chemically , it is a hydrophilic base which exists at physiological ph as the cationic species . this drug was observed to reduce hypergly - cemia , improve glucose utilization , reduce free fatty acid utilization , gluconeogenesis , serum lipids , insulin , insulin - like growth factor 1 ( igf-1 ) , reduce body weight , and decrease metabolic immunodepression both in humans and rodents.[14 ] various studies revealed that mf is more than a simple antidiabetic agent . indeed , for an equivalent effect on glycemic control after a long time of treatment , mf was found to be obviously superior to other therapeutic measures for reducing vessel diseases and all - cause related mortality . it was suggested that insulin and mf treatments , y improving glycoxidative , inflammatory , and fibrotic renal damage markers , play a key role in the prevention of diabetic nephropathy . the role of mitochondria in programmed cell death is associated with the release of apoptotic signaling molecules . the production of reactive oxygen species ( ros ) by mitochondria also contributes to cell degradation process . mitochondria are believed to have a role in a various renal diseases , and mitochondrial dysfunction could have a major role in nephrotoxicity . gentamicin ( gm ) is probably the most the commonly used and studied of all the aminoglycosides . hence , gm - enhanced ros formation in isolated cortical mitochondria[1117 ] and ros - induced cell death were found to have a role in gm - mediated acute renal failure . apart from the superiority of mf to other antidiabetic drugs , various investigations strongly suggest that this antidiabetic agent prevented oxidative stress - induced death in several cell types through a mechanism dependent on the mitochondrial permeability transition pore ( ptp ) opening.[68131719 ] thus , mf may afford protection against gm - induced tubular injury by affecting the mitochondria through a mechanism dependent on the mitochondrial ptp opening . however , less data are present regarding the renoprotective effects of mf , and previous studies on attenuation of gm tubular toxicity by mf need to be re - tested . therefore , we aimed to test the potential properties of mf to protect the kidney from gm - induced acute renal failure . also , finding whether delayed treatment with mf exerts similar benefits on gm - induced renal toxicity in rats was the second aim of this study . mf ( metformin hexal ; germany ) was supplied as a white powder , soluble in distilled water , and freshly prepared as an aqueous solution to be given as a single daily oral dose of 100 mg / kg / day . gm treatment protocols used in the present study have been reported earlier . in a preclinical study , 50 male wistar rats with a weight range of 200250 g , purchased from jundishapur university of medical sciences , ahvaz , iran , were transferred to the animal house of shahrekord university of medical sciences , shahrekord , iran . animals were housed at an environment of controlled temperature ( 25  3c ) and humidity ( 5060% ) with a 12-hour dark  light cycle ( lights on at 7 am ) and free access to pelleted diet and tap water for 2 weeks to acclimatize to the new environment . the animal experimentation was conducted in accordance with the national institute of health guide for the careful use of laboratory animals . the animals were divided into five groups ( 10 rats each ) as follows : on the first day ( before experiment ) and the final day ( day of sacrificing ) , serum samples were obtained to measure blood urea nitrogen ( bun ) and serum creatinine ( cr ) for all the rats . bun and cr levels were measured by a colorimetric method using commercial kits on an autoanalyzer . the kidneys of each animal were dissected out , then fixed in buffered formalin for 12 h and processed for histopathologic examination . three - micrometer - thick paraffin sections were stained with hematoxylin and eosin ( h and e ) for light microscope examination using conventional protocol . slides were coded and examined by a histopathologist who was blinded to the treatment groups . all specimens were examined for six morphologic parameters including epithelial cell vacuolization , degeneration , tubular cell flattening , hyaline cast , tubular dilatation , and debris materials in tubular lumen on a semi - quantitative score from 1 to 5 , while the score of zero was assigned to the normal tissue without damage . the t - paired test was used to compare the serum bun and cr levels before and after the experiments . one - way analysis of variance ( anova ) was applied to compare the serum bun and cr levels between the groups . to compare the pathology damage score between the groups , kruskal mf ( metformin hexal ; germany ) was supplied as a white powder , soluble in distilled water , and freshly prepared as an aqueous solution to be given as a single daily oral dose of 100 mg / kg / day . in a preclinical study , 50 male wistar rats with a weight range of 200250 g , purchased from jundishapur university of medical sciences , ahvaz , iran , were transferred to the animal house of shahrekord university of medical sciences , shahrekord , iran . animals were housed at an environment of controlled temperature ( 25  3c ) and humidity ( 5060% ) with a 12-hour dark  light cycle ( lights on at 7 am ) and free access to pelleted diet and tap water for 2 weeks to acclimatize to