diabetic nephropathy is one of the most important complications in diabetes and is also the leading cause of renal failure in adults . ages are a heterogeneous group of products in which protein and lipids are covalently bound to sugar residues under hyperglycemic and oxidative stress situations , which is proposed to play a major role in the pathogenesis of diabetic nephropathy . however , the mechanisms involving the pathogenesis of renal damage induced by ages were poorly understood . the na / h exchanger ( nhe ) is a protein that is expressed in many mammalian cell types , exchanging one intracellular h for an extracellular na . in this way , it regulates intracellular ph value and cell volume . to date , nine isoforms ( nhe1 - 9 ) have been identified . nhe1 is ubiquitously distributed in most tissues , which is localized in the membrane and sensitive to amiloride . our previous studies have indicated that hyperactivity of nhe1 exchanger is related to the vascular injury associated with high glucose or hyperglycemia [ 57 ] . cariporide , similar to amiloride , as a selective nhe1 inhibitor , prevents the process of vasculopathy in diabetic rats [ 8 , 9 ] . although its role in diabetic vascular complication has been extensively investigated , whether nhe1 mediates diabetic nephropathy and the pathogenic mechanism remain unclear . it has been reported that ages - induced hyperglycemic memory phenomenon [ 1012 ] is very similar to the persistent nhe1 activation in diabetic nephropathy . these findings suggest that the mass accumulation of ages may involve activation of nhe1 in the pathogenesis of nephropathy . our previous studies have also demonstrated that ages activate nhe1 to induce proliferation of vascular smooth muscle cell . therefore , we hypothesized that activation of nhe1 may be a critical step in the signal transduction of ages - induced renal damage . our results demonstrate that cariporide , via inhibition of nhe1 , normalized the redox status to protect renal function in rats injected with ages . cariporide , n - acetylcysteine ( nac ) , bovine serum albumin ( bsa , cat . a1933 , reagent  98% ) , and d - glucose were purchased from sigma company . antibody to ages receptor ( ab - rage ) was purchase from santa cruz company . male sprague - dawley rats ( 8  2 weeks old , 180  20  g ) were purchased from the center of experiment animals , central south university ( changsha , china ) . all rats were housed individually in cages at a room temperature of 21  1c with a 12 h light / dark cycle and were given free access to food and water . at the end of the experiments , rats were placed in individual metabolic cages and 24-hour urine samples for three consecutive days before sacrifice were collected . after fasting for 12  h , the rats in each group were anesthetized with sodium pentobarbitone ( 30  mg / kg , i.p . ) and exsanguinated . the right kidneys were collected after perfusion with 40  ml of ice - cold pbs and stored at 80c . this study was carried out in strict accordance with the recommendations in the guide for the care and use of laboratory animals of the national institutes of health . the protocol was approved by the committee on the ethics of animal experiments of the university of central south . briefly , bsa ( 50  mg / ml ) was incubated with d - glucose ( 0.5  m ) in pbs supplemented with penicillin ( 100  u / l ) and streptomycin ( 100 mg / l ) for 12 weeks at 37c under sterile environments and darkness . after incubation , the solutions were dialyzed against pbs ( ph 7.4 ) at 4c for 48  h to remove free glucose , following separation of ages into aliquots , and stored at 20c . age - specific fluorescence was determined using 370  nm excitation and 440  nm emission wavelengths by using a spectrofluorometer ( shimadzu , beijing beyond technology development co ) . bsa was incubated in the same conditions without d - glucose and served as control of ages . as described in details previously , the isolated kidneys were immediately placed in 5  ml ice - cold krebs buffer and kept on ice . the slices were rinsed two times in 5  ml oxygenated krebs buffer each for 3  min at 25c in an oxygen environment with constant shaking and then transferred to 3  ml oxygenated krebs in designated erlenmeyer flasks and equilibrated for 10  min at 37c prior to different treatments . the creatinine levels in both serum and urine were detected using alkaline picric acid method under the guidance of commercial kits ( nan jing jian cheng bioengineering institute , china ) . the creatinine clearance was calculated on the basis of urinary creatinine , serum creatinine , urine volume , and body weight as described previously . blood urea nitrogen level was measured using urea enzymatic colorimetric kit ( nan jing jian - cheng bioengineering institute , china ) . 