acute kidney injury ( aki ) pathogenesis is complex , and promoting events may be completely different ( ischemia or toxins are major factors that precipitate in the injury ) , but similar pathways may be involved in subsequent injury responses . for this , reason to study aki models , various methods were defined for each specific situation . gentamicin derived from gram - positive bacteria called micromonospora purpurea present in soil and water having potential in treating aerobic gram - negative bacteria . accumulation of gentamicin in proximal renal tubules may cause nephrotoxicity which leads to brush border network damage . the nephrotoxicity involves renal free radical production and accumulation , consumption of antioxidant defense mechanisms , glomerular congestion , and acute tubular necrosis [ 25 ] , leading to diminished creatinine clearance and renal dysfunction . the pathological mechanisms also involve elevation of endothelin-1 , upregulation of transforming growth factor - beta ( tgf- ) , significant increase in monocyte / macrophage infiltration into the renal cortex and medulla , augmentation of oxidative stress , and apoptosis and also necrosis [ 69 ] . its primary sites of action are still considered to be the intestine , bone , and kidneys . a number of studies have shown positive therapeutic efficacy of vitamin d and analogs to reduce proteinuria [ 1113 ] . recently , a large randomized placebo - controlled clinical trial ( the vital study , n = 281 ) confirmed that paricalcitol was able to reduce albuminuria and blood pressure in patients with diabetic nephropathy who were already on renin - angiotensin system inhibitor therapy . together , these clinical data provide a strong case to argue for the use of vitamin d analogs as a complementary therapy for treatment of proteinuria . given the importance of podocytes in the regulation of glomerular filtration , it is speculated that podocytes are important antiproteinuric targets of vitamin d   although tubular effect of vitamin d is still debate . the aim was to investigate whether vitamin d might be a useful therapeutic agent for gentamicin - induced aki model in rats . up to now vitamin d related protection mechanisms on aki remain to be fully proven . given the complexity of the disease and the pleiotropic nature of the agent activity , the protective effect would be expected and be of a multifactorial nature . thirty nonuremic wistar albino male rats ( n = 30 ; weight 180220  g ) were divided into three equal groups . they were housed in polycarbonate cages under 24c room temperature with a 12-hour light / dark cycle and fed a standard laboratory diet . the three groups of rats consisted of the following : control group , 1  ml saline intramuscular ( i m ) daily ; genta group , gentamicin 100  mg / kg / day ( i m ) ; genta + vitamin d , gentamicin 100  mg / kg / day ( i m ) in addition to 1 , 25 ( oh)2d3 0.4 systolic blood pressure was measured in conscious rats by the indirect tail cuff method , using an electrosphygmomanometer and pneumatic pulse transducer ( may nibp200-a , ankara , turkey ) , and 24-hour urine was collected in metabolic cages . after 1 hour , ketamine hcl anesthesia ( 60  ml / kg body weight ) was applied , and immediately , blood samples were collected through direct cardiac puncture in sacrificed rats . semiquantitative assessment of kidneys was carried out by the same pathologist who was unaware of the groups . serum urea and creatinine levels and urine gamma glutamine transferase ( ggt ) activity were determined using commercial available kits spectrophotometrically ( biolabo reagents , maizy france ) . urine ( lipocalin-2)-neutrophil gelatinase - associated lipocalin ( ngal ) ( boster biological technology co , ltd . ) , and kidney injury molecule-1 ( kim-1 ) ( adipo bioscience , inco , usa ) levels were measured with elisa kits . urine glutathione ( gsh ) levels were determined by a commercially available kit ( cayman chemical company , ann arbor , mi , usa ) . four percent formalin was used for fixation of kidney samples which were embedded into paraffin wax . paraffin blocks were divided into sections 5  m in thickness , and hematoxylin - eosin and masson 's were trichrome used for staining . all samples were examined by the same pathologist who was unaware of which samples originated from which group . tubular degeneration , necrosis , tubule interstitial nephritis , and total histological scores were evaluated semiquantitatively from 0 to 3 . tubular degeneration ( td ) : in the cytoplasm of the proximal tubule epithelial cells , stained bodies of various sizes and vacuolization containing acidophilus were considered as td . scoring :  absence of td ; 0  mild td : small and a few focus td in immediately beneath the capsule ( 0%10 ) ; 1  moderate td : for a few focal focus td and along the tubular segment ( 10%25 ) ; 2  severe td : diffuse and significant td along the tubular segment ( % 2550 ) ; 3  very severe td : td was greater than 50% ; 4 mild td : small and a few focus td in immediately beneath the capsule ( 0%10 ) ; 1   moderate td : for a few focal focus td and along the tubular segment ( 10%25 ) ; 2   severe td : diffuse and significant td along the tubular segment ( % 2550 ) ; 3   very severe td : td was greater than 50% ; 4 tubular necrosis ( tn ) : defined as loss of epithelial cells of the nucleus , dark acidophilic cytoplasm , loss of tubular epithelial cells into tubular lumen , and acellular sections of tubules . scoring :  absence of tn ; 0  mild tn : small and a few focus tn in