diabetic kidney disease ( dkd ) can be seen in approximately 30 to 40% of both type 1 and type 2 diabetic patients . according to american diabetes association ( ada ) data , there have been 17.5 million diagnosed and 6.6 million undiagnosed diabetics in the united states , and this situation was found to be closely associated with both cardiovascular morbidity and mortality in this population . despite improvements in their chemical structure , contrast dye agents are still the third leading cause of hospital - acquired acute kidney injury . reduction in the incidence of contrast - induced nephropathy ( cin ) can lead to a decrease in morbidity , mortality ( up to 36% ) , and length of hospital stay . diabetic patients are found to be more prone to cin , which might further enhance the progression of dkd . the exact pathophysiologic mechanisms of cin are not fully understood ; however , hypoxia and oxidative stress ( oxs ) constitute the main responsible pathways of this situation , especially in dkd . other important molecules that are closely associated with dkd and cin are inflammatory cells such as leukocytes , monocytes , and macrophages - derived inflammatory cytokines , including interleukin- ( il- ) 1 , il-6 , and il-18 . recently , a new member of the il-1 family , il-33 , is presented as an alarmin molecule that invites inflammatory cells to the kidney in cisplatin - induced nephropathy . accumulating evidence suggests that il-33 is one of the most important cytokines in the pathogenesis of various disorders , including autoimmune diseases , myocardial infarction , heart failure , and allergic pulmonary diseases ; however , in the literature , the data regarding the role of il-33 in dkd and cin are scant . if a causative role of il-33 is confirmed , preventing such inflammation and/or the immediate consequences of such inflammation may represent the key target to prevent dkd . although melatonin ( mt ) is one of the attempted molecules in dkd , the role of this drug in diabetic patients with cin remains unclear . hence , we sought to determine the levels of il-33 along with other inflammatory cytokines and oxs . the second aim of the present study was to determine whether mt has a preventive effect on kidneys in diabetic rats ( drs ) with cin treatment . the study protocol was approved by the medical ethics committee of erzincan university ( mengucek gazi training and research hospital , erzincan , turkey ) . thirty male sprague - dawley rats were obtained from ataturk university medical experimental application and research center . the rats were housed in a standard environment with a room temperature of 22  2c , humidity level of 4060% , and 12/12  h of light - dark cycle . before starting the experimental procedure , after completion of the acclimation period , the rats were randomly allocated into five groups . the first group was comprised of healthy rats ( hrs ) , whereas the other four groups were made up of drs , diabetic rats with contrast - induced nephropathy ( cin + drs ) , melatonin - treated diabetic rats ( mtdrs ) , and melatonin - treated cin + diabetic rats ( mtcin + drs ) . all groups except the hrs received 50  mg / kg / day streptozotocin ( stz ) ( sigma , st . louis , usa ) within 0.1  m sodium citrate solution ( ph : 4.50 ) . daily blood sugar measurements were performed through tail - end puncture using a glucomax ultra td 4227 ( germany ) blood sugar measurement device . in all stz - administered groups , the blood sugar level was > 400  mg / dl at the end of the 5th day . drs did not receive any other treatment , whereas cin + drs and mtcin + drs were given 1.5  mg / kg of intravenous radiocontrast dye ( omnipaque , amersham , ireland ) on the 35th day . in addition , mtdrs and mtcin + drs were given 20  mg / kg / day of intraperitoneal injection of mt ( sigma , st . louis , usa ) starting on the 28th day for seven days as described previously by gazi et al . . all rats were sacrificed under thiopental sodium ( 5  mg / kg ) anesthesia on the 36th day using blood evacuation via cardiac puncture and cervical dislocation . each kidney was fixed in 10% formalin solution for 4855  h , dehydrated in a graded alcohol series , embedded in paraffin wax , and serially sectioned using a microtome ( leica rm2125rt ) . for light microscope histological examination , thin sections of 5  m thickness were taken from the same paraffin blocks . slides were covered , and photographs were taken using a light microscope with a camera attachment ( nikon eclipse e600 , japan ) . for electron microscopy , kidneys were fixed in buffered 3% glutaraldehyde in 0.1  m phosphate solution , postfixed in 1% osmium tetroxide , dehydrated in a graded acetone series , and transferred to propylene oxide . sections were cut , using an ultratome ( nova lkb bromma , sweden ) , into 7080  nm thin sections for histological evaluation at the ultrastructural