagagcattgacagc-3 ; gapdh forward 5-acagcaacagggtgg tggac-3and reverse 5-tttgagggtgcagcgaactt-3. gapdh was used as an internal control . quantification of relative gene expression was calculated with 2 method as described by the manufacturer . data were analysed by one - way anova , followed by a post hoc test . aqp2 immunofluorescent labeling was largely dispersed along the cytoplasm of primary cultured imcd cells in the absence of 1-desamino-8-d - arginine vasopressin ( ddavp ) stimulation in control group . in contrast , treatment with 100 nm avp for 30 clearly increased the aqp2 expression in the plasma membrane and decreased it in the cytoplasm ( figure 1a ) , indicating that the imcd cells were correctly cultured . to study the effect of tnf- on aqp2 expression , the imcd cells were treated with tnf- ( 10 , 20 and 40 g / l ) for 24 h , and the expression of aqp2 protein and mrna was determined by western blot analysis and qrt - pcr , respectively . the results showed that tnf- significantly decreased the expression of aqp2 mrna and protein in a concentration - dependent manner ( figure 1b , 1c ) . we further investigate the time courses of aqp2 stimulated with tnf- ( 20 g / l ) for 4 , 8 , 12 and 24 h. as shown in figure 1d,1e , tnf- time - dependently reduced the expression of aqp2 mrna and protein in imcd cells . we next investigated the effect of tnf- on sirt1 expression in imcd cells , the imcd cells were treated with tnf- ( 10 , 20 and 40 g / l ) for 24 h. the results showed that tnf- down - regulated sirt1 expression at protein and mrna level in imcd cells in a concentration - dependent manner ( figure 2a , 2b ) . to study the time courses of sirt1 expression , the imcd cells were treated with tnf- ( 20 g / l ) for 4 , 8 , 12 and 24 h. the results revealed that tnf- inhibited the protein and mrna expression of sirt1 in a time - dependent manner ( figure 2c , 2d ) . to identify the role of sirt1 in the expression of aqp2 in tnf--induced imcd cells , we overexpressed the expressions of sirt1 in imcd cells . after treatment with the sirt1 activator srt1720 ( 10 m ) or the pcdna3.1(+)-sirt1 plasmid , increases in sirt1 expression were observed in imcd cells compared with the control group ( figure 3a , 3e , 3f ) . then the imcd cells were pretreated with srt1720 for 1 h or pcdna3.1(+)-sirt1 for 48 h , and then stimulated with tnf- ( 20g / l ) for 24 h. sirt1 activator srt1720 obviously increased the expression of sirt1 and aqp2 in tnf--induced imcd cells ( figure 3b3d ) . overexpression of sirt1 is further supported the results by inducing expression of aqp2 compared with the tnf- group ( figure 3 g , 3h ) . treatment of imcd cells with the sirt1 inhibitors ex527 ( 1 m ) and sirtinol ( 10 m ) or sirt1 sirna strikingly reduced the expression of sirt1 ( figure 3a , 3l ) . moreover , the imcd cells were pretreated with ex527 or sirtinol for 1 h or sirt1 sirna for 48 h , and then stimulated with tnf- ( 20 g / l ) for 24 h. the results showed that sirt1 inhibitors ex527 and sirtinol decreased the expression of sirt1 and aqp2 in tnf--induced imcd cells ( figure 3i3k ) . in addition , knockdown of sirt1 remarkably decreased aqp2 mrna and protein expression in imcd cells exposed to tnf- ( figure 3 m , 3n ) . to assess the acetylation of nf-bp65 in tnf--induced imcd cells , western blot was performed . the results showed that treatment of imcd cells with tnf- ( 10 , 20 and 40 g / l ) for 24 h significantly increased ac - p65 protein expression in a concentration - dependent manner ( figure 4a ) . we further studied the time courses of ac - p65 expression in imcd cells by treating with tnf- ( 20 g / l ) for 4 , 8 , 12 and 24 h. the results showed that tnf- up - regulated ac - p65 protein expression in a time - dependent manner ( figure 4b ) . to further assess whether nf-b pathway was associated with the expression of aqp2 in tnf--induced imcd cells , we silenced nf-b pathway . pretreatment with the nf-b inhibitor pdtc ( 10 m ) or nf-bp65 sirna strikingly decreased ac - p65 protein expression ( figure 4c , 4f ) . then the imcd cells were treated with pdtc for 1 h or nf-bp65 sirna for 48 h , and then stimulated with tnf- ( 20g / l ) for 24 h. the results showed that pdtc visibly