24-hour urinary protein was determined by the bca method ( beyotime institute of biotechnology , china ) . as described in details previously , the severity of renal damage was estimated by the following parameters : ( 1 ) total glomerular surface area and ( 2 ) mesangial matrix injury score expressed by mesangial surface area / glomerular total surface area . kidney was homogenized using an electromotion glass homogenizer ( ningbo scientz biotechnology co. , china ) as describe previously . after centrifugation , the supernatant was kept under 80c before determinations of mda , ldh , and gsh . the protocols of gsh and mda measurements were remanded by commercial kits ( nan jing jian cheng bioengineering institute , china ) . urinary tgf-1 was quantified by elisa using commercial kits ( biosource , camarillo , ca , usa ) according to the manufacturer 's instructions . total rna was extracted from each renal cortex tissue using 1  ml trizol reagent ( gibco , usa ) according to the manufacturer 's protocol . rna concentrations were determined by the a260/280 ratio using a spectrophotometer and the quality was assessed on a 1.5% ethidium bromide - agarose gel . three micrograms of total rna were reverse transcribed with oligo - dt primer and m - mlv reverse transcriptase ( tiangen , china ) . one microgram of the reaction mixture was used in each pcr containing a pair of specific primers for rat nhe1 , tgf-1 , and gapdh . the sequences of the nhe1 primers specific for rats were sense 5-cacgctgtggaatgct-3 and antisense 5-gaagatgtccgagatgc-3. pcr product was 289  bp . sequences of the tgf-1 specific for rats were sense 5-gccaagaccctaaca-3 and antisense 5-cactgaagtccaccaa-3. pcr product was 381  bp . gapdh mrna was codetected with sense 5-caatgtatccgttgtgg-3 and antisense 5-gtccagggtttcttactc-3. pcr product was 307  bp . target sequences were amplified at 1c below tm using the same amount of cdna for all primer sets , and the cycle number was adjusted between 30 and 35 to yield visible products within the linear amplification range . densities of bands were measured by scanning densitometry with image j analysis system software and normalized to gapdh in the same sample . as described previously , tissues were homogenized on ice in cell - lysis buffer ( 20  mm tris - hcl , ph 7.5 , 150  mm nacl , 1  mm na2edta , 1  mm egta , 1% triton , 2.5  mm sodium pyrophosphate , 1  mm beta - glycerophosphate , 1  mm na3vo4 , and 1  g / ml leupeptin ) and 1  mm pmsf . the protein content was assayed by bca protein assay reagent ( pierce , usa ) . 20  g proteins were loaded to sds - page and then transferred to membrane . membrane was incubated with a 1  :  1000 dilution of primary antibody , followed by a 1  :  2000 dilution of horseradish peroxidase - conjugated secondary antibody . the intensity ( area x density ) of the individual bands on western blots was measured by densitometry ( model gs-700 , imaging densitometer ; bio - rad ) . group 1 included the following : slices incubated with culture medium of dmem / f12 ; group 2 included the following : slices incubated with culture medium containing bsa ( 200  g / ml ) ; group 3 included the following : slices incubated with culture medium containing cariporide ( 1  m , h - car ) ; group 4 included the following : slices incubated with culture medium containing ages ( 200  g / ml ) ; group 5 included the following : slices incubated with culture medium containing ages plus cariporide ( 0.1  m , l - car ) ; group 6 included the following : slices incubated with culture medium containing ages plus cariporide ( 1  m ) ; group 7 included the following : slices incubated with culture medium containing ages plus antibody of ages receptor ( 5  g / ml , ab - rage ) . sd rats were divided into 5 groups : group 1 : control group ; group 2 : bsa - injected group ; group 3 : ages - injected group ; group 4 : ages - injected plus nac treatment group ; group 5 : ages - injected plus cariporide treatment group . rats in group 2 received tail vein injection of bsa ( 100  mg / kg / day ) . rats in group 3 received tail vein injection of ages ( 100  mg / kg / day ) . rats in group 4 received tail vein injection of ages plus gavage with nac ( 200  mg / kg / day ) . rats in group 5 received tail vein injection of ages plus gavage with cariporide ( 1  mg / kg / day ) . all treatments were performed for 12 consecutive weeks in rats fed with regular diet and tap water . the results were carried out by one - way analysis of variance ( anova ) , followed by the student - newman - keuls test for multiple comparisons with spss 11.5 . in order to test the hypothesis , we firstly investigated whether ages caused renal dysfunction in rats by measuring ldh leakage in isolated renal cortex from rats , which is an indicator of loss of membrane integrity . as shown in figure 1(a ) , incubation of rat renal cortex slice with ages ( 100  g / ml ) from 20 to 160 minutes increased ldh leakage in a time - dependent manner . g / ml for 2 hours , ldh leakage significantly began to increase when the concentration