level . after staining in 2% uranyl acetate and 0.4% lead citrate , sections were examined using a transmission electron microscope ( jeol 100 sx , tokyo , japan ) . also , semithin sections ( 1  m ) obtained with an ultramicrotome from the same araldite blocks were stained with toluidine blue and histopathologically examined using a light microscope . the kidneys were examined by light microscopy for histological evaluation of the following parameters : h&e and toluidine staining for inflammatory cell infiltrates , armanni - ebstein lesions , apoptotic cell death , glomerular changes and ultrastructural evaluation by electron microscopy for changes in glomerular basement membrane , podocytes and their pedicels , capillary structure , and mesangial cells . blood - containing tubes ( vacutainer sst , becton dickinson , usa ) were centrifuged for 7  min at 3,000  rpm . serum samples were placed in 2  ml eppendorf tubes ( eppendorf , germany ) and stored at 80c until the time of analysis . il-6 ( ebioscience , bms625 , usa ) , il-33 ( cusabio , csb - e14077r , china ) , and il-1 ( ebioscience , bms630 , usa ) levels were measured using elisa kits appropriate to the species in both kidney tissue and serum samples . serum blood - urea nitrogen , creatinine , glucose , sodium , potassium , and calcium levels were measured in an autoanalyzer ( au 5800 , beckman coulter , japan ) using original kits . the level of lipid peroxidation ( lpo ) in kidney tissues was determined by estimating malondialdehyde ( mda ) using the thiobarbituric acid test . the kidney tissues were dissected , weighed , and homogenized in 10  ml of 100  g / l kcl . the homogenate ( 0.5  ml ) was added to a solution containing 0.2  ml of 80  g / l sodium lauryl sulphate , 1.5  ml of 200 g / l acetic acid , 1.5  ml of 8  g / l 2-thiobarbiturate , and 0.3  ml of distilled water . the mixture was incubated at 98c for 1  h. upon cooling , 5  ml of n - butanol  :  pyridine ( 15  :  1 ) was added . the mixture was vortexed for 1  min and centrifuged at 4,000  g for 30  min . myeloperoxidase ( mpo ) activity was measured according to the modified method of bradley et al . . the homogenized samples were frozen and thawed three times and then centrifuged at 1,500  g for 10  min at 4c . mpo activity in the supernatant was determined by adding 100  ml of the supernatant to 1.9  ml of 10  mmol / l phosphate buffer ( ph 6.0 ) and 1  ml of 1.5  mmol / l o - dianisidine hydrochloride containing 0.0005%  ( wt / vol ) hydrogen peroxide . the changes in absorbance at 450  nm of each sample were recorded on a uv - vis spectrophotometer . mpo activity in tissues was expressed as micromoles per min per milligram of tissue ( mol / min / mg tissue ) . sod estimation was based on the generation of superoxide radicals produced by xanthine and xanthine oxidase , which react with nitroblue tetrazolium ( nbt ) to form formazan . sod activity was then measured at 560  nm by the degree of inhibition of this reaction and was expressed as mmol / min / mg of tissue . decomposition of h2o2 in the presence of catalase ( cat ) was measured at 240  nm according to aebi . the cat activity was defined as the amount of enzymes required to decompose 1  mmol of h2o2 per min at 25c at ph 7.8 . the amount of total glutathione ( gsh ) in kidney tissues was measured according to the method described by sedlak and lindsay , with some modifications . the kidney tissues were homogenized in 2  ml of 50 mmtris - hcl buffer containing 20  mm edta at ph 7.5 . after adding 2  ml ethanol ( to precipitate the proteins ) , the supernatant was used to determine the gsh level using 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb ) . subsequently , the gsh level of the kidney was expressed as nmol / g of tissue . all analyses were performed with the statistica software program version 7.0 ( statsoft , tulsa , ok , usa ) . in the present study , an lpo level which is accepted as an indicator of tissue damage as well as oxs was studied . in addition , the activities of sod , cat , and myeloperoxidase ( mpx ) enzymes and gsh levels were also measured to understand how the defense mechanisms were affected in kidney tissues . kidney lpo levels of drs were significantly increased when compared with hrs ( 40.1  0.6 versus 34.7  0.6 , p < 0.05 , resp . ) ( figure 1(a ) ) . when contrast dye was administered to drs , kidney lpo levels were further increased when compared with hrs and drs ( 44.1  2.6 versus 40.1  0.6 and 34.7  0.6 , p < 0.05 for both , resp . ) ( figure 1(a ) ) . in the present study , the kidney lpo levels were significantly decreased in both mtdrs and mtcin + drs when compared to drs without mt treatment ( 37.7  1.3 and 37.2  0.3 versus 40.1  0.6 , p < 0.05 , resp . ) ( figure 1(a ) ) . however , there was no statistically significant difference between mtdrs and mtcin + drs ( 37.7  1.3 versus 37.2  0.3 , p > 0.05 , resp . ) mpx