increased aqp2 mrna and protein expression in tnf--induced imcd cells ( figure 4d , 4e ) . knockdown of nf-bp65 was further supported the above results by increasing the expression of aqp2 in imcd cells induce by tnf- ( figure 4 g , 4h ) . to prove the relationship between sirt1 and nf-b in imcd cells , the expression of ac - p65 was assessed . we found that the sirt1 activator srt1720 significantly reduced ac - p65 expression , which was reversed by the sirt1 inhibitors ex527 and sirtinol in tnf--induced imcd cells ( figure 5a ) . moreover , the sirt1 overexpressing or knockdown cells were used to further verify the above results . the results revealed that sirt1 overexpression significantly decreased tnf--induced ac - p65 expression ( figure 5b ) , while knockdown of sirt1 obviously increased ac - p65 expression in imcd cells expose to tnf- ( figure 5c ) . to further clarify the underlying mechanism involved in sirt1-regulated aqp2 expression , nf-b signaling was included . the results revealed that the expression of aqp2 was correspondingly increased after preconditioning of nf-bp65 knockdown cells with pcdna3.1(+)-sirt1 , compared with nf-bp65 sirna or pcdna3.1(+)-sirt1 single treatment in tnf--induced imcd cells ( figure 5d ) . these results imply that sirt1 can regulate aqp2 expression in imcd cells stimulated with tnf- via suppression of nf-b signalling . aqp2 immunofluorescent labeling was largely dispersed along the cytoplasm of primary cultured imcd cells in the absence of 1-desamino-8-d - arginine vasopressin ( ddavp ) stimulation in control group . in contrast , treatment with 100 nm avp for 30 clearly increased the aqp2 expression in the plasma membrane and decreased it in the cytoplasm ( figure 1a ) , indicating that the imcd cells were correctly cultured . to study the effect of tnf- on aqp2 expression , the imcd cells were treated with tnf- ( 10 , 20 and 40 g / l ) for 24 h , and the expression of aqp2 protein and mrna was determined by western blot analysis and qrt - pcr , respectively . the results showed that tnf- significantly decreased the expression of aqp2 mrna and protein in a concentration - dependent manner ( figure 1b , 1c ) . we further investigate the time courses of aqp2 stimulated with tnf- ( 20 g / l ) for 4 , 8 , 12 and 24 h. as shown in figure 1d,1e , tnf- time - dependently reduced the expression of aqp2 mrna and protein in imcd cells . we next investigated the effect of tnf- on sirt1 expression in imcd cells , the imcd cells were treated with tnf- ( 10 , 20 and 40 g / l ) for 24 h. the results showed that tnf- down - regulated sirt1 expression at protein and mrna level in imcd cells in a concentration - dependent manner ( figure 2a , 2b ) . to study the time courses of sirt1 expression , the imcd cells were treated with tnf- ( 20 g / l ) for 4 , 8 , 12 and 24 h. the results revealed that tnf- inhibited the protein and mrna expression of sirt1 in a time - dependent manner ( figure 2c , 2d ) . to identify the role of sirt1 in the expression of aqp2 in tnf--induced imcd cells , we overexpressed the expressions of sirt1 in imcd cells . after treatment with the sirt1 activator srt1720 ( 10 m ) or the pcdna3.1(+)-sirt1 plasmid , increases in sirt1 expression were observed in imcd cells compared with the control group ( figure 3a , 3e , 3f ) . then the imcd cells were pretreated with srt1720 for 1 h or pcdna3.1(+)-sirt1 for 48 h , and then stimulated with tnf- ( 20g / l ) for 24 h. sirt1 activator srt1720 obviously increased the expression of sirt1 and aqp2 in tnf--induced imcd cells ( figure 3b3d ) . overexpression of sirt1 is further supported the results by inducing expression of aqp2 compared with the tnf- group ( figure 3 g , 3h ) . treatment of imcd cells with the sirt1 inhibitors ex527 ( 1 m ) and sirtinol ( 10 m ) or sirt1 sirna strikingly reduced the expression of sirt1 ( figure 3a , 3l ) . moreover , the imcd cells were pretreated with ex527 or sirtinol for 1 h or sirt1 sirna for 48 h , and then stimulated with tnf- ( 20 g / l ) for 24 h. the results showed that sirt1 inhibitors ex527 and sirtinol decreased the expression of sirt1 and aqp2 in tnf--induced imcd cells ( figure 3i3k ) . in addition , knockdown of sirt1 remarkably decreased aqp2 