the genesis of diabetic nephropathy ( dn ) in type 2 diabetes mellitus is clearly multifactorial , involving hypertension , hyperglycemia ,   hyperinsulinemia , and hyperlipidemia [ 16 ] . recent studies have further highlighted the role of obesity in the renal damage observed not only in patients with obesity - related glomerulopathy but also in overweight subjects with type 2 diabetes [ 610 ] . in several previous studies , we have explored a diabetic rat model , that is , the spontaneously hypertensive / nih - corpulent rat ( shr / ndmcr - cp ) , to unravel factors implicated in dn . this rat strain has the same hypertensive background as shr but also a genetic mutation in the leptin receptor gene , which leads to hyperphagia with an attendant wide range of metabolic abnormalities , that is , high body weight , hyperglycemia , hyperlipidemia , and hyperinsulinemia . it develops proteinuria , and glomerular and tubulointerstitial damages , mimicking human diabetic nephropathy , for example , focal and segmental glomerular sclerosis , mesangial expansion , and tubulointerstitial fibrosis [ 11 , 12 ] . previously , we demonstrated that a low caloric diet reduced both metabolic and renal alterations independently of blood pressure , lipid , glucose , and insulin levels . the observation that a high - carbohydrate / low - fat diet reduces effectively body weight without calorie restriction [ 13 , 14 ] led us to use a similar isocaloric diet in our rat model to better identify factors mediating the weight - related factors involved in the genesis of dn . shr / ndmcr - cp rats given a normal middle - carbohydrate / middle - fat diet ( mc / mf group ) were thus compared with similar rats fed a high - carbohydrate / low - fat diet ( hc / lf group ) . to our expectation , the latter diet induced significant less weight gain without calorie restriction . despite the equivalent degrees of hypertension , hyperglycemia , hyperlipidemia , hyperinsulinemia , and even a poorer glycemic control , the hc / lf group had a significantly lower proteinuria and less severe renal histological abnormalities . the mediators of the specific weight effect on the kidney appear to be an obesity - related inflammation , aggravated by a lowered anti - inflammatory adiponectin level , an increased oxidative stress and advanced glycation , and an enhanced tgf - beta expression , all of which might constitute promising therapeutic targets . animal experiments were performed in accordance with the guidelines of the committee on ethical animal care and use of tokai university . male spontaneously hypertensive / nih - corpulent rats ( shr / ndmcr - cp ) and male wistar - kyoto rats ( wky ) were purchased from slc ( shizuoka , japan ) . they were housed in individual cages , in a temperature- and light - controlled environment in an accredited animal care . shr / ndmcr - cp rats , aged 5 weeks , were randomly divided into two groups and given for 12 weeks either a normal diet ( ce-2 , clea japan inc . , tokyo , japan ) with tap water ( mc / mf group , 10 rats ) , or a high carbohydrate / low fat diet ( ce-2 with tap water containing 30% sucrose ) ( hc / lf group , 10 rats ) . five wky rats on a normal diet ( ce-2 ) served as a control group ( wky group , 5 rats ) . all rats were allowed unlimited access to diet and water , and each rat 's daily dietary intake was determined thrice weekly from the amount of actually consumed food and fluid . systolic blood pressure was determined in conscious rats by the tail - cuff method at the beginning of the study , 2  wk , 4  wk , and every 4  wk subsequently until euthanasia . rats were housed in metabolic cages for overnight collection of urine , and blood samples were obtained at the same time intervals . plasma triglycerides and urinary protein concentration were determined with an automatic analyzer ( hitachi automatic clinical analyzer 7170 , hitachi science systems , ibaraki , japan ) . the following methods were used : plasma insulin by a commercially available kit ( morinaga biochemistry lab , tokyo , japan ) , hba1c by the dca2000 ( bayer diagnostics , pittsburgh , pa ) , plasma