uses hydrogen peroxide and chloride ions for production of hypochlorous acid that is a defense of neutrophil against bacteria and pathogens . in this study , the kidney tissue mpx activity was heightened in drs and cin + drs when compared with hrs ( 0.0080  001 and 0.015  0.001 versus 0.003  0.0006 , p mpx levels of mtdrs and mtcin + drs were markedly decreased when compared with drs and cin + drs ( 0.0034  0.0014 and 0.012  0.001 versus 0.0083  0.001 and 0.015  0.001 , p < 0.05 for both , resp . ) ( figure 1(a ) ) . the sod enzyme forms hydrogen peroxide by using superoxide radicals for elimination of these harmful radicals . formed hydrogen peroxide is converted to water by the gsh peroxidase enzyme , which uses the gsh as a substrate . the increments or decrements of sod activity were found to be closely related to the intensity of superoxide radicals . in the present study , the kidney levels of sod activity were significantly raised in both drs and cin + drs when compared with hrs ( 1.35  0.02 and 1.44  0.02 versus 1.28  0.01 , p < 0.05 , resp . ) . kidney levels of gsh were significantly diminished in drs and cin + drs when compared with hrs ( 0.81  0.01 and 0.78  0.06 versus 097  0.04 , p < 0.05 , resp . ) . gsh levels of mtdrs and mtcin + drs were markedly increased when compared with drs and cin + drs ( 0.89 0.01 and 0.85  0.03 versus 0.81  0.01 and 0.78  0.06 , p < 0.05 for both , resp . ) . kidney tissue cat activity was increased in drs and cin + drs when compared with hrs ( 0.008  0.001 and 0.015  0.001 versus 0.003  0.0006 , p < 0.05 for both , resp . ) . however , cat activities of mtdrs and mtcin + drs significantly decreased compared with drs and cin + drs ( 81.7  3.6 and 84.6  3.8 versus 90.9  3.7 and 94.2  3.3 , p < 0.05 for all , resp . ) . kidney tissue il-33 levels as well as il-6 and il-1 levels were significantly increased in drs and cin + drs when compared with hrs ( table 1 , figure 1(b ) ) ( p < 0.05 , for all ) . il-6 and il-33 levels were found to be diminished in mtdrs and mtcin + drs when compared with drs and cin + drs ( p < 0.05 , for all ) ( table 1 , figure 1(b ) ) . in accordance with kidney tissue levels of ils , serum levels of il-33 , il-6 , and il-1 were found to be increased in drs ; moreover , contrast dye markedly boosted this rise ( table 1 ) ( p < 0.05 , for all ) . mt treatment significantly decreased serum il-33 levels in both drs and cin + drs ( p < 0.05 , for all ) ( table 1 ) . serum creatinine ( scr ) levels were observed to increase significantly in drs and cin + drs compared to hrs ( 0.35  0.05 and 0.43  0.05 versus 0.20  0.06 , resp . mt treatment significantly diminished scr in both drs and cin + drs ( 0.33  0.05 and 0.37  0.05 , resp . , p < 0.05 , for both ) . in the ultrastructural and histopathological sections of kidney tissues , mesangial cells and capillaries of the glomerulus , podocytes ( visceral epithelial cells ) and their pedicels in bowman 's capsule , and cortical and medullary tubules appeared normal ( figures 2 and 3 ) . renal tissues of the drs showed histopathological changes as demolition in some glomeruli , inflammatory cell infiltration , transparent tubules that show glucogenic vacuolization ( armanni - ebstein lesions ) , apoptotic cell death of renal tubules ( figures 2 and 3 ) and ultrastructural changes as closure of the filtration slits with atypic pedicels and basement membrane thickening ( figures 2(a)2(d ) ) , and mesangial hypertrophy in some glomeruli . in addition , electron - dense vesicle accumulation within endothelial cells was also observed ( figure 2(b ) ) . apoptosis , necrotic changes , and armanni - ebstein lesions were found to be diminished in mtdrs ( figures 4(a)4(d ) ) . also , basement membranes , pedicel , and endothelial cells generally appeared normal ( figures 2(a)2(d ) ) . however , local interstitial inflammatory cells were observed in mtdrs , although they were found to be less than drs ( figure 4 ) . in contrast , we observed less regenerative changes in mtcin + drs ( figures 2 and 4 ) . first , kidney tissue and serum levels of il-33 as well as other inflammatory cytokines and oxs markers were found to be significantly increased in drs , and further increase was also determined in contrast - applied drs . second , both kidney tissue and serum levels of oxs markers , inflammatory cytokines , and il-33 were found to be significantly diminished in mtcin + drs compared with the groups without mt treatment . third , these findings are also supported by the histopathological findings determined by electron and light microscope . this study is the first to demonstrate the possible role of il-33 in the pathogenesis of drs with cin and the preventive effect of mt in this situation . increased serum glucose levels along with chronic low grade inflammation , oxs , advanced glycation end products ( age ) , sorbitol accumulation , hexosamine , and protein