diabetes mellitus is a serious social problem . according to the world health organization ( who ) , in 2030 the number of people with diabetes worldwide will increase to 360 million , representing 4.5% of the global population . diabetic nephropathy is the most frequent complication of diabetes that develops in up to 3040% of patients . the main change of diabetic nephropathy is a thickening of the glomerular basement membrane and expansion of ecm proteins . hyperglycemia results in the activation of tissue angiotensin ii , which plays an important role in the pathogenesis of kidney disease , through inflammation , fibrosis , vascular wall remodeling , and oxidative stress . it was shown that blocking the at1 receptor and angiotensin ii - converting enzyme reduces the levels of inflammatory factors ( nf - kb , il-6 , and tnf-1 ) and is responsible for the processes of fibrosis ( ctgf and tgf-1 ) [ 3 , 4 ] . fibronectin is a glycoprotein present in the extracellular matrix , basal membranes , plasma , and other body fluids . the gene expression of fn in tissues of healthy adults is generally low and increases in areas of wound healing and damaged tissue . hyperglycemia causes increased expression of genes responsible for synthesis of fn [ 5 , 6 ] . 22.1 ) belongs to a class of cysteine proteinases and has optimum activity in acidic environment but can also be active in other ph ranges . it degrades structural proteins and enzymes in the cell , degrades the main elements of the basement membrane , and activates proenzymes , hormones , and growth factors involved in the induction phase and the executive apoptosis . it has been shown that the decrease of the activity of proteolytic enzymes in the states of hyperglycemia is genetically determined as type 2 diabetes in rats and in isolated glomeruli of diabetic rats [ 9 , 10 ] . the aim of this study was to assess the impact of multilevel blocking of the raas using an ace inhibitor , at1 receptor antagonist , administered separately and together , an inhibitor of aldosterone on the activity of cathepsin b , and fibronectin accumulation in the glomerular in the course of diabetic nephropathy in the diabetes model in rats . this study was performed in accordance with the ethical committee affairs experiments on animals of the medical university of warsaw ( opinion number 5/2006 ) . rats in the 4 groups were treated with inhibitors of the raa system , drugs were administered in drinking water in the morning : en : enalapril 3.2  mg / kg / day ; los : losartan 15  mg / kg / day ; en + los : enalapril and losartan , respectively , 3.2 and 7.5  mg / kg / day ; spir : spironolactone 15  mg / kg / day . another group received dih , dihydralazine , 2.7  mg / kg / day and the dm group did not receive any drug . the animals were followed up for six weeks ( five weeks , the duration of action of drugs ) , and blood glucose was regularly analyzed . when blood glucose was above 700  mg / dl , insulin was administered at a dose of 0.251.0  iu / day . in the last week of the experiment , rats ' blood pressure was measured using a pressure sensor apm mk-9301 ( mk - design , usa ) . a 24  hr urine collection was obtained at the end of the 6-week study using metabolic cages and urinary creatinine and microalbumin were measured . one of the kidneys was stored in saline in an ice bath until the isolation of glomeruli . the plasma biochemical tests performed are the following : glucose , total protein , albumin , creatinine , urea , and bicarbonate . isolated glomeruli were homogenized with a homogenizer labsonic u ( b braun , usa ) . dna in homogenates of glomeruli was determined using the fluorometric reagent bisbenzimide h 33258 ( hoechst , germany ) as previously described . protein in homogenates of glomeruli was determined spectrophotometrically using the bca assay ( bicinchoninic acid ) , protein assay reagent ( pierce , beijerland , netherlands ) . cathepsin b activity in glomerular homogenates , urine , and plasma was measured fluorometrically using the synthetic substrate z - arg - arg - amc ( n - co2-l - arginyl - arginine-7-amino-4-methylocoumarin salt ) ( bachem , biochemica gmbh , heidelberg , germany ) as previously described . fn concentration was determined by elisa ( enzyme - linked immunosorbent assay ; immunoenzyme test ) as previously described [ 15 , 16 ] . immunohistochemical staining was performed in paraffin sections of kidney with an antibody against fibronectin ( chemicon international , usa ) . immunohistochemical analysis began with the assessment of the entire tissue section to determine representative areas . we evaluated 20 subsequent glomeruli within the renal cortex of representative areas . in relation to the glomerular vascular loops , each immunohistochemical reaction assessment was based on an analysis of two features : the intensity of the antibody reaction and the percentage of immunopositive vascular loops . immunohistochemical expression was evaluated semiquantitatively using a 4-point scale :  ( 0 ) no reaction or very weak and focal ( < 1% ) expression ;  ( 1 + ) low intensity reaction , with moderate intensity expression of < 50% of vascular loops and high intensity expression being segmental and in < 25% of vascular loops ;  ( 2 + ) moderate intensity expression involving > 50% of vascular loops and high - intensity expression being segmental or continuous involving 2575% of vascular loops ;  ( 3 + ) high intensity expression that is continuous and includes > 75% of vascular loops.immunohistochemical expression of