adiponectin by a commercially available elisa kit ( adipogen inc . , seoul , korea ) . briefly , glomerulosclerosis was assessed in 4-m thick sections fixed in methyl - carnoy 's solution and stained with pas . glomerular sclerosis was graded according to the damaged area expressed as a percentage of total glomerular area : ( 0 , no lesions ; 1 + , 1% to 25% ; 2 + , 25% to 50% ; 3 + , 50% to 75% ; 4 + , 75% to 100% ) . tubulointerstitial fibrosis was evaluated in 4-m thick sections fixed in 10% neutral buffered formaldehyde and stained with masson 's trichrome . interstitial trichrome - positive areas were counted using the capacity of software image - pro plus ( planetron , inc . , mouse anti - rat macrophage / monocyte antibody ( anti - ed-1 , serotec , oxford , uk ) was used to stain 4-m thick renal sections fixed in 10% neutral buffered formaldehyde . ed -1-positive cells infiltrating the tubulointerstitium were counted in 10 randomly selected fields in the cortex ( 400 magnification ) . real - time pcr was performed by a previously described method   relying on one step rt - pcr kit ( takara bio inc . , shiga , japan ) , sybr green i reagent ( cambrex bio science , rockland , maine ) , and icycler pcr system ( bio - rad laboratories , hercules , ca ) , to evaluate the mrna expression of transforming growth factor ( tgf)-beta , connective tissue growth factor ( ctgf ) , plasminogen activator inhibitor ( pai)-1 , nox2 , p47 , tumor necrosis factor ( tnf)-alpha , intercellular adhesion molecule ( icam)-1 , vascular cell adhesion molecule ( vcam)-1 , and glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) . irptc is a cell line originating from proximal tubular cells of male wistar rats , immortalized by transformation with origin - defective sv 40 dna . irptcs were cultured in dulbecco 's modified eagle 's medium ( dmem ) ( invitrogen , carlsbad , ca ) , supplemented with 10% fbs . they were subsequently washed and incubated with serum - starved dmem containing 0.01 , 0.1 , 1  ng / ml human tnf - alpha ( sigma - aldrich , inc . , st . total rna was isolated from irptcs with rneasy mini kit according to the manufacturer 's instructions . briefly , a 20-l aliquot of a renal tissue acid hydrolysate diluted by pbs was injected into a reverse - phase hplc system and separated on a c18 reverse - phase column ( waters , tokyo , japan ) . pentosidine was measured in its effluent monitored with a fluorescence detector ( rf-10a ; shimadzu , kyoto , japan ) at an excitation - emission wavelength of 335/385  nm . statistical analysis was performed with spss for windows version 15.0 ( spss , chicago , il ) . for multiple comparisons , one - way analysis of variance ( anova ) and dunnett t test daily caloric intake ranged from 100 to 120  kcal per animal and was virtually identical in both groups throughout the study ( figure 1(a ) ) . daily intake of carbohydrate , fat , protein , and sodium was around 16.7  g , 1.5  g , 7.0  g , and 100  mg / day , respectively , in the mc / mf group and 22.6  g , 0.5  g , 4.0  g , and 35  mg / day , respectively , in the hc / lf group ( figures 1(b)1(e ) , p < .001 ) .      despite a virtually identical caloric intake , the rats gained less weight in the hc / lf than in the mc / mf group ( p < .01 ) : 505  21 versus 577  16  g at the end of the study ( figure 2(a ) ) . both groups of shr / ndmcr - cp rats gradually developed a similar degree of hypertension , as a consequence of their shr genetic background . compared to control wky rats , both the mc / mf and the hc / lf groups suffered a wide range of hyperphagia induced metabolic abnormalities , that is , high body weight , hyperglycemia ( as evidenced by hba1c levels ) , hyperinsulinemia , and hypertriglyceridemia .        