kinase c ( pkc ) pathway activation are well - known features of diabetes mellitus . diabetic patients might need further investigations with contrast using methods such as computed tomography imaging for clarifying the situations that are commonly faced . contrast agents have direct toxic effects on renal tubular cells and renal hemodynamics , and based on previous studies , oxygen radicals might play a major role as the primary physiological insult [ 16 , 17 ] . infusion of contrast agents increases in osmotic load and viscosity , and thus hypoxemia of the renal medulla and renal free radical production through postischemic oxs occur . in the present study , we found that markers of oxs are increased and antioxidants are decreased in drs and drs with cin , in accordance with the current literature [ 7 , 18 , 19 ] . previous clinical studies have established an association between a rise in inflammatory markers , including il-6 , il-18 , and tnf- , and dkd . among several cytokines that may be relevant to the development of dkd , it has been demonstrated that serum il-18 levels are markedly elevated in patients with type 2 diabetes with microalbuminuria when compared to those with normoalbuminuria , while il-6 rises at a later time , when clinical macroalbuminuria is present . according to a hypothesis adopted by many authors , oxs might be the main trigger system for the initiation and progression of dkd . another novel concept is that chronic inflammation may result in local disruption of insulin signaling , leading to the initial development of both micro- and macrovascular complications of diabetes . hence , further experimental data are needed to characterize the causative role of cytokines in the development of dkd . in this regard , we hypothesized that il-33 , also named alarmin , might be a candidate as a trigger cytokine in drs with cin . il-33 is a proinflammatory cytokine that can be released from necrotic cells , binds the st2r on immune cells , and increases secretion of cytokines , with resultant inflammation [ 23 , 24 ] . for instance , balb / c mice were found to be resistant to the induction of t cell - mediated diabetes when multiple low doses of stz were administered . additionally , balb / c mice with the st2 gene deleted were also found to be more prone to stz - induced diabetes . recently , karatas et al .   showed that il-33 expression was typically located in the peritubular and intraglomerular capillaries . they also found 6-fold ( diabetic mice at the 4th month versus healthy mice , p < 0.01 ) and 5.8-fold ( diabetic mice at 6th month versus healthy mice , p < 0.01 ) increases in il-33 protein expression in western blots of whole kidney tissue , and assessment of il-33 with elisa confirmed their results ( il-33 levels 0.409  pg/g diabetic mice at the 6th month versus 0.164  pg/g healthy mice , p < 0.01 ) . these results also support our hypothesis that il-33 may participate in the initiation and progression of dkd . the exact function of il-33 in the nucleus has not been established ; however , il-33 can activate the transcription factor ap-1 as well as nuclear factor - kappa b ( nf-b ) . recently , akcay et al .   clearly demonstrated that il-33 plays a central role in the pathogenesis of cisplatin - induced acute kidney injury via invoking cd4 t lymphocyte to the kidney . the major proinflammatory effect of il-33 is to induce cd4 th2 cell responses via the st2r . the present study is also unique in that it shows an effect of a nonimmune stimulus , contrast dye , on il-33 . in this regard , our study demonstrates that il-33 is expressed in the kidneys of both drs and contrast - applied drs . inflammatory mediators , including cytokines , have important roles during the recruitment of inflammatory cells ; these drive the fibrogenic process . recent studies also demonstrated that inhibition of inflammatory cell infiltration might contribute to the control of renal tubulointerstitial fibrosis . for instance , melatonin was found to be beneficial in terms of leukocyte infiltration , via reduction in the expression of nf-b in a rat model of unilateral ureteral obstruction . in another rat model of carbon tetrachloride nephrotoxicity , interstitial mononuclear cell infiltration and fibrosis of kidney were ameliorated by melatonin treatment . in this context , according to the present study results , it is wise to consider that melatonin treatment might reduce tubulointerstitial fibrosis via il-33 related pathways . previous studies have reported that mt can prevent drug - induced nephropathy including stz and contrast dye [ 10 , 18 ] . since mt has both highly lipophilic and hydrophilic properties , it passes rapidly through all biological membranes and enters the cells and their subcellular compartments when administered intraperitoneally or intravenously . the widespread subcellular distribution of mt may allow it to reduce