mrna and protein expression in imcd cells exposed to tnf- ( figure 3 m , 3n ) . to assess the acetylation of nf-bp65 in tnf--induced imcd cells , western blot was performed . the results showed that treatment of imcd cells with tnf- ( 10 , 20 and 40 g / l ) for 24 h significantly increased ac - p65 protein expression in a concentration - dependent manner ( figure 4a ) . we further studied the time courses of ac - p65 expression in imcd cells by treating with tnf- ( 20 g / l ) for 4 , 8 , 12 and 24 h. the results showed that tnf- up - regulated ac - p65 protein expression in a time - dependent manner ( figure 4b ) . to further assess whether nf-b pathway was associated with the expression of aqp2 in tnf--induced imcd cells , we silenced nf-b pathway . pretreatment with the nf-b inhibitor pdtc ( 10 m ) or nf-bp65 sirna strikingly decreased ac - p65 protein expression ( figure 4c , 4f ) . then the imcd cells were treated with pdtc for 1 h or nf-bp65 sirna for 48 h , and then stimulated with tnf- ( 20g / l ) for 24 h. the results showed that pdtc visibly increased aqp2 mrna and protein expression in tnf--induced imcd cells ( figure 4d , 4e ) . knockdown of nf-bp65 was further supported the above results by increasing the expression of aqp2 in imcd cells induce by tnf- ( figure 4 g , 4h ) . to prove the relationship between sirt1 and nf-b in imcd cells , the expression of ac - p65 was assessed . we found that the sirt1 activator srt1720 significantly reduced ac - p65 expression , which was reversed by the sirt1 inhibitors ex527 and sirtinol in tnf--induced imcd cells ( figure 5a ) . moreover , the sirt1 overexpressing or knockdown cells were used to further verify the above results . the results revealed that sirt1 overexpression significantly decreased tnf--induced ac - p65 expression ( figure 5b ) , while knockdown of sirt1 obviously increased ac - p65 expression in imcd cells expose to tnf- ( figure 5c ) . to further clarify the underlying mechanism involved in sirt1-regulated aqp2 expression , nf-b signaling was included . the results revealed that the expression of aqp2 was correspondingly increased after preconditioning of nf-bp65 knockdown cells with pcdna3.1(+)-sirt1 , compared with nf-bp65 sirna or pcdna3.1(+)-sirt1 single treatment in tnf--induced imcd cells ( figure 5d ) . these results imply that sirt1 can regulate aqp2 expression in imcd cells stimulated with tnf- via suppression of nf-b signalling . emerging evidence has suggested that sirt1 expresses in medullary tubular cells , and protects and maintains kidney cell function . our present study found that treatment of imcd cells with tnf- significantly decreased sirt1 expression at mrna and protein levels in a time - and concentration - dependent manner . it has also revealed that high glucose obviously decreased sirt1 mrna and protein expression in rat mesangial cells . other studies have reported that sirt1 expression was decreased in rats with gentamicin or cisplatin - induced nephrotoxicity . moreover , reduced sirt1 expression has been found in mouse cortical collecting duct cells , in human kidney proximal tubule epithelial cells , and in rats with aki induced by severe burns   and renal ischaemia - reperfusion injury . therefore , these findings imply that the decrease in sirt1 expression is involved in renal injury processes in vivo and in vitro . it has been shown that the abundance of aqp2 is decreased in collecting duct cells in a variety of water balance disorders as demonstrated in many studies . the present study found that the expression of aqp2 mrna and protein was time- and concentration- dependently decreased in imcd cells in response to tnf-. hasler et al . revealed that lps decreased aqp2 expression in cultured collecting duct principal cells . in addition to in vitro experiment , the in vivo study also found that aqp2 expression was significantly reduced in the renal cortex and papillas in rats with sepsis - induced acute renal failure . conversely , aqp2 mrna expression was significantly up - regulated in the kidneys of cirrhotic rats . these different results imply that aqp2 might play different roles in types of renal - related disease in response to different stimuli . more importantly , the