compared with the mc / mf group , the hc / lf group gained significantly less weight ( p < .01 ) , and had a significantly worse hba1c level ( p < .001 ) . other metabolic abnormalities , that is , hypertriglyceridemia and hyperinsulinemia , were equivalent between the two groups . the progressive rise in proteinuria observed in the mc / mf group was markedly attenuated in the hc / lf group throughout the study ( figure 3 ) . at the end of the study , proteinuria reached 59.8  7.2  mg / day in the mc / mf group versus 19.6  2.2  mg / day in the hc / lf group ( p < .001 ) , values to be compared to 9.8  0.6  mg / day in the wky rats ( p < .001 ) . representative periodic acid - schiff- ( pas- ) or masson 's trichrome-(mt- ) stained renal pictures obtained at the end of the study in each animal group are shown in figure 4 . glomerular sclerosis ( figures 4(a)4(c ) ) and tubulointerstitial fibrosis ( figures 4(d)4(f ) ) were visible in both groups but less in the hc / lf group than in the mc / mf group . on semiquantitative analysis ( table 2 ) , glomerulosclerosis and interstitial fibrosis were significantly more severe in both experimental groups than in the wky group ( p < .001 and p < .05 , resp . ) but were milder in the hc / lf group than in the mc / mf group ( p < .001 and p < .05 , resp . ) . obesity - related cytokines ( e.g. , adiponectin and tnf - alpha ) levels , inflammation , transforming growth factor ( tgf)-beta , oxidative stress , and advanced glycation were assessed to ascertain the biological mechanisms involved in the better renal outcome in the hc / lf group . macrophage infiltration was milder in the hc / lf than in the mc / mf group ( figures 4(g ) and 4(h ) ) . on semiquantitative analysis , tubulointerstitial infiltration was significantly ( p < .05 ) milder in the hc / lf group than in the mc / mf group ( figure 5(a ) ) . tnf - alpha mrna expression in kidney tissues was significantly ( p < .05 ) lower in the hc / lf than in the mc / lf group with a parallel decrease in the expression of intercellular adhesion molecule ( icam)-1 and vascular cell adhesion molecule ( vcam)-1 ( figure 5(b ) ) . adiponectin is an anti - inflammatory cytokine secreted by adipocytes [ 17 , 18 ] . its plasma level was significantly ( p < .01 ) higher in the hc / lf than in the mc / mf group ( figure 5(c ) ) . interestingly , plasma adiponectin level , determined in all shr / ndmcr - cp rats , was inversely correlated with body weight ( r = 0.602 , p < .01 ) and with proteinuria ( r = 0.654 , p < .01 ) . of note , body weight was also significantly correlated with proteinuria in all experimental rats ( p < .001 , r = 0.708 ) . tgf - beta has been implicated in the development of diabetic renal injury [ 19 , 20 ] . tgf - beta mrna expression in kidney tissue was significantly ( p < .01 ) lower in the hc / lf than in the mc / mf group , with a parallel decrease in the expression of tgf - beta - related genes , that is , connective tissue growth factor ( ctgf ) and plasminogen activator inhibitor ( pai)-1 ( figure 6(a ) ) . the interrelation between tgf - beta and tnf - alpha expression was further tested in vitro in cultured rat proximal tubular cells ( irptc ) . incubation for 4 hours in a tnf - alpha fortified medium significantly ( p < .05 ) increased mrna expression of tgf - beta in a dose - dependent manner ( figure 6(b ) ) . advanced glycation and its protein modifications , that is , advanced glycation end products ( ages ) , have been implicated in diabetic renal injury [ 2325 ] . at the end of the study , renal pentosidine content , a surrogate marker of advanced glycation , was markedly ( p < .01 ) lower in the lc / hf group than in the mc / mf group ( figure 7(a ) ) . oxidative stress , a mediator of diabetic renal injury , implicates nadph oxidase , one of the major sources of cellular reactive oxygen species ( ros ) . the mrna expressions of two subunits of nadph oxidase , nox2 and p47 , measured in renal tissues , were significantly ( p < .05 , p < .01 , resp . ) lower in the hc / lf than in the mc / mf group ( figure 7(b ) ) .   