the new environment . the animal experimentation was conducted in accordance with the national institute of health guide for the careful use of laboratory animals . the animals were divided into five groups ( 10 rats each ) as follows : on the first day ( before experiment ) and the final day ( day of sacrificing ) , serum samples were obtained to measure blood urea nitrogen ( bun ) and serum creatinine ( cr ) for all the rats . bun and cr levels were measured by a colorimetric method using commercial kits on an autoanalyzer . the kidneys of each animal were dissected out , then fixed in buffered formalin for 12 h and processed for histopathologic examination . three - micrometer - thick paraffin sections were stained with hematoxylin and eosin ( h and e ) for light microscope examination using conventional protocol . slides were coded and examined by a histopathologist who was blinded to the treatment groups . all specimens were examined for six morphologic parameters including epithelial cell vacuolization , degeneration , tubular cell flattening , hyaline cast , tubular dilatation , and debris materials in tubular lumen on a semi - quantitative score from 1 to 5 , while the score of zero was assigned to the normal tissue without damage . the t - paired test was used to compare the serum bun and cr levels before and after the experiments . one - way analysis of variance ( anova ) was applied to compare the serum bun and cr levels between the groups . to compare the pathology damage score between the groups , kruskal  wallis and mann  whitney u - test were applied . the levels of bun in groups ii , iv , and v , and the serum levels of cr in groups ii and v were increased significantly after the experiment ( p < 0.05 ) . the serum levels of bun and cr before and after the experiment in five groups of animals . group i , sham group ; group ii , positive control group treated with gentamicin ; group iii , treated with metformin ; group iv , treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days ; and group v , co - administration of metformin and gentamicin for 10 days . the symbols ( * ) and (  ) stand for significant difference before the experiment ( p < 0.05 ) and significant difference from positive control group ( p < 0.05 ) , respectively the increase in serum bun and cr levels was highly significant in group 2 versus groups 4 and 5 , therefore mf has a protective effect . the pathology damage score indicated a higher score for all gm treated groups , which was significantly different from non - gm treated groups ( p < 0.05 ) . however , post - administration of mf after 10 days of gm treatment ( group iv ) and co - administration of mf and gm for 10 days attenuated the damage score significantly ( p < 0.05 ) , when compared with group ii . as it is demonstrated in figure 2 , co - administration of gm and mf for 10 days or post - treatment with mf after 10 days of treatment with gm could reduce the damage induced by gm . the pathology damage score in five groups of animals . group i , sham group ; group ii , positive control group treated with gentamicin ; group iii , treated with metformin ; group iv , treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days ; and group v , co - administration of metformin ; and gentamicin for 10 days . the symbols ( # ) and (  ) stand for significant difference from group i ( p < 0.05 ) and group ii ( p < 0.05 ) , respectively the levels of bun in groups ii , iv , and v , and the serum levels of cr in groups ii and v were increased significantly after the experiment ( p < 0.05 ) . the serum levels of bun and cr before and after the experiment in five groups of animals . group i , sham group ; group ii , positive control group treated with gentamicin ; group iii , treated with metformin ; group iv , treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days ; and group v , co - administration of metformin and gentamicin for 10 days . the symbols ( * ) and (  ) stand for significant difference before the experiment ( p < 0.05 ) and significant difference from positive control group ( p < 0.05 ) , respectively the increase in serum bun and cr levels was highly significant in group 2 versus groups 4 and 5 , therefore mf has a protective effect . the pathology damage score indicated a higher score for all gm treated groups , which was significantly different from non - gm treated groups ( p < 0.05 ) . however , post - administration of mf after 10 days of gm treatment ( group iv ) and co - administration of mf and gm for 10 days attenuated the damage score significantly ( p < 0.05 ) , when compared with group ii . as it is demonstrated in figure 2 , co - administration of gm and mf for 10 days or post - treatment with mf after 10 days of treatment with gm could reduce the damage induced by gm . the pathology damage score in five groups of animals . group i , sham group ; group ii , positive control group treated with gentamicin ; group iii , treated with metformin ; group iv , treated with gentamicin for 10 days and post - treatment with metformin for the next 10 days ; and group v , co - administration of metformin ; and gentamicin for 10 days . the symbols ( # ) and (  ) stand for significant difference from group i ( p < 0.05 ) and group ii ( p < 0.05 ) , respectively in this study , we found that mf could ameliorate gm - induced kidney tissue toxicity . we also found that mf was still effective when the drug was administered after progression of tubular damage by gm . mf is used for the treatment of diabetes as a sugar - lowering agent . in addition , mf is recommended as the drug of first choice in type 2 diabetes.