all markers tested within the mesangium was evaluated in a similar fashion . the final evaluation for each specimen was the average ( rounded to unity ) from the analysis of 20 glomeruli . ( 0 ) no reaction or very weak and focal ( < 1% ) expression ; ( 1 + ) low intensity reaction , with moderate intensity expression of < 50% of vascular loops and high intensity expression being segmental and in < 25% of vascular loops ; ( 2 + ) moderate intensity expression involving > 50% of vascular loops and high - intensity expression being segmental or continuous involving 2575% of vascular loops ; ( 3 + ) high intensity expression that is continuous and includes > 75% of vascular loops . the results obtained were analyzed using statistica , version 9.0 which is available at the medical university of warsaw . statistical nonparametric tests were used : anova rank kruskal - wallis . obtaining a result of analysis of the level of significance p < 0.05 was an indication for the use of post hoc test , duncan . the value of kidney weight given as a percentage of final body weight in the group of untreated diabetic rats was 0.60  0.06% and was significantly higher compared to the group of diabetic rats treated dihydralazine ( 0.54  0.05% , p = 0.01 ) . in addition , statistically significant differences were demonstrated between the group of diabetic rats treated dihydralazine and groups with diabetes treated enalapril ( p = 0.01 ) , losartan ( p = 0.0001 ) , enalapril and losartan in combination ( p = 0.01 ) , and spironolactone ( p = 0.04 ) , and between group of diabetic rats treated with spironolactone and losartan ( p = 0.03 ) ( table 3 , figure 1 ) . the coefficient protein / dna in homogenates of glomeruli in the group of untreated diabetic rats was 23.3  6.50  g/g and was significantly higher compared to a group of diabetic rats treated losartan 15.68  5.71  g/g , p = 0.01 , and a group of rats treated spironolactone 17.31  4.2  g/g , p = 0.045 ( table 3 , figure 2 ) . activity of cathepsin b in terms of microgram of dna in homogenates of glomeruli in the group of untreated diabetic rats was 30.60  9.65  iu/g and was lower compared to the group of diabetic rats treated with enalapril and losartan in combination ( p = 0.04 ) and a group of rats diabetes treated with spironolactone ( ns ) and , respectively , was 51.79  20.37 and 44.88  32.18  iu/g . in the groups , diabetic rats treated with enalapril , losartan , and dihydralazine cathepsin b activity per  g of dna were insignificantly lower compared with untreated diabetic rats and were , respectively , 25.99  16.75 , 21.71  14.32 , and 22.70  14.97  iu/g . in addition , statistically significant differences were demonstrated between the group of diabetic rats treated with enalapril and losartan together , a group of diabetic rats treated with enalapril ( p = 0.01 ) , losartan ( p = 0.01 ) , and dihydralazine ( p = 0.01 ) , and groups of diabetic rats treated with spironolactone and enalapril ( p = 0.05 ) , losartan ( p = 0.02 ) , and dihydralazine ( p = 0.03 ) . similar results were obtained converting activity of cathepsin b in homogenates of glomeruli in microgram of protein , table 3 , figure 3 . fibronectin concentration values per 1  g of protein in homogenates of glomeruli in the untreated diabetic rats and the other treated diabetic rats showed no statistically significant differences . similar results were obtained converting the concentration of fibronectin in glomerular homogenates for the presence of dna ( table 3 , figure 4 ) . the content of fibronectin in kidney glomeruli evaluated immunohistochemical staining in the untreated diabetic rats was 2.33  0.52 score and was higher compared to the other groups examined , including significantly higher ( p = 0.003 ) compared to the diabetic rats treated spironolactone 0.90  0.99 score . additionally , statistically significant differences have been shown between groups of diabetic rats treated with spironolactone and enalapril ( p = 0.02 ) and diabetic rats treated with spironolactone and losartan and enalapril in combination ( p = 0.003 ) ( table 3 , figures 5 and 6 ) . our study , performed on an animal model of diabetes mellitus induced by streptozotocin , aimed at assessing the impact of raas blocking on the activity of cathepsin b and the accumulation of fn . rats of groups 15 were treated with an ace inhibitor , at1 receptor antagonist , administered separately and together , and an inhibitor of aldosterone and dihydralazine . the rats in group number 6 were not treated and they constituted the control group . in the renal cortex of diabetic rats treated and untreated , performed immunohistochemical evaluation of fibronectin was observed . the activity of cathepsin b and the concentration of fn were assessed in homogenates of glomeruli , in 24 hour urine and plasma . fibronectin is a protein of the extracellular matrix , produced by the cells and endothelial mesangium [ 17 , 18 ] . in mild and moderate diabetic nephropathy , accumulation of fn in the matrix mesangium has been shown and increased excretion in the urine does appear up earlier than microalbuminuria [ 17 , 19 ] . in previous studies , the correlation between the accumulation fn and cathepsin b activity in the kidney glomerulus in diabetic rats has been shown . the genetically determined type ii diabetes in rats showed an increased accumulation of fibronectin ( fn ) in the kidney glomerulus . cumulative increase in ecm proteins in glomeruli depends on the increase of synthesis and/or reduces protein degradation . proteolytic enzymes are responsible for maintaining the balance of synthesis and degradation of components of the extracellular matrix ( ecm ) . .   