in all diabetic rats , renal nox2 and 47 mrnas expression levels were significantly correlated with tnf - alpha ( r = 0.450 , p < .05 ; r = 0.727 , p < .001 , resp . ) , with tgf - beta ( r = 0.764 , p < .001 ; r = 0.699 , p < .01 , respectively ) , and with renal pentosidine content ( r = 0.719 , p < .001 ; r = 0.565 , p < .01 , resp . ) . the present study highlights body weight control as a marked contributor to the slowing of the progression of dn . it identifies inflammation , oxidative stress , advanced glycation , tgf - beta , and adiponectin as possible mediators of this effect . use of the same rat model in our several previous studies [ 11 , 12 , 2729 ] has provided a detailed picture of the features involved in the renal disorder observed in shr / ndmcr - cp given an ad libitum access to a standard diet . in the present study , the control group of rats on a middle - carbohydrate / middle - fat diet exhibits the same spectrum of abnormalities , that is , high body weight , hypertension , hyperglycemia with hyperinsulinemia , elevated lipids and tgf - beta expression together with proteinuria , glomerular and interstitial fibrosis with evidence of intrarenal advanced glycation and oxidative stress . unraveling the sequence of events leading to dn in this model is of importance as it might offer a clue to a better prevention and treatment of the renal disease complicating the outcome of diabetic patients . the present data demonstrate that body weight control by a high - carbohydrate / low - fat diet plays a role in the slowing of the development of dn , independently of calorie intake . in agreement with our findings , in human , a high - carbohydrate / low - fat diet reduces effectively body weight without calorie restriction [ 13 , 14 ] . the mechanism for body weight control by a high - carbohydrate / low - fat diet still remains unknown . its mitigation in the hc / lf group leads lower proteinuria and less severe renal lesions , despite an equivalent caloric intake and similar degrees of hypertension , hyperlipidemia , hyperinsulinemia , and an even poorer glycemic control . in a previous study , a similar effect of body weight control , achieved by a lower caloric intake in the same rat model , provided the same renoprotection , again independently of blood pressure , glucose , insulin and lipid levels . the benefits of body weight control appear thus independent of the latter factors as well as of caloric intake . of importance , renoprotection was achieved in both iso- and hypocaloric rat models together with a parallel reduction of intrarenal advanced glycation and oxidative stress , both of which clearly play a critical role in the development of dn . tgf - beta and its regulated products , ctgf and pai-1 , might be such agents as they have been previously implicated in dn [ 19 , 20 , 3032 ] . in the same rat strain , we have previously adduced evidence that insulin raises tgf - beta expression and that this rise was associated with renal damage . we now show that the lower body weight induced in the hc / lf group reduces tgf - beta expression , and attendant ctgf and pai-1 expressions . interestingly , tgf - beta expression fell despite unchanged insulin levels , suggesting that body weight control modified its expression independently of glucose and insulin levels . obesity triggers an inflammatory reaction which has been incriminated in the development of dn [ 16 , 3335 ] . the kidneys of the mc / mf group   reported in this as well as in previous studies   are infiltrated by macrophages . the lower body weight hc / lf rats are characterized by a decreased renal infiltration by macrophages and a parallel reduction in the mrna expression of tnf - alpha and its related proteins , icam-1 and vcam-1 . interestingly , we demonstrate , in cultured rat proximal tubular cells , that tnf - alpha and tgf - beta are interrelated : tnf - alpha per se upregulates tgf - beta mrna , a previously reported observation [ 21 , 22 ] . the correlation observed between both cytokine levels confirms this conclusion . adiponectin is an anti - inflammatory , anti - atherogenic , and anti - oxidative peptide downregulated in obese patients . its plasma level correlates inversely with proteinuria in diabetes [ 36 , 37 ] , a finding corroborated by the present study . plasma adiponectin levels were higher in the hc / lf group which exhibited slower progression of dn , suggesting that body weight control augmented the adiponectin plasma level , its anti - inflammatory effect , and thus renoprotection . this hypothesis is supported by ohashi et al . 