immediately beneath the capsule ( 0%10 ) ; 1  moderate tn : for a few focal focus tn and along the tubular segment ( 10%25 ) ; 2  severe td : diffuse and significant tn along the tubular segment ( % 2550 ) ; 3  very severe tn : tn was greater than 50% ; 4 mild tn : small and a few focus tn in immediately beneath the capsule ( 0%10 ) ; 1   moderate tn : for a few focal focus tn and along the tubular segment ( 10%25 ) ; 2   severe td : diffuse and significant tn along the tubular segment ( % 2550 ) ; 3   very severe tn : tn was greater than 50% ; 4 tubulointerstitial inflammation ( tin ) : defined as infiltration of inflammatory cells in perivascular and interstitial areas . scoring :  absence of tin ; 0  mild tin : a few pieces of infiltration concentrated on perivascular area ( 05% ) ; 1  moderate tin : usually infiltrations involved in cortical interstitial and many focal areas ( 510% ) ; 2  severe tin : diffuse and significant infiltration areas ( 1525% ) ; 3  very severe tin : tin was greater than 50% ; 4 mild tin : a few pieces of infiltration concentrated on perivascular area ( 05% ) ; 1   moderate tin : usually infiltrations involved in cortical interstitial and many focal areas ( 510% ) ; 2   severe tin : diffuse and significant infiltration areas ( 1525% ) ; 3   very severe tin : tin was greater than 50% ; 4 total histologic score ( ths ) : td/2 + tn + tin/2 , respectively . normal ths : 02  mild ths : 25  moderate ths : 58  severe th : > 8 study results are presented as mean  standard error of the mean ( sem ) . nonparametric tests ( kruskal wallis , man - whitney u test ) were performed as statistical evaluation , and p < 0.05 was considered as significant . systolic blood pressure in control group was 120  6 and decreased to 112  13  mmhg ( figure 1 ) , and urine volume increased in genta + vit d group ( 3.4  0.5  cc ) compared to control group ( 3  0.5  cc ) ( p < 0.05 ) ( figure 2 ) . in control group , urea and creatinine levels were 91  6 and 0.74  0.03 and increased to 137  6  mg / dl and 1.1  0.1  mg / dl in genta group , also 149  5  mg / dl and 1.6  0.3  mg / dl in genta + vit d , indicating acute kidney injury ( figure 3 ) . neutrophil gelatinase - associated lipocalin ( ngal ) was 49.5  7 significantly and increased to 390  143  ng / ml in genta group and decreased to 247  112  ng / ml in genta + vit d group . glutathione and gamma glutamine transferase were 0.4  0.13 and 1.3  0.35 and increased to 0.6  0.1  nmol / ml and 59  19  u / l in genta + vit d group ( figure 4 ) . kidney injury molecule 1 ( kim-1 ) level was 0.64  0.05 in control group and increased significantly in both genta and genta + vit d groups ( 4.7  0.6 and 6  0.5  ng / ml ) ( table 1 ) . histological scores of tubular degeneration ( td ) , tubular necrosis ( tn ) , tubulointerstitial nephritis ( tin ) , and total histological score ( ths ) all increased significantly in genta and genta + vit d groups compared to control group ( figures 5 and 6 ) . tin and ths scores also significantly were higher in genta + vit d group compared to genta group ( table 1 ) . gentamicin is a positively charged chemical that strongly binds to the acidic phosphoinositide components of the brush border membrane which is a negatively charged portion of the proximal tubule , and they mainly act on the cationic drug receptor , megalin , located deeply at the base of the brush border villi . the receptor - drug complex thus formed is rapidly internalized by a pinocytosis process and checked up by lysosomes , where lysosomal phospholipidosis occurs that disrupts a number of renal intracellular processes [ 16 , 17 ] . renin angiotensin system ( ras ) in the kidney is a mandatory mediator of renal injury . vitamin d hormone has a negative regulatory effect on ras by suppressing renin expression [ 18 , 19 ] . it is shown that vitamin d receptor - absent mutant mice develop more severe renal damage ( e.g. , interstitial fibrosis , increased albuminuria , and glomerulosclerosis ) than wild - type counterparts in diabetic state   or under postrenal acute kidney injury , because of enhanced activation of the ras in the kidney . in 5/6 nephrectomised rats given paricalcitol treatment attenuated tubulointerstitial and glomerular injury and decreased blood pressure and albuminuria by inhibiting the activation of the locally produced ras in the remnant kidneys . doxercalciferol had an effect on modulating fat - induced renal injury by targeting the ras and lipid metabolism . other studies proved that combination therapy with one ras inhibitor ( ace inhibitor or arb ) and one vitamin d analog ( paricalcitol or doxercalciferol ) leads to additive or synergistic therapeutic effects in blocking renal injury in experimental rat models of type 1 and type 2 diabetes mellitus [ 2427 ] . the renal protection of the combination therapy is the inhibition of the compensatory renin induction usually encountered in the use of both ras inhibitors and the vitamin d analogues . renin induction inhibition and accumulation of angiotensin ii within the kidney leads to excellent therapeutic results . the combination strategy in these studies explains why vitamin d analogs are still effective in reducing albuminuria in ckd patients who are already receiving ras inhibitors [ 29 , 30 ] . in present study , we found that sbp is decreased in genta + vitamin d group compared to control group . this indicates that the ras blocking effect of vitamin d was still strong enough even in the presence of acute kidney injury . increased urine volume in this group also may be attributed to ras