| Background: Gentamicin is aminoglycoside antibiotic which hasbeen widely used for the clinical treatment of Gram-negative bacterialinfection. However, the major limitations in clinical use are partially dueto nephrotoxicity and ototoxicity. Among several side effects induced bygentamicin, nephrotoxicity has been reported widely. Although a largenumber of studies have been focused on the side effects induced bygentamicin, the mechanism of nephrotoxicity induced by gentamicin isnot completely known and remains to be studied further. It has beendemonstrated that gentamicin administration increases renal cortical lipidperoxidation, nitric oxide generation and mitochondria hydrogen peroxideproduction. Recent studies have postulated that reactive oxygen speciesparticularly superoxide anion radical are involved in this process.Prohibitin is a ubiquitous and highly conserved protein and has beenshown to play different functions by its special molecular chaperoneprotein. A large number of reports have been shown that prohibitin caninhibit tumor cell proliferation and apoptosis through regulating genetranscription and maintaining mitochondrial protein stability. In addition,studies have demonstrated that prohibitin plays an important role ininflammatory effects and myocardial ischemia reperfusion by protectedthe mitochondria from oxidative stress-induced injury. Recent studieshave indicated that prohibitin protein is involved in tubulointerstitialfibrosis and negatively correlated with the degrees of tubulointerstitiallesions, which suggested that prohibitin protein might be a potentialtherapeutic target for renal injury. However, there are few reports about its role in nephrotoxicity induced by gentamicin. Based on this, weevaluated the potential role of prohibitin in Gentamicin-inducednephrotoxicity in rats.Sodium butyrate, a potent inhibitor of histone deacetylase, caninduce a nonspecific increase in gene expression. A large number ofreports have been demonstrated that it could provide antidepressant-likeeffect, improve memory function, protect against myocardial ischemiaand reperfusion injury and attenuate acute lung injury and reduceinflammatory cytokines. Recent studies indicated that enhancement ofhistone acetylation induced by sodium butyrate is correlated with increaseof the activities of several antioxidant enzymes, which has been revealedto result in gentamicin-induced nephrotoxicity. Moreover, it has beenreported that sodium butyrate decreases the activation of NF-κB in thekidney of rats subjected to contrast-induced nephropathy. However, theinformation on the effects of sodium butyrate on renal injury induced bygentamicin is lack. Thus, it is necessary to examine our hypothesis thatsodium butyrate may protect against gentamicin-induced renal injury.Objective：The major purpose in our study was to investigate theeffects of sodium butyrate (NaBu) on renal injury induced by gentamicinin rats and determine further whether the protective effect is mediated bymodulation of prohibitin protein expression.Method：Gentamicin was injected intraperitoneally (150mg/kg,100mg/kg,50mg/kg,25mg/kg,) once daily for8days to induce renal injury.To observe the effects of acute and chronic treatment of sodium butyrateon gentamicin-induced renal injury, rats received different doses ofsodium butyrate (50,100,200mg/kg, i.p.)30min prior to the gentamicin(100mg/kg, i.p.) injection on the last day and for8consecutive days,respectively. Blood and urine were collected24h after the last gentamicininjection. Then animals were sacrificed by anesthesia and kidneys wereharvested and rinsed in saline for further serum biochemical andhistopathological analysis. In addition, for each group, food intake,drinking water and body weight were recorded daily, respectively. Theaverage and accumulative consumption amounts of food and drinkingwater of each rat were calculated.Blood was collected24h after the last gentamicin injection fromdifferent treatment groups. Serum urea nitrogen and creatinine weremeasured by an automatic Clinical Analyzer.Urine was collected under cooled conditions and stored untilmeasurement of the urinary NGAL and Kim-1. Urinary NGAL andKim-1were determined using the enzyme-linked immunosorbent assay(ELISA).Kidneys were collected24h after the last gentamicin injection andfixed in10%formalin solution before being embedded in paraffin forsections (3μ mole thick). Sections were stained with hematoxylin andeosin (H&E). A semi-quantitative evaluation of renal tissues wasaccomplished by scoring the degree of severity. Tubular lesions andhyperplasia were observed under bright field using a Carl Zeiss Axioscopmicroscope.Immunohistochemistry was performed as follows. Kidneys werecollected and processed by a standard pathology procedure. The tissuesections were deparaffinized by placing the slides in an oven at60°C for10min and then rinsed twice in xylene for10min each. The slides werethen hydrated in a graded ethanol series and then finally indouble-distilled water for10min. Next, endogenous peroxidase activitywas blocked using3%hydrogen peroxide solution (for10min at room temperature). Subsequently, the sections were incubated sequentially withthe primary antibody, rabbit antimouse prohibitin (Sigma;1:500dilution,overnight at4°C). After incubation, the tissue sections were rinsed withTBS containing0.05%Tween20twice and incubated with secondaryantibody (poly-HRP anti-rabbit IgG,1:2000dilution,1h at4°C.). Thetissue sections were observed for brown color formation under brightfield.Kidney was homogenized and then centrifuged as described(Manikandan et al.,2011). The concentration of protein extracts wasdetermined using BCA kit (Pierce, Rockford, IL, USA) and30μg proteinlysates were separated on12%SDA-PAGE followed by being transferredto nitrocellular membrane. After mounting with TBST (Tris-bufferedsaline+0.05%Tween) containing non-fat dry milk (5%w/v) for2h, themembrane was incubated with the primary antibody, rabbit antimouseprohibitin (Sigma;1:500dilution, overnight at4°C). After washing withTBST3times, the membrane was incubated with secondary antibody(poly-HRP anti-rabbit IgG,1:2000dilution,1h at4°C.). After washing3times with TBST, the blots were developed using a chemiluminescencedetection kit. The image was scanned using a gel image analysis system,and the grey scale values of the experimental points were compared withthat of the internal reference β-actin, and statistically were analyzed withKodak Software.Result：The present study demonstrated that gentamicin treatmentfor8consecutive days significantly increased in the levels of blood ureanitrogen (BUN), creatinine (CRE), kidney injury molecule (KIM-1) andneutrophil gelatinase-associated lipocalin (NGAL) which indicated renalinjury induced by gentamicin. In addition, chronic treatment with NaBusignificantly attenuated gentamicin-induced renal injury. Our results showed that it was not acute treatment of sodium butyrate on the last day,but systematic administration of sodium butyrate for8consecutive dayssignificantly decreased the gentamicin-induced increase of blood ureanitrogen and creatinine when compared to gentamicin/saline treatmentgroup. Urinary NGAL and Kim-1levels in rat with gentamicin-inducedrenal injury were attenuated after chronic treatment of sodium butyrate.Moreover, chronic treatment of sodium butyrate improves the bodyweight, food and water intake in rats with gentamicin-induced renalinjury.To study whether the protective effects of sodium butyrate ongentamicin-induced renal injury could be related to the activation ofprohibitin protein, the expression of prohibitin in the kidney of rats incontrol and experimental group is examined by immunohistochemistryand western blot analysis. The immunohistochemistry and western blotanalysis revealed that sodium butyrate prevented gentamicin-inducedrenal injury by increasing prohibitin expression.Conclusion：We have confirmed that sodium butyrate plays aprotective role against renal injury induced by gentamicin exposure.According to our biochemical findings, which were supported by westernblot and immunohistochemistry analysis, chronic administration ofsodium butyrate reduced gentamicin-induced renal injury. Therefore, wepropose that sodium butyrate might be a potential candidate agent againstgentamicin-induced renal injury. |
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