of ages is 100  g / ml or above ( figure 1(b ) ) . these pieces of data indicate that ages induce ldh leakage in isolated renal cortex , which is time / dose dependent . cariporide , a selective nhe1 inhibitor , which has been identified by us and others [ 7 , 21 ] , was used to inhibit nhe1 activity in this section of the present study . as indicated in figure 2(a ) , ages , but not bsa , dramatically increased nhe1 activity in cultured renal cortex cells . though cariporide did not inhibit nhe1 activity in cells without ages treatment , it significantly reduced nhe1 activity in ages - treated cells at low or high dose , indicating that ages activate nhe1 , which is cariporide sensitive , consistent with our previous report . the effects of cariporide on ages - induced nhe1 activation were mirrored by blocking receptor of ages by using specific antibody ( ab - rage ) , further supporting that ages via its receptor activate nhe1 . as depicted in figure 2(b ) , the increased ldh leakage by ages was abolished by cariporide at low or high dose and ab - rage , demonstrating that ages - induced ldh leakage enhancement is possibly related to its receptor and subsequent nhe1 activation . inhibition of nhe1 has been reported to suppress oxidative stress in vascular system [ 22 , 23 ] . thus , we hypothesized that cariporide may inhibit ages - induced oxidative stress to maintain membrane integrity in isolated renal cortex . we evaluated the levels of oxidative stress by determinations of mda and gsh , two markers of oxidative stress in cells . in figures 2(c ) and 2(d ) , either cariporide or ab - rage reduced the ages - increased mda and ages - decreased gsh levels in renal cortex , suggesting that inhibition of nhe1 by cariporide normalizes the redox state in ages - treated rat renal cortex . ex vivo experiments indicated the beneficial effects of cariporide on ahges - induced membrane disruption of renal cortex . we next investigated the in vivo effects of cariporide on ages - induced renal dysfunction . as shown in table 1 , 12-week injection of ages via tail vein did not affect the body weight and blood sugar levels in rats . however , ages dramatically increased the weight of kidney , as indicated by kidney index . treatment of cariporide failed to alter body weight and blood glucose levels but inhibited the hypertrophy of kidney . the hypertrophy of kidney induced by ages was further supported by morphological analysis by he staining . as indicated in figure 3(a ) , compared to bsa - treated rats , widespread glomerular sclerosis was observed in ages alone challenged rats , as well as increased mesangial matrix injury score ( figure 3(b ) ) , cell numbers ( figure 3(c ) ) , and glomerular volume ( figure 3(d ) ) by quantitative analysis . in contrast , all these effects induced by ages were corrected by cariporide intervention . inhibition of oxidative stress by nac also mimicked these effects induced by cariporide in ages - injected rats . taking these data together , it suggests that inhibition of nhe1 protects kidney structure in ages - injected rats , which is related to suppression of oxidative stress . the protective effects of cariporide in kidney of ages - injected rat were further confirmed by analysis of renal function . compared to bsa - injected rats , ages remarkably caused renal dysfunction as increased serum creatinine ( figure 4(a ) ) , blood urea nitrogen ( figure 4(b ) ) , and urinary albumin excretion ( figure 4(c ) ) and decreased clearance of creatinine ( figure 4(d ) ) . as expected , cariporide reversed ages - induced enhancements of serum creatinine , blood urea nitrogen , and urinary albumin excretion and reduction of clearance of creatinine . these data suggest that activation of nhe1 and oxidative stress are key steps contributing to ages - induced renal dysfunction . in order to establish the relationship between nhe1 activation and oxidative stress in ages - induced renal damage , we examined the levels of oxidative stress in cariporide - treated ages - injected rats . as indicated in figures 5(a ) and 5(b ) , both increased mda and decreased gsh were induced by ages in rats , which were reduced by cariporide or nac , indicating cariporide via inhibition of nhe1 suppress oxidative stress in ages - injected rats . we finally determined the effects of cariporide on tgf-1 , which is a key mediator for diabetic nephropathy . compared with bsa - treated rats , ages increased nhe1 and tgf-1 gene expressions , as determined by rt - pcr ( figure 6(a ) ) . cariporide obviously downregulated nhe1 and tgf-1 gene expressions to the level of bsa - treated group . the reductions of nhe1 and tgf-1 gene expressions induced by nac were weaker than cariporide . the effects of cariporide and nac on tgf-1 gene expression were further confirmed by assaying urinary tgf-1 excretion ( figure 6(b ) ) . collectively , it indicates that nhe1 is a potential target of cariporide to prevent