showed a decrease of fn immunoreactivity in the kidney glomerulus in rats with hypertension that have blocked aldosterone and angiotensin ii . in the present study the contents of the fn , determined by immunohistochemical staining in renal glomeruli of diabetic rats treated with enalapril , losartan , spironolactone , and dihydralazine were lower compared to the amount of fn in the renal glomeruli of rats with untreated diabetes . the largest decrease in the content fn was obtained in the group of diabetic rats receiving spironolactone ( p = 0.003 ) . no fn obtained reduction in kidney glomerulus of diabetic rats was treated with enalapril and losartan together . the concentration of fibronectin in glomerular homogenates , based on dna , in groups of diabetic rats treated has only a slight decrease compared to untreated diabetic rats . similarly to the fibronectin excretion in the urine per mg creatinine , it has slightly decreased in the groups treated diabetic rats compared to untreated diabetic rats , while the concentration of fn in plasma of rats in all groups examined were in similar extent . rao et al .   giving the rats streptozotocin diabetes acei and at1 blocker received separately and together decrease the concentration of fn in the plasma . the reduction ratio kidney weight / body weight , reflecting the development of kidney hypertrophy , was obtained only in the group of diabetic rats treated with dihydralazine . the coefficient of protein / dna , reflecting glomerulosclerosis , was decreased in the group with diabetic rats treated with losartan ( p = 0.01 ) and spironolactone ( p = 0.045 ) . in the other groups treated diabetic rats have shown a downward trend compared to the group of untreated diabetic rats . a significant reduction of albuminuria has been shown in diabetic rats treated with dihydralazine compared to untreated diabetic rats . in other groups of diabetic rats receiving treatment , a decreasing trend of albuminuria has been shown in relation to untreated diabetic rats . rao et al .   demonstrated reduction in daily urinary albumin excretion in treated diabetic rats compared to untreated diabetic rats . angiotensin ii stimulates the synthesis of ecm proteins and causes a decrease activity of proteolytic enzymes . 22.1 ) belongs to a class of cysteine proteinases and plays many functions in physiological and pathological processes . reduction of the activity of proteolytic enzymes in the states of hyperglycemia in the genetically determined type 2 diabetes in rats and in streptozotocin diabetic rats has been shown [ 9 , 10 , 24 ] . multilevel blocking of the raa system significantly increased activity of cathepsin b in homogenates of glomerular in diabetic rats treated with spironolactone ( p = 0.02 ) and in combination with therapy with losartan and enalapril ( p = 0.001 ) compared to the untreated diabetic rats . dihydralazine administration , enalapril , or losartan in rats with diabetes did not lead to the expected results . showed a beneficial effect of treatment acei to restore the activity of cathepsin b in the heart and kim et al .   demonstrated that combination therapy acei and arb ( angiotensin - ii receptor blocker ) can effectively prevent or reverse myocardial fibrosis . .   showed that the development of an end - stage renal failure and change micro- to macroalbuminuria were significantly reduced after treatment of acei versus placebo and arb versus placebo . this effect is not present in combined therapy ( acei + arb ) versus monotherapy . assessing the activity of cathepsin b in 24-hour urine has shown a downward trend in the values of individual treatment groups compared to diabetic rats which are not treated . the values of cathepsin b in the plasma of rats in all groups studied were similar and showed no tendency . recently it is proved that the process of autophagy and activity of cathepsins are strictly linked to the process of inflammation .
hyperglycemia results in the activation of tissue angiotensin ii . 
 angiotensin ii stimulates the synthesis of ecm proteins and causes a decrease activity of proteolytic enzymes . 
 the aim of this study was to assess the impact of multilevel blocking of the raas , cathepsin b activity , and fibronectin accumulation in the glomerular in the rats diabetes model . 
 sixty male wistar rats were initially included . 
 diabetes was induced by intravenous administration of streptozotocin . 
 the animals were randomized to six groups of ten rats in group . 
 rats in the four groups were treated with inhibitors of the raas : enalapril ( en ) , losartan ( los ) , enalapril plus losartan ( en + los ) , and spironolactone ( spir ) ; another group received dihydralazine ( dih ) and the diabetic rats ( dm ) did not receive any drug . 
 after six weeks , we evaluated blood pressure , 24  h urine collection , and blood for biochemical parameters and kidneys . in this study , 
 fluorometric , elisa , and immunohistochemical methods were used . 
 administration of en + los increased activity of cathepsin b in homogenates of glomeruli compared to dm . 
 losartan treatment resulted in reduction of the ratio kidney weight / body weight compared to untreated diabetic rats . 
 spir resulted in the increase activity of cathepsin b in the homogenate of glomeruli . 
 the values of cathepsin b in the plasma of rats in all studied groups were similar and showed no tendency .