present study found that sirt1 overexpression or activation by srt1720 increased aqp2 expression in imcd cells treated with tnf-. the corresponding changes were also observed when silencing sirt1 expression by sirt1 sirna or he inhibitors ex527 and sirtinol . this is consistent with the results of the latest study showing that resveratrol increased the expression of aqp2 in mouse cortical collecting duct cells induced by 4-hydroxy-2-hexenal , a inducer for tubular injury , by activating sirt1 pathway . these results indicate that sirt1 increased aqp2 expression which was involved in water balance disorders - associated diseases . it is well recognized that the renoprotective effect of sirt1 is due to deacetylating its target proteins , such as nf-b , a master transcription factor involved in the development of renal disease . the transcriptional activity of nf-b requires acetylation at lysine 310 . in the present study , tnf- significantly increased the acetylation of nf-bp65 ( lys 310 ) expression , which was reversed by sirt1 overexpression or the activator srt1720 , and augmented by sirt1 sirna or the inhibitors ex527 and sirtinol in imcd cells . other study further supported that tnf- increased the acetylation of nf-b at lysine 310 in fibroblasts , while this effect was inhibited by resveratrol via a sirt1-dependent manner . these findings show that sirt1 plays renoprotective role in antagonizing inflammatory renal injury in imcd cells through deacetylation of nf-bp65 at lysine 310 . furthermore , it has been revealed that activation of nf-b observed in renal diseases most likely accounts for the observed decrease in aqp2 expression . the present study found that knockdown of nf-b or the nf-b inhibitor pdtc remarkably antagonized the downregulation of aqp2 induced by tnf- in imcd cells . our results further found that knockdown of nf-bp65 significantly augmented the effect of sirt1 on increased expression of aqp2 in imcd cells exposed to tnf-. similarly , sirt1 activator resveratrol significantly increased aqp2 expression by suppressing the nuclear localization of the nf-bp65 subunit in vitro . taken together , these findings suggest that sirt1-mediated rescuing of aqp2 expression was due , at least in part , to inhibited nf-b signalling in imcd cells . these findings might be important to understand the potential role of sirt1 activation in water balance disorders - related diseases .
backgroundaquaporin-2 ( aqp2 ) plays a major role in water reabsorption in the renal collecting duct , and is involved in a variety of renal disease . 
 recent studies have indicate that sirtuin1 ( sirt1 ) exerts renoprotective properties against kidney diseases . 
 this study aimed to determine the potential role of sirt1 in aqp2 expression induced by tumor necrosis factor - alpha ( tnf- ) and to disclose the underlying mechanism in renal inner medullary collecting duct ( imcd ) cells.material/methodsquantitative real - time pcr and western blotting were respectively identified mrna and protein expression . 
 immunofluorescence staining was used to detect the localization of aqp2 . 
 small - interfering rna ( sirna ) was carried out for mechanism study.resultsresults showed that aqp2 was clearly increased in the plasma membrane and decreased in the cytoplasm of imcd cells treated with avp . 
 tnf- treatment in imcd cells significantly reduced sirt1 and aqp2 expression , and increased acetylated nf-bp65 protein level in time- and concentration - dependent manners . 
 moreover , sirt1 overexpression or the activator srt1720 augmented aqp2 expression and reduced the acetylation of nf-bp65 , which was reversed by sirt1 sirna or the inhibitors ex527 and sirtinol in tnf--induced imcd cells . 
 knockdown of nf-bp65 or nf-bp65 inhibition by pyrrolidine dithiocarbamate ( pdtc ) enhanced aqp2 expression in imcd cells exposed to tnf-. importantly , knockdown of nf-bp65 augmented the up - regulation of sirt1 on aqp2 expression in imcd cells induced by tnf-.conclusionsthese findings indicate that sirt1 increases aqp2 expression in tnf--induced imcd cells via the nf-b - dependent signalling pathway , which might provide novel insight to understanding the renoprotective effects of sirt1 in kidney diseases .