blocking effect of vitamin d therapy . functionally , gentamicin - related nephrotoxicity is characterized by a decrease in glomerular filtration rate and high levels of serum creatinine and blood urea , indicating renal dysfunction [ 3137 ] . in the present study , genta - induced experimental aki model is formed and proven by an increase of these renal function tests appropriately . unfortunately , they were still higher in genta + vitamin d group than in control group . in the literature , there are increasing multifactorial mechanisms suggested as the leading cause of gentamicin nephrotoxicity . lysosomal apoptosis and phospholipidosis have been suggested to play a pivotal role in gentamicin - induced nephrotoxicity [ 3840 ] . in the past , gentamicin was shown to increase reactive oxygen species ( ros ) like superoxide anions , hydroxyl radicals and hydrogen peroxides , and reactive nitrogen species generation in the renal cortex that eventually lead to renal structural and functional deterioration [ 4144 ] . further , it is linked with marked increases in lipid peroxidation levels , nitrotyrosine formation   and protein oxidation . in our study , we demonstrated that in genta group although it did not reach statistical significance a little gsh decrease occurred . on the other hand , genta + vit d group had a statistically significant gsh increase . in the literature , gentamicin has been also shown to cause changes in the composition of lipid membranes executed by free radicals mediated lipid peroxidation . furthermore , gentamicin - administered rat kidneys are more susceptible to ros damage because of the induction of deficiency in antioxidant defense enzymes like superoxide dismutase and catalase [ 49 , 50 ] . here in our study , vitamin d might have some beneficial effect on gentamicin - induced aki by increasing gsh levels and acting as an antioxidant mechanism , and also ngal levels were not increased unlike to genta group . structurally , gentamicin - related nephrotoxicity is associated with the edema of proximal tubular cells , glomerular hypertrophy , perivascular edema , inflammation , glomerular congestion , cellular desquamation , glomerular atrophy , tubular necrosis , and tubular fibrosis [ 40 , 5157 ] . gentamicin causes macrophage infiltration and higher transforming growth factor- which may lead to progression of tin . in the present study , tin scores were significantly higher in genta group , but surprisingly in genta + vit d group , histological scores were even higher than genta group . tubular histological parameters all were increased in genta group indicating experimental aki model occurred but unfortunately all these parameters were not decreased in genta + vit d group . acut kidney injury as a result of gentamicin - induced tubular necrosis stimulates inflammatory events by recruiting intercellular adhesion molecule-1 and monocyte chemo - attractant protein-1 at the site of injury that enhance the migration of monocytes and macrophages to the site of tissue damage , ultimately leading to renal pathogenesis [ 58 , 59 ] . in present study we demonstrated that ggt levels in genta + vit d group and kim-1 levels in both genta and genta + vit d groups were increased indicating that renal tubular damage occurred in genta groups and also even using vit d did not prevent progression of injury . in the past , vitamin d was shown as an effective drug on podocytes preventing proteinuria , regulate bone remodeling , regulate cell cycles , and the renin - angiotensin system . the present study indicates that the progression of gentamicin - induced aki was not stopped by vitamin d treatment shown by histological findings although it probably has some beneficial effects on the ras system via blood pressure lowering and increase of urine volume and a promising effect on antioxidant system . as a result given the various overlapping pathways involved in aki pathogenesis , intended therapies may need to use vitamin d in addition to other therapeutical approaches to target diverse pathways in order to achieve success .
introduction . acute kidney injury ( aki ) 
 pathogenesis is complex . 
 findings of gentamicin nephrotoxicity are seen in 30% of the aki patients . 
 vitamin d has proven to be effective on renin expression , inflammatory response , oxidative stress , apoptosis , and atherosclerosis . we aimed to investigate the effect of vitamin d in an experimental rat model of gentamicin - induced aki . 
 methods . 
 thirty nonuremic wistar albino rats were divided into 3 groups : control group , 1  ml saline intramuscular ( i m ) daily ; genta group , gentamicin 100  mg / kg / day ( i m ) ; genta  +  vitamin d , gentamicin 100  mg / kg / day ( i m ) in addition to 1 , 25 ( oh)2d3 0.4 
 mcg / kg / day subcutaneously for 8 days . 
 blood pressures and 24-hour urine were measured . 
 blood urea and creatinine levels and urine tubular injury markers were measured . 
 renal histology was semiquantitatively assessed . 
 results . 
 urea , creatinine and urine neutrophil gelatinase - associated lipocalin , and kidney injury molecule-1 were all increased in genta group indicating aki model . 
 systolic blood pressure decreased , but urine volume and glutathione increased in genta  +  vit d group compared to control group . 
 histological scores indicating tubular injury increased in genta and genta  +  vit d groups . conclusions . 
 vitamin d does not seem to be effective on histological findings although it has some beneficial effects via ras system and a promising effect on antioxidant system .