oxidative damage in both the lipid and aqueous environments of the cell . this is an advantage of mt over some other antioxidants , which penetrate cells more slowly . however , there has been no data regarding whether mt is preventive against both dkd and cin situations together . in our daily practice , the physicians use diagnostic tools such as computed tomography with contrast dye or do angiography when needed in patients with dkd . the risk of morbidity and mortality was found to be higher in patients with dkd when exposed to contrast dye . our study is unique in that it has demonstrated that pretreatment with mt significantly improved cin in patients with dkd . structural features of mt , including lipophilicity , hydrophilicity , and electron - richness , provide additional beneficial effects of this molecule in terms of antioxidant capacity and insulin secretion effect via the mt1 receptor . mt activates several antioxidative enzymes , including gsh peroxidase , sod , and lipid peroxidase . mt also easily crosses cellular membranes and provides on - site protection against free radical - mediated damage to biomolecules . although the exact mechanism of cin is not very well known , deleterious factors such as vasoconstriction , tubular obstruction , mitochondrial injury , and plasma membrane toxicity have been implicated . hence , mt might be a promising agent to protect renal functions in dkd and cin via its antioxidant and antihyperglycemic effect . between 1990 and 2000 , many studies have demonstrated the possible interaction between mt and the immune system [ 35 , 36 ] . mt 's immunoenhancing effect depends upon its ability to enhance the production of cytokines as well as its antiapoptotic and antioxidant action . mt has been proposed to regulate the immune system by affecting cytokine release from immunocompetent cells . additionally , mt was found to augment cd4 + lymphocytes and decrease cd8 + lymphocytes in rat submaxillary lymph nodes . mt enhances the production of il2 , ifn- , and il-6 by cultured human mononuclear cells . mt might also increase the production of il-1 , il-6 , and il-12 via activating monocytes . repetitive stimulation of t helper ( th ) cells via mt might force th cells to switch to th1 lymphocytes that enhance the release of il-2 and ifn-. these latter cytokines can particularly invite macrophages and other phagocytes to the inflammation site . in addition , mt supplementation was found to increase cd4 th production . besides the release of proinflammatory th-1 cytokines , raghavendra et al .   also demonstrated that mt might activate an anti - inflammatory th-2-like immune response . collectively , these data indicate that mt possesses important immunoenhancing properties and suggest that mt may favor both th-1 and th-2 responses . il-33 stimulation was found to be crucial for the transformation of cd4 t cells into the th-2 subgroup . we did not measure the levels of cd4 + lymphocytes before and after mt treatment . however , the beneficial effect that we show in the mtdrs and mtcin + drs might depend on a cd4 + lymphocytes - il-33-mt circle . in summary inhibition of il-33 with mt provides functional and histologic protection against cin , demonstrating that il-33 might be a candidate for mediating cin in dkd . inhibition of il-33 and oxidants with mt pretreatment offers therapeutic potential in contrast dye - applied diabetic patients .
background . inflammation and oxidative stress ( oxs ) contribute to the pathogenesis of diabetic kidney disease ( dkd ) and contrast - induced nephropathy ( cin ) . 
 patients with dkd were found to be more prone to cin . 
 interleukin-33 ( il-33 ) is a proinflammatory cytokine , but its role in dkd and cin is unknown . methods . 
 thirty male sprague - dawley rats were enrolled . 
 the first group was comprised of healthy rats ( hrs ) , whereas the other four groups were made up of diabetic rats ( drs ) , diabetic rats with contrast - induced nephropathy ( cin + drs ) , melatonin - treated diabetic rats ( mtdrs ) , and melatonin - treated cin + drs ( mtcin + drs ) . 
 all groups except the hrs received 50  mg / kg / day streptozotocin ( stz ) . 
 cin + drs were constituted by administrating 1.5  mg / kg of intravenous radiocontrast dye on the 35th day . 
 mtdrs and mtcin + drs were given 20  mg / kg / day of intraperitoneal injection of melatonin ( mt ) from the 28th day for the constitutive seven days . 
 results . 
 we observed increased il-33 in the kidney tissue following induction of cin in drs . to determine whether mt is effective in preventing cin , we administered mt in cin + drs and demonstrated that kidney tissue levels of oxs markers , inflammatory cytokines , and il-33 were significantly diminished in mtcin + drs compared with other groups without mt treatment ( p < 0.05 ) . conclusion . 
 inhibition of il-33 with mt provides therapeutic potential in dkd with cin .