renal function in ages - injected rats . the present study demonstrates that ages ex vivo or in vivo cause glomerular sclerosis and renal dysfunction , which is abrogated by nhe1 inhibition , blockage of ages receptor , and suppression of oxidative stress . mechanistically , the detrimental effects of ages on kidney function might be related to activation of its receptor and sequent activation of nhe1 , resulting in upregulation of oxidative stress . in this way , cariporide , a selective nhe1 inhibitor , normalizes the redox state in renal cortex and functions . ages can accumulate in diverse biological settings , such as diabetes , inflammation , renal failure , and aging . many studies support that interactions between ages and its receptor are involved in the pathogenesis of diabetic complications , in particular , nephropathy [ 1 , 25 , 28 ] . the formation and accumulation of ages adducts in various tissues are associated with altered protein structure and function . in addition , ages are able to activate intracellular signaling by binding to specific receptors . a number of ages inhibitors and crosslink breakers , such as aminoguanidine and alt-711 , have been shown to prevent the formation and break the crosslink of ages [ 30 , 31 ] . however , they could not affect the cellular interactions of the existing ages with its receptor . in this present study , we injected exogenous ages into rats to mimic diabetic nephropathy and examined the effects of nhe1 selective inhibitor cariporide on kidney function . we observed that ages alone resulted in the increase of glomerular sclerosis and renal dysfunction , which are reversed by cariporide . another important discovery is that nhe1 inhibition reduces oxidative stress induced by ages in diabetic nephropathy . ages may be linked to the increased reactive oxygen species ( ros ) by decreasing antioxidative enzyme including superoxide dismutase and catalase , diminishing glutathione stores . our study indicated that ages significantly increased production of mda and lower gsh content , while treatment with cariporide and nac dramatically decreased mda concentration and restored gsh level . our previous study has showed that cariporide could prevent oxidative stress reaction mediated by high glucose [ 6 , 33 ] . these findings confirmed that there was a close relationship between the oxidative stress and the changed activity of nhe1 . during the pathologic process of ages in renal damage , nhe1 may be also activated by ros due to age - rage reaction because ros is also a well - known activator of nhe1 . it should be noted that this study has been examined only in animal experiment ; the precise mechanism remains to be determined in cell studies . it stimulates the synthesis of ecm such as collagen and laminin and blocks ecm degradation through inhibition of matrix metalloproteinase . it is considered to be the strongest cytokine to glomerulosclerosis in diabetic nephropathy and a last major mediator induced by a variety of damaging factors in the pathological process . therefore , we tentatively put forward that nhe1 function as a platform to link ages with tgf-1 pathway . in this study , ages treated alone rats showed widespread fibrosis in renal glomerulus , parallel increase of tgf-1 expression in the renal cortex , and urinary tgf-1 excretion . cariporide treatment congruously decreased the above index and prevented renal glomerulus fibrosis , suggesting that nhe1 plays a decisive role in activation of tgf-1 pathway of renal injury induced by ages . it is worth more detailed studies in the future to further confirm the precise pathway . in conclusion , we have identified that inhibiting nhe1 with cariporide exhibited marked protection from ages - mediated renal damage . nhe1 may function as a structural scaffold to link ages with tgf-1 signaling in renal damage . these findings suggest that nhe1 is a promising target for the treatment of diabetic nephropathy and makes cariporide a promising drug for the future treatment of diabetic nephropathy .
it has been recognized that sodium hydrogen exchanger 1 ( nhe1 ) is involved in the development of diabetic nephropathy . 
 the role of nhe1 in kidney dysfunction induced by advanced glycation end products ( ages ) remains unknown . 
 renal damage was induced by ages via tail vein injections in rats . 
 function and morphology of kidney were determined . 
 compared to vehicle- or bsa - treated rats , ages caused abnormalities of kidney structures and functions in rats , accompanied with higher mda level and lower gsh content . 
 gene expressions of nhe1 gene and tgf-1 in the renal cortex and urine were also increased in ages - injected rats . 
 importantly , all these detrimental effects induced by ages were reversed by inhibition of nhe1 or suppression of oxidative stress . 
 these pieces of data demonstrated that ages may activate nhe1 to induce renal damage , which is related to tgf-1 .