[1619 ] there are potential survival benefits associated with the use of mf , in addition to the benefits in respect to cardiovascular outcomes and metabolic parameters as suggested by recent studies.[25 ] mf exerts its metabolic activity through the induction of the adenosine monophosphate ( amp)-activated protein kinase ( ampk ) pathway which acts as a sensor detecting variations of intracellular energy levels . it was suggested that hypoxia - induced hypoxia - inducible factor ( hif)-1 accumulation in diabetic nephropathy could be suppressed by gm through the repression of oxygen consumption.[92327 ] hif-1 plays an important role in chronic hypoxia and tubulointerstitial fibrosis , which are presently considered to be the common pathways for various progressive kidney diseases , including diabetic nephropathy.[92327 ] alterations in epithelial cell polarity and in the subcellular distributions of epithelial ion transport proteins are key molecular consequences of acute kidney injury and intracellular energy depletion . ampk , a cellular energy sensor , is rapidly activated in response to renal ischemia , and ampk activity may influence the maintenance or recovery of epithelial cell organization in mammalian renal epithelial cells subjected to energy depletion . at a molecular level , energy deprivation causes key energy - dependent membrane proteins to become displaced and dysfunctional . specially , in the proximal tubule , the na  k - atpase is internalized from the basolateral membrane , disrupting the cell 's capacity to maintain normal transepithelial sodium transport . preservation of a polarized plasma membrane distribution of na  k - atpase in renal epithelia is essential for the maintenance of both solute reabsorption and volume homeostasis . atp depletion also perturbs the distribution of tight junction proteins , further disrupting epithelial cell polarity and organization and leading to back leak of extracellular fluid into the urinary space . mf treatment increases detectable p - ampk in a dose - dependent manner , and mf - induced ampk activation occurs in proximal tubules as well as in distal segments . mitochondria represents one of the major cellular sources of ros generation and mitochondrial toxicity can also be mediated by ros . indeed , after gm treatment , intracellular ros content can reach a toxic level , thus causing cell death and malfunctioning of the organ.[113033 ] literature review shows that the renal protective effects of mf in diabetic nephropathy have not been fully evaluated . meanwhile , in accordance with our results , prevention of histologic changes due to gm toxicity by mf was shown by morales et al . they found that control and mf - treated rats showed no structural alterations in renal tissues , while massive and diffuse cell necrosis was observed in the proximal tubules of kidneys from rats injected with gm . they could show that tubular lumen was frequently filled with hyaline casts or heterogeneous cellular debris , while in rats treated with gm and mf , most of the proximal tubules were saved and manifestations of necrosis were observed in less than 10% of cells . based on the above in vivo results , it was concluded that mf ameliorates gm - induced acute renal failure . also , the present study shows that delayed treatment of mf for gm - induced acute renal failure was still effective as when mf was given in combination with the gm . hence , mf is a nephroprotective drug to prevent or attenuate the tubular damage caused by gm or other nephrotoxic agents which act through the same mechanisms as this aminoglycoside .
background : the aim of this study was to test the potential properties of metformin ( mf ) to protect the kidney from gentamicin ( gm)-induced renal toxicity.materials and methods : in this preclinical study , 50 male wistar rats were randomly divided into five groups of 10 rats in each . in the first group ( group i ) , they were kept in the same condition as others without receiving drugs for 10 days . in group 
 ii , the rats were injected intraperitoneally with 100 mg / kg / day of gm for 10 consecutive days . 
 group iii rats received 100 mg / kg / day mf orally for 10 days . in group iv , 
 the rats received gm ( 100 mg / kg ; intraperitoneally ) for 10 days and 100 mg / kg / day mf orally for the next 10 days . in the last group ( group v ) , 
 the rats received a combination of gm 100 mg / kg / day intraperitoneally and mf 100 mg / kg / day orally for 10 days simultaneously . 
 serum blood urea nitrogen ( bun ) and creatinine ( cr ) values were measured and renal tissues of the animals were processed for light microscope examination.results:the levels of bun in groups ii , iv , and v , and also the serum level of cr in groups ii and v were increased significantly after the experiment . 
 furthermore , post - treatment with mf or co - treatment with mf could prevent the elevation of serum bun and cr induced by gm and also attenuates the damage score ( p < 0.05).conclusions : mf may prevent or ameliorate gm - induced acute renal failure , and therefore it might be beneficial in patients under treatment with this medicine .