's observation in adiponectin knockout mice with subtotal nephrectomy : adiponectin deficiency exacerbated albuminuria and renal lesions together with an increased inflammation , tgf - beta levels , and oxidative stress . we previously demonstrated in the same diabetic rat strain that pioglitazone , a class of thiazolidinediones ( tzds ) , protected the diabetic kidney by improving hyperinsulinemia and tgf - beta expression . in this context , it is of note that tzds upregulate plasma adiponectin level , raising the possibility of tzds ' renoprotection might be attributed , at least in part , to the specific induction of adiponectin [ 17 , 37 ] . indeed several age inhibitors , for example , pyridoxamine , alt-711 , and lr-90 , prevent the development of diabetic nephropathy [ 3942 ] , suggesting that accumulation of ages is an important risk factor for diabetic renal injury . angiotensin ii receptor blockers ( arbs ) are also known to protect the diabetic kidney , at least in part , via inhibition of advanced glycation and oxidative stress , independently of body weight [ 11 , 29 ] . the lowered renal pentosidine content observed in the hc / lf group confirms this contention . it is of special interest as it occurs despite higher plasma glucose levels than in the mc / mf control group and might thus reflect a lower oxidative stress . tgf - beta , and proinflammatory cytokines generate reactive oxygen species ( ros ) via nadph oxidase which , in turn , amplifies ages formation , tgf - beta expression , and inflammation [ 26 , 4345 ] . in the hc / lf group , both advanced glycation , assessed by the renal pentosidine content , and oxidative stress , evaluated by the renal mrna expressions of two subunits of nadph oxidase , nox2 and p47 , decreased significantly . in our model , in vivo changes in local oxidative stress and advanced glycation we can not rule out that the underlying hypertension , diabetes , and obesity are not playing a role in this model . furthermore , the intake of fat is implicated in the progression of renal damage via lipid accumulation , inflammation , and renal hemodynamics change [ 46 , 47 ] . the beneficial changes observed in the hc / lf group could be attributed to the low fat content of diet , more than to changes in body weight . admittedly , the protein contents in diet also differ in our two experimental rat groups . although this possibility can not be formally ruled out , it appears unlikely as previous studies by others have shown in shr rats that dietary protein content did not affect the development of proteinuria and renal lesions .   in conclusion , body weight control by a high - carbohydrate / low - fat diet slows the progression of diabetic nephropathy through the suppression of inflammation , sclerotic change , and oxidative stress . they identify adiponectin , tnf - alpha , tgf - beta , and ages as potential therapeutic targets for the prevention of the renal consequences of type 2 diabetes .
obesity is one of several factors implicated in the genesis of diabetic nephropathy ( dn ) . 
 obese , hypertensive , type 2 diabetic rats shr / ndmcr - cp were given , for 12 weeks , either a normal , middle - carbohydrate / middle - fat diet ( mc / mf group ) or a high - carbohydrate / low - fat diet ( hc / lf group ) . daily caloric intake was the same in both groups . 
 nevertheless , the hc / lf group gained less weight . despite equivalent degrees of hypertension , hyperglycemia , hyperlipidemia , hyperinsulinemia , and even a poorer glycemic control , the hc / lf group had less severe renal histological abnormalities and a reduced intrarenal advanced glycation and oxidative stress . 
 mediators of the renoprotection , specifically linked to obesity and body weight control , include a reduced renal inflammation and tgf - beta expression , together with an enhanced level of adiponectin . 
 altogether , these data identify a specific role of body weight control by a high - carbohydrate / low - fat diet in the progression of dn . 
 body weight control thus impacts on local intrarenal advanced glycation and oxidative stress through inflammation and adiponectin levels .